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An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity Shimon Ben-Shabat a , Ester Fride a , Tzviel Sheskin a , Tsippy Tamiri b , Man-Hee Rhee c , Zvi Vogel c , Tiziana Bisogno d , Luciano De Petrocellis e , Vincenzo Di Marzo d , Raphael Mechoulam a,) a Department of Natural Products, The Hebrew UniÕersity Medical Faculty, Ein Kerem Campus, Jerusalem 91120, Israel b DiÕision of Identification and Forensic Science, Israel Police Headquarters, Jerusalem, Israel c Department of Neurobiology, The Weizmann Institute of Science, RehoÕot 76100, Israel d Istituto per la Chimica di Molecole di Interesse Biologico, CNR, Via Toiano, 6, 80072, Arco Felice, Naples, Italy e Istituto Di Cibernetica, CNR, Via Toiano, 6, 80072, Arco Felice, Naples, Italy Received 29 January 1998; revised 11 May 1998; accepted 15 May 1998 Abstract 2-Arachidonoyl-glycerol 2-Ara-Gl has been isolated from various tissues and identified as an endogenous ligand for both Ž . cannabinoid receptors, CB and CB .

 

Here we report that in spleen, as in brain and gut, 2-Ara-Gl is accompanied by several 1 2 2-acyl-glycerol esters, two major ones being 2-linoleoyl-glycerol 2-Lino-Gl and 2-palmitoyl-glycerol 2-Palm-Gl . These two esters do Ž. Ž . not bind to the cannabinoid receptors, nor do they inhibit adenylyl cyclase via either CB or CB ; however, they significantly potentiate 1 2 the apparent binding of 2-Ara-Gl and its apparent capacity to inhibit adenylyl cyclase. Together these esters also significantly potentiate 2-Ara-Gl inhibition of motor behavior, immobility on a ring, analgesia on a hot plate and hypothermia caused by 2-Ara-Gl in mice. 2-Lino-Gl, but not 2-Palm-Gl, significantly inhibits the inactivation of 2-Ara-Gl by neuronal and basophilic cells. These data indicate that the biological activity of 2-Ara-Gl can be increased by related, endogenous 2-acyl-glycerols, which alone show no significant activity in any of the tests employed. This effect ‘entourage effect’ may represent a novel route for molecular regulation of endogenous Ž . cannabinoid activity. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Anandamide; Endocannabinoid; Cannabinoid receptor; 2-Arachidonoyl-glycerol inactivation 1. Introduction We have identified anandamide arachidonoy- Ž lethanolamide in porcine brain and 2-arachidonoyl- . glycerol 2-Ara-Gl in canine gut Devane et al., 1992b; Ž. Ž Mechoulam et al., 1995 . Both ligands bind to the CB and . 1 CB cannabinoid receptors and exhibit cannabinoid-type 2 activities. Later Sugiura et al. 1995 and Stella et al. Ž . Ž . 1997 reported the presence of 2-Ara-Gl in brain, while Bisogno et al. 1997b found that 2-Ara-Gl is biosynthe- Ž . ) Corresponding author. Tel.: q972-2-6758634; Fax: q972-2- 6410740; E-mail: mechou@yam-suff.cc.huji.ac.il sized and released in a Ca2q-dependent fashion by mouse neuroblastoma cells. 2-Ara-Gl inhibits forskolin-stimulated adenylyl cyclase in mouse spleen cells Mechoulam et al., Ž 1995 and rat neurons Stella et al., 1997 . In mice, .Ž. 2-Ara-Gl is active in a tetrad of assays, which together have been shown to be highly predictive of cannabinoidinduced activity Mechoulam et al., 1995; Fride and Me- Ž choulam, 1993; Martin et al., 1991 .. In view of the identification of CB cannabinoid recep- 2 tor in immune cells Munro et al., 1993 and of the Ž . inhibition by 2-Ara-Gl of T- and B-cell proliferation Lee Ž et al., 1995 , we decided to look for the presence of active . endogenous ligands in the spleen, an organ with well established immune functions, using a fractionation guided by a binding assay. 0014-2999r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S0014-2999 98 00392-6 Ž . 24 S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) 2. Materials and methods 2.1. Isolation of fatty acid esters of glycerol Mouse spleen tissue 280 mg from three mice was Ž . homogenized in chloroformrmethanol 2:1 v Ž . rv with a Kontex glass tissue grinder. The homogenate was filtered via a sintered glass and the residue reextracted. The chloroform layer, which contained the extracted lipids, was partitioned against 0.8% aqueous NaCl, dried under a stream of nitrogen and redissolved in 1 ml of chloroform. Ten volumes of acetone were added to the solution and after 20 min at Ž . y208C the mixture was centrifuged at 3500=g for 10 min. The supernatant was evaporated to dryness and the residue was dissolved in 1 ml chloroform, 100 ml was spotted on a thin layer chromatography TLC Ž . plate silica gel 60, Merck and developed in hexane Ž . rdiethyletherracetoneracetic acid 40:20:30:1 v Ž . rvrvrv . The TLC plate was divided into five bands, which were eluted from the TLC plate with a solvent mixture of chloroformrmethanol 9:1 v Ž . rv . Activity was assayed by inhibition of binding of the high affinity cannabinoid w 3 ligand H HU-243 to rat brain synaptosomal membranes x Ž .Ž . CB cannabinoid receptors Devane et al., 1992a , and to 1 membranes of Chinese hamster ovary CHO cells tran- Ž . siently transfected with CB cannabinoid receptor see Ž 2 below . The only TLC band that showed cannabinoid . binding activity had an R of 0.5. This sample was f analysed by gas chromatography–mass spectrometry GC– Ž MS for the presence of anandamide and acylglycerols . Ž . Devane et al., 1992b; Mechoulam et al., 1995 . 2.2. Gas chromatography–mass spectrometry GC–MS ( ) GC–MS analyses were carried out with a Finnigan TSQ 700 mass spectrometer coupled to a Varian 3400 gas chromatograph. Chromatographic separation were performed on a cross-linked methyl silicone DB-5MS capil- Ž . lary column length, 15 m; i.d. 0.25 mm; film thickness, Ž 0.25 mm ; column temperature was programmed to in- . crease from 150 to 2808C at a rate of 258Crmin following a 5 min holding time at 2808C. Helium was used as the carrier gas at a head pressure of 6 psi. Injection temperature was 2208C in the splitless mode. Mass spectra were obtained in electron impact EI mode with electron energy Ž . of 70 eV. Ion source and transfer-line temperatures were 1508C and 2808C, respectively. The quadrupole was scanned in the mrz range 50–550 at 1 scanrs. Silylation of the endogenous compounds was made by adding bis-trimethylsilyl TMS trifluoroacetamide to the dry sample. Ž . After 30 min of incubation at room temperature, the silylated material was injected into the GC–MS. For quantitative analysis, an internal standard, 1 3 - Ž . eicosanoyl-glycerol 25 nmol Nu-Check Prep, Elysian, Ž .Ž MN , was added during homogenization and the same . procedure described above was followed. The isolated fraction was reacted with 50 ml of bis-TMS trifluoroacetamide 30 min at room temperature , dried under nitrogen, Ž . and resuspended in 50 ml chloroform. The sample was analysed by GC–MS in a Hewlett-Packard G 1800 A GCD system. The capillary column HP5MS, 30 m Ž =0.25 mm i.d. was temperature programmed from 150–280 . 8C at 508Crmin, using selective ion monitoring. The calibration curve area ratio vs. weight ratio of 2-Ara-Gl to 1 3 eico- Ž Ž. sanoyl-glycerol was linear. . [ 3 2.3. Binding of H HU-2432 to CB or CB cannabinoid ] 1 2 receptors The monoacylglycerols 2-Ara-Gl, 2-linoleoyl-glycerol Ž. Ž . 2-Lino-Gl and 2-palmitoyl-glycerol 2-Palm-Gl were asw 3 sayed for competition in binding H HU-243 to the CB - x 2 receptor in CHO cells in a centrifugation based ligand binding assay which has been described in detail in previous publications Devane et al., 1992a,b; Rhee et al., Ž 1997 . To measure the binding to CB and CB receptors . 1 2 in African green monkey kidney cells COS-7 cells , the Ž . latter were transiently transfected using DEAE-dextran Ž method with plasmids 5 .Ž . mgr100 mm dish encoding CB or CB . Two days later the cells were washed with 1 2 phosphate-buffered saline, scraped, pelleted, and stored at y808C. Cell pellets were homogenized in 50 mM Tris– HCl, 5 mM MgCl , and 2.5 mM EDTA, pH 7.4, and 50 2 w 3 mg protein aliquots were assayed for binding of H HU- x 243 in this buffer supplemented with 10 mM CaCl . The 2 w 3 final concentration of H HU-243 in the binding mixture x was 300 pM. For more detailed information on the assay and the calculation of the K values see Devane et al. i Ž . Ž. 1992a and Rhee et al. 1997 . 2.4. Adenylyl cyclase assay In brief, COS-7 cells in 100 mm dishes were cotransfected with adenylyl cyclase type V and either CB or CB 1 2 cDNAs. The cells were replated in 24-well plates, labelled w 3 with H adenine for 2 h and the adenylyl cyclase was x stimulated with 1 mM forskolin in the presence of the Ž tested compounds for 10 min at 37 . 8C. Incubation was stopped by the addition of perchloric acid followed by w 3 neutralization and the amount of H cAMP formed was x assayed by a two column separation procedure. For more details see Rhee et al. 1997 and Bayewitch et al. 1996 . Ž. Ž. 2.5. Pharmacological tests in mice Female mice C57BL Ž . r6, 2–3 months old were injected i.p. with 2-Ara-Gl, 2-Lino-Gl and 2-Palm-Gl 1, 10 Ž and 5 mgrkg, respectively , in a vehicle consisting of . ethanol:emulphor:saline 1:1:18 , with each drug alone, or Ž . together. We employed a tetrad of tests commonly used to demonstrate cannabinoid activity Martin et al., 1991; Fride Ž and Mechoulam, 1993 . These tests include ambulation in . S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) 25 an open field for 8 min; immobility on a ring of 5.5 cm diameter; change in rectal temperature measured with a telethermometer Yellow Springs Instrument Yellow Ž Springs, OH and analgesia on a hot plate Columbus . Ž Instruments, Columbus, OH . The effects in mice were . observed 15 min after injections. Data from the tetrad of observations were analysed by one-way analyses of variance with Newman–Keuls multiple comparison tests. 2.6. Effect of monoacylglycerols on the hydrolysisr inactiÕation of 2-Ara-Gl Experiments on 2-Ara-Gl and 1-Ara-Gl hydrolysis were performed with both particulate fractions and intact cells. Particulate fractions 10 000 Ž . =g from N18TG2 and RBL2H3 cells were prepared as previously described Bisogno Ž et al., 1997a,b . These fractions 0.05–0.1 mg total pro- . Ž teins were incubated for 30 min at 37 . 8C in 0.25 ml of a 50 w 3 mM Tris–HCl buffer, pH 7.4 with 10 000 cpm H 2-Ara- x w 3 Gl or H 1-Ara-Gl 8.0 x Ž . mM , and with increasing concentrations 0, 10, 50, 100 and Ž . ror 250 mM of various monoacylglycerols or with unlabelled 1- or 2-Ara-Gl 100 Ž mM . In a separate set of experiments, the effect of a . mixture of 2-Palm-Gl and 2-Lino-Gl on the hydrolysis of w 3 x w3 x w3 H 2-Ara-Gl or H 1-Ara-Gl was also studied. H -Ara- x w 3 chidonic acid produced from the hydrolysis of H 2-Ara- x w 3 Gl or H 1-Ara-Gl was quantified by TLC carried out as x described previously Bisogno et al., 1997b . For experi- Ž . ments with intact cells Di Marzo et al., 1994 , confluent Ž . RBL-2H3 or N18TG2 cells in 6-well dishes were washed three times with serum-free minimal essential medium and then incubated for increasing periods of time at 378C with 0.5 ml serum-free minimal essential medium containing w 3 x w3 10 000 cpm of H 2- or H 1-Ara-Gl 4.0 x Ž . mM with or without 1- or 2-Lino-Gl 100 Ž. Ž mM or 1- or 2-Palm-Gl 100 mM . In a separate set of experiments, RBL-243 cells were . incubated for 30 min in the presence of a mixture of 2-Palm-Gl and 2-Lino-Gl in a 5:12:1 molar ratio with Ž w 3 H 2-Ara-Gl . After the incubation, the media were ex- x . tracted with chloroformrmethanol 2:1 vŽ . rv and the organic phase was analysed as described previously Bi- Ž sogno et al., 1997b . After three washings with 2 ml of . serum minimal essential medium containing 1% bovine serum albumin, cells from each well were extracted by sonication with chloroformrmethanolr50 mM Tris –HCl buffer pH 7.4, 2:1:1 vŽ . rvrv . The organic phase w 3 was then analysed for the presence of H 2-Ara-Gl x w 3 and H -arachidonic acid as described above. x 3. Results 3.1. Isolation and identification Mouse spleen was extracted with methanolrchloroform Ž . 1:2 and the extract was chromatographed to yield a fraction that was found to bind to both CB and CB 1 2 cannabinoid receptors. The active fraction was silylated with bis-TMS trifluoroacetamide and the resulting mixture was analysed by GC–MS. Several of the single peaks observed before silylation were transformed into pairs of compounds which, on the basis of our previous work, are the silylated derivatives of 1- and 2- monoacylglycerols Ž .Ž . Mechoulam et al., 1995 Fig. 1 . The 1-acyl-glycerols Fig. 1. Capillary gas chromatography analysis of the endogenous compounds after silylation with bis-TMS trifluoroacetamide performed on DB-5MS capillary column 15 m Ž . Ž. Ž. Ž. =0.25 mm . Peak identities: tetramethylsilyl ethers of the glycerol esters of a myristic acid; b palmitic acid; c linoleic acid; Ž . d arachidonic acid. 26 S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) derivatives are formed by rearrangement from the 2-acylglycerol derivatives Mechoulam et al., 1995 . The molec- Ž . ular weights of the compounds in each pair, as determined by MS, were identical, being 450, 474, 498 and 522. As monoacylglycerols form bis-TMS ethers, the molecular weights recorded represent the molecular weight of the original monoacylglycerols plus 144 units as each bis-TMS Ž moiety has a molecular weight of 72 . On silylation we . thus obtained the bis-TMS ethers of the glycerol esters of myristic 14:0 , palmitic 16:0 , linoleic 18:2, Ž. Ž. Ž . ny6 , and arachidonic 20:4, Ž. Ž ny6 acids, respectively approximate ratio of peaks 1:5–6:10–12:1 Fig. 1 . The EI spectra of .Ž . the endogenous bis-silylated compounds were compared with those of the corresponding synthetic bis-TMS-monoacylglycerols and were found to be identical. For a mass spectrometric figure comparing endogenous 2-Ara-Gl with synthetic 2-Ara-Gl as their bis-TMS derivatives see Me- Ž . choulam et al. 1995 . The amounts of 2-Ara-Gl, 2-Lino-Gl Ž . and 2-Palm-Gl see Section 2 were 5.0 Ž . "1.3, 60.0"6.5 and 23.0"6.5 nmolrg wet weight of spleen tissue, respectively. 3.2. Binding to CB and CB 1 2 In view of the high levels of 2-Lino-Gl and of 2-Palm-Gl present together with 2-Ara-Gl, as recorded here in spleen and previously in gut Mechoulam et al., 1995 and brain Ž . Ž . Sugiura et al., 1995 , we carried out a series of experiments aimed at assessing the possible biological role of these two esters. In binding assays, based on competition w 3 with the binding of H HU-243 using membranes of CHO x cells transfected with CB , 2-Palm-Gl and 2-Lino-Gl were 2 found to be inactive up to 20 mM, while 2-Ara-Gl, in the same system, had a K of 1640"260 nM. These three i esters were then mixed in molar ratio of 5:12:1, for Ž 2-Palm-Gl, 2-Lino-Gl and 2-Ara-Gl, respectively, as present in the spleen and the mixture assayed for its effects . on cannabinoid binding. The mixture competes with w 3 H HU-243 for CB with an apparent x 2 i K value of 273" 22 nM see Fig. 2a calculated for the concentration of Ž . 2-Ara-Gl in the mixture. The apparent K values for i 2-Ara-Gl in mixtures with each of the two esters were 476"23 nM for 2-Ara-Gl with 2-Lino-Gl ratio 1:12 , and Ž . 339"5 nM for 2-Ara-Gl with 2-Palm-Gl ratio 1:5 . The Ž . potentiation by 2-Palm-Gl was thus significantly higher than that induced by 2-Lino-Gl Ž . P-0.01 despite its lower concentration in the reaction mixture. Further binding experiments with various ratios of the ‘entourage compounds’ to 2-Ara-Gl, indicated that there is a range of concentrations in which 2-Palm-Gl and 2-Lino-Gl can potentiate 2-Ara-Gl binding. Thus, we found apparent Ki values close to those reported above, with ratios of 2-AraGl to 2-Lino-Gl varying from 1:5 to 1:12 and of 2-Ara-Gl to 2-Palm-Gl varying from 1:2 to 1:5 data not shown . Ž . The K values, or apparent K values, resulting from all i i combinations of compounds 2-Ara-Gl alone; 2-Ara-Gl Ž q 2-Lino-Gl; 2-Ara-Glq2-Palm-Gl; all three were first . compared with analysis of variance. Individual post hoc comparisons were subsequently made using Newman–Keuls multiple comparison test. Using COS-7 cells transfected with CB , 2-Ara-Gl had 2 a Ki of 145"39 nM. A mixture of 2-Palm, 2-Lino-Gl and 2-Ara-Gl applied in the same molar ratio as described Ž . w 3 above was shown to compete with H HU-243 with an x apparent K value of 58"14 nM calculated for the Ž i concentration of 2-Ara-Gl . The differences in . K values i observed for the two cell lines are probably due to the different methodologies used in the assays. Ž . w 3 Fig. 2. Binding of 2-Ara-Gl to CB and CB cannabinoid receptors. a Binding of H HU-243 to membranes of CB cannabinoid receptor transfected x 1 2 2 CHO cells was assayed in the presence of the indicated concentrations of 2-Ara-Gl and in the absence or the presence of the inactive acyl-glycerols Ž . Ž. w 3 2-Lino-Gl and 2-Palm-Gl in a ratio of 1:12:5 . Data are the means"S.E. of three experiments performed in triplicate. b–c Binding of H HU-243 to x Ž . w 3 membranes of CB cannabinoid receptor transfected COS-7 cells was determined as in a except that the concentration of H HU-243 was raised to 300 x 1 pM and 10 mM CaCl were added to the assay mixture. When indicated, the membranes were preincubated for 10 min with 2 mM PMSF final Ž 2 concentration of PMSF in the assay 0.2 mM . Data are represented as the means . "S.E. of three experiments performed in triplicate. The PMSF treatment w 3 as well as those of 2-Lino-Gl and 2-Palm-Gl did not affect the binding of H HU-243. x S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) 27 The above mixture of 2-Lino-Gl and 2-Palm-Gl also potentiated the binding of 2-Ara-Gl to the CB receptor in 1 COS cells Fig. 2b . The apparent Ž . K value observed for i 2-Ara-Gl was shifted from 58.3"10.7 nM for 2-Ara-Gl Ž alone to 13.9 . "2.1 nM when assayed in the presence of the two other glycerol esters. Pretreatment of the membranes with phenyl-methylsulphonyl fluoride PMSF , a Ž . non-selective inhibitor of a variety of esterases, as well as of 2-Ara-Gl enzymatic hydrolysis Bisogno et al., 1997b , Ž . potentiated the binding of 2-Ara-Gl to the CB receptor, 1 but to a lesser extent reaching an apparent Ž Ki of 34.6"5.6 nM , compared with the mixture of the two esters Fig. . Ž 2c . The final concentration of PMSF in the assay was 0.2 . mM. The combination of PMSF with the two esters yielded essentially the same apparent K value 13.5 Ž . "5.1 nM as i observed with the two esters alone, indicating the high efficacy of the esters in enhancing the activity of 2-Ara-Gl. 3.3. Inhibition of adenylyl cyclase COS-7 cells were transfected with plasmids encoding either CB or CB and adenylyl cyclase type V Fig. Ž 1 2 3a–d . The addition of 2-Ara-Gl to the cells transfected . with CB led to inhibition of adenylyl cyclase activity with 1 IC of 1463"170 nM Fig. 3a . Pretreatment of the Ž . 50 membranes with PMSF with a final concentration of 0.2 Ž mM during the assay reduced the IC to 428 . 50 "45 nM Ž. Ž Fig. 3b , while the addition of the two fatty acid esters in the ratio described above reduced the IC to 307 . 50 "51 nM. The addition of the two esters to PMSF treated cells yielded a similar IC value 385 Ž . "26 mM . A similar, 50 albeit lower, potentiation of 2-Ara-Gl inhibition of adenylyl cylcase by 2-Lino-Gl and 2-Palm-Gl was found in cells transfected with CB Fig. 3c,d . The addition of these two Ž . 2 esters reduced the apparent IC of 2-Ara-Gl from 2724 50 " 371 to 794"111 mM and in cells treated with PMSF from 1884"297 to 784"197 mM demonstrating again the entourage effect of the two esters on the activity of 2-AraGl Fig. 3c,d . Ž . 3.4. In ÕiÕo assays The 2-acyl-glycerols were tested in mice in the tetrad of tests which together are generally considered to reflect Fig. 3. Inhibition by 2-Ara-Gl of adenylyl cyclase activity. COS-7 cells were transfected with plasmids encoding adenylycl cyclase type V and either CB1 Ž. Ž. a,b or CB c,d receptors. Two days later, the effect of 2-Ara-Gl on forskolin-stimulated adenylyl cyclase activity was determined in the absence or 2 presence of the acyl glycerols 2-Lino-Gl and 2-Palm-Gl. These glycerol esters had no effect by themselves on adenylyl cyclase activity in the range tested Ž . up to 10 mM . In a second experiment, the cells were preincubated with 0.2 mM PMSF for 5 min before being assayed for adenylyl cyclase activity in the presence of the same concentration of PMSF. PMSF treatment by itself did not affect adenyl cyclase activity. Data presented are from a representative experiment out of three experiments performed in triplicates. 28 S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) Fig. 4. Mice were tested on the tetrad of tests for cannabinoid-induced effects including ambulation in the open field a , immobility on a ring b , Ž. Ž. analgesia on a hot place c and hypothermia d . Data were analysed by one-way analyses of variance. Individual treatment groups were compared with Ž. Ž. post hoc Newman–Keuls multiple comparison tests. Injections of 2-Ara-Gl, 1 mgrkg Ž . Ž. ns10 , 2-Lino-Gl, 10 mgrkg ns5 or 2-Palm-Gl, 5 mgrkg Ž . ns5 by themselves did not produce any significant effect. However, when 2-Ara-Gl, 2-Lino-Gl and 2-Palm-Gl were injected together, significant effects were obtained in each of the four tests. Moreover, the effects induced by these triple injections differed significantly from each single injection vŽ ehicle, 2-Ara-Gl alone, 2-Palm-Gl alone, 2-Lino-Gl alone .. ) p-0.05, )) p-0.01, ))) p-0.001. cannabinoid-induced activity Mechoulam et al., 1995; Ž Fride and Mechoulam, 1993; Martin et al., 1991 . We . investigated whether, similarly to the above described in vitro findings, the presence of 2-Lino-Gl andror 2-PalmGl, potentiates the effects of 2-Ara-Gl. A dose of 2-Ara-Gl Ž . 1 mgrkg at the low end of the dose–response curve was administered, yielding barely observable effects. This low dose was chosen in order to be able to discern any potentiation effects, if present. Administration of 2-Lino-Gl Ž . Ž. 10 mgrkg or 2-Palm-Gl 5 mgrkg alone produced no significant effects Fig. 4 . No effect was observed when Ž . the two esters were administered together, at the above dose levels not shown . Further, injections of 2-Ara-Gl Ž . with either 2-Lino-Gl or 2-Palm-Gl at the above indicated doses, produced no significant effects data not shown . Ž . However, injections of 2-Ara-Gl together with both 2- Lino-Gl and 2-Palm-Gl 1, 10, 5 mg Ž . rkg, respectively produced significant inhibition of motor behavior, immobility on the ring, hypothermia and analgesia Fig. 4 . The Ž . potentiating effect was especially pronounced in the ring immobility test Fig. 4b . Since a high dose 20 mg Ž. Ž . rkg of either 2-Lino-Gl or 2-Palm-Gl did not produce significant effects, except for reduction in motor activity in the openfield by 2-Palm-Gl data not shown , we conclude that the Ž . potentiation of 2-Ara-Gl induced effects by combined injections of 2-Lino-Gl and 2-Palm-Gl is not the result of a cumulative effect of the low doses of these compounds. 3.5. Inhibition of 2-Ara-Gl enzymatic hydrolysis by 2-LinoGl and 2-Palm-Gl We assessed whether the facilitatory action of the inactive 2-Lino-Gl and 2-Palm-Gl could be due to protection of 2-Ara-Gl against metabolic inactivation. The possible inhibitory action of the two monoglycerides on 2-Ara-Gl hydrolysis was tested by using two cell lines, i.e., mouse neuroblastoma N18TG2 and rat basophilic leukaemia Ž . RBL-2H3 cells, previously shown to express selectively CB and CB cannabinoid receptors, respectively How- Ž 1 2 lett, 1995; Facci et al., 1995 . Subcellular fractions from . both cell types have been shown to contain enzymatic S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) 29 activities for catalyzing 2-Ara-Gl hydrolysis to arachidonic acid AA Bisogno et al., 1997b; Di Marzo et al., 1998 . Ž .Ž . Fig. 5a,b show the effect of 2-Palm-Gl and 2-Lino-Gl on w 3 H 2-Ara-Gl hydrolysis by the RBL-2H3 and N18TG2 x cell particulate fractions. Significant inhibitory effects were obtained with increasing doses of the two compounds. Fig. 5. Effect of monoacylglycerols on 2-Ara-Gl inactivation by RBL-2H3 andror N18TG2 cells. a and b : effect of 1-Lino-, 2-Lino-, 1-Palm-, 2-Palm- Ž. Ž. w 3 and 2-Ara-Gl on the hydrolysis of H 2-Ara-Gl by 10,000 x =g pellet fractions from RBL-2H3 a and N18TG2 b cells. In some cases only the effects of Ž. Ž. Ž . w 3 the doses exerting the maximal inhibition are shown. Data are means"S.D. of three experiments. c–e Clearance and hydrolysis of H 2-Ara-Gl by x intact RBL-2H3 cells, at 378C or 48C c , and in the absence c or presence of either 100 Ž. Ž. Ž. Ž. mM 2-Lino-Gl d or 100 mM 2-Palm-Gl e in the incubation Ž . w 3 media. f Clearance and hydrolysis of H 2-Ara-Gl by intact N18TG2 cells in the absence or presence of 100 x mM 2-Lino-Gl. No significant inhibitory effect was obtained with 100 mM 2-Palm-Gl not shown for the sake of clarity . Data in c–f are means of duplicates and representative of two separate Ž . Ž. experiments. Occasional differences between the radioactivity cleared from media and that found associated with cells or AA was due to non-specific w 3 binding of H -2-Ara-Gl to plastic, and found in the 1% bovine serum albumin washes see Section 2 . x Ž . 30 S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) However, only the effects of 2-Lino-Gl were comparable with the inhibitory action observed with 2-Ara-Gl, whereas 2-Palm-Gl was much less effective. A significant inhibiw 3 tion of H 2-Ara-Gl hydrolysis by RBL-2H3 membranes x was also exerted by a mixture of 2-Palm-Gl, 2-Lino-Gl and 2-Ara-Gl in a molar ratio identical to that observed in the spleen 5:12:1, i.e., 20, 48 and 4 Ž . mM, respectively . The inhibitory effect in this case 42.7 Ž "3.3%, means" S.E.M., ns3 , however, was not higher than that exerted . by 2-Lino-Gl alone, suggesting that the active component of the mixture responsible for the inhibition was the latter monoglyceride. 3.6. Inhibition of 2-Ara-Gl inactiÕation by liÕing intact cells It was of interest to determine if the protection of 2-Ara-Gl levels by 2-Lino-Gl could also be observed in living intact cells. In RBL-2H3 cells, we found that, without the addition of 2-Lino-Gl and 2-Palm-Gl, 2-Ara-Gl disappeared from the culture media in a temperature-dependent fashion with a half life at 378C of 6"2 min Ž .Ž . means"S.E.M., ns4 Fig. 5c . This clearance was mostly the result of hydrolysis by and of diffusion into intact cells, as shown by the time-dependent formation of AA and by the finding of increasing amounts of cell-assow 3 ciated H 2-Ara-Gl with increasing incubation times Fig. x Ž 5c . Some 2-Ara-Gl was also incorporated into phospho- . lipids Di Marzo et al., 1998 . 2-Lino-Gl, but not 2-Palm- Ž . Gl, prevented this loss of 2-Ara-Gl from the medium Fig. Ž 5d,e . An eight-fold increase of 2-Ara-Gl levels was ob- . served after 30 min incubation. This was accompanied by a reduction in the amount of 2-Ara-Gl associated with the cell fraction as well as with a reduction in the amount of w 3 x w3 H -arachidonic acid derived from the hydrolysis of H 2- x Ara-Gl. A similar result was obtained with N18TG2 cells Ž . Fig. 5f and data not shown . A mixture of 2-Palm-, 2-Lino- and 2-Ara-Gl in a 5:12:1 molar ratio 20, 48 and 4 Ž mM, respectively also inhibited the clearance of the latter . from the incubation medium, as well as its inactivation by intact cells, leading to an overall elevation of 2-Ara-Gl levels 260 Ž "40%, after 30 min incubations, means"S.D., ns2 . This value however, was not significantly higher . than that observed with 2-Lino-Gl alone 210 Ž . "30% . 1-Lino-Gl and 1-Palm-Gl did not differ significantly in their inhibition of the inactivation of 2-Ara-Gl, from 2- Lino-Gl and 2-Palm-Gl Fig. 5a,b and data not shown . Ž . 4. Discussion The above results indicate that in spleen, as in canine gut and rat brain Mechoulam et al., 1995; Sugiura et al., Ž 1995 , 2-Ara-Gl is present together with additional 2-acyl- . glycerols, two of which, 2-Lino-Gl and 2-Palm-Gl, showed neither binding activity to CB or CB cannabinoid recep- 1 2 tors in membranes of CHO andror COS-7 cells nor in vivo cannabinoid effects in mice. However, both 2-Lino-Gl and 2-Palm-Gl separately or together in the ratio present Ž in the spleen potentiated the apparent binding of 2-Ara-Gl . to CB and CB . The mixture of the three monoglycerides 1 2 is also more potent than 2-Ara-Gl in the inhibition of adenylyl cyclase in COS-7 cells transfected for either CB1 or CB cannabinoid receptors. The same type of ‘entour- 2 age’ effect was observed in several in vivo tests which are commonly used with cannabinoids. The ‘entourage’ effects reported above are only in part due to inhibition of 2-Ara-Gl inactivation by cells. In fact, of the two monoacylglycerols tested, only 2-Lino-Gl efficiently inhibited 2-Ara-Gl inactivation by either neuronal or leukocyte cell types used in this study, thus increasing the amounts of 2-Ara-Gl available for cannabinoid receptor activation. 2-Palm-Gl, which was more active than 2-Lino-Gl in the potentiation of 2-Ara-Gl binding to CB2 receptors, did not significantly counteract the inactivation of 2-Ara-Gl by whole cells. These observations suggest that endogenous, inactive 2-acyl-glycerols enhance 2-AraGl activity through inhibition of 2-Ara-Gl inactivation and possibly through other, as yet unknown, mechanisms such as potentiation of binding to CB receptors or inhibition of binding to plasma proteins. Previously, we and others have shown that fatty acid amides, which have no affinity for CB receptors, inhibit anandamide metabolism by several 1 types of intact cells Maurelli et al., 1995; Di Tomaso et Ž al., 1996; Bisogno et al., 1997a; Mechoulam et al., 1997 ,. thus potentially leading to increased levels of endogenous anandamide available for CB rCB cannabinoid receptor 1 2 activation. This inhibitory effect may underlie the in vivo cannabimimetic actions observed by us for the fatty acid amide oleamide Mechoulam et al., 1997 , a putative sleep Ž . factor. Competition for the binding site of metabolic enzymes also explains, for example, why vy3 fatty acids inhibit some of the actions of vy6 fatty acids by counteracting the formation of vy6 fatty acid derivatives Ž . Okuyama et al., 1997 . These results may have considerable specific biological importance. The observation that the potency of 2-Ara-Gl can be modified by related 2- acyl-glycerols present with it could represent a novel route for molecular regulation of endogenous cannabinoid activity. The results reported now may also be of general importance. Biologically active natural products, from either plant or animal origin, are in many instances accompanied by chemically related, though biologically inactive, constituents. Very seldom is the biological activity of the active constituent assayed together with the inactive ‘entourage’ compounds. In view of the results described above investigations of the effect of the active component in the presence of its ‘entourage’ compounds may lead to observations of effects closer to those in Nature than investigations with the active component only. S. Ben-Shabat et al.rEuropean Journal of Pharmacology 353 1998 23–31 ( ) 31 Acknowledgements This work was supported by NIDA grant DA 9789 toŽ R.M. , a grant by the Israel Science Foundation to Z.V. . Ž and R.M. , and a grant RG26 . r95 from The Human Frontier Science Program Organization to V.D.M. . R.M. is Ž . associated with the David Bloom Centre for Pharmacy at the Hebrew University. References Bayewitch, M., Rhee, M.-H., Avidor-Reiss, T., Breuer, A., Mechoulam, Ž . 9 R., Vogel, Z., 1996. y -D -Tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated inhibition of adenylyl cyclase. J. Biol. Chem. 271, 9902–9905. Bisogno, T., Maurelli, S., Melck, D., De Petrocellis, L., Di Marzo, V., 1997a. Biosynthesis, uptake and degradation of anandamide and palmitoyl-ethanolamide in leukocytes. J. Biol. Chem. 272, 3315–3323. Bisogno, T., Sepe, N., Melck, D., Maurelli, S., De Petrocellis, L., Di Marzo, V., 1997b. Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoyl-glycerol in mouse neuroblastoma cells. Biochem. J. 322, 671–677. Devane, W.A., Breuer, A., Sheskin, T., Jarbe, T.U.C., Eisen, M., Me- ¨ choulam, R., 1992a. A novel probe for the cannabinoid receptor. J. Med. Chem. 35, 2065–2069. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., ˇ Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A., Mechoulam, R., 1992b. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946–1949. Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J.-C., Piomelli, D., 1994. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686–691. Di Marzo, V., Bisogno, T., Sugiura, T., Melck, D., De Petrocellis, L., 1998. The novel endogenous cannabinoid 2-arachidonoyl glycerol is inactivated by neuro and basophil-like cells: connections with anandamide. Biochem. J. 331, 15–19. Di Tomaso, E., Beltramo, M., Piomelli, D., 1996. Brain cannabinoids in chocolate. Nature 382, 677–678. Facci, L., Dal-Toso, R., Romanello, S., Buriani, A., Skaper, S.D., Leon, A., 1995. Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc. Natl. Acad. Sci. USA 92, 3376–3380. Fride, E., Mechoulam, R., 1993. Pharmacological activity of the cannabinoid agonist anandamide, a brain constituent. Eur. J. Pharmacol. 231, 313–314. Howlett, A., 1995. Pharmacology of cannabinoid receptors. Annu. Rev. Pharmacol. Toxicol. 35, 607–634. Lee, M., Yang, K.H., Kaminski, N.E., 1995. Effects of putative cannabinoid receptor ligands, anandamide and 2-arachidonyl-glycerol, on immune function in B6C3F1 mouse splenocytes. J. Pharmacol. Exp. Ther. 275, 529–536. Martin, B.R., Compton, D.R., Thomas, B.F., Prescot, W.R., Little, P.J., Razdan, R.K., Johnson, M.R., Melvin, L.S., Mechoulam, R., Ward, S.J., 1991. Behavioral, biochemical and molecular modeling evaluations of cannabinoid analogs. Pharmacol. Biochem. Behav. 40, 471– 478. Maurelli, S., Bisogno, T., De Petrocellis, L., Di Luccia, A., Marino, G., Di Marzo, V., 1995. Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma anandamide amidohydrolase. FEBS Lett. 377, 82–86. Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, ˇ N.E., Schatz, A.R., Gopher, A., Almog, S., Martin, B.R., Compton, D.R., Pertwee, R.G., Griffin, G., Bayewitch, M., Barg, J., Vogel, Z., 1995. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50, 83–90. Mechoulam, R., Fride, E., Hanus, L., Sheskin, T., Bisogno, T., Di Marzo, ˇ V., Bayewitch, M., Vogel, Z., 1997. Anandamide may mediate sleep induction. Nature 389, 25–26. Munro, S., Thomas, K.L., Abu-Shaar, M., 1993. Molecular characterization of a peripheral receptor for cannabinoids. Nature 365, 61–65. Okuyama, H., Kobayshi, Watanabe, S., 1997. Dietary fatty acids—the ny6rny3 balance and chronic elderly diseases. Excess linoleic acid and relative ny3 deficiency syndrome seen in Japan. Prog. Lipid Res. 35, 409–457. Rhee, M.-H., Vogel, Z., Barg, J., Bayewitch, M., Levy, R., Hanus, L., ˇ Breuer, A., Mechoulam, R., 1997. Cannabinol derivatives: binding to cannabinoid receptors and inhibition of adenylyl cyclase. J. Med. Chem. 40, 3228–3233. Stella, N., Schweitzer, P., Piomelli, D., 1997. A second endogenous cannabinoid that modulates long-term potentiation. Nature 388, 773– 778. Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K., Yamashita, A., Waku, K., 1995. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor in the brain. Biochem. Biophys. Res. Commun. 215, 89–97.

 

Themed Issue: Cannabinoids in Biology and Medicine, Part I REVIEWbph_1238 1344..1364 Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects Ethan B Russo GW Pharmaceuticals, Salisbury, Wiltshire, UK Correspondence Ethan Russo, MD, 20402 81st Avenue SW, Vashon, WA 98070, USA. E-mail: ethanrusso@comcast.net ---------------------------------------------------------------- Keywords cannabinoids; terpenoids; essential oils; THC; CBD; limonene; pinene; linalool; caryophyllene; phytotherapy ---------------------------------------------------------------- Received 19 November 2010 Revised 29 December 2010 Accepted 12 January 2011 Tetrahydrocannabinol (THC) has been the primary focus of cannabis research since 1964, when Raphael Mechoulam isolated and synthesized it. More recently, the synergistic contributions of cannabidiol to cannabis pharmacology and analgesia have been scientifically demonstrated. Other phytocannabinoids, including tetrahydrocannabivarin, cannabigerol and cannabichromene, exert additional effects of therapeutic interest. Innovative conventional plant breeding has yielded cannabis chemotypes expressing high titres of each component for future study. This review will explore another echelon of phytotherapeutic agents, the cannabis terpenoids: limonene, myrcene, a-pinene, linalool, b-caryophyllene, caryophyllene oxide, nerolidol and phytol. Terpenoids share a precursor with phytocannabinoids, and are all flavour and fragrance components common to human diets that have been designated Generally Recognized as Safe by the US Food and Drug Administration and other regulatory agencies. Terpenoids are quite potent, and affect animal and even human behaviour when inhaled from ambient air at serum levels in the single digits ng·mL-1 . They display unique therapeutic effects that may contribute meaningfully to the entourage effects of cannabis-based medicinal extracts. Particular focus will be placed on phytocannabinoid-terpenoid interactions that could produce synergy with respect to treatment of pain, inflammation, depression, anxiety, addiction, epilepsy, cancer, fungal and bacterial infections (including methicillin-resistant Staphylococcus aureus). Scientific evidence is presented for non-cannabinoid plant components as putative antidotes to intoxicating effects of THC that could increase its therapeutic index. Methods for investigating entourage effects in future experiments will be proposed. Phytocannabinoid-terpenoid synergy, if proven, increases the likelihood that an extensive pipeline of new therapeutic products is possible from this venerable plant. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7 Abbreviations 2-AG, 2-arachidonoylglycerol; 5-HT, 5-hydroxytryptamine (serotonin); AD, antidepressant; AEA, arachidonoylethanolamide (anandamide); AI, anti-inflammatory; AMPA, a-amino-3-hydroxyl-5-methyl-4- isoxazole-propionate; Ca++, calcium ion; CB1/CB2, cannabinoid receptor 1 or 2; CBC, cannabichromene; CBCA, cannabichromenic acid; CBD, cannabidiol; CBDA, cannabidiolic acid; CBDV, cannabidivarin; CBG, cannabigerol; CBGA, cannabigerolic acid; CBGV, cannabigerivarin; CNS, central nervous system; COX, cyclo-oxygenase; DAGL, diacylglycerol lipase; ECS, endocannabinoid system; EO, essential oil; FAAH, fatty acid amidohydrolase; FDA, US Food and Drug Administration; FEMA, Food and Extract Manufacturers Association; fMRI, functional magnetic resonance imaging; GABA, gamma aminobutyric acid; GPCR, G-protein coupled receptor; GPR, G-protein coupled receptor; HEK, human embryonic kidney; IC50, 50% inhibitory concentration; i.p., intraperitoneal; MAGL, monoacylglycerol lipase; MIC, minimum inhibitory concentration; MS, multiple sclerosis; NGF, nerve growth factor; NIDA, US National Institute on Drug Abuse; PG, prostaglandin; PTSD, post-traumatic stress disorder; RCT, randomized clinical trial; SPECT, single photon emission computed tomography; SSADH, succinic semialdehyde dehydrogenase; Sx, symptoms; T1/2, half-life; TCA, tricyclic antidepressant; THC, tetrahydrocannabinol; THCA, tetrahydrocannabinolic acid; THCV, tetrahydrocannabivarin; TNF-a, tumour necrosis factor-alpha, TRPV, transient receptor potential vanilloid receptor BJP British Journal of Pharmacology DOI:10.1111/j.1476-5381.2011.01238.x www.brjpharmacol.org 1344 British Journal of Pharmacology (2011) 163 1344–1364 © 2011 The Author British Journal of Pharmacology © 2011 The British Pharmacological Society The roots of cannabis synergy Cannabis has been a medicinal plant of unparalleled versatility for millennia (Mechoulam, 1986; Russo, 2007; 2008), but whose mechanisms of action were an unsolved mystery until the discovery of tetrahydrocannabinol (THC) (Gaoni and Mechoulam, 1964a), the first cannabinoid receptor, CB1 (Devane et al., 1988), and the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) (Devane et al., 1992) and 2-arachidonoylglycerol (2-AG) (Mechoulam et al., 1995; Sugiura et al., 1995). While a host of phytocannabinoids were discovered in the 1960s: cannabidiol (CBD) (Mechoulam and Shvo, 1963), cannabigerol (CBG) (Gaoni and Mechoulam, 1964b), cannabichromene (CBC) (Gaoni and Mechoulam, 1966), cannabidivarin (CBDV) (Vollner et al., 1969) and tetrahydrocannabivarin (THCV) (Gill et al., 1970), the overwhelming preponderance of research focused on psychoactive THC. Only recently has renewed interest been manifest in THC analogues, while other key components of the activity of cannabis and its extracts, the cannabis terpenoids, remain understudied (McPartland and Russo, 2001b; Russo and McPartland, 2003). The current review will reconsider essential oil (EO) agents, their peculiar pharmacology and possible therapeutic interactions with phytocannabinoids. Nomenclature follows conventions in Alexander et al. (2009). Phytocannabinoids and terpenoids are synthesized in cannabis, in secretory cells inside glandular trichomes (Figure 1) that are most highly concentrated in unfertilized female flowers prior to senescence (Potter, 2004; Potter, 2009). Geranyl pyrophosphate is formed as a precursor via the deoxyxylulose pathway in cannabis (Fellermeier et al., 2001), and is a parent compound to both phytocannabinoids and terpenoids (Figure 2). After coupling with either olivetolic acid or divarinic acid, pentyl or propyl cannabinoid acids are produced, respectively, via enzymes that accept either substrate (de Meijer et al., 2003), a manifestation of Mechoulam’s postulated ‘Nature’s Law of Stinginess’. Although having important biochemical properties in their own right, acid forms of phytocannabinoids are most commonly decarboxylated via heat to produce the more familiar neutral phytocannabinoids (Table 1). Alternatively, geranyl pyrophosphate may form limonene and other monoterpenoids in secretory cell plastids, or couple with isopentenyl pyrophosphate in the cytoplasm to form farnesyl pyrophosphate, parent compound to the sesquiterpenoids, that co-localizes with transient receptor potential vanilloid receptor (TRPV) 1 in human dorsal root ganglion, suggesting a role in sensory processing of noxious stimuli (Bradshaw et al., 2009), and which is the most potent endogenous ligand to date on the G-protein coupled receptor (GPR) 92 (Oh et al., 2008). An obvious question pertains to the chemical ecology of such syntheses that require obvious metabolic demands on the plant (Gershenzon, 1994), and these will be considered. Is cannabis merely a crude vehicle for delivery of THC? Might it rather display herbal synergy (Williamson, 2001) encompassing potentiation of activity by active or inactive components, antagonism (evidenced by the ability of CBD to reduce side effects of THC; Russo and Guy, 2006), summation, pharmacokinetic and metabolic interactions? Recently, four basic mechanisms of synergy have been proposed (Wagner and Ulrich-Merzenich, 2009): (i) multi-target effects; (ii) pharmacokinetic effects such as improved solubility or bioavailability; (iii) agent interactions affecting bacterial resistance; and (iv) modulation of adverse events. Cannabis was cited as an illustration. Could phytocannabinoids function analogously to the endocannabinoid system (ECS) with its combination of active and ‘inactive’ synergists, first described as an entourage (Ben-Shabat et al., 1998), with subsequent refinement (Mechoulam and Ben-Shabat, 1999) and qualification (p. 136): ‘This type of synergism may play a role in the widely held (but not experimentally based) view that in some cases plants are better drugs than the natural products isolated from them’. Support derives from studies in which cannabis extracts demonstrated effects two to four times greater than THC (Carlini et al., 1974); unidentified THC antagonists and synergists were claimed (Fairbairn and Pickens, 1981), anticonvulsant activity was observed beyond the cannabinoid fraction (Wilkinson et al., 2003), and extracts of THC and CBD modulated effects in hippocampal neurones distinctly from pure compounds (Ryan et al., 2006). Older literature also presented refutations: no observed differences were noted by humans ingesting or smoking pure THC versus herbal cannabis (Wachtel et al., 2002); pure THC seemed to account for all tetrad-type effects in mice (Varvel et al., 2005); and smoked cannabis with varying CBD or CBC content failed to yield subjective differences combined with THC (Ilan et al., 2005). Explanations include that the cannabis employed by Wachtel yielded 2.11% THC, but with only 0.3% cannabinol (CBN) and 0.05% CBD (Russo and McPartland, 2003), and Ilan’s admission that CBN and CBD content might be too low to modulate THC. Another factor is apparent in that terpenoid yields from vaporization of street cannabis were 4.3–8.5 times of those from US National Institute on Drug Abuse cannabis (Bloor et al., 2008). It is undisputed that the black market cannabis in the UK (Potter et al., 2008), Continental Europe (King et al., 2005) and the USA (Mehmedic et al., 2010) has become almost exclusively a high-THC preparation to the almost total exclusion of other phytocannabinoids. If – as many consumers and experts maintain (Clarke, 2010) – there are biochemical, pharmacological and Figure 1 Cannabis capitate glandular (EBR by permission of Bedrocan BV, Netherlands). BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1345 phenomenological distinctions between available cannabis ‘strains’, such phenomena are most likely related to relative terpenoid contents and ratios. This treatise will assess additional evidence for putative synergistic phytocannabinoidterpenoid effects exclusive of THC, to ascertain whether this botanical may fulfil its promise as, ‘a neglected pharmacological treasure trove’ (Mechoulam, 2005). Phytocannabinoids, beyond THC: a brief survey Phytocannabinoids are exclusively produced in cannabis (vide infra for exception), but their evolutionary and ecological raisons d’être were obscure until recently. THC production is maximized with increased light energy (Potter, 2009). It has been known for some time that CBG and CBC are mildly antifungal (ElSohly et al., 1982), as are THC and CBD against a cannabis pathogen (McPartland, 1984). More pertinent, however, is the mechanical stickiness of the trichomes, capable of trapping insects with all six legs (Potter, 2009). Tetrahydrocannabinolic acid (THCA) and cannabichromenic acid (Morimoto et al., 2007), as well as cannabidiolic acid and cannabigerolic acid (CBGA; Shoyama et al., 2008) produce necrosis in plant cells. Normally, the cannabinoid acids are sequestered in trichomes away from the flower tissues. Any trichome breakage at senescence may contribute to natural pruning of lower fan leaves that otherwise utilize energy that the plant preferentially diverts to the flower, in continued efforts to affect fertilization, generally in vain when subject to human horticulture for pharmaceutical production. THCA and CBGA have also proven to be insecticidal in their own right (Sirikantaramas et al., 2005). Over 100 phytocannabinoids have been identified (Brenneisen, 2007; Mehmedic et al., 2010), but many are artefacts of analysis or are produced in trace quantities that have not permitted thorough investigation. The pharmacology of the more accessible phytocannabinoids has received excellent recent reviews (Pertwee et al., 2007; Izzo et al., 2009; De Petrocellis and Di Marzo, 2010; De Petrocellis et al., 2011), and will be summarized here, with emphasis on activities with particular synergistic potential. Geranylphosphate: olivetolate geranyltransferase HO OH COOH cannabigerolic acid O OH COOH delta-9-tetrahydrocannabinolic acid OH OH COOH cannabidiolic acid O OH COOH cannabichromenenic acid HO OH COOH cannabigerovarinic acid Geranylphosphate: olivetolate geranyltransferase O OH COOH tetrahydrocannabivarinic acid OH OH COOH cannabidivarinic acid O OH COOH cannabichromevarinic acid PPO dimethylallyl pyrophosphate (DMAPP) OPP isopentenyl pyrophosphate (IPP) GPP synthase + + + THCA synthaseCBDA synthase CBCA synthase THCA synthaseCBDA synthase CBCA synthase H limonene OPO3OPO3 farnesyl pyrophosphate OPO3OPO3 geranyl pyrophosphate x3 Sesquiterpenoids FPP synthase Limonene synthase Monoterpenoids HO OH COOH divarinic acid (5-propyl resorcinolic acid HO OH COOH olivetolic acid (5-pentyl resorcinolic acid) ) Phytocannabinoid Acids Figure 2 Phytocannabinoid and cannabis terpenoid biosynthesis. BJP EB Russo 1346 British Journal of Pharmacology (2011) 163 1344–1364 Table 1 Phytocannabinoid activity table Phytocannabinoid structure Selected pharmacology (reference) Synergistic terpenoids O OH delta-9-tetrahydrocannabinol (THC) Analgesic via CB1 and CB2 (Rahn and Hohmann, 2009) Various AI/antioxidant (Hampson et al., 1998) Limonene et al. Bronchodilatory (Williams et al., 1976) Pinene ↓ Sx. Alzheimer disease (Volicer et al., 1997; Eubanks et al., 2006) Limonene, pinene, linalool Benefit on duodenal ulcers (Douthwaite, 1947) Caryophyllene, limonene Muscle relaxant (Kavia et al., 2010) Linalool? Antipruritic, cholestatic jaundice (Neff et al., 2002) Caryophyllene? OH OH cannabidiol AI/antioxidant (Hampson et al., 1998) Limonene et al. Anti-anxiety via 5-HT1A (Russo et al., 2005) Linalool, limonene Anticonvulsant (Jones et al., 2010) Linalool Cytotoxic versus breast cancer (Ligresti et al., 2006) Limonene ↑ adenosine A2A signalling (Carrier et al., 2006) Linalool Effective versus MRSA (Appendino et al., 2008) Pinene Decreases sebum/sebocytes (Biro et al., 2009) Pinene, limonene, linalool Treatment of addiction (see text) Caryophyllene O OH cannabichromene Anti-inflammatory/analgesic (Davis and Hatoum, 1983) Various Antifungal (ElSohly et al., 1982) Caryophyllene oxide AEA uptake inhibitor (De Petrocellis et al., 2011) – Antidepressant in rodent model (Deyo and Musty, 2003) Limonene HO OH cannabigerol TRPM8 antagonist prostate cancer (De Petrocellis et al., 2011) Cannabis terpenoids GABA uptake inhibitor (Banerjee et al., 1975) Phytol, linalool Anti-fungal (ElSohly et al., 1982) Caryophyllene oxide Antidepressant rodent model (Musty and Deyo, 2006); and via 5-HT1A antagonism (Cascio et al., 2010) Limonene Analgesic, a-2 adrenergic blockade (Cascio et al., 2010) Various ↓ keratinocytes in psoriasis (Wilkinson and Williamson, 2007) adjunctive role? Effective versus MRSA (Appendino et al., 2008) Pinene O OH tetrahydrocannabivarin AI/anti-hyperalgesic (Bolognini et al., 2010) Caryophyllene et al.... Treatment of metabolic syndrome (Cawthorne et al., 2007) – Anticonvulsant (Hill et al., 2010) Linalool OH OH cannabidivarin Inhibits diacylglycerol lipase (De Petrocellis et al., 2011) – Anticonvulsant in hippocampus (Hill et al., 2010) Linalool O OH cannabinol (CBN) Sedative (Musty et al., 1976) Nerolidol, myrcene Effective versus MRSA (Appendino et al., 2008) Pinene TRPV2 agonist for burns (Qin et al., 2008) Linalool ↓ keratinocytes in psoriasis (Wilkinson and Williamson, 2007) adjunctive role? ↓ breast cancer resistance protein (Holland et al., 2008) Limonene 5-HT, 5-hydroxytryptamine (serotonin); AEA, arachidonoylethanolamide (anandamide); AI, anti-inflammatory; CB1/CB2, cannabinoid receptor 1 or 2; GABA, gamma aminobutyric acid; TRPV, transient receptor potential vanilloid receptor; MRSA, methicillin-resistant Staphylococcus aureus; Sx, symptoms. BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1347 THC (Table 1) is the most common phytocannabinoid in cannabis drug chemotypes, and is produced in the plant via an allele co-dominant with CBD (de Meijer et al., 2003). THC is a partial agonist at CB1 and cannabinoid receptor 2 (CB2) analogous to AEA, and underlying many of its activities as a psychoactive agent, analgesic, muscle relaxant and antispasmodic (Pacher et al., 2006). Additionally, it is a bronchodilator (Williams et al., 1976), neuroprotective antioxidant (Hampson et al., 1998), antipruritic agent in cholestatic jaundice (Neff et al., 2002) and has 20 times the antiinflammatory power of aspirin and twice that of hydrocortisone (Evans, 1991). THC is likely to avoid potential pitfalls of either COX-1 or COX-2 inhibition, as such activity is only noted at concentrations far above those attained therapeutically (Stott et al., 2005). CBD is the most common phytocannabinoid in fibre (hemp) plants, and second most prevalent in some drug chemotypes. It has proven extremely versatile pharmacologically (Table 1) (Pertwee, 2004; Mechoulam et al., 2007), displaying the unusual ability to antagonize CB1 at a low nM level in the presence of THC, despite having little binding affinity (Thomas et al., 2007), and supporting its modulatory effect on THC-associated adverse events such as anxiety, tachycardia, hunger and sedation in rats and humans (Nicholson et al., 2004; Murillo-Rodriguez et al., 2006; Russo and Guy, 2006). CBD is an analgesic (Costa et al., 2007), is a neuroprotective antioxidant more potent than ascorbate or tocopherol (Hampson et al., 1998), without COX inhibition (Stott et al., 2005), acts as a TRPV1 agonist analogous to capsaicin but without noxious effect (Bisogno et al., 2001), while also inhibiting uptake of AEA and weakly inhibiting its hydrolysis. CBD is an antagonist on GPR55, and also on GPR18, possibly supporting a therapeutic role in disorders of cell migration, notably endometriosis (McHugh et al., 2010). CBD is anticonvulsant (Carlini and Cunha, 1981; Jones et al., 2010), anti-nausea (Parker et al., 2002), cytotoxic in breast cancer (Ligresti et al., 2006) and many other cell lines while being cyto-preservative for normal cells (Parolaro and Massi, 2008), antagonizes tumour necrosis factor-alpha (TNF-a) in a rodent model of rheumatoid arthritis (Malfait et al., 2000), enhances adenosine receptor A2A signalling via inhibition of an adenosine transporter (Carrier et al., 2006), and prevents prion accumulation and neuronal toxicity (Dirikoc et al., 2007). A CBD extract showed greater anti-hyperalgesia over pure compound in a rat model with decreased allodynia, improved thermal perception and nerve growth factor levels and decreased oxidative damage (Comelli et al., 2009). CBD also displayed powerful activity against methicillin-resistant Staphylococcus aureus (MRSA), with a minimum inhibitory concentration (MIC) of 0.5–2 mg·mL-1 (Appendino et al., 2008). In 2005, it was demonstrated that CBD has agonistic activity at 5-hydroxytryptamine (5-HT)1A at 16 mM (Russo et al., 2005), and that despite the high concentration, may underlie its anti-anxiety activity (Resstel et al., 2009; Soares Vde et al., 2010), reduction of stroke risk (Mishima et al., 2005), anti-nausea effects (Rock et al., 2009) and ability to affect improvement in cognition in a mouse model of hepatic encephalopathy (Magen et al., 2009). A recent study has demonstrated that CBD 30 mg·kg-1 i.p. reduced immobility time in the forced swim test compared to imipramine (P < 0.01), an effect blocked by pre-treatment with the 5-HT1A antagonist WAY100635 (Zanelati et al., 2010), supporting a prospective role for CBD as an antidepressant. CBD also inhibits synthesis of lipids in sebocytes, and produces apoptosis at higher doses in a model of acne (vide infra). One example of CBD antagonism to THC would be the recent observation of lymphopenia in rats (CBD 5 mg·kg-1 ) mediated by possible CB2 inverse agonism (Ignatowska-Jankowska et al., 2009), an effect not reported in humans even at doses of pure CBD up to 800 mg (Crippa et al., 2010), possibly due to marked interspecies differences in CB2 sequences and signal transduction. CBD proved to be a critical factor in the ability of nabiximols oromucosal extract in successfully treating intractable cancer pain patients unresponsive to opioids (30% reduction in pain from baseline), as a high-THC extract devoid of CBD failed to distinguish from placebo (Johnson et al., 2010). This may represent true synergy if the THC–CBD combination were shown to provide a larger effect than a summation of those from the compounds separately (Berenbaum, 1989). CBC (Table 1) was inactive on adenylate cyclase inhibition (Howlett, 1987), but showed activity in the mouse cannabinoid tetrad, but only at 100 mg·kg-1 , and at a fraction of THC activity, via a non-CB1, non-CB2 mechanism (Delong et al., 2010). More pertinent are anti-inflammatory (Wirth et al., 1980) and analgesic activity (Davis and Hatoum, 1983), its ability to reduce THC intoxication in mice (Hatoum et al., 1981), antibiotic and antifungal effects (ElSohly et al., 1982), and observed cytotoxicity in cancer cell lines (Ligresti et al., 2006). A CBC-extract displayed pronounced antidepressant effect in rodent models (Deyo and Musty, 2003). Additionally, CBC was comparable to mustard oil in stimulating TRPA1- mediated Ca++ in human embryonic kidney 293 cells (50– 60 nM) (De Petrocellis et al., 2008). CBC recently proved to be a strong AEA uptake inhibitor (De Petrocellis et al., 2011). CBC production is normally maximal, earlier in the plant’s life cycle (de Meijer et al., 2009a). An innovative technique employing cold water extraction of immature leaf matter from selectively bred cannabis chemotypes yields a high-CBC ‘enriched trichome preparation’ (Potter, 2009). CBG (Table 1), the parent phytocannabinoid compound, has a relatively weak partial agonistic effect at CB1 (Ki 440 nM) and CB2 (Ki 337 nM) (Gauson et al., 2007). Older work supports gamma aminobutyric acid (GABA) uptake inhibition greater than THC or CBD (Banerjee et al., 1975) that could suggest muscle relaxant properties. Analgesic and anti-erythemic effects and the ability to block lipooxygenase were said to surpass those of THC (Evans, 1991). CBG demonstrated modest antifungal effects (ElSohly et al., 1982). More recently, it proved to be an effective cytotoxic in high dosage on human epithelioid carcinoma (Baek et al., 1998), is the next most effective phytocannabinoid against breast cancer after CBD (Ligresti et al., 2006), is an antidepressant in the rodent tail suspension model (Musty and Deyo, 2006) and is a mildly anti-hypertensive agent (Maor et al., 2006). Additionally, CBG inhibits keratinocyte proliferation suggesting utility in psoriasis (Wilkinson and Williamson, 2007), it is a relatively potent TRPM8 antagonist for possible application in prostate cancer (De Petrocellis and Di Marzo, 2010) and detrusor over-activity and bladder pain (Mukerji et al., 2006). It is a strong AEA uptake inhibitor (De Petrocellis et al., 2011) and a powerful agent against MRSA (Appendino et al., 2008; vide infra). Finally, CBG behaves as a potent a-2 adrenorecepBJP EB Russo 1348 British Journal of Pharmacology (2011) 163 1344–1364 tor agonist, supporting analgesic effects previously noted (Formukong et al., 1988), and moderate 5-HT1A antagonist suggesting antidepressant properties (Cascio et al., 2010). Normally, CBG appears as a relatively low concentration intermediate in the plant, but recent breeding work has yielded cannabis chemotypes lacking in downstream enzymes that express 100% of their phytocannabinoid content as CBG (de Meijer and Hammond, 2005; de Meijer et al., 2009a). THCV (Table 1) is a propyl analogue of THC, and can modulate intoxication of the latter, displaying 25% of its potency in early testing (Gill et al., 1970; Hollister, 1974). A recrudescence of interest accrues to this compound, which is a CB1 antagonist at lower doses (Thomas et al., 2005), but is a CB1 agonist at higher doses (Pertwee, 2008). THCV produces weight loss, decreased body fat and serum leptin concentrations with increased energy expenditure in obese mice (Cawthorne et al., 2007; Riedel et al., 2009). THCV also demonstrates prominent anticonvulsant properties in rodent cerebellum and pyriform cortex (Hill et al., 2010). THCV appears as a fractional component of many southern African cannabis chemotypes, although plants highly predominant in this agent have been produced (de Meijer, 2004). THCV recently demonstrated a CB2-based ability to suppress carageenan-induced hyperalgesia and inflammation, and both phases of formalin-induced pain behaviour via CB1 and CB2 in mice (Bolognini et al., 2010). CBDV (Table 1), the propyl analogue of CBD, was first isolated in 1969 (Vollner et al., 1969), but formerly received little investigation. Pure CBDV inhibits diacylglycerol lipase [50% inhibitory concentration (IC50) 16.6 mM] and might decrease activity of its product, the endocannabinoid, 2-AG (De Petrocellis et al., 2011). It is also anticonvulsant in rodent hippocampal brain slices, comparable to phenobarbitone and felbamate (Jones et al., 2010). Finally, CBN is a non-enzymatic oxidative by-product of THC, more prominent in aged cannabis samples (Merzouki and Mesa, 2002). It has a lower affinity for CB1 (Ki 211.2 nM) and CB2 (Ki 126.4 nM) (Rhee et al., 1997); and was judged inactive when tested alone in human volunteers, but produced greater sedation combined with THC (Musty et al., 1976). CBN demonstrated anticonvulsant (Turner et al., 1980), anti-inflammatory (Evans, 1991) and potent effects against MRSA (MIC 1 mg·mL-1 ). CBN is a TRPV2 (highthreshold thermosensor) agonist (EC 77.7 mM) of possible interest in treatment of burns (Qin et al., 2008). Like CBG, it inhibits keratinocyte proliferation (Wilkinson and Williamson, 2007), independently of cannabinoid receptor effects. CBN stimulates the recruitment of quiescent mesenchymal stem cells in marrow (10 mM), suggesting promotion of bone formation (Scutt and Williamson, 2007) and inhibits breast cancer resistance protein, albeit at a very high concentration (IC50 145 mM) (Holland et al., 2008). Cannabis terpenoids: neglected entourage compounds? Terpenoids are EO components, previously conceived as the quintessential fifth element, ‘life force’ or spirit (Schmidt, 2010), and form the largest group of plant chemicals, with 15–20 000 fully characterized (Langenheim, 1994). Terpenoids, not cannabinoids, are responsible for the aroma of cannabis. Over 200 have been reported in the plant (Hendriks et al., 1975; 1977; Malingre et al., 1975; Davalos et al., 1977; Ross and ElSohly, 1996; Mediavilla and Steinemann, 1997; Rothschild et al., 2005; Brenneisen, 2007), but only a few studies have concentrated on their pharmacology (McPartland and Pruitt, 1999; McPartland and Mediavilla, 2001a; McPartland and Russo, 2001b). Their yield is less than 1% in most cannabis assays, but they may represent 10% of trichome content (Potter, 2009). Monoterpenes usually predominate (limonene, myrcene, pinene), but these headspace volatiles (Hood et al., 1973), while only lost at a rate of about 5% before processing (Gershenzon, 1994), do suffer diminished yields with drying and storage (Turner et al., 1980; Ross and ElSohly, 1996), resulting in a higher relative proportion of sesquiterpenoids (especially caryophyllene), as also often occurs in extracts. A ‘phytochemical polymorphism’ seems operative in the plant (Franz and Novak, 2010), as production favours agents such as limonene and pinene in flowers that are repellent to insects (Nerio et al., 2010), while lower fan leaves express higher concentrations of bitter sesquiterpenoids that act as anti-feedants for grazing animals (Potter, 2009). Evolutionarily, terpenoids seem to occur in complex and variable mixtures with marked structural diversity to serve various ecological roles. Terpenoid composition is under genetic control (Langenheim, 1994), and some enzymes produce multiple products, again supporting Mechoulam’s ‘Law of Stinginess’. The particular mixture of mono- and sesquiterpenoids will determine viscosity, and in cannabis, this certainly is leveraged to practical advantage as the notable stickiness of cannabis exudations traps insects (McPartland et al., 2000), and thus, combined with the insecticidal phytocannabinoid acids (Sirikantaramas et al., 2005), provides a synergistic mechano-chemical defensive strategy versus predators. As observed for cannabinoids, terpenoid production increases with light exposure, but decreases with soil fertility (Langenheim, 1994), and this is supported by the glasshouse experience that demonstrates higher yields if plants experience relative nitrogen lack just prior to harvest (Potter, 2004), favouring floral over foliar growth. EO composition is much more genetically than environmentally determined, however (Franz and Novak, 2010), and while cannabis is allogamous and normally requires repeat selective breeding for maintenance of quality, this problem may be practically circumvented by vegetative propagation of high-performance plants under controlled environmental conditions (light, heat and humidity) (Potter, 2009), and such techniques have proven to provide notable consistency to tight tolerances as Good Manufacturing Practice for any pharmaceutical would require (Fischedick et al., 2010). The European Pharmacopoeia, Sixth Edition (2007), lists 28 EOs (Pauli and Schilcher, 2010). Terpenoids are pharmacologically versatile: they are lipophilic, interact with cell membranes, neuronal and muscle ion channels, neurotransmitter receptors, G-protein coupled (odorant) receptors, second messenger systems and enzymes (Bowles, 2003; Buchbauer, 2010). All the terpenoids discussed herein are Generally Recognized as Safe, as attested by the US Food and Drug AdminBJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1349 istration as food additives, or by the Food and Extract Manufacturers Association and other world regulatory bodies. Germane is the observation (Adams and Taylor, 2010) (p. 193), ‘With a high degree of confidence one may presume that EOs derived from food are likely to be safe’. Additionally, all the current entries are non-sensitizing to skin when fresh (Tisserand and Balacs, 1995; Adams and Taylor, 2010), but may cause allergic reactions at very low rates when oxidized (Matura et al., 2005). For additional pharmacological data on other common cannabis terpenoids not discussed herein (1,8-cineole, also known as eucalyptol, pulegone, a-terpineol, terpineol-4-ol, r-cymene, borneol and D-3-carene), please see McPartland and Russo (2001b). Are cannabis terpenoids actually relevant to the effects of cannabis? Terpenoid components in concentrations above 0.05% are considered of pharmacological interest (Adams and Taylor, 2010). Animal studies are certainly supportive (Buchbauer et al., 1993). Mice exposed to terpenoid odours inhaled from ambient air for 1 h demonstrated profound effects on activity levels, suggesting a direct pharmacological effect on the brain, even at extremely low serum concentrations (examples: linalool with 73% reduction in motility at 4.22 ng·mL-1 , pinene 13.77% increase at trace concentration, terpineol 45% reduction at 4.7 ng·mL-1 ). These levels are comparable to those of THC measured in humans receiving cannabis extracts yielding therapeutic effects in pain, or symptoms of multiple sclerosis in various randomized controlled trials (RCTs) (Russo, 2006; Huestis, 2007). Positive effects at undetectable serum concentrations with orange terpenes (primarily limonene, 35.25% increase in mouse activity), could be explainable on the basis of rapid redistribution and concentration in lipophilic cerebral structures. A similar rationale pertains to human studies (Komori et al., 1995), subsequently discussed. Limonene is highly bioavailable with 70% human pulmonary uptake (Falk-Filipsson et al., 1993), and a figure of 60% for pinene with rapid metabolism or redistribution (Falk et al., 1990). Ingestion and percutaneous absorption is also well documented in humans (Jäger et al., 1992): 1500 mg of lavender EO with 24.7% linalool (total 372 mg) was massaged into the skin of a 60 kg man for 10 min, resulting in a peak plasma concentration of 100 ng·mL-1 at 19 min, and a half-life of 13.76 min in serum (Jäger et al., 1992). EO mixtures (including limonene and pinene) also increase permeation of estradiol through mouse skin (Monti et al., 2002). Government-approved cannabis supplied to patients in national programmes in the Netherlands and Canada is gamma-irradiated to sterilize coliform bacteria, but the safety of this technique for a smoked and inhaled product has never been specifically tested. Gamma-radiation significantly reduced linalool titres in fresh cilantro (Fan and Sokorai, 2002), and myrcene and linalool in orange juice (Fan and Gates, 2001). D-limonene, common to the lemon and other citrus EOs (Table 2), is the second most widely distributed terpenoid in nature (Noma and Asakawa, 2010), and is the precursor to other monoterpenoids (Figure 2) through species-specific synthetic schemes. Unfortunately, these pathways have not yet been investigated in cannabis. The ubiquity of limonene serves, perhaps, as a demonstration of convergent evolution that supports an important ecological role for this monoterpene. Studies with varying methodology and dosing in citrus oils in mice suggest it to be a powerful anxiolytic agent (Carvalho-Freitas and Costa, 2002; Pultrini Ade et al., 2006), with one EO increasing serotonin in the prefrontal cortex, and dopamine (DA) in hippocampus mediated via 5-HT1A (Komiya et al., 2006). Compelling confirmatory evidence in humans was provided in a clinical study (Komori et al., 1995), in which hospitalized depressed patients were exposed to citrus fragrance in ambient air, with subsequent normalization of Hamilton Depression Scores, successful discontinuation of antidepressant medication in 9/12 patients and serum evidence of immune stimulation (CD4/8 ratio normalization). Limonene also produces apoptosis of breast cancer cells, and was employed at high doses in Phase II RCTs (Vigushin et al., 1998). Subsequent investigation in cancer treatment has centred on its immediate hepatic metabolite, perillic acid, which demonstrates anti-stress effects in rat brain (Fukumoto et al., 2008). A patent has been submitted, claiming that limonene effectively treats gastro-oesophageal reflux (Harris, 2010). Citrus EOs containing limonene proved effective against dermatophytes (Sanguinetti et al., 2007; Singh et al., 2010), and citrus EOs with terpenoid profiles resembling those in cannabis demonstrated strong radical scavenging properties (Choi et al., 2000). As noted above, limonene is highly bioavailable (Falk-Filipsson et al., 1993), and rapidly metabolized, but with indications of accumulation and retention in adipose tissues (e.g. brain). It is highly non-toxic (estimated human lethal dose 0.5–5 g·kg-1 ) and non-sensitizing (Von Burg, 1995) b-Myrcene is another common monoterpenoid in cannabis (Table 2) with myriad activities: diminishing inflammation via prostaglandin E-2 (PGE-2) (Lorenzetti et al., 1991), and blocking hepatic carcinogenesis by aflatoxin (DeOliveira et al., 1997). Interestingly, myrcene is analgesic in mice, but this action can be blocked by naloxone, perhaps via the a-2 adrenoreceptor (Rao et al., 1990). It is nonmutagenic in the Ames test (Gomes-Carneiro et al., 2005). Myrcene is a recognized sedative as part of hops preparations (Humulus lupulus), employed to aid sleep in Germany (Bisset and Wichtl, 2004). Furthermore, myrcene acted as a muscle relaxant in mice, and potentiated barbiturate sleep time at high doses (do Vale et al., 2002). Together, these data would support the hypothesis that myrcene is a prominent sedative terpenoid in cannabis, and combined with THC, may produce the ‘couch-lock’ phenomenon of certain chemotypes that is alternatively decried or appreciated by recreational cannabis consumers. a-Pinene is a bicyclic monoterpene (Table 2), and the most widely encountered terpenoid in nature (Noma and Asakawa, 2010). It appears in conifers and innumerable plant EOs, with an insect-repellent role. It is anti-inflammatory via PGE-1 (Gil et al., 1989), and is a bronchodilator in humans at low exposure levels (Falk et al., 1990). Pinene is a major component of Sideritis spp. (Kose et al., 2010) and Salvia spp. EOs (Ozek et al., 2010), both with prominent activity against MRSA (vide infra). Beyond this, it seems to be a broadspectrum antibiotic (Nissen et al., 2010). a-Pinene forms the biosynthetic base for CB2 ligands, such as HU-308 (Hanus et al., 1999). Perhaps most compelling, however, is its activity as an acetylcholinesterase inhibitor aiding memory (Perry et al., 2000), with an observed IC50 of 0.44 mM (Miyazawa BJP EB Russo 1350 British Journal of Pharmacology (2011) 163 1344–1364 Table 2 Cannabis Terpenoid Activity Table Terpenoid Structure Commonly encountered in Pharmacological activity (Reference) Synergistic cannabinoid Limonene H Lemon Potent AD/immunostimulant via inhalation (Komori et al., 1995) CBD Anxiolytic (Carvalho-Freitas and Costa, 2002; Pultrini Ade et al., 2006) via 5-HT1A (Komiya et al., 2006) CBD Apoptosis of breast cancer cells (Vigushin et al., 1998) CBD, CBG Active against acne bacteria (Kim et al., 2008) CBD Dermatophytes (Sanguinetti et al., 2007; Singh et al., 2010) CBG Gastro-oesophageal reflux (Harris, 2010) THC a-Pinene Pine Anti-inflammatory via PGE-1 (Gil et al., 1989) CBD Bronchodilatory in humans (Falk et al., 1990) THC Acetylcholinesterase inhibitor, aiding memory (Perry et al., 2000) THC?, CBD b-Myrcene Hops Blocks inflammation via PGE-2 (Lorenzetti et al., 1991) CBD Analgesic, antagonized by naloxone (Rao et al., 1990) CBD, THC Sedating, muscle relaxant, hypnotic (do Vale et al., 2002) THC Blocks hepatic carcinogenesis by aflatoxin (de Oliveira et al., 1997) CBD, CBG Linalool HO Lavender Anti-anxiety (Russo, 2001) CBD, CBG? Sedative on inhalation in mice (Buchbauer et al., 1993) THC Local anesthetic (Re et al., 2000) THC Analgesic via adenosine A2A (Peana et al., 2006) CBD Anticonvulsant/anti-glutamate (Elisabetsky et al., 1995) CBD, THCV, CBDV Potent anti-leishmanial (do Socorro et al., 2003) ? b-Caryophyllene Pepper AI via PGE-1 comparable phenylbutazone (Basile et al., 1988) CBD Gastric cytoprotective (Tambe et al., 1996) THC Anti-malarial (Campbell et al., 1997) ? Selective CB2 agonist (100 nM) (Gertsch et al., 2008) THC Treatment of pruritus? (Karsak et al., 2007) THC Treatment of addiction? (Xi et al., 2010) CBD Caryophyllene Oxide O Lemon balm Decreases platelet aggregation (Lin et al., 2003) THC Antifungal in onychomycosis comparable to ciclopiroxolamine and sulconazole (Yang et al., 1999) CBC,CBG Insecticidal/anti-feedant (Bettarini et al., 1993) THCA, CBGA Nerolidol OH Orange Sedative (Binet et al., 1972) THC, CBN Skin penetrant (Cornwell and Barry, 1994) – Potent antimalarial (Lopes et al., 1999, Rodrigues Goulart et al., 2004) ? Anti-leishmanial activity (Arruda et al., 2005) ? Phytol OH Green tea Breakdown product of chlorophyll – Prevents Vitamin A teratogenesis (Arnhold et al., 2002) – ↑GABA via SSADH inhibition (Bang et al., 2002) CBG Representative plants containing each terpenoid are displayed as examples to promote recognition, but many species contain them in varying concentrations. 5-HT, 5-hydroxytryptamine (serotonin); AD, antidepressant; AI, anti-inflammatory; CB1/CB2, cannabinoid receptor 1 or 2; GABA, gamma aminobutyric acid; PGE-1/PGE-2, prostaglandin E-1/prostaglandin E-2; SSADH, succinic semialdehyde dehydrogenase. BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1351 and Yamafuji, 2005). This feature could counteract short-term memory deficits induced by THC intoxication (vide infra). D-Linalool is a monoterpenoid alcohol (Table 2), common to lavender (Lavandula angustifolia), whose psychotropic anxiolytic activity has been reviewed in detail (Russo, 2001). Interestingly, linalyl acetate, the other primary terpenoid in lavender, hydrolyses to linalool in gastric secretions (Bickers et al., 2003). Linalool proved sedating to mouse activity on inhalation (Buchbauer et al., 1991; Jirovetz et al., 1992). In traditional aromatherapy, linalool is the likely suspect in the remarkable therapeutic capabilities of lavender EO to alleviate skin burns without scarring (Gattefosse, 1993). Pertinent to this, the local anaesthetic effects of linalool (Re et al., 2000) are equal to those of procaine and menthol (Ghelardini et al., 1999). Another explanation would be its ability to produce hot-plate analgesia in mice (P < 0.001) that was reduced by administration of an adenosine A2A antagonist (Peana et al., 2006). It is also anti-nociceptive at high doses in mice via ionotropic glutamate receptors (Batista et al., 2008). Linalool demonstrated anticonvulsant and antiglutamatergic activity (Elisabetsky et al., 1995), and reduced seizures as part of Ocimum basilicum EO after exposure to pentylenetetrazole, picrotoxin and strychnine (Ismail, 2006). Furthermore, linalool decreased K+ -stimulated glutamate release and uptake in mouse synaptosomes (Silva Brum et al., 2001). These effects were summarized (Nunes et al., 2010, p. 303): ‘Overall, it seems reasonable to argue that the modulation of glutamate and GABA neurotransmitter systems are likely to be the critical mechanism responsible for the sedative, anxiolytic and anticonvulsant properties of linalool and EOs containing linalool in significant proportions’. Linalool also proved to be a powerful anti-leishmanial agent (do Socorro et al., 2003), and as a presumed lavender EO component, decreased morphine opioid usage after inhalation versus placebo (P = 0.04) in gastric banding in morbidly obese surgical patients (Kim et al., 2007). b-Caryophyllene (Table 2) is generally the most common sesquiterpenoid encountered in cannabis (Mediavilla and Steinemann, 1997), wherein its evolutionary function may be due to its ability to attract insect predatory green lacewings, while simultaneously inhibiting insect herbivory (Langenheim, 1994). It is frequently the predominant terpenoid overall in cannabis extracts, particularly if they have been processed under heat for decarboxylation (Guy and Stott, 2005). Caryophyllene is common to black pepper (Piper nigrum) and Copaiba balsam (Copaifera officinalis) (Lawless, 1995). It is anti-inflammatory via PGE-1, comparable in potency to the toxic phenylbutazone (Basile et al., 1988), and an EO containing it was on par with etodolac and indomethacin (Ozturk and Ozbek, 2005). In contrast to the latter agents, however, caryophyllene was a gastric cytoprotective (Tambe et al., 1996), much as had been claimed in the past in treating duodenal ulcers in the UK with cannabis extract (Douthwaite, 1947). Caryophyllene may have contributed to antimalarial effects as an EO component (Campbell et al., 1997). Perhaps the greatest revelation regarding caryophyllene has been its demonstration as a selective full agonist at CB2 (100 nM), the first proven phytocannabinoid beyond the cannabis genus (Gertsch et al., 2008). Subsequent work has demonstrated that this dietary component produced antiinflammatory analgesic activity at the lowest dose of 5 mg·kg-1 in wild-type, but not CB2 knockout mice (Gertsch, 2008). Given the lack of attributed psychoactivity of CB2 agonists, caryophyllene offers great promise as a therapeutic compound, whether systemically, or in dermatological applications such as contact dermatitis (Karsak et al., 2007). Sensitization reactions are quite rare, and probably due to oxidized product (Skold et al., 2006). Nerolidol is a sesquiterpene alcohol with sedative properties (Binet et al., 1972), present as a low-level component in orange and other citrus peels (Table 2). It diminished experimentally induced formation of colon adenomas in rats (Wattenberg, 1991). It was an effective agent for enhancing skin penetration of 5-fluorouracil (Cornwell and Barry, 1994). This could be a helpful property in treating fungal growth, where it is also an inhibitor (Langenheim, 1994). It seems to have anti-protozoal parasite control benefits, as a potent antimalarial (Lopes et al., 1999; Rodrigues Goulart et al., 2004) and anti-leishmanial agent (Arruda et al., 2005). Nerolidol is nontoxic and non-sensitizing (Lapczynski et al., 2008). Caryophyllene oxide (Table 2) is a sesquiterpenoid oxide common to lemon balm (Melissa officinalis), and to the eucalyptus, Melaleuca stypheloides, whose EO contains 43.8% (Farag et al., 2004). In the plant, it serves as an insecticidal/ anti-feedant (Bettarini et al., 1993) and as broad-spectrum antifungal in plant defence (Langenheim, 1994). Analogously, the latter properties may prove therapeutic, as caryophyllene oxide demonstrated antifungal efficacy in a model of clinical onychomycosis comparable to ciclopiroxalamine and sulconazole, with an 8% concentration affecting eradication in 15 days (Yang et al., 1999). Caryophyllene oxide is non-toxic and non-sensitizing (Opdyke, 1983). This agent also demonstrates anti-platelet aggregation properties in vitro (Lin et al., 2003). Caryophyllene oxide has the distinction of being the component responsible for cannabis identification by drug-sniffing dogs (Stahl and Kunde, 1973). Phytol (Table 2) is a diterpene (McGinty et al., 2010), present in cannabis extracts, as a breakdown product of chlorophyll and tocopherol. Phytol prevented vitamin A-induced teratogenesis by inhibiting conversion of retinol to a harmful metabolite, all-trans-retinoic acid (Arnhold et al., 2002). Phytol increased GABA expression via inhibition of succinic semialdehyde dehydrogenase, one of its degradative enzymes (Bang et al., 2002). Thus, the presence of phytol could account for the alleged relaxing effect of wild lettuce (Lactuca sativa), or green tea (Camellia sinensis), despite the latter’s caffeine content. Selected possibilities for phytocannabinoid-terpenoid synergy Cannabis and acne AEA simulates lipid production in human sebocytes of sebaceous glands at low concentrations, but induces apoptosis at higher levels, suggesting that this system is under ECS control (Dobrosi et al., 2008). CBD 10–20 mM did not affect basal lipid synthesis in SZ95 sebocytes, but did block such stimulation by AEA and arachidonate (Biro et al., 2009). Higher doses of CBD (30–50 mM) induced sebocyte apoptosis, which was augmented in the presence of AEA. The effect of CBD to increase BJP EB Russo 1352 British Journal of Pharmacology (2011) 163 1344–1364 Ca++ was blocked by ruthenium red, a TRP-inhibitor. RNAmediated silencing of TRPV1 and TRPV3 failed to attenuate CBD effects, but experiments did support the aetiological role of TRPV4, a putative regulator of systemic osmotic pressure (T. Bíró, 2010, pers. comm.). Given the observed ability of CBD to be absorbed transcutaneously, it offers great promise to attenuate the increased sebum production at the pathological root of acne. Cannabis terpenoids could offer complementary activity. Two citrus EOs primarily composed of limonene inhibited Propionibacterium acnes, the key pathogen in acne (MIC 0.31 mL·mL-1 ), more potently than triclosan (Kim et al., 2008). Linalool alone demonstrated an MIC of 0.625 mL·mL-1 . Both EOs inhibited P. acnes-induced TNF-a production, suggesting an adjunctive anti-inflammatory effect. In a similar manner, pinene was the most potent component of a tea-tree eucalyptus EO in suppression of P. acnes and Staph spp. in another report (Raman et al., 1995). Considering the known minimal toxicities of CBD and these terpenoids and the above findings, new acne therapies utilizing whole CBD-predominant extracts, via multitargeting (Wagner and Ulrich-Merzenich, 2009), may present a novel and promising therapeutic approach that poses minimal risks in comparison to isotretinoin. MRSA MRSA accounted for 10% of cases of septicaemia and 18 650 deaths in the USA in 2005, a number greater than that attributable to human immunodeficiency virus/acquired immunodeficiency syndrome (Bancroft, 2007). Pure CBD and CBG powerfully inhibit MRSA (MIC 0.5–2 mg·mL-1 ) (Appendino et al., 2008). Amongst terpenoids, pinene was a major component of Sideritis erythrantha EO that was as effective against MRSA and other antibiotic-resistant bacterial strains as vancomycin and other agents (Kose et al., 2010). A Salvia rosifolia EO with 34.8% pinene was also effective against MRSA (MIC 125 mg·mL-1 ). The ability of monoterpenoids to enhance skin permeability and entry of other drugs may further enhance antibiotic benefits (Wagner and Ulrich-Merzenich, 2009). Given that CBG can be produced in selected cannabis chemotypes (de Meijer and Hammond, 2005; de Meijer et al., 2009a), with no residual THC as a possible drug abuse liability risk, a whole plant extract of a CBG-chemotype also expressing pinene would seem to offer an excellent, safe new antiseptic agent. Psychopharmacological applications: depression, anxiety, insomnia, dementia and addiction Scientific investigation of the therapeutic application of terpenoids in psychiatry has been hampered by methodological concerns, subjective variability of results and a genuine dearth of appropriate randomized controlled studies of high quality (Russo, 2001; Bowles, 2003; Lis-Balchin, 2010). The same is true of phytocannabinoids (Fride and Russo, 2006). Abundant evidence supports the key role of the ECS in mediating depression (Hill and Gorzalka, 2005a,b), as well as anxiety, whether induced by aversive stimuli, such as posttraumatic stress disorder (Marsicano et al., 2002) or pain (Hohmann et al., 2005), and psychosis (Giuffrida et al., 2004). With respect to the latter risk, the presence of CBD in smoked cannabis based on hair analysis seems to be a mitigating factor reducing its observed incidence (Morgan and Curran, 2008). A thorough review of cannabis and psychiatry is beyond the scope of this article, but several suggestions are offered with respect to possible therapeutic synergies operative with phytocannabinoids-terpenoid combinations. While the possible benefits of THC on depression remain controversial (Denson and Earleywine, 2006), much less worrisome would be CBD- or CBG-predominant preparations. Certainly the results obtained in human depression solely with a citrus scent (Komori et al., 1995), strongly suggest the possibility of synergistic benefit of a phytocannabinoid-terpenoid preparation. Enriched odour exposure in adult mice induced olfactory system neurogenesis (Rochefort et al., 2002), an intriguing result that could hypothetically support plasticity mechanisms in depression (Delgado and Moreno, 1999), and similar hypotheses with respect to the ECS in addiction treatment (Gerdeman and Lovinger, 2003). Phytocannabinoidterpenoid synergy might theoretically apply. The myriad effects of CBD on 5-HT1A activity provide a strong rationale for this and other phytocannabinoids as base compounds for treatment of anxiety. Newer findings, particularly imaging studies of CBD in normal individuals in anxiety models (Fusar-Poli et al., 2009; 2010; Crippa et al., 2010) support this hypothesis. Even more compelling is a recent randomized control trial of pure CBD in patients with social anxiety disorder with highly statistical improvements over placebo in anxiety and cognitive impairment (Crippa et al., 2011). Addition of anxiolytic limonene and linalool could contribute to the clinical efficacy of a CBD extract. THC was demonstrated effective in a small crossover clinical trial versus placebo in 11 agitated dementia patients with Alzheimer’s disease (Volicer et al., 1997). THC was also observed to be an acetylcholinesterase inhibitor in its own right, as well as preventing amyloid b-peptide aggregation in that disorder (Eubanks et al., 2006). Certainly, the antianxiety and anti-psychotic effects of CBD may be of additional benefit (Zuardi et al., 1991; 2006; Zuardi and Guimaraes, 1997). A recent study supports the concept that CBD, when present in significant proportion to THC, is capable of eliminating induced cognitive and memory deficits in normal subjects smoking cannabis (Morgan et al., 2010b). Furthermore, CBD may also have primary benefits on reduction of b-amyloid in Alzheimer’s disease (Iuvone et al., 2004; Esposito et al., 2006a,b). Psychopharmacological effects of limonene, pinene and linalool could putatively extend benefits in mood in such patients. The effects of cannabis on sleep have been reviewed (Russo et al., 2007), and highlight the benefits that can accrue in this regard, particularly with respect to symptom reduction permitting better sleep, as opposed to a mere hypnotic effect. Certainly, terpenoids with pain-relieving, anti-anxiety or sedative effects may supplement such activity, notably, caryophyllene, linalool and myrcene. BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1353 The issue of cannabis addiction remains controversial. Some benefit of oral THC has been noted in cannabis withdrawal (Hart et al., 2002; Haney et al., 2004). More intriguing, perhaps, are claims of improvement on other substance dependencies, particularly cocaine (Labigalini et al., 1999; Dreher, 2002). The situation with CBD is yet more promising. CBD and THC at doses of 4 mg·kg-1 i.p. potentiated extinction of cocaine- and amphetamine-induced conditioned place preference in rats, and CBD produced no hedonic effects of its own (Parker et al., 2004). CBD 5 mg·kg-1 ·d-1 in rats attenuated heroin-seeking behaviour by conditioned stimuli, even after a lapse of 2 weeks (Ren et al., 2009). A suggested mechanism of CBD relates to its ability to reverse changes in a-amino-3-hydroxyl-5-methyl-4- isoxazole-propionate glutamate and CB1 receptor expression in the nucleus accumbens induced by heroin. The authors proposed CBD as a treatment for heroin craving and addiction relapse. A recent study demonstrated the fascinating result that patients with damage to the insula due to cerebrovascular accident were able to quit tobacco smoking without relapse or urges (Naqvi et al., 2007), highlighting this structure as a critical neural centre mediating addiction to nicotine. Further study has confirmed the role of the insula in cocaine, alcohol and heroin addiction (Naqvi and Bechara, 2009; Naqvi and Bechara, 2010). In a provocative parallel, CBD 600 mg p.o. was demonstrated to deactivate functional magnetic resonance imaging (fMRI) activity in human volunteers in the left insula versus placebo (P < 0.01) without accompanying sedation or psychoactive changes (Borgwardt et al., 2008), suggesting the possibility that CBD could act as a pharmaceutical surrogate for insular damage in exerting an anti-addiction therapeutic benefit. Human studies have recently demonstrated that human volunteers smoking cannabis with higher CBD content reduced their liking for drugrelated stimuli, including food (Morgan et al., 2010a). The authors posited that CBD can modulate reinforcing properties of drugs of abuse, and help in training to reduce relapse to alcoholism. A single case report of a successful withdrawal from cannabis dependency utilizing pure CBD treatment was recently published (Crippa et al., 2010). Perhaps terpenoids can provide adjunctive support. In a clinical trial, 48 cigarette smokers inhaling vapour from an EO of black pepper (Piper nigrum), a mint-menthol mixture or placebo (Rose and Behm, 1994). Black pepper EO reduced nicotine craving significantly (P < 0.01), an effect attributed to irritation of the bronchial tree, simulating the act of cigarette smoking, but without nicotine or actual burning of material. Rather, might not the effect have been pharmacological? The terpenoid profile of black pepper suggests possible candidates: myrcene via sedation, pinene via increased alertness, or especially caryophyllene via CB2 agonism and a newly discovered putative mechanism of action in addiction treatment. CB2 is expressed in dopaminergic neurones in the ventral tegmental area and nucleus accumbens, areas mediating addictive phenomena (Xi et al., 2010). Activation of CB2 by the synthetic agonist JWH144 administered systemically, intranasally, or by microinjection into the nucleus accumbens in rats inhibited DA release and cocaine selfadministration. Caryophyllene, as a high-potency selective CB2 agonist (Gertsch et al., 2008), would likely produce similar effects, and have the advantage of being a nontoxic dietary component. All factors considered, CBD, with caryophyllene, and possibly other adjunctive terpenoids in the extract, offers significant promise in future addiction treatment. Taming THC: cannabis entourage compounds as antidotes to intoxication Various sources highlight the limited therapeutic index of pure THC, when given intravenously (D’Souza et al., 2004) or orally (Favrat et al., 2005), especially in people previously naïve to its effects. Acute overdose incidents involving THC or THC-predominant cannabis usually consist of self-limited panic reactions or toxic psychoses, for which no pharmacological intervention is generally necessary, and supportive counselling (reassurance or ‘talking down’) is sufficient to allow resolution without sequelae. CBD modulates the psychoactivity of THC and reduces its adverse event profile (Russo and Guy, 2006), highlighted by recent results above described. Could it be, however, that other cannabis components offer additional attenuation of the less undesirable effects of THC? History provides some clues. In 10th century Persia, Al-Razi offered a prescription in his Manafi al-agdhiya wa-daf madarri-ha (p. 248), rendered (Lozano, 1993, p. 124; translation EBR) ‘ – and to avoid these harms {from ingestion of cannabis seeds or hashish}, one should drink fresh water and ice or eat any acid fruits’. This concept was repeated in various forms by various authorities through the ages, including ibn Sina (ibn Sina (Avicenna), 1294), and Ibn al-Baytar (ibn al-Baytar, 1291), until O’Shaughnessy brought Indian hemp to Britain in 1843 (O’Shaughnessy, 1843). Robert Christison subsequently cited lemon (Figure 3A) as an antidote to acute intoxication in numerous cases (Christison, 1851) and this excerpt regarding morning-after residua (Christison, 1848) (p. 973): Next morning there was an ordinary appetite, much torpidity, great defect and shortness of memory, extreme apparent protraction of time, but no peculiarity of articulation or other effect; and these symptoms lasted until 2 P.M., when they ceased entirely in a few minutes after taking lemonade. Literary icons on both sides of the Atlantic espoused similar support for the citrus cure in the 19th century, notably Bayard Taylor after travels in Syria (Taylor, 1855), and Fitzhugh Ludlow after his voluntary experiments with ever higher cannabis extract doses in the USA (Ludlow, 1857). The sentiment was repeated by Calkins (1871), who noted the suggestion of a friend in Tunis that lemon retained the confidence of cure of overdoses by cannabis users in that region. This is supported by the observation that lemon juice, which normally contains small terpenoid titres, is traditionally enhanced in North Africa by the inclusion in drinks of the limonene-rich rind, as evidenced by the recipe for Agua Limón from modern Morocco (Morse and Mamane, 2001). In his comprehensive review of cannabis in the first half of the 20th century, Walton once more supported its prescription (Walton, 1938). BJP EB Russo 1354 British Journal of Pharmacology (2011) 163 1344–1364 Another traditional antidote to cannabis employing Acorus calamus (Figure 3B) is evident from the Ayurvedic tradition of India (Lad, 1990, p. 131): Calamus root is the best antidote for the ill effects of marijuana. . . . if one smokes a pinch of calamus root powder with the marijuana, this herb will completely neutralize the toxic side effects of the drug. This claim has gained credence, not only through force of anecdotal accounts that abound on the Internet, but with formal scientific case reports and scientific analysis (McPartland et al., 2008) documenting clearer thinking and improved memory with the cannabis–calamus combination, and with provision of a scientific rationale: calamus contains beta-asarone, an acetylcholinesterase inhibitor with 10% of the potency of physotigmine (Mukherjee et al., 2007). Interestingly, the cannabis terpenoid, a-pinene, also has been characterized as a potent inhibitor of that enzyme (Miyazawa and Yamafuji, 2005), bolstering the hypothesis of a second antidote to THC contained in cannabis itself. Historical precedents also support pinene in this pharmacological role. In the firstt century, Pliny wrote of cannabis in his Natural History, Book XXIV (Pliny, 1980, p. 164): The gelotophyllis [‘leaves of laughter’ = cannabis] grows in Bactria and along the Borysthenes. If this be taken in myrrh and wine all kinds of phantoms beset the mind, causing laughter which persists until the kernels of pinenuts are taken with pepper and honey in palm wine. Of the components, palm wine is perhaps the most mysterious. Ethanol does not reduce cannabis intoxication (Mello and Mendelson, 1978). However, ancient wines were stored in clay pots or goatskins, and required preservation, usually with addition of pine tar or terebinth resin (from Pistacia spp.; McGovern et al., 2009). Pine tar is rich in pinene, as is terebinth resin (from Pistacia terebinthus; Tsokou et al., 2007), while the latter also contains limonene (Duru et al., 2003). Likewise, the pine nuts (Figure 3C) prescribed by Pliny the Elder harbour pinene, along with additional limonene (Salvadeo et al., 2007). Al-Ukbari also suggested pistachio nuts as a cannabis antidote in the 13th century (Lozano, 1993), and the ripe fruits of Pistacia terebinthus similarly contain pinene (Couladis et al., 2003). The black pepper (Figure 3D), might offer the mental clarity afforded by pinene, sedation via myrcene and helpful contributions by b-caryophyllene. The historical suggestions for cannabis antidotes are thus supported by modern scientific rationales for the claims, and if proven experimentally would provide additional evidence of synergy (Berenbaum, 1989; Wagner and Ulrich-Merzenich, 2009). Conclusions and suggestions for future study Considered ensemble, the preceding body of information supports the concept that selective breeding of cannabis chemotypes rich in ameliorative phytocannabinoid and terpenoid content offer complementary pharmacological activities that may strengthen and broaden clinical applications and improve the therapeutic index of cannabis extracts containing THC, or other base phytocannabinoids. Psychopharmacological and dermatological indications show the greatest promise. Figure 3 Ancient cannabis antidotes. (A) Lemon (Citrus limon). (B) Calamus plant roots (Acorus calamus). (C) Pine nuts (Pinus spp.). (D) Black pepper (Piper nigrum). BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1355 One important remaining order of business is the elucidation of mono- and sesquiterpenoid biosynthetic pathways in cannabis, as has been achieved previously in other species of plants (Croteau, 1987; Gershenzon and Croteau, 1993; Bohlmann et al., 1998; Turner et al., 1999; Trapp and Croteau, 2001). Various cannabis component combinations or cannabis extracts should be examined via high throughput pharmacological screening where not previously accomplished. Another goal is the investigation of the biochemical targets of the cannabis terpenoids, along with their mechanisms of action, particularly in the central nervous system. Possible techniques for such research include radio-labelling of select agents in animals with subsequent necropsy. On a molecular level, investigation of terpenoid changes to phytocannabinoid signal transduction and trafficking may prove illuminating. While it is known that terpenoids bind to odorant receptors in the nasal mucosa (Friedrich, 2004) and proximal olfactory structures (Barnea et al., 2004), it would be essential to ascertain if direct effects in limbic or other cerebral structures are operative. Given that farnesyl pyrophosphate is a sesquiterpenoid precursor and the most potent endogenous agonist yet discovered for GPR92 (McHugh et al., 2010), in silico studies attempting to match minor cannabinoids and terpenoids to orphan GPCRs may prove fruitful. Behavioural assays of agents in animal models may also provide clues. Simple combinations of phytocannabinoids and terpenoids may demonstrate synergy as antibiotics if MICs are appreciable lowered (Wagner and Ulrich-Merzenich, 2009). Ultimately, fMRI and single photon emission computed tomography studies in humans, with simultaneous drug reaction questionnaires and psychometric testing employing individual agents and phytocannabinoid-terpenoid pairings via vaporization or oromucosal application, would likely offer safe and effective methods to investigate possible interactions and synergy. Should positive outcomes result from such studies, phytopharmaceutical development may follow. The development of zero-cannabinoid cannabis chemotypes (de Meijer et al., 2009b) has provided extracts that will facilitate discernment of the pharmacological effects and contributions of different fractions. Breeding work has already resulted in chemotypes that produce 97% of monoterpenoid content as myrcene, or 77% as limonene (E. de Meijer, pers. comm.). Selective cross-breeding of high-terpenoid- and highphytocannabinoid-specific chemotypes has thus become a rational target that may lead to novel approaches to such disorders as treatment-resistant depression, anxiety, drug dependency, dementia and a panoply of dermatological disorders, as well as industrial applications as safer pesticides and antiseptics. A better future via cannabis phytochemistry may be an achievable goal through further research of the entourage effect in this versatile plant that may help it fulfil its promise as a pharmacological treasure trove. Acknowledgements The author offers appreciation to the following individuals, who provided materials and/or consultation: David Potter, Etienne de Meijer, John McPartland, David Watson, Rob Clarke, Indalecio Lozano, Támas Bíró, José Crippa, Roger Pertwee, Colin Stott, Vincenzo Di Marzo, Luciano De Petrocellis, Patrick McGovern, John Riddle and Elisaldo Carlini. Most of all, I would like to thank Raphael Mechoulam for his example, guidance, friendship, a life of good works and for listening to many ‘crazy ideas’. Conflict of Interest The author is a Senior Medical Advisor to GW Pharmaceuticals and serves as a consultant. References Adams TB, Taylor SV (2010). Safety evaluation of essential oils: a constituent-based approach. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 185–208. Alexander SP, Mathie A, Peters JA (2009). Guide to Receptors and Channels (GRAC), 4th edition. Br J Pharmacol 158 (Suppl. 1): S1–254. Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M et al. (2008). Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. J Nat Prod 71: 1427–1430. Arnhold T, Elmazar MM, Nau H (2002). Prevention of vitamin A teratogenesis by phytol or phytanic acid results from reduced metabolism of retinol to the teratogenic metabolite, all-trans-retinoic acid. Toxicol Sci 66: 274–282. Arruda DC, D’Alexandri FL, Katzin AM, Uliana SR (2005). Antileishmanial activity of the terpene nerolidol. Antimicrob Agents Chemother 49: 1679–1687. Baek SH, Kim YO, Kwag JS, Choi KE, Jung WY, Han DS (1998). Boron trifluoride etherate on silica-A modified Lewis acid reagent (VII). Antitumor activity of cannabigerol against human oral epitheloid carcinoma cells. Arch Pharm Res 21: 353–356. Bancroft EA (2007). Antimicrobial resistance: it’s not just for hospitals. JAMA 298: 1803–1804. Banerjee SP, Snyder SH, Mechoulam R (1975). Cannabinoids: influence on neurotransmitter uptake in rat brain synaptosomes. J Pharmacol Exp Ther 194: 74–81. Bang MH, Choi SY, Jang TO, Kim SK, Kwon OS, Kang TC et al. (2002). Phytol, SSADH inhibitory diterpenoid of Lactuca sativa. Arch Pharm Res 25: 643–646. Barnea G, O’Donnell S, Mancia F, Sun X, Nemes A, Mendelsohn M et al. (2004). Odorant receptors on axon termini in the brain. Science 304: 1468. Basile AC, Sertie JA, Freitas PC, Zanini AC (1988). Anti-inflammatory activity of oleoresin from Brazilian Copaifera. J Ethnopharmacol 22: 101–109. Batista PA, Werner MF, Oliveira EC, Burgos L, Pereira P, Brum LF et al. (2008). Evidence for the involvement of ionotropic glutamatergic receptors on the antinociceptive effect of (-)-linalool in mice. Neurosci Lett 440: 299–303. Ben-Shabat S, Fride E, Sheskin T, Tamiri T, Rhee MH, Vogel Z et al. (1998). An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity. Eur J Pharmacol 353: 23–31. BJP EB Russo 1356 British Journal of Pharmacology (2011) 163 1344–1364 Berenbaum MC (1989). What is synergy? Pharmacol Rev 41: 93–141. Bettarini F, Borgonovi GE, Fiorani T, Gagliardi I, Caprioli V, Massardo P et al. (1993). Antiparasitic compounds from East African plants: isolation and biological activtiry of anonaine, matricarianol, canthin-6-one, and caryophyllene oxide. Insect Sci Appl 14: 93–99. Bickers D, Calow P, Greim H, Hanifin JM, Rogers AE, Saurat JH et al. (2003). A toxicologic and dermatologic assessment of linalool and related esters when used as fragrance ingredients. Food Chem Toxicol 41: 919–942. Binet L, Binet P, Miocque M, Roux M, Bernier A (1972). Recherches sur les proprietes pharmcodynamiques (action sedative et action spasmolytique) de quelques alcools terpeniques aliphatiques. Ann Pharm Fr 30: 611–616. Biro T, Olah A, Toth BI, Czifra G, Zouboulis CC, Paus R (2009). Cannabidiol as a novel anti-acne agent? Cannabidiol inhibits lipid synthesis and induces cell death in human sebaceous gland-derived sebocytes. Proceedings 19th Annual Conference on the Cannabinoids. International Cannabinoid Research Society: Pheasant Run, St. Charles, IL, p. 28. Bisogno T, Hanus L, De Petrocellis L, Tchilibon S, Ponde DE, Brandi I et al. (2001). Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Br J Pharmacol 134: 845–852. Bisset NG, Wichtl M (2004). Herbal Drugs and Phytopharmaceuticals: A Handbook for Practice on A Scientific Basis, 3rd edn. Medpharm Scientific Publishers: Stuttgart; CRC Press: Boca Raton, FL. Bloor RN, Wang TS, Spanel P, Smith D (2008). Ammonia release from heated ‘street’ cannabis leaf and its potential toxic effects on cannabis users. Addiction 103: 1671–1677. Bohlmann J, Meyer-Gauen G, Croteau R (1998). Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95: 4126–4133. Bolognini D, Costa B, Maione S, Comelli F, Marini P, Di Marzo V et al. (2010). The plant cannabinoid Delta9-tetrahydrocannabivarin can decrease signs of inflammation and inflammatory pain in mice. Br J Pharmacol 160: 677–687. Borgwardt SJ, Allen P, Bhattacharyya S, Fusar-Poli P, Crippa JA, Seal ML et al. (2008). Neural basis of Delta-9-tetrahydrocannabinol and cannabidiol: effects during response inhibition. Biol Psychiatry 64: 966–973. Bowles EJ (2003). The Chemistry of Aromatherapeutic Oils, 3rd edn. Allen & Unwin: Crow’s Nest, NSW. Bradshaw HB, Lee SH, McHugh D (2009). Orphan endogenous lipids and orphan GPCRs: a good match. Prostaglandins Other Lipid Mediat 89: 131–134. Brenneisen R (2007). Chemistry and analysis of phytocannabinoids and other Cannabis constituents. In: Elsohly M (ed.). Marijuana and the Cannabinoids. Humana Press: Totowa, NY, pp. 17–49. Buchbauer G (2010). Biological activities of essential oils. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 235–280. Buchbauer G, Jirovetz L, Jager W, Dietrich H, Plank C (1991). Aromatherapy: evidence for sedative effects of the essential oil of lavender after inhalation. Z Naturforsch [C] 46: 1067–1072. Buchbauer G, Jirovetz L, Jager W, Plank C, Dietrich H (1993). Fragrance compounds and essential oils with sedative effects upon inhalation. J Pharm Sci 82: 660–664. Calkins A (1871). Opium and the Opium-Appetite: with Notices of Alcoholic Beverages, Cannabis Indica, Tobacco and Coca, and Tea and Coffee, in Their Hygienic Aspects and Pathologic Relationships. J.B. Lippincott: Philadelphia, PA. Campbell WE, Gammon DW, Smith P, Abrahams M, Purves TD (1997). Composition and antimalarial activity in vitro of the essential oil of Tetradenia riparia. Planta Med 63: 270–272. Carlini EA, Cunha JM (1981). Hypnotic and antiepileptic effects of cannabidiol. J Clin Pharmacol 21 (Suppl.): 417S–427S. Carlini EA, Karniol IG, Renault PF, Schuster CR (1974). Effects of marihuana in laboratory animals and in man. Br J Pharmacol 50: 299–309. Carrier EJ, Auchampach JA, Hillard CJ (2006). Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. Proc Natl Acad Sci USA 103: 7895–7900. Carvalho-Freitas MI, Costa M (2002). Anxiolytic and sedative effects of extracts and essential oil from Citrus aurantium L. Biol Pharm Bull 25: 1629–1633. Cascio MG, Gauson LA, Stevenson LA, Ross RA, Pertwee RG (2010). Evidence that the plant cannabinoid cannabigerol is a highly potent alpha2-adrenoceptor agonist and moderately potent 5HT1A receptor antagonist. Br J Pharmacol 159: 129–141. Cawthorne MA, Wargent E, Zaibi M, Stott C, Wright S (2007). The CB1 antagonist, delta-9-tetrahydrocannabivarin (THCV) has antioebesity activity in dietary-induced obese (DIO) mice. Proceedings 17th Annual Symposium on the Cannabinoids. International Cannabinoid Research Society: Saint-Sauveur, QC, p. 141. Choi HS, Song HS, Ukeda H, Sawamura M (2000). Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2-picrylhydrazyl. J Agric Food Chem 48: 4156–4161. Christison R (1848). A Dispensatory Or Commentary on the Pharmacopoeias of Great Britain and the United States. Lea and Blanchard: Philadelphia, PA. Christison A (1851). On the natural history, action, and uses of Indian hemp. Monthly J Med Sci Edinburgh, Scotland 13: 26–45. 117-121. Clarke RC (2010). Hashish!, 2nd edn. Red Eye Press: Los Angeles, CA. Comelli F, Bettoni I, Colleoni M, Giagnoni G, Costa B (2009). Beneficial effects of a Cannabis sativa extract treatment on diabetes-induced neuropathy and oxidative stress. Phytother Res 23: 1678–1684. Cornwell PA, Barry BW (1994). Sesquiterpene components of volatile oils as skin penetration enhancers for the hydrophilic permeant 5-fluorouracil. J Pharm Pharmacol 46: 261–269. Costa B, Trovato AE, Comelli F, Giagnoni G, Colleoni M (2007). The non-psychoactive cannabis constituent cannabidiol is an orally effective therapeutic agent in rat chronic inflammatory and neuropathic pain. Eur J Pharmacol 556: 75–83. Couladis M, Ozcan M, Tzakou O, Akgul A (2003). Comparative essential oil compostion of various parts of the turpentine tree (Pistacia terebinthus) growing wild in Turkey. J Sci Food Agric 83: 136–138. BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1357 Crippa JA, Zuardi AW, Hallak JE (2010). [Therapeutical use of the cannabinoids in psychiatry]. Rev Bras Psiquiatr 32 (Suppl. 1): S56–S66. Crippa JA, Derenusson GN, Ferrari TB, Wichert-Ana L, Duran F, Marti NSRO et al. (2011). Neural basis of anxiolytic effects of cannabidiol (CBD) in generalized social anxiety disorder: a preliminary report. J Psychopharmacol 25: 121–130. Croteau R (1987). Biosynthesis and catabolism of monoterpenoids. Chem Rev 87: 929–954. De Oliveira AC, Ribeiro-Pinto LF, Paumgartten JR (1997). In vitro inhibition of CYP2B1 monooxygenase by beta-myrcene and other monoterpenoid compounds. Toxicol Lett 92: 39–46. D’Souza DC, Perry E, MacDougall L, Ammerman Y, Cooper T, Wu YT et al. (2004). The psychotomimetic effects of intravenous delta-9-tetrahydrocannabinol in healthy individuals: implications for psychosis. Neuropsychopharmacology 29: 1558–1572. Davalos SD, Fournier G, Boucher F, Paris M (1977). [Contribution to the study of Mexican marihuana. Preliminary studies: cannabinoids and essential oil (author’s transl)]. J Pharm Belg 32: 89–99. Davis WM, Hatoum NS (1983). Neurobehavioral actions of cannabichromene and interactions with delta 9-tetrahydrocannabinol. Gen Pharmacol 14: 247–252. De Oliveira AC, Ribeiro-Pinto LF, Paumgartten JR (1997). In vitro inhibition of CYP2B1 monooxygenase by beta-myrcene and other monoterpenoid compounds. Toxicol Lett 92: 39–46. De Petrocellis L, Di Marzo V (2010). Non-CB1, non-CB2 receptors for endocannabinoids, plant cannabinoids, and synthetic cannabimimetics: focus on G-protein-coupled receptors and transient receptor potential channels. J Neuroimmune Pharmacol 5: 103–121. De Petrocellis L, Vellani V, Schiano-Moriello A, Marini P, Magherini PC, Orlando P et al. (2008). Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8. J Pharmacol Exp Ther 325: 1007–1015. De Petrocellis L, Ligresti A, Moriello AS, Allara M, Bisogno T, Petrosino S et al. (2011). Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes. Br J Pharmacol DOI:10.1111/j.1476-5381.2010.0166.x Delgado P, Moreno F (1999). Antidepressants and the brain. Int Clin Psychopharmacol 14 (Suppl. 1): S9–16. Delong GT, Wolf CE, Poklis A, Lichtman AH (2010). Pharmacological evaluation of the natural constituent of Cannabis sativa, cannabichromene and its modulation by Delta(9)-tetrahydrocannabinol. Drug Alcohol Depend 112: 126–133. Denson TF, Earleywine M (2006). Decreased depression in marijuana users. Addict Behav 31: 738–742. Devane WA, Dysarz FA 3rd, Johnson MR, Melvin LS, Howlett AC (1988). Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol 34: 605–613. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G et al. (1992). Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258: 1946–1949. Deyo R, Musty R (2003). A cannabichromene (CBC) extract alters behavioral despair on the mouse tail suspension test of depression. Proceedings 2003 Symposium on the Cannabinoids. International Cannabinoid Research Society: Cornwall, ON, p. 146. Dirikoc S, Priola SA, Marella M, Zsurger N, Chabry J (2007). Nonpsychoactive cannabidiol prevents prion accumulation and protects neurons against prion toxicity. J Neurosci 27: 9537–9544. Dobrosi N, Toth BI, Nagy G, Dozsa A, Geczy T, Nagy G et al. (2008). Endocannabinoids enhance lipid synthesis and apoptosis of human sebocytes via cannabinoid receptor-2-mediated signaling. FASEB J 22: 3685–3695. Douthwaite AH (1947). Choice of drugs in the treatment of duodenal ulcer. Br Med J 2: 43–47. Dreher M (2002). Crack heads and roots daughters: the therapeutic use of cannabis in Jamaica. J Cannabis Therap 2: 121–133. Duru ME, Cakir A, Kordali S, Zengin H, Harmandar M, Izumi S et al. (2003). Chemical composition and antifungal properties of essential oils of three Pistacia species. Fitoterapia 74: 170–176. Elisabetsky E, Marschner J, Souza DO (1995). Effects of Linalool on glutamatergic system in the rat cerebral cortex. Neurochem Res 20: 461–465. ElSohly HN, Turner CE, Clark AM, ElSohly MA (1982). Synthesis and antimicrobial activities of certain cannabichromene and cannabigerol related compounds. J Pharm Sci 71: 1319–1323. Esposito G, De Filippis D, Carnuccio R, Izzo AA, Iuvone T (2006a). The marijuana component cannabidiol inhibits beta-amyloid-induced tau protein hyperphosphorylation through Wnt/beta-catenin pathway rescue in PC12 cells. J Mol Med 84: 253–258. Esposito G, De Filippis D, Maiuri MC, De Stefano D, Carnuccio R, Iuvone T (2006b). Cannabidiol inhibits inducible nitric oxide synthase protein expression and nitric oxide production in beta-amyloid stimulated PC12 neurons through p38 MAP kinase and NF-kappaB involvement. Neurosci Lett 399: 91–95. Eubanks LM, Rogers CJ, Beuscher AE 4th, Koob GF, Olson AJ, Dickerson TJ et al. (2006). A molecular link between the active component of marijuana and Alzheimer’s disease pathology. Mol Pharm 3: 773–777. Evans FJ (1991). Cannabinoids: the separation of central from peripheral effects on a structural basis. Planta Med 57: S60–S67. Fairbairn JW, Pickens JT (1981). Activity of cannabis in relation to its delta’-trans-tetrahydro-cannabinol content. Br J Pharmacol 72: 401–409. Falk AA, Hagberg MT, Lof AE, Wigaeus-Hjelm EM, Wang ZP (1990). Uptake, distribution and elimination of alpha-pinene in man after exposure by inhalation. Scand J Work Environ Health 16: 372–378. Falk-Filipsson A, Lof A, Hagberg M, Hjelm EW, Wang Z (1993). d-limonene exposure to humans by inhalation: uptake, distribution, elimination, and effects on the pulmonary function. J Toxicol Environ Health 38: 77–88. Fan X, Gates RA (2001). Degradation of monoterpenes in orange juice by gamma radiation. J Agric Food Chem 49: 2422–2426. Fan X, Sokorai KJ (2002). Changes in volatile compounds of gamma-irradiated fresh cilantro leaves during cold storage. J Agric Food Chem 50: 7622–7626. Farag RS, Shalaby AS, El-Baroty GA, Ibrahim NA, Ali MA, Hassan EM (2004). Chemical and biological evaluation of the essential oils of different Melaleuca species. Phytother Res 18: 30–35. Favrat B, Menetrey A, Augsburger M, Rothuizen L, Appenzeller M, Buclin T et al. (2005). Two cases of ‘cannabis acute psychosis’ following the administration of oral cannabis. BMC Psychiatry 5: 17. BJP EB Russo 1358 British Journal of Pharmacology (2011) 163 1344–1364 Fellermeier M, Eisenreich W, Bacher A, Zenk MH (2001). Biosynthesis of cannabinoids. Incorporation experiments with (13)C-labeled glucoses. Eur J Biochem 268: 1596–1604. Fischedick JT, Hazekamp A, Erkelens T, Choi YH, Verpoorte R (2010). Metabolic fingerprinting of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. Phytochem 71: 2058–2073. Formukong EA, Evans AT, Evans FJ (1988). Analgesic and antiinflammatory activity of constituents of Cannabis sativa L. Inflammation 12: 361–371. Franz C, Novak J (2010). Sources of essential oils. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 39–82. Fride E, Russo EB (2006). Neuropsychiatry: Schizophrenia, depression, and anxiety. In: Onaivi E, Sugiura T, Di Marzo V (eds). Endocannabinoids: The Brain and Body’s Marijuana and beyond. Taylor & Francis: Boca Raton, FL, pp. 371–382. Friedrich RW (2004). Neurobiology: odorant receptors make scents. Nature 430: 511–512. Fukumoto S, Morishita A, Furutachi K, Terashima T, Nakayama T, Yokogoshi H (2008). Effect of flavour components in lemon essential oil on physical or psychological stress. Stress Health 24: 3–12. Fusar-Poli P, Allen P, Bhattacharyya S, Crippa JA, Mechelli A, Borgwardt S et al. (2010). Modulation of effective connectivity during emotional processing by Delta9-tetrahydrocannabinol and cannabidiol. Int J Neuropsychopharmacol 13: 421–432. Fusar-Poli P, Crippa JA, Bhattacharyya S, Borgwardt SJ, Allen P, Martin-Santos R et al. (2009). Distinct effects of {delta}9-tetrahydrocannabinol and cannabidiol on neural activation during emotional processing. Archiv Gen Psychiatr 66: 95–105. Gaoni Y, Mechoulam R (1964a). Isolation, structure and partial synthesis of an active constituent of hashish. J Am Chem Soc 86: 1646–1647. Gaoni Y, Mechoulam R (1964b). The structure and function of cannabigerol, a new hashish constituent. Proc Chem Soc 1: 82. Gaoni Y, Mechoulam R (1966). Cannabichromene, a new active principle in hashish. Chem Commun 1: 20–21. Gattefosse R-M (1993). Gatefosse’s Aromatherapy. C.W. Daniel: Essex, MD. Gauson LA, Stevenson LA, Thomas A, Baillie GL, Ross RA, Pertwee RG (2007). Cannabigerol behaves as a partial agonist at both CB1 and CB2 receptors. Proceedings 17th Annual Symposium on the Cannabinoids. International Cannabinoid Research Society: Saint-Sauveur, QC, p. 206. Gerdeman GL, Lovinger DM (2003). Emerging roles for endocannabinoids in long-term synaptic plasticity. Br J Pharmacol 140: 781–789. Gershenzon J (1994). Metabolic costs of terpenoid accumulation in higher plants. J Chem Ecol 20: 1281–1328. Gershenzon J, Croteau R (1993). Terepenoid Biosynthesis: the basic pathway and formation of monoterpenes, sequiterpenes, and diterpenes. In: Moore TS (ed.). Lipid Metabolism in Plants. CRC Press: Boca Raton, FL, pp. 339–388. Gertsch J (2008). Anti-inflammatory cannabinoids in diet: towards a better understanding of CB(2) receptor action? Commun Integr Biol 1: 26–28. Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ et al. (2008). Beta-caryophyllene is a dietary cannabinoid. Proc Natl Acad Sci USA 105: 9099–9104. Ghelardini C, Galeotti N, Salvatore G, Mazzanti G (1999). Local anaesthetic activity of the essential oil of Lavandula angustifolia. Planta Med 65: 700–703. Gil ML, Jimenez J, Ocete MA, Zarzuelo A, Cabo MM (1989). Comparative study of different essential oils of Bupleurum gibraltaricum Lamarck. Pharmazie 44: 284–287. Gill EW, Paton WD, Pertwee RG (1970). Preliminary experiments on the chemistry and pharmacology of cannabis. Nature 228: 134–136. Giuffrida A, Leweke FM, Gerth CW, Schreiber D, Koethe D, Faulhaber J et al. (2004). Cerebrospinal anandamide levels are elevated in acute schizophrenia and are inversely correlated with psychotic symptoms. Neuropsychopharmacol 29: 2108–2114. Gomes-Carneiro MR, Viana ME, Felzenszwalb I, Paumgartten FJ (2005). Evaluation of beta-myrcene, alpha-terpinene and (+)- and (-)-alpha-pinene in the Salmonella/microsome assay. Food Chem Toxicol 43: 247–252. Guy GW, Stott CG (2005). The development of Sativex- a natural cannabis-based medicine. In: Mechoulam R (ed.). Cannabinoids As Therapeutics. Birkhäuser Verlag: Basel, pp. 231–263. Hampson AJ, Grimaldi M, Axelrod J, Wink D (1998). Cannabidiol and (-)Delta9-tetrahydrocannabinol are neuroprotective antioxidants. Proc Natl Acad Sci USA 95: 8268–8273. Haney M, Hart CL, Vosburg SK, Nasser J, Bennett A, Zubaran C et al. (2004). Marijuana withdrawal in humans: effects of oral THC or divalproex. Neuropsychopharmacol 29: 158–170. Hanus L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M et al. (1999). HU-308: a specific agonist for CB(2), a peripheral cannabinoid receptor. Proc Natl Acad Sci USA 96: 14228–14233. Harris B (2010). Phytotherapeutic uses of essential oils. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 315–352. Hart CL, Haney M, Ward AS, Fischman MW, Foltin RW (2002). Effects of oral THC maintenance on smoked marijuana self-administration. Drug Alcohol Depend 67: 301–309. Hatoum NS, Davis WM, Elsohly MA, Turner CE (1981). Cannabichromene and delta 9-tetrahydrocannabinol: interactions relative to lethality, hypothermia and hexobarbital hypnosis. Gen Pharmacol 12: 357–362. Hendriks H, Malingré TM, Batterman S, Bos R (1975). Mono- and sesqui-terpene hydrocarbons of the eseential oil of Cannabis sativa. Phytochem 14: 814–815. Hendriks H, Malingré TM, Batterman S, Bos R (1977). Alkanes of the essential oil of Cannabis sativa. Phytochem 16: 719–721. Hill MN, Gorzalka BB (2005a). Is there a role for the endocannabinoid system in the etiology and treatment of melancholic depression? Behav Pharmacol 16: 333–352. Hill MN, Gorzalka BB (2005b). Pharmacological enhancement of cannabinoid CB1 receptor activity elicits an antidepressant-like response in the rat forced swim test. Eur Neuropsychopharmacol 15: 593–599. Hill AJ, Weston SE, Jones NA, Smith I, Bevan SA, Williamson EM et al. (2010). Delta-Tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult rats. Epilepsia 51: 1522–1532. BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1359 Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D, Mangieri R et al. (2005). An endocannabinoid mechanism for stress-induced analgesia. Nature 435: 1108–1112. Holland ML, Allen JD, Arnold JC (2008). Interaction of plant cannabinoids with the multidrug transporter ABCC1 (MRP1). Eur J Pharmacol 591: 128–131. Hollister LE (1974). Structure-activity relationships in man of cannabis constituents, and homologs and metabolites of delta9-tetrahydrocannabinol. Pharmacol 11: 3–11. Hood LV, Dames ME, Barry GT (1973). Headspace volatiles of marijuana. Nature 242: 402–403. Howlett AC (1987). Cannabinoid inhibition of adenylate cyclase: relative activity of constituents and metabolites of marihuana. Neuropharmacol 26: 507–512. Huestis MA (2007). Human Cannabinoid Pharmacokinetics. Chem Biodivers 4: 1770–1804. ibn al-Baytar (1291) Kitab al-Yami’ li-mufradat al-adwiya wa-l-agdiya. Bulaq: Egypt. ibn Sina (Avicenna) (1294). Al-Qanun fi l-tibb (Canon of medicine). Bulaq: Egypt. Ignatowska-Jankowska B, Jankowski M, Glac W, Swiergel AH (2009). Cannabidiol-induced lymphopenia does not involve NKT and NK cells. J Physiol Pharmacol 60 (Suppl. 3): 99–103. Ilan AB, Gevins A, Coleman M, ElSohly MA, de Wit H (2005). Neurophysiological and subjective profile of marijuana with varying concentrations of cannabinoids. Behav Pharmacol 16: 487–496. Ismail M (2006). Central properties and chemcial composition of Ocimum basilicum essential oil. Pharm Biol 44: 619–626. Iuvone T, Esposito G, Esposito R, Santamaria R, Di Rosa M, Izzo AA (2004). Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on beta-amyloid-induced toxicity in PC12 cells. J Neurochem 89: 134–141. Izzo AA, Borrelli F, Capasso R, Di Marzo V, Mechoulam R (2009). Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends Pharmacol Sci 30: 515–527. Jäger W, Buchbauer G, Jirovetz L, Fritzer M (1992). Percutaneous absorption of lavender oil from a massage oil. J Soc Cosmet Chem 43 (Jan/Feb): 49–54. Jirovetz L, Buchbauer G, Jager W, Woidich A, Nikiforov A (1992). Analysis of fragrance compounds in blood samples of mice by gas chromatography, mass spectrometry, GC/FTIR and GC/AES after inhalation of sandalwood oil. Biomed Chromatogr 6: 133–134. Johnson JR, Burnell-Nugent M, Lossignol D, Ganae-Motan ED, Potts R, Fallon MT (2010). Multicenter, double-blind, randomized, placebo-controlled, parallel-group study of the efficacy, safety, and tolerability of THC:CBD extract and THC extract in patients with intractable cancer-related pain. J Pain Symptom Manage 39: 167–179. Jones NA, Hill AJ, Smith I, Bevan SA, Williams CM, Whalley BJ et al. (2010). Cannabidiol displays antiepileptiform and antiseizure properties in vitro and in vivo. J Pharmacol Exp Ther 332: 569–577. Karsak M, Gaffal E, Date R, Wang-Eckhardt L, Rehnelt J, Petrosino S et al. (2007). Attenuation of allergic contact dermatitis through the endocannabinoid system. Science 316: 1494–1497. Kavia R, De Ridder D, Constantinescu C, Stott C, Fowler C (2010). Randomized controlled trial of Sativex to treat detrusor overactivity in multiple sclerosis. Mult Scler 16: 1349–1359. Kim JT, Ren CJ, Fielding GA, Pitti A, Kasumi T, Wajda M et al. (2007). Treatment with lavender aromatherapy in the post-anesthesia care unit reduces opioid requirements of morbidly obese patients undergoing laparoscopic adjustable gastric banding. Obes Surg 17: 920–925. Kim SS, Baik JS, Oh TH, Yoon WJ, Lee NH, Hyun CG (2008). Biological activities of Korean Citrus obovoides and Citrus natsudaidai essential oils against acne-inducing bacteria. Biosci Biotechnol Biochem 72: 2507–2513. King LA, Carpentier C, Griffiths P (2005). Cannabis potency in Europe. Addiction 100: 884–886. Komiya M, Takeuchi T, Harada E (2006). Lemon oil vapor causes an anti-stress effect via modulating the 5-HT and DA activities in mice. Behav Brain Res 172: 240–249. Komori T, Fujiwara R, Tanida M, Nomura J, Yokoyama MM (1995). Effects of citrus fragrance on immune function and depressive states. Neuroimmunomodulation 2: 174–180. Kose EO, Deniz IG, Sarikurkcu C, Aktas O, Yavuz M (2010). Chemical composition, antimicrobial and antioxidant activities of the essential oils of Sideritis erythrantha Boiss. and Heldr. (var. erythrantha and var. cedretorum P.H. Davis) endemic in Turkey. Food Chem Toxicol 48: 2960–2965. Labigalini E Jr, Rodrigues LR, Da Silveira DX (1999). Therapeutic use of cannabis by crack addicts in Brazil. J Psychoactive Drugs 31: 451–455. Lad V (1990). Ayurveda: the Science of Self-Healing: A Practical Guide. Lotus Light Publications: Milwaukee, WI. Langenheim JH (1994). Higher plant terpenoids: a phytocentric overview of their ecological roles. J Chem Ecol 20: 1223–1279. Lapczynski A, Bhatia SP, Letizia CS, Api AM (2008). Fragrance material review on nerolidol (isomer unspecified). Food Chem Toxicol 46 (Suppl. 11): S247–S250. Lawless J (1995). The Illustrated Encyclopedia of Essential Oils : the Complete Guide to the Use of Oils in Aromatherapy and Herbalism. Element: Shaftesbury, Dorset, [England]; Rockport, MA. Ligresti A, Moriello AS, Starowicz K, Matias I, Pisanti S, De Petrocellis L et al. (2006). Antitumor activity of plant cannabinoids with emphasis on the effect of cannabidiol on human breast carcinoma. J Pharmacol Exp Ther 318: 1375–1387. Lin WY, Kuo YH, Chang YL, Teng CM, Wang EC, Ishikawa T et al. (2003). Anti-platelet aggregation and chemical constituents from the rhizome of Gynura japonica. Planta Med 69: 757–764. Lis-Balchin M (2010). Aromatherapy with essential oils. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 549–584. Lopes NP, Kato MJ, Andrade EH, Maia JG, Yoshida M, Planchart AR et al. (1999). Antimalarial use of volatile oil from leaves of Virola surinamensis (Rol.) Warb. by Waiapi Amazon Indians. J Ethnopharmacol 67: 313–319. Lorenzetti BB, Souza GE, Sarti SJ, Santos Filho D, Ferreira SH (1991). Myrcene mimics the peripheral analgesic activity of lemongrass tea. J Ethnopharmacol 34: 43–48. Lozano I (1993). Estudios Y Documentos Sobre La Historia Del Cáñamo Y Del Hachís En El Islam Medievaldoctoral Dissertation. Universidad de Granada: Granada. BJP EB Russo 1360 British Journal of Pharmacology (2011) 163 1344–1364 Ludlow FH (1857). The Hasheesh Eater: Being Passages Form the Life of A Pythagorean. Harper: New York. McGinty D, Letizia CS, Api AM (2010). Fragrance material review on phytol. Food Chem Toxicol 48 (Suppl. 3): S59–S63. McGovern PE, Mirzoian A, Hall GR (2009). Ancient Egyptian herbal wines. Proc Natl Acad Sci USA 106: 7361–7366. McHugh D, Hu SS, Rimmerman N, Juknat A, Vogel Z, Walker JM et al. (2010). N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor. BMC Neurosci 11: 44. McPartland J (1984). Pathogenicity of Phomopsis ganjae on Cannabis sativa and the fungistatic effect of cannabinoids produced by the host. Mycopathologia 87: 149–153. McPartland JM, Pruitt PL (1999). Side effects of pharmaceuticals not elicited by comparable herbal medicines: the case of tetrahydrocannabinol and marijuana. Altern Ther Health Med 5: 57–62. McPartland JM, Mediavilla V (2001a). Non-cannabinoids in cannabis. In: Grotenhermen F, Russo EB (eds). Cannabis and Cannabinoids. NY: Haworth Press: Binghamton, NY, pp. 401–409. McPartland JM, Russo EB (2001b). Cannabis and cannabis extracts: greater than the sum of their parts? J Cannabis Therap 1: 103–132. McPartland JM, Clarke RC, Watson DP (2000). Hemp Diseases and Pests: Management and Biological Control. CABI: Wallingford. McPartland JM, Blanchon DJ, Musty RE (2008). Cannabimimetic effects modulated by cholinergic compounds. Addict Biol 13: 411–415. Magen I, Avraham Y, Ackerman Z, Vorobiev L, Mechoulam R, Berry EM (2009). Cannabidiol ameliorates cognitive and motor impairments in mice with bile duct ligation. J Hepatol 51: 528–534. Malfait AM, Gallily R, Sumariwalla PF, Malik AS, Andreakos E, Mechoulam R et al. (2000). The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc Natl Acad Sci USA 97: 9561–9566. Malingre T, Hendriks H, Batterman S, Bos R, Visser J (1975). The essential oil of Cannabis sativa. Planta Med 28: 56–61. Maor Y, Gallily R, Mechoulam R (2006). The relevance of the steric factor in the biological activity of CBD derivaties-a tool in identifying novel molecular target for cannabinoids. In: Symposium on the Cannabinoids. International Cannabinoid Research Society: Tihany, Hungary, p. 1. Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, Cascio MG et al. (2002). The endogenous cannabinoid system controls extinction of aversive memories. Nature 418: 530–534. Matura M, Skold M, Borje A, Andersen KE, Bruze M, Frosch P et al. (2005). Selected oxidized fragrance terpenes are common contact allergens. Contact Dermatitis 52: 320–328. de Meijer E (2004). The breeding of cannabis cultivars for pharmaceutical end uses. In: Guy GW, Whittle BA, Robson P (eds). Medicinal Uses of Cannabis and Cannabinoids. Pharmaceutical Press: London, pp. 55–70. de Meijer EPM, Hammond KM (2005). The inheritance of chemical phenotype in Cannabis sativa L. (II): cannabigerol predominant plants. Euphytica 145: 189–198. de Meijer EP, Bagatta M, Carboni A, Crucitti P, Moliterni VM, Ranalli P et al. (2003). The inheritance of chemical phenotype in Cannabis sativa L. Genetics 163: 335–346. de Meijer EPM, Hammond KM, Micheler M (2009a). The inheritance of chemical phenotype in Cannabis sativa L. (III): variation in cannabichromene proportion. Euphytica 165: 293–311. de Meijer EPM, Hammond KM, Sutton A (2009b). The inheritance of chemical phenotype in Cannabis sativa L. (IV): cannabinoid-free plants. Euphytica 168: 95–112. Mechoulam R (1986). The pharmacohistory of Cannabis sativa. In: Mechoulam R (ed.). Cannabinoids As Therapeutic Agents. CRC Press: Boca Raton, FL, pp. 1–19. Mechoulam R (2005). Plant cannabinoids: a neglected pharmacological treasure trove. Br J Pharmacol 146: 913–915. Mechoulam R, Ben-Shabat S (1999). From gan-zi-gun-nu to anandamide and 2-arachidonoylglycerol: the ongoing story of cannabis. Nat Prod Rep 16: 131–143. Mechoulam R, Shvo Y (1963). Hashish-I. The structure of cannabidiol. Tetrahedron 19: 2073–2078. Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR et al. (1995). Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 50: 83–90. Mechoulam R, Peters M, Murillo-Rodriguez E, Hanus LO (2007). Cannabidiol – recent advances. Chem Biodivers 4: 1678–1692. Mediavilla V, Steinemann S (1997). Essential oil of Cannabis sativa L. strains. J Intl Hemp Assoc 4: 82–84. Mehmedic Z, Chandra S, Slade D, Denham H, Foster S, Patel AS et al. (2010). Potency trends of delta(9)-THC and other cannabinoids in confiscated cannabis preparations from 1993 to 2008. J Forensic Sci 55: 1209–1217. Mello NK, Mendelson JH (1978). Marihuana, alcohol, and polydrug use: human self-administration studies. NIDA Res Monogr 20: 93–127. Merzouki A, Mesa JM (2002). Concerning kif, a Cannabis sativa L. preparation smoked in the Rif mountains of northern Morocco. J Ethnopharmacol 81: 403–406. Mishima K, Hayakawa K, Abe K, Ikeda T, Egashira N, Iwasaki K et al. (2005). Cannabidiol prevents cerebral infarction via a serotonergic 5-hydroxytryptamine1A receptor-dependent mechanism. Stroke 36: 1077–1082. Miyazawa M, Yamafuji C (2005). Inhibition of acetylcholinesterase activity by bicyclic monoterpenoids. J Agric Food Chem 53: 1765–1768. Monti D, Chetoni P, Burgalassi S, Najarro M, Saettone MF, Boldrini E (2002). Effect of different terpene-containing essential oils on permeation of estradiol through hairless mouse skin. Int J Pharm 237: 209–214. Morgan CJ, Curran HV (2008). Effects of cannabidiol on schizophrenia-like symptoms in people who use cannabis. Br J Psychiatry 192: 306–307. Morgan CJ, Freeman TP, Schafer GL, Curran HV (2010a). Cannabidiol attenuates the appetitive effects of Delta 9-tetrahydrocannabinol in humans smoking their chosen cannabis. Neuropsychopharmacology 35: 1879–1885. Morgan CJ, Schafer G, Freeman TP, Curran HV (2010b). Impact of cannabidiol on the acute memory and psychotomimetic effects of smoked cannabis: naturalistic study. Br J Psychiatry 197: 285–290. Morimoto S, Tanaka Y, Sasaki K, Tanaka H, Fukamizu T, Shoyama Y et al. (2007). Identification and characterization of cannabinoids that induce cell death through mitochondrial permeability transition in Cannabis leaf cells. J Biol Chem 282: 20739–20751. BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1361 Morse K, Mamane D (2001). The Scent of Orange Blossoms : Sephardic Cuisine from Morocco. Ten Speed Press: Berkeley, CA. Mukerji G, Yiangou Y, Corcoran SL, Selmer IS, Smith GD, Benham CD et al. (2006). Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations. BMC Urol 6: 6. Mukherjee PK, Kumar V, Mal M, Houghton PJ (2007). In vitro acetylcholinesterase inhibitory activity of the essential oil from Acorus calamus and its main constituents. Planta Med 73: 283–285. Murillo-Rodriguez E, Millan-Aldaco D, Palomero-Rivero M, Mechoulam R, Drucker-Colin R (2006). Cannabidiol, a constituent of Cannabis sativa, modulates sleep in rats. FEBS Lett 580: 4337–4345. Musty R, Deyo R (2006). A cannabigerol extract alters behavioral despair in an animal model of depression. Proceedings June 26; Symposium on the Cannabinoids. International Cannabinoid Research Society: Tihany, p. 32. Musty RE, Karniol IG, Shirikawa I, Takahashi RN, Knobel E (1976). Interactions of delta-9-tetrahydrocannabinol and cannabinol in man. In: Braude MC, Szara S (eds). The Pharmacology of Marihuana, Vol. 2. Raven Press: New York, pp. 559–563. Naqvi NH, Bechara A (2009). The hidden island of addiction: the insula. Trends Neurosci 32: 56–67. Naqvi NH, Bechara A (2010). The insula and drug addiction: an interoceptive view of pleasure, urges, and decision-making. Brain Struct Funct 214: 435–450. Naqvi NH, Rudrauf D, Damasio H, Bechara A (2007). Damage to the insula disrupts addiction to cigarette smoking. Science 315: 531–534. Neff GW, O’Brien CB, Reddy KR, Bergasa NV, Regev A, Molina E et al. (2002). Preliminary observation with dronabinol in patients with intractable pruritus secondary to cholestatic liver disease. Am J Gastroenterol 97: 2117–2119. Nerio LS, Olivero-Verbel J, Stashenko E (2010). Repellent activity of essential oils: a review. Bioresour Technol 101: 372–378. Nicholson AN, Turner C, Stone BM, Robson PJ (2004). Effect of delta-9-tetrahydrocannabinol and cannabidiol on nocturnal sleep and early-morning behavior in young adults. J Clin Psychopharmacol 24: 305–313. Nissen L, Zatta A, Stefanini I, Grandi S, Sgorbati B, Biavati B et al. (2010). Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.). Fitoterapia 81: 413–419. Noma Y, Asakawa Y (2010). Biotransformation of monoterpenoids by microorganisms, insects, and mammals. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 585–736. Nunes DS, Linck VM, da Silva AL, Figueiro M, Elisabetsky E (2010). Psychopharmacology of essential oils. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 297–314. O’Shaughnessy WB (1843). Indian hemp. Prov Med J Retrosp Med Sci 5: 397–398. Oh DY, Yoon JM, Moon MJ, Hwang JI, Choe H, Lee JY et al. (2008). Identification of farnesyl pyrophosphate and N-arachidonylglycine as endogenous ligands for GPR92. J Biol Chem 283: 21054–21064. Opdyke DLJ (1983). Caryophyllene oxide. Food Chem Toxicol 21: 661–662. Ozek G, Demirci F, Ozek T, Tabanca N, Wedge DE, Khan SI et al. (2010). Gas chromatographic-mass spectrometric analysis of volatiles obtained by four different techniques from Salvia rosifolia Sm., and evaluation for biological activity. J Chromatog 1217: 741–748. Ozturk A, Ozbek H (2005). The anti-inflammatory activity of Eugenia caryophyllata essential oil: an animal model of anti-inflammatory activity. Eur J Gen Med 2: 159–163. Pacher P, Batkai S, Kunos G (2006). The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 58: 389–462. Parker LA, Mechoulam R, Schlievert C (2002). Cannabidiol, a non-psychoactive component of cannabis and its synthetic dimethylheptyl homolog suppress nausea in an experimental model with rats. Neuroreport 13: 567–570. Parker LA, Burton P, Sorge RE, Yakiwchuk C, Mechoulam R (2004). Effect of low doses of Delta(9)-tetrahydrocannabinol and cannabidiol on the extinction of cocaine-induced and amphetamine-induced conditioned place preference learning in rats. Psychopharmacol (Berl) 175: 360–366. Parolaro D, Massi P (2008). Cannabinoids as potential new therapy for the treatment of gliomas. Expert Rev Neurother 8: 37–49. Pauli A, Schilcher H (2010). In vitro antimicrobial activities of essential oils monographed in the European Pharmacopoeia 6th Edition. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 353–548. Peana AT, Rubattu P, Piga GG, Fumagalli S, Boatto G, Pippia P et al. (2006). Involvement of adenosine A1 and A2A receptors in (-)-linalool-induced antinociception. Life Sci 78: 2471–2474. Perry NS, Houghton PJ, Theobald A, Jenner P, Perry EK (2000). In-vitro inhibition of human erythrocyte acetylcholinesterase by salvia lavandulaefolia essential oil and constituent terpenes. J Pharm Pharmacol 52: 895–902. Pertwee RG (2004). The pharmacology and therapeutic potential of cannabidiol. In: DiMarzo V (ed.). Cannabinoids. Kluwer Academic Publishers: Dordrecht, pp. 32–83. Pertwee RG, Thomas A, Stevenson LA, Ross RA, Varvel SA, Lichtman AH, Martin BR, Razdan RK (2007). The psychoactive plant cannabinoid, Delta9-tetrahydrocannabinol, is antagonized by Delta8- and Delta9-tetrahydrocannabivarin in mice in vivo. Br J Pharmacol 150: 586–594. Pertwee RG (2008). The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin. Br J Pharmacol 153: 199–215. Pliny (1980). Natural History, Books XXIV-XXVII., Vol. 7. Harvard University Press: Cambridge, MA. Potter D (2004). Growth and morphology of medicinal cannabis. In: Guy GW, Whittle BA, Robson P (eds). Medicinal Uses of Cannabis and Cannabinoids. Pharmaceutical Press: London, pp. 17–54. Potter DJ (2009). The propagation, characterisation and optimisation of Cannabis sativa L. as a phytopharmaceutical. PhD, King’s College, London, 2009. Potter DJ, Clark P, Brown MB (2008). Potency of delta 9-THC and other cannabinoids in cannabis in England in 2005: implications for psychoactivity and pharmacology. J Forensic Sci 53: 90–94. BJP EB Russo 1362 British Journal of Pharmacology (2011) 163 1344–1364 Pultrini Ade M, Galindo LA, Costa M (2006). Effects of the essential oil from Citrus aurantium L. in experimental anxiety models in mice. Life Sci 78: 1720–1725. Qin N, Neeper MP, Liu Y, Hutchinson TL, Lubin ML, Flores CM (2008). TRPV2 is activated by cannabidiol and mediates CGRP release in cultured rat dorsal root ganglion neurons. J Neurosci 28: 6231–6238. Rahn EJ, Hohmann AG (2009). Cannabinoids as pharmacotherapies for neuropathic pain: from the bench to the bedside. Neurotherapeutics 6: 713–737. Raman A, Weir U, Bloomfield SF (1995). Antimicrobial effects of tea-tree oil and its major components on Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes. Lett Appl Microbiol 21: 242–245. Rao VS, Menezes AM, Viana GS (1990). Effect of myrcene on nociception in mice. J Pharm Pharmacol 42: 877–878. Re L, Barocci S, Sonnino S, Mencarelli A, Vivani C, Paolucci G et al. (2000). Linalool modifies the nicotinic receptor-ion channel kinetics at the mouse neuromuscular junction. Pharmacol Res 42: 177–182. Ren Y, Whittard J, Higuera-Matas A, Morris CV, Hurd YL (2009). Cannabidiol, a nonpsychotropic component of cannabis, inhibits cue-induced heroin seeking and normalizes discrete mesolimbic neuronal disturbances. J Neurosci 29: 14764–14769. Resstel LB, Tavares RF, Lisboa SF, Joca SR, Correa FM, Guimaraes FS (2009). 5-HT1A receptors are involved in the cannabidiol-induced attenuation of behavioural and cardiovascular responses to acute restraint stress in rats. Br J Pharmacol 156: 181–188. Rhee MH, Vogel Z, Barg J, Bayewitch M, Levy R, Hanus L et al. (1997). Cannabinol derivatives: binding to cannabinoid receptors and inhibition of adenylylcyclase. J Med Chem 40: 3228–3233. Riedel G, Fadda P, McKillop-Smith S, Pertwee RG, Platt B, Robinson L (2009). Synthetic and plant-derived cannabinoid receptor antagonists show hypophagic properties in fasted and non-fasted mice. Br J Pharmacol 156: 1154–1166. Rochefort C, Gheusi G, Vincent JD, Lledo PM (2002). Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory. J Neurosci 22: 2679–2689. Rock EM, Limebeer CL, Mechoulam R, Parker LA (2009). Cannabidiol (the non-psychoactive component of cannabis) may act as a 5-HT1A auto-receptor agonist to reduce toxin-induced nausea and vomiting. Proceedings 19th Annual Symposium on the Cannabinoids. International Cannabinoid Research Society: St. Charles, IL, p. 29. Rodrigues Goulart H, Kimura EA, Peres VJ, Couto AS, Aquino Duarte FA, Katzin AM (2004). Terpenes arrest parasite development and inhibit biosynthesis of isoprenoids in Plasmodium falciparum. Antimicrobial Agents Chemother 48: 2502–2509. Rose JE, Behm FM (1994). Inhalation of vapor from black pepper extract reduces smoking withdrawal symptoms. Drug Alcohol Depend 34: 225–229. Ross SA, ElSohly MA (1996). The volatile oil composition of fresh and air-dried buds of Cannabis sativa. J Nat Prod 59: 49–51. Rothschild M, Bergstrom G, Wangberg S-A (2005). Cannabis sativa: volatile compounds from pollen and entire male and female plants of two variants, Northern Lights and Hawaian Indica. Bot J Linn Soc 147: 387–397. Russo EB (2001). Handbook of Psychotropic Herbs: A Scientific Analysis of Herbal Remedies for Psychiatric Conditions. Haworth Press: Binghamton, NY. Russo EB (2006). The solution to the medicinal cannabis problem. In: Schatman ME (ed.). Ethical Issues in Chronic Pain Management. Taylor & Francis: Boca Raton, FL, pp. 165–194. Russo EB (2007). History of cannabis and its preparations in saga, science and sobriquet. Chem Biodivers 4: 2624–2648. Russo EB, Guy GW (2006). A tale of two cannabinoids: the therapeutic rationale for combining tetrahydrocannabinol and cannabidiol. Med Hypotheses 66: 234–246. Russo EB, McPartland JM (2003). Cannabis is more than simply Delta(9)-tetrahydrocannabinol. Psychopharmacol (Berl) 165: 431–432. Russo EB, Burnett A, Hall B, Parker KK (2005). Agonistic properties of cannabidiol at 5-HT-1a receptors. Neurochem Res 30: 1037–1043. Russo EB, Guy GW, Robson PJ (2007). Cannabis, pain, and sleep: lessons from therapeutic clinical trials of Sativex, a cannabis-based medicine. Chem Biodivers 4: 1729–1743. Russo EB, Jiang HE, Li X, Sutton A, Carboni A, del Bianco F et al. (2008). Phytochemical and genetic analyses of ancient cannabis from Central Asia. J Exp Bot 59: 4171–4182. Ryan D, Drysdale AJ, Pertwee RG, Platt B (2006). Differential effects of cannabis extracts and pure plant cannabinoids on hippocampal neurones and glia. Neurosci Lett 408: 236–241. Salvadeo P, Boggia R, Evangelisti F, Zunin P (2007). Analysis of the volatile fraction of ‘Pesto Genovese’ by headspace sorptive extraction (HSSE). Food Chem 105: 1228–1235. Sanguinetti M, Posteraro B, Romano L, Battaglia F, Lopizzo T, De Carolis E et al. (2007). In vitro activity of Citrus bergamia (bergamot) oil against clinical isolates of dermatophytes. J Antimicrob Chemother 59: 305–308. Schmidt E (2010). Production of essential oils. In: Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science, Technology, and Applications. CRC Press: Boca Raton, FL, pp. 83–120. Scutt A, Williamson EM (2007). Cannabinoids stimulate fibroblastic colony formation by bone marrow cells indirectly via CB2 receptors. Calcif Tissue Int 80: 50–59. Shoyama Y, Sugawa C, Tanaka H, Morimoto S (2008). Cannabinoids act as necrosis-inducing factors in Cannabis sativa. Plant Signal Behav 3: 1111–1112. Silva Brum LF, Emanuelli T, Souza DO, Elisabetsky E (2001). Effects of linalool on glutamate release and uptake in mouse cortical synaptosomes. Neurochem Res 26: 191–194. Singh P, Shukla R, Prakash B, Kumar A, Singh S, Mishra PK et al. (2010). Chemical profile, antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, DL-limonene. Food Chem Toxicol 48: 1734–1740. Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y, Morimoto S, Shoyama Y (2005). Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes. Plant Cell Physiol 46: 1578–1582. Skold M, Karlberg AT, Matura M, Borje A (2006). The fragrance chemical beta-caryophyllene-air oxidation and skin sensitization. Food Chem Toxicol 44: 538–545. BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1363 Soares Vde P, Campos AC, Bortoli VC, Zangrossi H Jr, Guimaraes FS, Zuardi AW (2010). Intra-dorsal periaqueductal gray administration of cannabidiol blocks panic-like response by activating 5-HT1A receptors. Behavioural Brain Res 213: 225–229. do Socorro SRMS, Mendonca-Filho RR, Bizzo HR, de Almeida Rodrigues I, Soares RM, Souto-Padron T et al. (2003). Antileishmanial activity of a linalool-rich essential oil from Croton cajucara. Antimicrob Agents Chemother 47: 1895–1901. Stahl E, Kunde R (1973). Die Leitsubstanzen der HaschischSuchhunde. Kriminalistik: Z Gesamte Kriminal Wiss Prax 27: 385–389. Stott CG, Guy GW, Wright S, Whittle BA (2005). The effects of cannabis extracts Tetranabinex and Nabidiolex on human cytochrome P450-mediated metabolism. In: Symposium on the Cannabinoids, June 27. International Cannabinoid Research Association, Clearwater, FL, p. 163. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K et al. (1995). 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun 215: 89–97. Tambe Y, Tsujiuchi H, Honda G, Ikeshiro Y, Tanaka S (1996). Gastric cytoprotection of the non-steroidal anti-inflammatory sesquiterpene, beta-caryophyllene. Planta Med 62: 469–470. Taylor B (1855). The Lands of the Saracens. G.P. Putnam & Sons: New York. Thomas A, Stevenson LA, Wease KN, Price MR, Baillie G, Ross RA et al. (2005). Evidence that the plant cannabinoid delta-9- tetrahydrocannabivarin is a cannabinoid CB1 and CB2 antagonist. Br J Pharmacol 146: 917–926. Thomas A, Baillie GL, Phillips AM, Razdan RK, Ross RA, Pertwee RG (2007). Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro. Br J Pharmacol 150: 613–623. Tisserand R, Balacs T (1995). Essential Oil Safety: A Guide for Health Care Professionals. Churchill Livingstone: Edinburgh. Trapp SC, Croteau RB (2001). Genomic organization of plant terpene synthases and molecular evolutionary implications. Genet 158: 811–832. Tsokou A, Georgopoulou K, Melliou E, Magiatis P, Tsitsa E (2007). Composition and enantiomeric analysis of the essential oil of the fruits and the leaves of Pistacia vera from Greece. Molecules 12: 1233–1239. Turner CE, Elsohly MA, Boeren EG (1980). Constituents of Cannabis sativa L. XVII. A review of the natural constituents. J Nat Prod 43: 169–234. Turner G, Gershenzon J, Nielson EE, Froehlich JE, Croteau R (1999). Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells. Plant Physiol 120: 879–886. do Vale TG, Furtado EC, Santos JG Jr, Viana GS (2002). Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (Mill.) n.e. Brown. Phytomed 9: 709–714. Varvel SA, Bridgen DT, Tao Q, Thomas BF, Martin BR, Lichtman AH (2005). Delta9-tetrahydrocannbinol accounts for the antinociceptive, hypothermic, and cataleptic effects of marijuana in mice. J Pharmacol Exp Ther 314: 329–337. Vigushin DM, Poon GK, Boddy A, English J, Halbert GW, Pagonis C et al. (1998). Phase I and pharmacokinetic study of d-limonene in patients with advanced cancer. Cancer Research Campaign Phase I/II Clinical Trials Committee. Cancer Chemother Pharmacol 42: 111–117. Volicer L, Stelly M, Morris J, McLaughlin J, Volicer BJ (1997). Effects of dronabinol on anorexia and disturbed behavior in patients with Alzheimer’s disease. Int J Geriatr Psychiatry 12: 913–919. Vollner L, Bieniek D, Korte F (1969). [Hashish. XX. Cannabidivarin, a new hashish constituent]. Tetrahedron Lett 3: 145–147. Von Burg R (1995). Toxicology update. Limonene. J Appl Toxicol 15: 495–499. Wachtel SR, ElSohly MA, Ross RA, Ambre J, de Wit H (2002). Comparison of the subjective effects of delta9-tetrahydrocannabinol and marijuana in humans. Psychopharmacol 161: 331–339. Wagner H, Ulrich-Merzenich G (2009). Synergy research: approaching a new generation of phytopharmaceuticals. Phytomed 16: 97–110. Walton RP (1938). Marihuana, America’s New Drug Problem. A Sociologic Question with Its Basic Explanation Dependent on Biologic and Medical Principles. J.B. Lippincott: Philadelphia, PA. Wattenberg LW (1991). Inhibition of azoxymethane-induced neoplasia of the large bowel by 3-hydroxy-3,7,11-trimethyl-1,6, 10-dodecatriene (nerolidol). Carcinogen 12: 151–152. Wilkinson JD, Williamson EM (2007). Cannabinoids inhibit human keratinocyte proliferation through a non-CB1/CB2 mechanism and have a potential therapeutic value in the treatment of psoriasis. J Dermatol Sci 45: 87–92. Wilkinson JD, Whalley BJ, Baker D, Pryce G, Constanti A, Gibbons S et al. (2003). Medicinal cannabis: is delta9-tetrahydrocannabinol necessary for all its effects? J Pharm Pharmacol 55: 1687–1694. Williams SJ, Hartley JP, Graham JD (1976). Bronchodilator effect of delta1-tetrahydrocannabinol administered by aerosol of asthmatic patients. Thorax 31: 720–723. Williamson EM (2001). Synergy and other interactions in phytomedicines. Phytomed 8: 401–409. Wirth PW, Watson ES, ElSohly M, Turner CE, Murphy JC (1980). Anti-inflammatory properties of cannabichromene. Life Sci 26: 1991–1995. Xi Z-X, Peng X-Q, Li X, Zhang H, Li JG, Gardner EL (2010). Brain cannabinoid CB2 receptors inhibit cocaine self-administration and cocaine-enhanced extracellular dopamine in mice. Proceedings 20th Annual Symposium on the Cannabinoids. International Cannabinoid Research Society: Lund, p. 32. Yang D, Michel L, Chaumont JP, Millet-Clerc J (1999). Use of caryophyllene oxide as an antifungal agent in an in vitro experimental model of onychomycosis. Mycopathologia 148: 79–82. Zanelati TV, Biojone C, Moreira FA, Guimaraes FS, Joca SR (2010). Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT1A receptors. Br J Pharmacol 159: 122–128. Zuardi AW, Guimaraes FS (1997). Cannabidiol as an anxiolytic and antipsychotic. In: Mathre ML (ed.). Cannabis in Medical Practice: A Legal, Historical and Pharmacological Overview of the Therapeutic Use of Marijuana. McFarland: Jefferson, NC, pp. 133–141. Zuardi AW, Rodrigues JA, Cunha JM (1991). Effects of cannabidiol in animal models predictive of antipsychotic activity. Psychopharmacol 104: 260–264. Zuardi AW, Crippa JA, Hallak JE, Moreira FA, Guimaraes FS (2006). Cannabidiol, a Cannabis sativa constituent, as an antipsychotic drug. Braz J Med Biol Res 39: 421–429. BJP EB Russo 1364 British Journal of Pharmacology (2011) 163 1344–1364

Themed Issue: Cannabinoids in Biology and Medicine, Part I
REVIEWbph_1238 1344..1364
Taming THC: potential
cannabis synergy and
phytocannabinoid-terpenoid
entourage effects
Ethan B Russo
GW Pharmaceuticals, Salisbury, Wiltshire, UK
Correspondence
Ethan Russo, MD, 20402 81st
Avenue SW, Vashon, WA 98070,
USA. E-mail:
ethanrusso@comcast.net
----------------------------------------------------------------
Keywords
cannabinoids; terpenoids;
essential oils; THC; CBD;
limonene; pinene; linalool;
caryophyllene; phytotherapy
----------------------------------------------------------------
Received
19 November 2010
Revised
29 December 2010
Accepted
12 January 2011
Tetrahydrocannabinol (THC) has been the primary focus of cannabis research since 1964, when Raphael Mechoulam isolated
and synthesized it. More recently, the synergistic contributions of cannabidiol to cannabis pharmacology and analgesia
have been scientifically demonstrated. Other phytocannabinoids, including tetrahydrocannabivarin, cannabigerol and
cannabichromene, exert additional effects of therapeutic interest. Innovative conventional plant breeding has yielded cannabis
chemotypes expressing high titres of each component for future study. This review will explore another echelon of
phytotherapeutic agents, the cannabis terpenoids: limonene, myrcene, a-pinene, linalool, b-caryophyllene, caryophyllene
oxide, nerolidol and phytol. Terpenoids share a precursor with phytocannabinoids, and are all flavour and fragrance
components common to human diets that have been designated Generally Recognized as Safe by the US Food and Drug
Administration and other regulatory agencies. Terpenoids are quite potent, and affect animal and even human behaviour
when inhaled from ambient air at serum levels in the single digits ng·mL-1
. They display unique therapeutic effects that may
contribute meaningfully to the entourage effects of cannabis-based medicinal extracts. Particular focus will be placed on
phytocannabinoid-terpenoid interactions that could produce synergy with respect to treatment of pain, inflammation,
depression, anxiety, addiction, epilepsy, cancer, fungal and bacterial infections (including methicillin-resistant Staphylococcus
aureus). Scientific evidence is presented for non-cannabinoid plant components as putative antidotes to intoxicating effects of
THC that could increase its therapeutic index. Methods for investigating entourage effects in future experiments will be
proposed. Phytocannabinoid-terpenoid synergy, if proven, increases the likelihood that an extensive pipeline of new
therapeutic products is possible from this venerable plant.
LINKED ARTICLES
This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit
http://dx.doi.org/10.1111/bph.2011.163.issue-7
Abbreviations
2-AG, 2-arachidonoylglycerol; 5-HT, 5-hydroxytryptamine (serotonin); AD, antidepressant; AEA,
arachidonoylethanolamide (anandamide); AI, anti-inflammatory; AMPA, a-amino-3-hydroxyl-5-methyl-4-
isoxazole-propionate; Ca++, calcium ion; CB1/CB2, cannabinoid receptor 1 or 2; CBC, cannabichromene; CBCA,
cannabichromenic acid; CBD, cannabidiol; CBDA, cannabidiolic acid; CBDV, cannabidivarin; CBG, cannabigerol;
CBGA, cannabigerolic acid; CBGV, cannabigerivarin; CNS, central nervous system; COX, cyclo-oxygenase; DAGL,
diacylglycerol lipase; ECS, endocannabinoid system; EO, essential oil; FAAH, fatty acid amidohydrolase; FDA, US Food
and Drug Administration; FEMA, Food and Extract Manufacturers Association; fMRI, functional magnetic resonance
imaging; GABA, gamma aminobutyric acid; GPCR, G-protein coupled receptor; GPR, G-protein coupled receptor; HEK,
human embryonic kidney; IC50, 50% inhibitory concentration; i.p., intraperitoneal; MAGL, monoacylglycerol lipase;
MIC, minimum inhibitory concentration; MS, multiple sclerosis; NGF, nerve growth factor; NIDA, US National Institute
on Drug Abuse; PG, prostaglandin; PTSD, post-traumatic stress disorder; RCT, randomized clinical trial; SPECT, single
photon emission computed tomography; SSADH, succinic semialdehyde dehydrogenase; Sx, symptoms; T1/2, half-life;
TCA, tricyclic antidepressant; THC, tetrahydrocannabinol; THCA, tetrahydrocannabinolic acid; THCV,
tetrahydrocannabivarin; TNF-a, tumour necrosis factor-alpha, TRPV, transient receptor potential vanilloid receptor
BJP British Journal of
Pharmacology
DOI:10.1111/j.1476-5381.2011.01238.x
www.brjpharmacol.org
1344 British Journal of Pharmacology (2011) 163 1344–1364 © 2011 The Author
British Journal of Pharmacology © 2011 The British Pharmacological Society
The roots of cannabis synergy
Cannabis has been a medicinal plant of unparalleled versatility for millennia (Mechoulam, 1986; Russo, 2007; 2008),
but whose mechanisms of action were an unsolved mystery
until the discovery of tetrahydrocannabinol (THC) (Gaoni
and Mechoulam, 1964a), the first cannabinoid receptor, CB1
(Devane et al., 1988), and the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) (Devane et al., 1992)
and 2-arachidonoylglycerol (2-AG) (Mechoulam et al., 1995;
Sugiura et al., 1995). While a host of phytocannabinoids were
discovered in the 1960s: cannabidiol (CBD) (Mechoulam and
Shvo, 1963), cannabigerol (CBG) (Gaoni and Mechoulam,
1964b), cannabichromene (CBC) (Gaoni and Mechoulam,
1966), cannabidivarin (CBDV) (Vollner et al., 1969) and
tetrahydrocannabivarin (THCV) (Gill et al., 1970), the
overwhelming preponderance of research focused on psychoactive THC. Only recently has renewed interest been manifest
in THC analogues, while other key components of the activity of cannabis and its extracts, the cannabis terpenoids,
remain understudied (McPartland and Russo, 2001b;
Russo and McPartland, 2003). The current review will reconsider essential oil (EO) agents, their peculiar pharmacology
and possible therapeutic interactions with phytocannabinoids. Nomenclature follows conventions in Alexander et al.
(2009).
Phytocannabinoids and terpenoids are synthesized in
cannabis, in secretory cells inside glandular trichomes
(Figure 1) that are most highly concentrated in unfertilized
female flowers prior to senescence (Potter, 2004; Potter,
2009). Geranyl pyrophosphate is formed as a precursor via
the deoxyxylulose pathway in cannabis (Fellermeier et al.,
2001), and is a parent compound to both phytocannabinoids
and terpenoids (Figure 2). After coupling with either olivetolic acid or divarinic acid, pentyl or propyl cannabinoid
acids are produced, respectively, via enzymes that accept
either substrate (de Meijer et al., 2003), a manifestation
of Mechoulam’s postulated ‘Nature’s Law of Stinginess’.
Although having important biochemical properties in their
own right, acid forms of phytocannabinoids are most commonly decarboxylated via heat to produce the more familiar
neutral phytocannabinoids (Table 1). Alternatively, geranyl
pyrophosphate may form limonene and other monoterpenoids in secretory cell plastids, or couple with isopentenyl
pyrophosphate in the cytoplasm to form farnesyl pyrophosphate, parent compound to the sesquiterpenoids, that
co-localizes with transient receptor potential vanilloid receptor (TRPV) 1 in human dorsal root ganglion, suggesting a role
in sensory processing of noxious stimuli (Bradshaw et al.,
2009), and which is the most potent endogenous ligand to
date on the G-protein coupled receptor (GPR) 92 (Oh et al.,
2008).
An obvious question pertains to the chemical ecology of
such syntheses that require obvious metabolic demands on
the plant (Gershenzon, 1994), and these will be considered.
Is cannabis merely a crude vehicle for delivery of THC?
Might it rather display herbal synergy (Williamson, 2001)
encompassing potentiation of activity by active or inactive
components, antagonism (evidenced by the ability of CBD to
reduce side effects of THC; Russo and Guy, 2006), summation,
pharmacokinetic and metabolic interactions? Recently, four
basic mechanisms of synergy have been proposed (Wagner
and Ulrich-Merzenich, 2009): (i) multi-target effects; (ii) pharmacokinetic effects such as improved solubility or bioavailability; (iii) agent interactions affecting bacterial resistance;
and (iv) modulation of adverse events. Cannabis was cited as
an illustration.
Could phytocannabinoids function analogously to the
endocannabinoid system (ECS) with its combination of
active and ‘inactive’ synergists, first described as an entourage
(Ben-Shabat et al., 1998), with subsequent refinement
(Mechoulam and Ben-Shabat, 1999) and qualification
(p. 136): ‘This type of synergism may play a role in the widely
held (but not experimentally based) view that in some cases
plants are better drugs than the natural products isolated
from them’. Support derives from studies in which cannabis
extracts demonstrated effects two to four times greater than
THC (Carlini et al., 1974); unidentified THC antagonists and
synergists were claimed (Fairbairn and Pickens, 1981), anticonvulsant activity was observed beyond the cannabinoid
fraction (Wilkinson et al., 2003), and extracts of THC and
CBD modulated effects in hippocampal neurones distinctly
from pure compounds (Ryan et al., 2006). Older literature
also presented refutations: no observed differences were
noted by humans ingesting or smoking pure THC versus
herbal cannabis (Wachtel et al., 2002); pure THC seemed to
account for all tetrad-type effects in mice (Varvel et al., 2005);
and smoked cannabis with varying CBD or CBC content
failed to yield subjective differences combined with THC (Ilan
et al., 2005). Explanations include that the cannabis
employed by Wachtel yielded 2.11% THC, but with only
0.3% cannabinol (CBN) and 0.05% CBD (Russo and McPartland, 2003), and Ilan’s admission that CBN and CBD content
might be too low to modulate THC. Another factor is apparent in that terpenoid yields from vaporization of street cannabis were 4.3–8.5 times of those from US National Institute
on Drug Abuse cannabis (Bloor et al., 2008). It is undisputed
that the black market cannabis in the UK (Potter et al., 2008),
Continental Europe (King et al., 2005) and the USA (Mehmedic et al., 2010) has become almost exclusively a high-THC
preparation to the almost total exclusion of other phytocannabinoids. If – as many consumers and experts maintain
(Clarke, 2010) – there are biochemical, pharmacological and
Figure 1
Cannabis capitate glandular (EBR by permission of Bedrocan BV,
Netherlands).
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1345
phenomenological distinctions between available cannabis
‘strains’, such phenomena are most likely related to relative
terpenoid contents and ratios. This treatise will assess additional evidence for putative synergistic phytocannabinoidterpenoid effects exclusive of THC, to ascertain whether this
botanical may fulfil its promise as, ‘a neglected pharmacological treasure trove’ (Mechoulam, 2005).
Phytocannabinoids, beyond THC:
a brief survey
Phytocannabinoids are exclusively produced in cannabis
(vide infra for exception), but their evolutionary and ecological raisons d’être were obscure until recently. THC production is maximized with increased light energy (Potter,
2009). It has been known for some time that CBG and CBC
are mildly antifungal (ElSohly et al., 1982), as are THC and
CBD against a cannabis pathogen (McPartland, 1984). More
pertinent, however, is the mechanical stickiness of the
trichomes, capable of trapping insects with all six legs
(Potter, 2009). Tetrahydrocannabinolic acid (THCA) and
cannabichromenic acid (Morimoto et al., 2007), as well as
cannabidiolic acid and cannabigerolic acid (CBGA; Shoyama
et al., 2008) produce necrosis in plant cells. Normally, the
cannabinoid acids are sequestered in trichomes away from
the flower tissues. Any trichome breakage at senescence may
contribute to natural pruning of lower fan leaves that otherwise utilize energy that the plant preferentially diverts to
the flower, in continued efforts to affect fertilization, generally in vain when subject to human horticulture for pharmaceutical production. THCA and CBGA have also proven
to be insecticidal in their own right (Sirikantaramas et al.,
2005).
Over 100 phytocannabinoids have been identified (Brenneisen, 2007; Mehmedic et al., 2010), but many are artefacts
of analysis or are produced in trace quantities that have not
permitted thorough investigation. The pharmacology of the
more accessible phytocannabinoids has received excellent
recent reviews (Pertwee et al., 2007; Izzo et al., 2009; De Petrocellis and Di Marzo, 2010; De Petrocellis et al., 2011), and
will be summarized here, with emphasis on activities with
particular synergistic potential.
Geranylphosphate: olivetolate geranyltransferase
HO
OH
COOH
cannabigerolic acid
O
OH
COOH
delta-9-tetrahydrocannabinolic acid
OH
OH
COOH
cannabidiolic acid
O
OH
COOH
cannabichromenenic acid
HO
OH
COOH
cannabigerovarinic acid
Geranylphosphate: olivetolate geranyltransferase
O
OH
COOH
tetrahydrocannabivarinic acid
OH
OH
COOH
cannabidivarinic acid
O
OH
COOH
cannabichromevarinic acid
PPO
dimethylallyl pyrophosphate (DMAPP)
OPP
isopentenyl pyrophosphate (IPP)
GPP synthase
+
+ +
THCA synthaseCBDA synthase CBCA synthase THCA synthaseCBDA synthase CBCA synthase
H
limonene
OPO3OPO3
farnesyl pyrophosphate
OPO3OPO3
geranyl pyrophosphate
x3
Sesquiterpenoids
FPP synthase
Limonene synthase Monoterpenoids
HO
OH
COOH
divarinic acid (5-propyl resorcinolic acid HO
OH
COOH
olivetolic acid (5-pentyl resorcinolic acid) )
Phytocannabinoid
Acids
Figure 2
Phytocannabinoid and cannabis terpenoid biosynthesis.
BJP EB Russo
1346 British Journal of Pharmacology (2011) 163 1344–1364
Table 1
Phytocannabinoid activity table
Phytocannabinoid structure Selected pharmacology (reference) Synergistic terpenoids
O
OH
delta-9-tetrahydrocannabinol (THC)
Analgesic via CB1 and CB2 (Rahn and Hohmann, 2009) Various
AI/antioxidant (Hampson et al., 1998) Limonene et al.
Bronchodilatory (Williams et al., 1976) Pinene
↓ Sx. Alzheimer disease (Volicer et al., 1997; Eubanks et al., 2006) Limonene, pinene, linalool
Benefit on duodenal ulcers (Douthwaite, 1947) Caryophyllene, limonene
Muscle relaxant (Kavia et al., 2010) Linalool?
Antipruritic, cholestatic jaundice (Neff et al., 2002) Caryophyllene?
OH
OH
cannabidiol
AI/antioxidant (Hampson et al., 1998) Limonene et al.
Anti-anxiety via 5-HT1A (Russo et al., 2005) Linalool, limonene
Anticonvulsant (Jones et al., 2010) Linalool
Cytotoxic versus breast cancer (Ligresti et al., 2006) Limonene
↑ adenosine A2A signalling (Carrier et al., 2006) Linalool
Effective versus MRSA (Appendino et al., 2008) Pinene
Decreases sebum/sebocytes (Biro et al., 2009) Pinene, limonene, linalool
Treatment of addiction (see text) Caryophyllene
O
OH
cannabichromene
Anti-inflammatory/analgesic (Davis and Hatoum, 1983) Various
Antifungal (ElSohly et al., 1982) Caryophyllene oxide
AEA uptake inhibitor (De Petrocellis et al., 2011) –
Antidepressant in rodent model (Deyo and Musty, 2003) Limonene
HO
OH
cannabigerol
TRPM8 antagonist prostate cancer (De Petrocellis et al., 2011) Cannabis terpenoids
GABA uptake inhibitor (Banerjee et al., 1975) Phytol, linalool
Anti-fungal (ElSohly et al., 1982) Caryophyllene oxide
Antidepressant rodent model (Musty and Deyo, 2006); and via
5-HT1A antagonism (Cascio et al., 2010)
Limonene
Analgesic, a-2 adrenergic blockade (Cascio et al., 2010) Various
↓ keratinocytes in psoriasis (Wilkinson and Williamson, 2007) adjunctive role?
Effective versus MRSA (Appendino et al., 2008) Pinene
O
OH
tetrahydrocannabivarin
AI/anti-hyperalgesic (Bolognini et al., 2010) Caryophyllene et al....
Treatment of metabolic syndrome (Cawthorne et al., 2007) –
Anticonvulsant (Hill et al., 2010) Linalool
OH
OH
cannabidivarin
Inhibits diacylglycerol lipase (De Petrocellis et al., 2011) –
Anticonvulsant in hippocampus (Hill et al., 2010) Linalool
O
OH
cannabinol (CBN)
Sedative (Musty et al., 1976) Nerolidol, myrcene
Effective versus MRSA (Appendino et al., 2008) Pinene
TRPV2 agonist for burns (Qin et al., 2008) Linalool
↓ keratinocytes in psoriasis (Wilkinson and Williamson, 2007) adjunctive role?
↓ breast cancer resistance protein (Holland et al., 2008) Limonene
5-HT, 5-hydroxytryptamine (serotonin); AEA, arachidonoylethanolamide (anandamide); AI, anti-inflammatory; CB1/CB2, cannabinoid receptor 1 or 2; GABA, gamma
aminobutyric acid; TRPV, transient receptor potential vanilloid receptor; MRSA, methicillin-resistant Staphylococcus aureus; Sx, symptoms.
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1347
THC (Table 1) is the most common phytocannabinoid in
cannabis drug chemotypes, and is produced in the plant via
an allele co-dominant with CBD (de Meijer et al., 2003). THC
is a partial agonist at CB1 and cannabinoid receptor 2 (CB2)
analogous to AEA, and underlying many of its activities as a
psychoactive agent, analgesic, muscle relaxant and antispasmodic (Pacher et al., 2006). Additionally, it is a bronchodilator (Williams et al., 1976), neuroprotective antioxidant
(Hampson et al., 1998), antipruritic agent in cholestatic jaundice (Neff et al., 2002) and has 20 times the antiinflammatory power of aspirin and twice that of
hydrocortisone (Evans, 1991). THC is likely to avoid potential
pitfalls of either COX-1 or COX-2 inhibition, as such activity
is only noted at concentrations far above those attained
therapeutically (Stott et al., 2005).
CBD is the most common phytocannabinoid in fibre
(hemp) plants, and second most prevalent in some drug
chemotypes. It has proven extremely versatile pharmacologically (Table 1) (Pertwee, 2004; Mechoulam et al., 2007), displaying the unusual ability to antagonize CB1 at a low nM
level in the presence of THC, despite having little binding
affinity (Thomas et al., 2007), and supporting its modulatory
effect on THC-associated adverse events such as anxiety,
tachycardia, hunger and sedation in rats and humans
(Nicholson et al., 2004; Murillo-Rodriguez et al., 2006; Russo
and Guy, 2006). CBD is an analgesic (Costa et al., 2007), is a
neuroprotective antioxidant more potent than ascorbate or
tocopherol (Hampson et al., 1998), without COX inhibition
(Stott et al., 2005), acts as a TRPV1 agonist analogous to
capsaicin but without noxious effect (Bisogno et al., 2001),
while also inhibiting uptake of AEA and weakly inhibiting its
hydrolysis. CBD is an antagonist on GPR55, and also on
GPR18, possibly supporting a therapeutic role in disorders of
cell migration, notably endometriosis (McHugh et al., 2010).
CBD is anticonvulsant (Carlini and Cunha, 1981; Jones et al.,
2010), anti-nausea (Parker et al., 2002), cytotoxic in breast
cancer (Ligresti et al., 2006) and many other cell lines while
being cyto-preservative for normal cells (Parolaro and Massi,
2008), antagonizes tumour necrosis factor-alpha (TNF-a) in a
rodent model of rheumatoid arthritis (Malfait et al., 2000),
enhances adenosine receptor A2A signalling via inhibition of
an adenosine transporter (Carrier et al., 2006), and prevents
prion accumulation and neuronal toxicity (Dirikoc et al.,
2007). A CBD extract showed greater anti-hyperalgesia over
pure compound in a rat model with decreased allodynia,
improved thermal perception and nerve growth factor levels
and decreased oxidative damage (Comelli et al., 2009). CBD
also displayed powerful activity against methicillin-resistant
Staphylococcus aureus (MRSA), with a minimum inhibitory
concentration (MIC) of 0.5–2 mg·mL-1 (Appendino et al.,
2008). In 2005, it was demonstrated that CBD has agonistic
activity at 5-hydroxytryptamine (5-HT)1A at 16 mM (Russo
et al., 2005), and that despite the high concentration, may
underlie its anti-anxiety activity (Resstel et al., 2009; Soares
Vde et al., 2010), reduction of stroke risk (Mishima et al.,
2005), anti-nausea effects (Rock et al., 2009) and ability to
affect improvement in cognition in a mouse model of hepatic
encephalopathy (Magen et al., 2009). A recent study has demonstrated that CBD 30 mg·kg-1 i.p. reduced immobility time
in the forced swim test compared to imipramine (P < 0.01), an
effect blocked by pre-treatment with the 5-HT1A antagonist
WAY100635 (Zanelati et al., 2010), supporting a prospective
role for CBD as an antidepressant. CBD also inhibits synthesis
of lipids in sebocytes, and produces apoptosis at higher doses
in a model of acne (vide infra). One example of CBD antagonism to THC would be the recent observation of lymphopenia in rats (CBD 5 mg·kg-1
) mediated by possible CB2 inverse
agonism (Ignatowska-Jankowska et al., 2009), an effect not
reported in humans even at doses of pure CBD up to 800 mg
(Crippa et al., 2010), possibly due to marked interspecies
differences in CB2 sequences and signal transduction. CBD
proved to be a critical factor in the ability of nabiximols
oromucosal extract in successfully treating intractable cancer
pain patients unresponsive to opioids (30% reduction in pain
from baseline), as a high-THC extract devoid of CBD failed to
distinguish from placebo (Johnson et al., 2010). This may
represent true synergy if the THC–CBD combination were
shown to provide a larger effect than a summation of those
from the compounds separately (Berenbaum, 1989).
CBC (Table 1) was inactive on adenylate cyclase inhibition (Howlett, 1987), but showed activity in the mouse cannabinoid tetrad, but only at 100 mg·kg-1
, and at a fraction of
THC activity, via a non-CB1, non-CB2 mechanism (Delong
et al., 2010). More pertinent are anti-inflammatory (Wirth
et al., 1980) and analgesic activity (Davis and Hatoum, 1983),
its ability to reduce THC intoxication in mice (Hatoum et al.,
1981), antibiotic and antifungal effects (ElSohly et al., 1982),
and observed cytotoxicity in cancer cell lines (Ligresti et al.,
2006). A CBC-extract displayed pronounced antidepressant
effect in rodent models (Deyo and Musty, 2003). Additionally,
CBC was comparable to mustard oil in stimulating TRPA1-
mediated Ca++ in human embryonic kidney 293 cells (50–
60 nM) (De Petrocellis et al., 2008). CBC recently proved to be
a strong AEA uptake inhibitor (De Petrocellis et al., 2011).
CBC production is normally maximal, earlier in the plant’s
life cycle (de Meijer et al., 2009a). An innovative technique
employing cold water extraction of immature leaf matter
from selectively bred cannabis chemotypes yields a high-CBC
‘enriched trichome preparation’ (Potter, 2009).
CBG (Table 1), the parent phytocannabinoid compound,
has a relatively weak partial agonistic effect at CB1 (Ki
440 nM) and CB2 (Ki 337 nM) (Gauson et al., 2007). Older
work supports gamma aminobutyric acid (GABA) uptake
inhibition greater than THC or CBD (Banerjee et al., 1975)
that could suggest muscle relaxant properties. Analgesic and
anti-erythemic effects and the ability to block lipooxygenase
were said to surpass those of THC (Evans, 1991). CBG demonstrated modest antifungal effects (ElSohly et al., 1982).
More recently, it proved to be an effective cytotoxic in high
dosage on human epithelioid carcinoma (Baek et al., 1998), is
the next most effective phytocannabinoid against breast
cancer after CBD (Ligresti et al., 2006), is an antidepressant in
the rodent tail suspension model (Musty and Deyo, 2006)
and is a mildly anti-hypertensive agent (Maor et al., 2006).
Additionally, CBG inhibits keratinocyte proliferation suggesting utility in psoriasis (Wilkinson and Williamson, 2007), it is
a relatively potent TRPM8 antagonist for possible application
in prostate cancer (De Petrocellis and Di Marzo, 2010) and
detrusor over-activity and bladder pain (Mukerji et al., 2006).
It is a strong AEA uptake inhibitor (De Petrocellis et al., 2011)
and a powerful agent against MRSA (Appendino et al., 2008;
vide infra). Finally, CBG behaves as a potent a-2 adrenorecepBJP EB Russo
1348 British Journal of Pharmacology (2011) 163 1344–1364
tor agonist, supporting analgesic effects previously noted
(Formukong et al., 1988), and moderate 5-HT1A antagonist
suggesting antidepressant properties (Cascio et al., 2010).
Normally, CBG appears as a relatively low concentration
intermediate in the plant, but recent breeding work has
yielded cannabis chemotypes lacking in downstream
enzymes that express 100% of their phytocannabinoid
content as CBG (de Meijer and Hammond, 2005; de Meijer
et al., 2009a).
THCV (Table 1) is a propyl analogue of THC, and can
modulate intoxication of the latter, displaying 25% of its
potency in early testing (Gill et al., 1970; Hollister, 1974). A
recrudescence of interest accrues to this compound, which is
a CB1 antagonist at lower doses (Thomas et al., 2005), but is a
CB1 agonist at higher doses (Pertwee, 2008). THCV produces
weight loss, decreased body fat and serum leptin concentrations with increased energy expenditure in obese mice
(Cawthorne et al., 2007; Riedel et al., 2009). THCV also demonstrates prominent anticonvulsant properties in rodent cerebellum and pyriform cortex (Hill et al., 2010). THCV appears
as a fractional component of many southern African cannabis chemotypes, although plants highly predominant in
this agent have been produced (de Meijer, 2004). THCV
recently demonstrated a CB2-based ability to suppress
carageenan-induced hyperalgesia and inflammation, and
both phases of formalin-induced pain behaviour via CB1 and
CB2 in mice (Bolognini et al., 2010).
CBDV (Table 1), the propyl analogue of CBD, was first
isolated in 1969 (Vollner et al., 1969), but formerly received
little investigation. Pure CBDV inhibits diacylglycerol lipase
[50% inhibitory concentration (IC50) 16.6 mM] and might
decrease activity of its product, the endocannabinoid, 2-AG
(De Petrocellis et al., 2011). It is also anticonvulsant in rodent
hippocampal brain slices, comparable to phenobarbitone and
felbamate (Jones et al., 2010).
Finally, CBN is a non-enzymatic oxidative by-product of
THC, more prominent in aged cannabis samples (Merzouki
and Mesa, 2002). It has a lower affinity for CB1 (Ki 211.2 nM)
and CB2 (Ki 126.4 nM) (Rhee et al., 1997); and was judged
inactive when tested alone in human volunteers, but produced greater sedation combined with THC (Musty et al.,
1976). CBN demonstrated anticonvulsant (Turner et al.,
1980), anti-inflammatory (Evans, 1991) and potent effects
against MRSA (MIC 1 mg·mL-1
). CBN is a TRPV2 (highthreshold thermosensor) agonist (EC 77.7 mM) of possible
interest in treatment of burns (Qin et al., 2008). Like CBG, it
inhibits keratinocyte proliferation (Wilkinson and Williamson, 2007), independently of cannabinoid receptor effects.
CBN stimulates the recruitment of quiescent mesenchymal
stem cells in marrow (10 mM), suggesting promotion of bone
formation (Scutt and Williamson, 2007) and inhibits breast
cancer resistance protein, albeit at a very high concentration
(IC50 145 mM) (Holland et al., 2008).
Cannabis terpenoids: neglected
entourage compounds?
Terpenoids are EO components, previously conceived as the
quintessential fifth element, ‘life force’ or spirit (Schmidt,
2010), and form the largest group of plant chemicals, with
15–20 000 fully characterized (Langenheim, 1994). Terpenoids, not cannabinoids, are responsible for the aroma of
cannabis. Over 200 have been reported in the plant (Hendriks
et al., 1975; 1977; Malingre et al., 1975; Davalos et al., 1977;
Ross and ElSohly, 1996; Mediavilla and Steinemann, 1997;
Rothschild et al., 2005; Brenneisen, 2007), but only a few
studies have concentrated on their pharmacology (McPartland and Pruitt, 1999; McPartland and Mediavilla, 2001a;
McPartland and Russo, 2001b). Their yield is less than 1% in
most cannabis assays, but they may represent 10% of trichome content (Potter, 2009). Monoterpenes usually predominate (limonene, myrcene, pinene), but these headspace
volatiles (Hood et al., 1973), while only lost at a rate of about
5% before processing (Gershenzon, 1994), do suffer diminished yields with drying and storage (Turner et al., 1980; Ross
and ElSohly, 1996), resulting in a higher relative proportion
of sesquiterpenoids (especially caryophyllene), as also often
occurs in extracts. A ‘phytochemical polymorphism’ seems
operative in the plant (Franz and Novak, 2010), as production
favours agents such as limonene and pinene in flowers that
are repellent to insects (Nerio et al., 2010), while lower fan
leaves express higher concentrations of bitter sesquiterpenoids that act as anti-feedants for grazing animals (Potter,
2009). Evolutionarily, terpenoids seem to occur in complex
and variable mixtures with marked structural diversity to
serve various ecological roles. Terpenoid composition is
under genetic control (Langenheim, 1994), and some
enzymes produce multiple products, again supporting
Mechoulam’s ‘Law of Stinginess’. The particular mixture of
mono- and sesquiterpenoids will determine viscosity, and in
cannabis, this certainly is leveraged to practical advantage as
the notable stickiness of cannabis exudations traps insects
(McPartland et al., 2000), and thus, combined with the insecticidal phytocannabinoid acids (Sirikantaramas et al., 2005),
provides a synergistic mechano-chemical defensive strategy
versus predators.
As observed for cannabinoids, terpenoid production
increases with light exposure, but decreases with soil fertility
(Langenheim, 1994), and this is supported by the glasshouse
experience that demonstrates higher yields if plants experience relative nitrogen lack just prior to harvest (Potter, 2004),
favouring floral over foliar growth. EO composition is much
more genetically than environmentally determined, however
(Franz and Novak, 2010), and while cannabis is allogamous
and normally requires repeat selective breeding for maintenance of quality, this problem may be practically circumvented by vegetative propagation of high-performance plants
under controlled environmental conditions (light, heat and
humidity) (Potter, 2009), and such techniques have proven to
provide notable consistency to tight tolerances as Good
Manufacturing Practice for any pharmaceutical would require
(Fischedick et al., 2010).
The European Pharmacopoeia, Sixth Edition (2007), lists 28
EOs (Pauli and Schilcher, 2010). Terpenoids are pharmacologically versatile: they are lipophilic, interact with cell membranes, neuronal and muscle ion channels, neurotransmitter
receptors, G-protein coupled (odorant) receptors, second
messenger systems and enzymes (Bowles, 2003; Buchbauer,
2010). All the terpenoids discussed herein are Generally Recognized as Safe, as attested by the US Food and Drug AdminBJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1349
istration as food additives, or by the Food and Extract
Manufacturers Association and other world regulatory
bodies. Germane is the observation (Adams and Taylor, 2010)
(p. 193), ‘With a high degree of confidence one may presume
that EOs derived from food are likely to be safe’. Additionally,
all the current entries are non-sensitizing to skin when fresh
(Tisserand and Balacs, 1995; Adams and Taylor, 2010), but
may cause allergic reactions at very low rates when oxidized
(Matura et al., 2005). For additional pharmacological data on
other common cannabis terpenoids not discussed herein
(1,8-cineole, also known as eucalyptol, pulegone, a-terpineol,
terpineol-4-ol, r-cymene, borneol and D-3-carene), please see
McPartland and Russo (2001b).
Are cannabis terpenoids actually relevant to the effects of
cannabis? Terpenoid components in concentrations above
0.05% are considered of pharmacological interest (Adams and
Taylor, 2010). Animal studies are certainly supportive (Buchbauer et al., 1993). Mice exposed to terpenoid odours inhaled
from ambient air for 1 h demonstrated profound effects on
activity levels, suggesting a direct pharmacological effect on
the brain, even at extremely low serum concentrations
(examples: linalool with 73% reduction in motility at
4.22 ng·mL-1
, pinene 13.77% increase at trace concentration,
terpineol 45% reduction at 4.7 ng·mL-1
). These levels are
comparable to those of THC measured in humans receiving
cannabis extracts yielding therapeutic effects in pain, or
symptoms of multiple sclerosis in various randomized controlled trials (RCTs) (Russo, 2006; Huestis, 2007). Positive
effects at undetectable serum concentrations with orange terpenes (primarily limonene, 35.25% increase in mouse activity), could be explainable on the basis of rapid redistribution
and concentration in lipophilic cerebral structures. A similar
rationale pertains to human studies (Komori et al., 1995),
subsequently discussed. Limonene is highly bioavailable with
70% human pulmonary uptake (Falk-Filipsson et al., 1993),
and a figure of 60% for pinene with rapid metabolism or
redistribution (Falk et al., 1990). Ingestion and percutaneous
absorption is also well documented in humans (Jäger et al.,
1992): 1500 mg of lavender EO with 24.7% linalool (total
372 mg) was massaged into the skin of a 60 kg man for
10 min, resulting in a peak plasma concentration of
100 ng·mL-1 at 19 min, and a half-life of 13.76 min in serum
(Jäger et al., 1992). EO mixtures (including limonene and
pinene) also increase permeation of estradiol through mouse
skin (Monti et al., 2002).
Government-approved cannabis supplied to patients in
national programmes in the Netherlands and Canada is
gamma-irradiated to sterilize coliform bacteria, but the safety
of this technique for a smoked and inhaled product has never
been specifically tested. Gamma-radiation significantly
reduced linalool titres in fresh cilantro (Fan and Sokorai,
2002), and myrcene and linalool in orange juice (Fan and
Gates, 2001).
D-limonene, common to the lemon and other citrus EOs
(Table 2), is the second most widely distributed terpenoid in
nature (Noma and Asakawa, 2010), and is the precursor to
other monoterpenoids (Figure 2) through species-specific
synthetic schemes. Unfortunately, these pathways have not
yet been investigated in cannabis. The ubiquity of limonene
serves, perhaps, as a demonstration of convergent evolution
that supports an important ecological role for this monoterpene. Studies with varying methodology and dosing in citrus
oils in mice suggest it to be a powerful anxiolytic agent
(Carvalho-Freitas and Costa, 2002; Pultrini Ade et al., 2006),
with one EO increasing serotonin in the prefrontal cortex,
and dopamine (DA) in hippocampus mediated via 5-HT1A
(Komiya et al., 2006). Compelling confirmatory evidence in
humans was provided in a clinical study (Komori et al., 1995),
in which hospitalized depressed patients were exposed to
citrus fragrance in ambient air, with subsequent normalization of Hamilton Depression Scores, successful discontinuation of antidepressant medication in 9/12 patients and serum
evidence of immune stimulation (CD4/8 ratio normalization). Limonene also produces apoptosis of breast cancer
cells, and was employed at high doses in Phase II RCTs
(Vigushin et al., 1998). Subsequent investigation in cancer
treatment has centred on its immediate hepatic metabolite,
perillic acid, which demonstrates anti-stress effects in rat
brain (Fukumoto et al., 2008). A patent has been submitted,
claiming that limonene effectively treats gastro-oesophageal
reflux (Harris, 2010). Citrus EOs containing limonene proved
effective against dermatophytes (Sanguinetti et al., 2007;
Singh et al., 2010), and citrus EOs with terpenoid profiles
resembling those in cannabis demonstrated strong radical
scavenging properties (Choi et al., 2000). As noted above,
limonene is highly bioavailable (Falk-Filipsson et al., 1993),
and rapidly metabolized, but with indications of accumulation and retention in adipose tissues (e.g. brain). It is highly
non-toxic (estimated human lethal dose 0.5–5 g·kg-1
) and
non-sensitizing (Von Burg, 1995)
b-Myrcene is another common monoterpenoid in cannabis (Table 2) with myriad activities: diminishing inflammation via prostaglandin E-2 (PGE-2) (Lorenzetti et al.,
1991), and blocking hepatic carcinogenesis by aflatoxin (DeOliveira et al., 1997). Interestingly, myrcene is analgesic in
mice, but this action can be blocked by naloxone, perhaps
via the a-2 adrenoreceptor (Rao et al., 1990). It is nonmutagenic in the Ames test (Gomes-Carneiro et al., 2005).
Myrcene is a recognized sedative as part of hops preparations (Humulus lupulus), employed to aid sleep in Germany
(Bisset and Wichtl, 2004). Furthermore, myrcene acted as a
muscle relaxant in mice, and potentiated barbiturate sleep
time at high doses (do Vale et al., 2002). Together, these
data would support the hypothesis that myrcene is a prominent sedative terpenoid in cannabis, and combined with
THC, may produce the ‘couch-lock’ phenomenon of certain
chemotypes that is alternatively decried or appreciated by
recreational cannabis consumers.
a-Pinene is a bicyclic monoterpene (Table 2), and the
most widely encountered terpenoid in nature (Noma and
Asakawa, 2010). It appears in conifers and innumerable plant
EOs, with an insect-repellent role. It is anti-inflammatory via
PGE-1 (Gil et al., 1989), and is a bronchodilator in humans at
low exposure levels (Falk et al., 1990). Pinene is a major component of Sideritis spp. (Kose et al., 2010) and Salvia spp. EOs
(Ozek et al., 2010), both with prominent activity against
MRSA (vide infra). Beyond this, it seems to be a broadspectrum antibiotic (Nissen et al., 2010). a-Pinene forms the
biosynthetic base for CB2 ligands, such as HU-308 (Hanus
et al., 1999). Perhaps most compelling, however, is its activity
as an acetylcholinesterase inhibitor aiding memory (Perry
et al., 2000), with an observed IC50 of 0.44 mM (Miyazawa
BJP EB Russo
1350 British Journal of Pharmacology (2011) 163 1344–1364
Table 2
Cannabis Terpenoid Activity Table
Terpenoid Structure
Commonly
encountered in Pharmacological activity (Reference)
Synergistic
cannabinoid
Limonene
H
Lemon
Potent AD/immunostimulant via inhalation
(Komori et al., 1995)
CBD
Anxiolytic (Carvalho-Freitas and Costa, 2002; Pultrini Ade et al.,
2006) via 5-HT1A (Komiya et al., 2006)
CBD
Apoptosis of breast cancer cells (Vigushin et al., 1998) CBD, CBG
Active against acne bacteria (Kim et al., 2008) CBD
Dermatophytes (Sanguinetti et al., 2007; Singh et al., 2010) CBG
Gastro-oesophageal reflux (Harris, 2010) THC
a-Pinene
Pine
Anti-inflammatory via PGE-1 (Gil et al., 1989) CBD
Bronchodilatory in humans (Falk et al., 1990) THC
Acetylcholinesterase inhibitor, aiding memory
(Perry et al., 2000)
THC?, CBD
b-Myrcene
Hops
Blocks inflammation via PGE-2 (Lorenzetti et al., 1991) CBD
Analgesic, antagonized by naloxone (Rao et al., 1990) CBD, THC
Sedating, muscle relaxant, hypnotic (do Vale et al., 2002) THC
Blocks hepatic carcinogenesis by aflatoxin
(de Oliveira et al., 1997)
CBD, CBG
Linalool HO
Lavender
Anti-anxiety (Russo, 2001) CBD, CBG?
Sedative on inhalation in mice (Buchbauer et al., 1993) THC
Local anesthetic (Re et al., 2000) THC
Analgesic via adenosine A2A (Peana et al., 2006) CBD
Anticonvulsant/anti-glutamate (Elisabetsky et al., 1995) CBD, THCV,
CBDV
Potent anti-leishmanial (do Socorro et al., 2003) ?
b-Caryophyllene
Pepper
AI via PGE-1 comparable phenylbutazone (Basile et al., 1988) CBD
Gastric cytoprotective (Tambe et al., 1996) THC
Anti-malarial (Campbell et al., 1997) ?
Selective CB2 agonist (100 nM) (Gertsch et al., 2008) THC
Treatment of pruritus? (Karsak et al., 2007) THC
Treatment of addiction? (Xi et al., 2010) CBD
Caryophyllene
Oxide O
Lemon balm
Decreases platelet aggregation (Lin et al., 2003) THC
Antifungal in onychomycosis comparable to
ciclopiroxolamine and sulconazole (Yang et al., 1999)
CBC,CBG
Insecticidal/anti-feedant (Bettarini et al., 1993) THCA, CBGA
Nerolidol
OH
Orange
Sedative (Binet et al., 1972) THC, CBN
Skin penetrant (Cornwell and Barry, 1994) –
Potent antimalarial (Lopes et al., 1999,
Rodrigues Goulart et al., 2004)
?
Anti-leishmanial activity (Arruda et al., 2005) ?
Phytol
OH
Green tea
Breakdown product of chlorophyll –
Prevents Vitamin A teratogenesis (Arnhold et al., 2002) –
↑GABA via SSADH inhibition (Bang et al., 2002) CBG
Representative plants containing each terpenoid are displayed as examples to promote recognition, but many species contain them in varying concentrations.
5-HT, 5-hydroxytryptamine (serotonin); AD, antidepressant; AI, anti-inflammatory; CB1/CB2, cannabinoid receptor 1 or 2; GABA, gamma aminobutyric acid;
PGE-1/PGE-2, prostaglandin E-1/prostaglandin E-2; SSADH, succinic semialdehyde dehydrogenase.
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1351
and Yamafuji, 2005). This feature could counteract short-term
memory deficits induced by THC intoxication (vide infra).
D-Linalool is a monoterpenoid alcohol (Table 2),
common to lavender (Lavandula angustifolia), whose psychotropic anxiolytic activity has been reviewed in detail (Russo,
2001). Interestingly, linalyl acetate, the other primary terpenoid in lavender, hydrolyses to linalool in gastric secretions
(Bickers et al., 2003). Linalool proved sedating to mouse activity on inhalation (Buchbauer et al., 1991; Jirovetz et al.,
1992). In traditional aromatherapy, linalool is the likely
suspect in the remarkable therapeutic capabilities of lavender
EO to alleviate skin burns without scarring (Gattefosse, 1993).
Pertinent to this, the local anaesthetic effects of linalool (Re
et al., 2000) are equal to those of procaine and menthol
(Ghelardini et al., 1999). Another explanation would be its
ability to produce hot-plate analgesia in mice (P < 0.001) that
was reduced by administration of an adenosine A2A antagonist (Peana et al., 2006). It is also anti-nociceptive at high
doses in mice via ionotropic glutamate receptors (Batista
et al., 2008). Linalool demonstrated anticonvulsant and antiglutamatergic activity (Elisabetsky et al., 1995), and reduced
seizures as part of Ocimum basilicum EO after exposure to
pentylenetetrazole, picrotoxin and strychnine (Ismail, 2006).
Furthermore, linalool decreased K+
-stimulated glutamate
release and uptake in mouse synaptosomes (Silva Brum et al.,
2001). These effects were summarized (Nunes et al., 2010,
p. 303): ‘Overall, it seems reasonable to argue that the modulation of glutamate and GABA neurotransmitter systems are
likely to be the critical mechanism responsible for the sedative, anxiolytic and anticonvulsant properties of linalool and
EOs containing linalool in significant proportions’. Linalool
also proved to be a powerful anti-leishmanial agent (do
Socorro et al., 2003), and as a presumed lavender EO component, decreased morphine opioid usage after inhalation
versus placebo (P = 0.04) in gastric banding in morbidly obese
surgical patients (Kim et al., 2007).
b-Caryophyllene (Table 2) is generally the most common
sesquiterpenoid encountered in cannabis (Mediavilla and
Steinemann, 1997), wherein its evolutionary function may be
due to its ability to attract insect predatory green lacewings,
while simultaneously inhibiting insect herbivory (Langenheim, 1994). It is frequently the predominant terpenoid
overall in cannabis extracts, particularly if they have been
processed under heat for decarboxylation (Guy and Stott,
2005). Caryophyllene is common to black pepper (Piper
nigrum) and Copaiba balsam (Copaifera officinalis) (Lawless,
1995). It is anti-inflammatory via PGE-1, comparable in
potency to the toxic phenylbutazone (Basile et al., 1988), and
an EO containing it was on par with etodolac and indomethacin (Ozturk and Ozbek, 2005). In contrast to the latter agents,
however, caryophyllene was a gastric cytoprotective (Tambe
et al., 1996), much as had been claimed in the past in treating
duodenal ulcers in the UK with cannabis extract (Douthwaite, 1947). Caryophyllene may have contributed to antimalarial effects as an EO component (Campbell et al., 1997).
Perhaps the greatest revelation regarding caryophyllene has
been its demonstration as a selective full agonist at CB2
(100 nM), the first proven phytocannabinoid beyond the
cannabis genus (Gertsch et al., 2008). Subsequent work has
demonstrated that this dietary component produced antiinflammatory analgesic activity at the lowest dose of
5 mg·kg-1 in wild-type, but not CB2 knockout mice (Gertsch,
2008). Given the lack of attributed psychoactivity of CB2
agonists, caryophyllene offers great promise as a therapeutic
compound, whether systemically, or in dermatological applications such as contact dermatitis (Karsak et al., 2007). Sensitization reactions are quite rare, and probably due to
oxidized product (Skold et al., 2006).
Nerolidol is a sesquiterpene alcohol with sedative properties (Binet et al., 1972), present as a low-level component in
orange and other citrus peels (Table 2). It diminished experimentally induced formation of colon adenomas in rats (Wattenberg, 1991). It was an effective agent for enhancing skin
penetration of 5-fluorouracil (Cornwell and Barry, 1994). This
could be a helpful property in treating fungal growth, where
it is also an inhibitor (Langenheim, 1994). It seems to have
anti-protozoal parasite control benefits, as a potent antimalarial (Lopes et al., 1999; Rodrigues Goulart et al., 2004) and
anti-leishmanial agent (Arruda et al., 2005). Nerolidol is nontoxic and non-sensitizing (Lapczynski et al., 2008).
Caryophyllene oxide (Table 2) is a sesquiterpenoid oxide
common to lemon balm (Melissa officinalis), and to the eucalyptus, Melaleuca stypheloides, whose EO contains 43.8%
(Farag et al., 2004). In the plant, it serves as an insecticidal/
anti-feedant (Bettarini et al., 1993) and as broad-spectrum
antifungal in plant defence (Langenheim, 1994). Analogously, the latter properties may prove therapeutic, as caryophyllene oxide demonstrated antifungal efficacy in a model
of clinical onychomycosis comparable to ciclopiroxalamine
and sulconazole, with an 8% concentration affecting eradication in 15 days (Yang et al., 1999). Caryophyllene oxide is
non-toxic and non-sensitizing (Opdyke, 1983). This agent
also demonstrates anti-platelet aggregation properties in vitro
(Lin et al., 2003). Caryophyllene oxide has the distinction of
being the component responsible for cannabis identification
by drug-sniffing dogs (Stahl and Kunde, 1973).
Phytol (Table 2) is a diterpene (McGinty et al., 2010),
present in cannabis extracts, as a breakdown product of chlorophyll and tocopherol. Phytol prevented vitamin A-induced
teratogenesis by inhibiting conversion of retinol to a harmful
metabolite, all-trans-retinoic acid (Arnhold et al., 2002).
Phytol increased GABA expression via inhibition of succinic
semialdehyde dehydrogenase, one of its degradative enzymes
(Bang et al., 2002). Thus, the presence of phytol could
account for the alleged relaxing effect of wild lettuce (Lactuca
sativa), or green tea (Camellia sinensis), despite the latter’s
caffeine content.
Selected possibilities for
phytocannabinoid-terpenoid synergy
Cannabis and acne
AEA simulates lipid production in human sebocytes of sebaceous glands at low concentrations, but induces apoptosis at
higher levels, suggesting that this system is under ECS control
(Dobrosi et al., 2008). CBD 10–20 mM did not affect basal lipid
synthesis in SZ95 sebocytes, but did block such stimulation
by AEA and arachidonate (Biro et al., 2009). Higher doses of
CBD (30–50 mM) induced sebocyte apoptosis, which was augmented in the presence of AEA. The effect of CBD to increase
BJP EB Russo
1352 British Journal of Pharmacology (2011) 163 1344–1364
Ca++ was blocked by ruthenium red, a TRP-inhibitor. RNAmediated silencing of TRPV1 and TRPV3 failed to attenuate
CBD effects, but experiments did support the aetiological role
of TRPV4, a putative regulator of systemic osmotic pressure
(T. Bíró, 2010, pers. comm.). Given the observed ability of
CBD to be absorbed transcutaneously, it offers great promise
to attenuate the increased sebum production at the pathological root of acne.
Cannabis terpenoids could offer complementary activity.
Two citrus EOs primarily composed of limonene inhibited
Propionibacterium acnes, the key pathogen in acne (MIC
0.31 mL·mL-1
), more potently than triclosan (Kim et al.,
2008). Linalool alone demonstrated an MIC of 0.625 mL·mL-1
.
Both EOs inhibited P. acnes-induced TNF-a production, suggesting an adjunctive anti-inflammatory effect. In a similar
manner, pinene was the most potent component of a tea-tree
eucalyptus EO in suppression of P. acnes and Staph spp. in
another report (Raman et al., 1995).
Considering the known minimal toxicities of CBD and
these terpenoids and the above findings, new acne therapies
utilizing whole CBD-predominant extracts, via multitargeting (Wagner and Ulrich-Merzenich, 2009), may present
a novel and promising therapeutic approach that poses
minimal risks in comparison to isotretinoin.
MRSA
MRSA accounted for 10% of cases of septicaemia and 18 650
deaths in the USA in 2005, a number greater than that attributable to human immunodeficiency virus/acquired immunodeficiency syndrome (Bancroft, 2007). Pure CBD and CBG
powerfully inhibit MRSA (MIC 0.5–2 mg·mL-1
) (Appendino
et al., 2008).
Amongst terpenoids, pinene was a major component of
Sideritis erythrantha EO that was as effective against MRSA and
other antibiotic-resistant bacterial strains as vancomycin and
other agents (Kose et al., 2010). A Salvia rosifolia EO with
34.8% pinene was also effective against MRSA (MIC
125 mg·mL-1
). The ability of monoterpenoids to enhance skin
permeability and entry of other drugs may further enhance
antibiotic benefits (Wagner and Ulrich-Merzenich, 2009).
Given that CBG can be produced in selected cannabis
chemotypes (de Meijer and Hammond, 2005; de Meijer et al.,
2009a), with no residual THC as a possible drug abuse liability
risk, a whole plant extract of a CBG-chemotype also expressing pinene would seem to offer an excellent, safe new antiseptic agent.
Psychopharmacological applications:
depression, anxiety, insomnia,
dementia and addiction
Scientific investigation of the therapeutic application of terpenoids in psychiatry has been hampered by methodological
concerns, subjective variability of results and a genuine
dearth of appropriate randomized controlled studies of high
quality (Russo, 2001; Bowles, 2003; Lis-Balchin, 2010). The
same is true of phytocannabinoids (Fride and Russo, 2006).
Abundant evidence supports the key role of the ECS in mediating depression (Hill and Gorzalka, 2005a,b), as well as
anxiety, whether induced by aversive stimuli, such as posttraumatic stress disorder (Marsicano et al., 2002) or pain
(Hohmann et al., 2005), and psychosis (Giuffrida et al., 2004).
With respect to the latter risk, the presence of CBD in smoked
cannabis based on hair analysis seems to be a mitigating
factor reducing its observed incidence (Morgan and Curran,
2008). A thorough review of cannabis and psychiatry is
beyond the scope of this article, but several suggestions are
offered with respect to possible therapeutic synergies operative with phytocannabinoids-terpenoid combinations. While
the possible benefits of THC on depression remain controversial (Denson and Earleywine, 2006), much less worrisome
would be CBD- or CBG-predominant preparations. Certainly
the results obtained in human depression solely with a citrus
scent (Komori et al., 1995), strongly suggest the possibility of
synergistic benefit of a phytocannabinoid-terpenoid preparation. Enriched odour exposure in adult mice induced olfactory system neurogenesis (Rochefort et al., 2002), an
intriguing result that could hypothetically support plasticity
mechanisms in depression (Delgado and Moreno, 1999), and
similar hypotheses with respect to the ECS in addiction treatment (Gerdeman and Lovinger, 2003). Phytocannabinoidterpenoid synergy might theoretically apply.
The myriad effects of CBD on 5-HT1A activity provide a
strong rationale for this and other phytocannabinoids as base
compounds for treatment of anxiety. Newer findings, particularly imaging studies of CBD in normal individuals in anxiety
models (Fusar-Poli et al., 2009; 2010; Crippa et al., 2010)
support this hypothesis. Even more compelling is a recent
randomized control trial of pure CBD in patients with social
anxiety disorder with highly statistical improvements over
placebo in anxiety and cognitive impairment (Crippa et al.,
2011). Addition of anxiolytic limonene and linalool could
contribute to the clinical efficacy of a CBD extract.
THC was demonstrated effective in a small crossover clinical trial versus placebo in 11 agitated dementia patients with
Alzheimer’s disease (Volicer et al., 1997). THC was also
observed to be an acetylcholinesterase inhibitor in its own
right, as well as preventing amyloid b-peptide aggregation in
that disorder (Eubanks et al., 2006). Certainly, the antianxiety and anti-psychotic effects of CBD may be of additional benefit (Zuardi et al., 1991; 2006; Zuardi and
Guimaraes, 1997). A recent study supports the concept that
CBD, when present in significant proportion to THC, is
capable of eliminating induced cognitive and memory deficits in normal subjects smoking cannabis (Morgan et al.,
2010b). Furthermore, CBD may also have primary benefits on
reduction of b-amyloid in Alzheimer’s disease (Iuvone et al.,
2004; Esposito et al., 2006a,b). Psychopharmacological effects
of limonene, pinene and linalool could putatively extend
benefits in mood in such patients.
The effects of cannabis on sleep have been reviewed
(Russo et al., 2007), and highlight the benefits that can accrue
in this regard, particularly with respect to symptom reduction
permitting better sleep, as opposed to a mere hypnotic effect.
Certainly, terpenoids with pain-relieving, anti-anxiety or
sedative effects may supplement such activity, notably, caryophyllene, linalool and myrcene.
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1353
The issue of cannabis addiction remains controversial.
Some benefit of oral THC has been noted in cannabis withdrawal (Hart et al., 2002; Haney et al., 2004). More intriguing,
perhaps, are claims of improvement on other substance
dependencies, particularly cocaine (Labigalini et al., 1999;
Dreher, 2002). The situation with CBD is yet more promising.
CBD and THC at doses of 4 mg·kg-1 i.p. potentiated extinction of cocaine- and amphetamine-induced conditioned
place preference in rats, and CBD produced no hedonic
effects of its own (Parker et al., 2004). CBD 5 mg·kg-1
·d-1 in
rats attenuated heroin-seeking behaviour by conditioned
stimuli, even after a lapse of 2 weeks (Ren et al., 2009).
A suggested mechanism of CBD relates to its ability
to reverse changes in a-amino-3-hydroxyl-5-methyl-4-
isoxazole-propionate glutamate and CB1 receptor expression
in the nucleus accumbens induced by heroin. The authors
proposed CBD as a treatment for heroin craving and addiction relapse. A recent study demonstrated the fascinating
result that patients with damage to the insula due to cerebrovascular accident were able to quit tobacco smoking
without relapse or urges (Naqvi et al., 2007), highlighting this
structure as a critical neural centre mediating addiction to
nicotine. Further study has confirmed the role of the insula in
cocaine, alcohol and heroin addiction (Naqvi and Bechara,
2009; Naqvi and Bechara, 2010). In a provocative parallel,
CBD 600 mg p.o. was demonstrated to deactivate functional
magnetic resonance imaging (fMRI) activity in human volunteers in the left insula versus placebo (P < 0.01) without
accompanying sedation or psychoactive changes (Borgwardt
et al., 2008), suggesting the possibility that CBD could act as
a pharmaceutical surrogate for insular damage in exerting an
anti-addiction therapeutic benefit. Human studies have
recently demonstrated that human volunteers smoking cannabis with higher CBD content reduced their liking for drugrelated stimuli, including food (Morgan et al., 2010a). The
authors posited that CBD can modulate reinforcing properties of drugs of abuse, and help in training to reduce relapse
to alcoholism. A single case report of a successful withdrawal
from cannabis dependency utilizing pure CBD treatment was
recently published (Crippa et al., 2010).
Perhaps terpenoids can provide adjunctive support. In a
clinical trial, 48 cigarette smokers inhaling vapour from an
EO of black pepper (Piper nigrum), a mint-menthol mixture or
placebo (Rose and Behm, 1994). Black pepper EO reduced
nicotine craving significantly (P < 0.01), an effect attributed
to irritation of the bronchial tree, simulating the act of cigarette smoking, but without nicotine or actual burning of
material. Rather, might not the effect have been pharmacological? The terpenoid profile of black pepper suggests possible candidates: myrcene via sedation, pinene via increased
alertness, or especially caryophyllene via CB2 agonism and a
newly discovered putative mechanism of action in addiction
treatment.
CB2 is expressed in dopaminergic neurones in the ventral
tegmental area and nucleus accumbens, areas mediating
addictive phenomena (Xi et al., 2010). Activation of CB2 by
the synthetic agonist JWH144 administered systemically,
intranasally, or by microinjection into the nucleus accumbens in rats inhibited DA release and cocaine selfadministration. Caryophyllene, as a high-potency selective
CB2 agonist (Gertsch et al., 2008), would likely produce
similar effects, and have the advantage of being a nontoxic dietary component. All factors considered, CBD, with
caryophyllene, and possibly other adjunctive terpenoids in
the extract, offers significant promise in future addiction
treatment.
Taming THC: cannabis entourage
compounds as antidotes
to intoxication
Various sources highlight the limited therapeutic index of
pure THC, when given intravenously (D’Souza et al., 2004) or
orally (Favrat et al., 2005), especially in people previously
naïve to its effects. Acute overdose incidents involving THC
or THC-predominant cannabis usually consist of self-limited
panic reactions or toxic psychoses, for which no pharmacological intervention is generally necessary, and supportive
counselling (reassurance or ‘talking down’) is sufficient to
allow resolution without sequelae. CBD modulates the psychoactivity of THC and reduces its adverse event profile
(Russo and Guy, 2006), highlighted by recent results above
described. Could it be, however, that other cannabis components offer additional attenuation of the less undesirable
effects of THC? History provides some clues.
In 10th century Persia, Al-Razi offered a prescription in his
Manafi al-agdhiya wa-daf madarri-ha (p. 248), rendered
(Lozano, 1993, p. 124; translation EBR) ‘ – and to avoid these
harms {from ingestion of cannabis seeds or hashish}, one
should drink fresh water and ice or eat any acid fruits’. This
concept was repeated in various forms by various authorities
through the ages, including ibn Sina (ibn Sina (Avicenna),
1294), and Ibn al-Baytar (ibn al-Baytar, 1291), until
O’Shaughnessy brought Indian hemp to Britain in 1843
(O’Shaughnessy, 1843). Robert Christison subsequently cited
lemon (Figure 3A) as an antidote to acute intoxication in
numerous cases (Christison, 1851) and this excerpt regarding
morning-after residua (Christison, 1848) (p. 973):
Next morning there was an ordinary appetite, much
torpidity, great defect and shortness of memory, extreme
apparent protraction of time, but no peculiarity of
articulation or other effect; and these symptoms lasted
until 2 P.M., when they ceased entirely in a few minutes
after taking lemonade.
Literary icons on both sides of the Atlantic espoused
similar support for the citrus cure in the 19th century,
notably Bayard Taylor after travels in Syria (Taylor, 1855), and
Fitzhugh Ludlow after his voluntary experiments with ever
higher cannabis extract doses in the USA (Ludlow, 1857). The
sentiment was repeated by Calkins (1871), who noted the
suggestion of a friend in Tunis that lemon retained the confidence of cure of overdoses by cannabis users in that region.
This is supported by the observation that lemon juice, which
normally contains small terpenoid titres, is traditionally
enhanced in North Africa by the inclusion in drinks of the
limonene-rich rind, as evidenced by the recipe for Agua Limón
from modern Morocco (Morse and Mamane, 2001). In his
comprehensive review of cannabis in the first half of the
20th century, Walton once more supported its prescription
(Walton, 1938).
BJP EB Russo
1354 British Journal of Pharmacology (2011) 163 1344–1364
Another traditional antidote to cannabis employing
Acorus calamus (Figure 3B) is evident from the Ayurvedic tradition of India (Lad, 1990, p. 131):
Calamus root is the best antidote for the ill effects of
marijuana. . . . if one smokes a pinch of calamus root
powder with the marijuana, this herb will completely
neutralize the toxic side effects of the drug.
This claim has gained credence, not only through force of
anecdotal accounts that abound on the Internet, but
with formal scientific case reports and scientific analysis
(McPartland et al., 2008) documenting clearer thinking and
improved memory with the cannabis–calamus combination,
and with provision of a scientific rationale: calamus contains
beta-asarone, an acetylcholinesterase inhibitor with 10% of
the potency of physotigmine (Mukherjee et al., 2007). Interestingly, the cannabis terpenoid, a-pinene, also has been
characterized as a potent inhibitor of that enzyme (Miyazawa
and Yamafuji, 2005), bolstering the hypothesis of a second
antidote to THC contained in cannabis itself. Historical precedents also support pinene in this pharmacological role.
In the firstt century, Pliny wrote of cannabis in his Natural
History, Book XXIV (Pliny, 1980, p. 164):
The gelotophyllis [‘leaves of laughter’ = cannabis] grows
in Bactria and along the Borysthenes. If this be taken in
myrrh and wine all kinds of phantoms beset the mind,
causing laughter which persists until the kernels of pinenuts are taken with pepper and honey in palm wine.
Of the components, palm wine is perhaps the most mysterious. Ethanol does not reduce cannabis intoxication (Mello
and Mendelson, 1978). However, ancient wines were stored in
clay pots or goatskins, and required preservation, usually with
addition of pine tar or terebinth resin (from Pistacia spp.;
McGovern et al., 2009). Pine tar is rich in pinene, as is terebinth resin (from Pistacia terebinthus; Tsokou et al., 2007),
while the latter also contains limonene (Duru et al., 2003).
Likewise, the pine nuts (Figure 3C) prescribed by Pliny the
Elder harbour pinene, along with additional limonene (Salvadeo et al., 2007). Al-Ukbari also suggested pistachio nuts as a
cannabis antidote in the 13th century (Lozano, 1993), and the
ripe fruits of Pistacia terebinthus similarly contain pinene (Couladis et al., 2003). The black pepper (Figure 3D), might offer
the mental clarity afforded by pinene, sedation via myrcene
and helpful contributions by b-caryophyllene. The historical
suggestions for cannabis antidotes are thus supported by
modern scientific rationales for the claims, and if proven
experimentally would provide additional evidence of synergy
(Berenbaum, 1989; Wagner and Ulrich-Merzenich, 2009).
Conclusions and suggestions for
future study
Considered ensemble, the preceding body of information
supports the concept that selective breeding of cannabis
chemotypes rich in ameliorative phytocannabinoid and terpenoid content offer complementary pharmacological activities that may strengthen and broaden clinical applications and
improve the therapeutic index of cannabis extracts containing
THC, or other base phytocannabinoids. Psychopharmacological and dermatological indications show the greatest promise.
Figure 3
Ancient cannabis antidotes. (A) Lemon (Citrus limon). (B) Calamus plant roots (Acorus calamus). (C) Pine nuts (Pinus spp.). (D) Black pepper
(Piper nigrum).
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1355
One important remaining order of business is the elucidation of mono- and sesquiterpenoid biosynthetic pathways
in cannabis, as has been achieved previously in other species
of plants (Croteau, 1987; Gershenzon and Croteau, 1993;
Bohlmann et al., 1998; Turner et al., 1999; Trapp and Croteau,
2001).
Various cannabis component combinations or cannabis
extracts should be examined via high throughput pharmacological screening where not previously accomplished. Another
goal is the investigation of the biochemical targets of the
cannabis terpenoids, along with their mechanisms of action,
particularly in the central nervous system. Possible techniques
for such research include radio-labelling of select agents in
animals with subsequent necropsy. On a molecular level,
investigation of terpenoid changes to phytocannabinoid
signal transduction and trafficking may prove illuminating.
While it is known that terpenoids bind to odorant receptors in
the nasal mucosa (Friedrich, 2004) and proximal olfactory
structures (Barnea et al., 2004), it would be essential to ascertain if direct effects in limbic or other cerebral structures are
operative. Given that farnesyl pyrophosphate is a sesquiterpenoid precursor and the most potent endogenous agonist yet
discovered for GPR92 (McHugh et al., 2010), in silico studies
attempting to match minor cannabinoids and terpenoids to
orphan GPCRs may prove fruitful. Behavioural assays of
agents in animal models may also provide clues. Simple combinations of phytocannabinoids and terpenoids may demonstrate synergy as antibiotics if MICs are appreciable lowered
(Wagner and Ulrich-Merzenich, 2009). Ultimately, fMRI and
single photon emission computed tomography studies in
humans, with simultaneous drug reaction questionnaires and
psychometric testing employing individual agents and
phytocannabinoid-terpenoid pairings via vaporization or oromucosal application, would likely offer safe and effective
methods to investigate possible interactions and synergy.
Should positive outcomes result from such studies, phytopharmaceutical development may follow. The development of zero-cannabinoid cannabis chemotypes (de Meijer
et al., 2009b) has provided extracts that will facilitate discernment of the pharmacological effects and contributions of
different fractions. Breeding work has already resulted in
chemotypes that produce 97% of monoterpenoid content as
myrcene, or 77% as limonene (E. de Meijer, pers. comm.).
Selective cross-breeding of high-terpenoid- and highphytocannabinoid-specific chemotypes has thus become a
rational target that may lead to novel approaches to such
disorders as treatment-resistant depression, anxiety, drug
dependency, dementia and a panoply of dermatological disorders, as well as industrial applications as safer pesticides
and antiseptics. A better future via cannabis phytochemistry
may be an achievable goal through further research of the
entourage effect in this versatile plant that may help it fulfil
its promise as a pharmacological treasure trove.
Acknowledgements
The author offers appreciation to the following individuals,
who provided materials and/or consultation: David Potter,
Etienne de Meijer, John McPartland, David Watson, Rob
Clarke, Indalecio Lozano, Támas Bíró, José Crippa, Roger
Pertwee, Colin Stott, Vincenzo Di Marzo, Luciano De Petrocellis, Patrick McGovern, John Riddle and Elisaldo Carlini.
Most of all, I would like to thank Raphael Mechoulam for his
example, guidance, friendship, a life of good works and for
listening to many ‘crazy ideas’.
Conflict of Interest
The author is a Senior Medical Advisor to GW Pharmaceuticals and serves as a consultant.
References
Adams TB, Taylor SV (2010). Safety evaluation of essential oils: a
constituent-based approach. In: Baser KHC, Buchbauer G (eds).
Handbook of Essential Oils: Science, Technology, and Applications.
CRC Press: Boca Raton, FL, pp. 185–208.
Alexander SP, Mathie A, Peters JA (2009). Guide to Receptors and
Channels (GRAC), 4th edition. Br J Pharmacol 158 (Suppl. 1):
S1–254.
Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M
et al. (2008). Antibacterial cannabinoids from Cannabis sativa: a
structure-activity study. J Nat Prod 71: 1427–1430.
Arnhold T, Elmazar MM, Nau H (2002). Prevention of vitamin A
teratogenesis by phytol or phytanic acid results from reduced
metabolism of retinol to the teratogenic metabolite,
all-trans-retinoic acid. Toxicol Sci 66: 274–282.
Arruda DC, D’Alexandri FL, Katzin AM, Uliana SR (2005).
Antileishmanial activity of the terpene nerolidol. Antimicrob
Agents Chemother 49: 1679–1687.
Baek SH, Kim YO, Kwag JS, Choi KE, Jung WY, Han DS (1998).
Boron trifluoride etherate on silica-A modified Lewis acid reagent
(VII). Antitumor activity of cannabigerol against human oral
epitheloid carcinoma cells. Arch Pharm Res 21: 353–356.
Bancroft EA (2007). Antimicrobial resistance: it’s not just for
hospitals. JAMA 298: 1803–1804.
Banerjee SP, Snyder SH, Mechoulam R (1975). Cannabinoids:
influence on neurotransmitter uptake in rat brain synaptosomes. J
Pharmacol Exp Ther 194: 74–81.
Bang MH, Choi SY, Jang TO, Kim SK, Kwon OS, Kang TC et al.
(2002). Phytol, SSADH inhibitory diterpenoid of Lactuca sativa.
Arch Pharm Res 25: 643–646.
Barnea G, O’Donnell S, Mancia F, Sun X, Nemes A, Mendelsohn M
et al. (2004). Odorant receptors on axon termini in the brain.
Science 304: 1468.
Basile AC, Sertie JA, Freitas PC, Zanini AC (1988).
Anti-inflammatory activity of oleoresin from Brazilian Copaifera. J
Ethnopharmacol 22: 101–109.
Batista PA, Werner MF, Oliveira EC, Burgos L, Pereira P, Brum LF
et al. (2008). Evidence for the involvement of ionotropic
glutamatergic receptors on the antinociceptive effect of (-)-linalool
in mice. Neurosci Lett 440: 299–303.
Ben-Shabat S, Fride E, Sheskin T, Tamiri T, Rhee MH, Vogel Z et al.
(1998). An entourage effect: inactive endogenous fatty acid glycerol
esters enhance 2-arachidonoyl-glycerol cannabinoid activity. Eur J
Pharmacol 353: 23–31.
BJP EB Russo
1356 British Journal of Pharmacology (2011) 163 1344–1364
Berenbaum MC (1989). What is synergy? Pharmacol Rev 41:
93–141.
Bettarini F, Borgonovi GE, Fiorani T, Gagliardi I, Caprioli V,
Massardo P et al. (1993). Antiparasitic compounds from East African
plants: isolation and biological activtiry of anonaine, matricarianol,
canthin-6-one, and caryophyllene oxide. Insect Sci Appl 14: 93–99.
Bickers D, Calow P, Greim H, Hanifin JM, Rogers AE, Saurat JH
et al. (2003). A toxicologic and dermatologic assessment of linalool
and related esters when used as fragrance ingredients. Food Chem
Toxicol 41: 919–942.
Binet L, Binet P, Miocque M, Roux M, Bernier A (1972). Recherches
sur les proprietes pharmcodynamiques (action sedative et action
spasmolytique) de quelques alcools terpeniques aliphatiques. Ann
Pharm Fr 30: 611–616.
Biro T, Olah A, Toth BI, Czifra G, Zouboulis CC, Paus R (2009).
Cannabidiol as a novel anti-acne agent? Cannabidiol inhibits lipid
synthesis and induces cell death in human sebaceous gland-derived
sebocytes. Proceedings 19th Annual Conference on the
Cannabinoids. International Cannabinoid Research Society:
Pheasant Run, St. Charles, IL, p. 28.
Bisogno T, Hanus L, De Petrocellis L, Tchilibon S, Ponde DE,
Brandi I et al. (2001). Molecular targets for cannabidiol and its
synthetic analogues: effect on vanilloid VR1 receptors and on the
cellular uptake and enzymatic hydrolysis of anandamide. Br J
Pharmacol 134: 845–852.
Bisset NG, Wichtl M (2004). Herbal Drugs and
Phytopharmaceuticals: A Handbook for Practice on A Scientific
Basis, 3rd edn. Medpharm Scientific Publishers: Stuttgart; CRC
Press: Boca Raton, FL.
Bloor RN, Wang TS, Spanel P, Smith D (2008). Ammonia release
from heated ‘street’ cannabis leaf and its potential toxic effects on
cannabis users. Addiction 103: 1671–1677.
Bohlmann J, Meyer-Gauen G, Croteau R (1998). Plant terpenoid
synthases: molecular biology and phylogenetic analysis. Proc Natl
Acad Sci USA 95: 4126–4133.
Bolognini D, Costa B, Maione S, Comelli F, Marini P, Di Marzo V
et al. (2010). The plant cannabinoid Delta9-tetrahydrocannabivarin
can decrease signs of inflammation and inflammatory pain in mice.
Br J Pharmacol 160: 677–687.
Borgwardt SJ, Allen P, Bhattacharyya S, Fusar-Poli P, Crippa JA,
Seal ML et al. (2008). Neural basis of Delta-9-tetrahydrocannabinol
and cannabidiol: effects during response inhibition. Biol Psychiatry
64: 966–973.
Bowles EJ (2003). The Chemistry of Aromatherapeutic Oils, 3rd
edn. Allen & Unwin: Crow’s Nest, NSW.
Bradshaw HB, Lee SH, McHugh D (2009). Orphan endogenous
lipids and orphan GPCRs: a good match. Prostaglandins Other
Lipid Mediat 89: 131–134.
Brenneisen R (2007). Chemistry and analysis of phytocannabinoids
and other Cannabis constituents. In: Elsohly M (ed.). Marijuana and
the Cannabinoids. Humana Press: Totowa, NY, pp. 17–49.
Buchbauer G (2010). Biological activities of essential oils. In:
Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science,
Technology, and Applications. CRC Press: Boca Raton, FL,
pp. 235–280.
Buchbauer G, Jirovetz L, Jager W, Dietrich H, Plank C (1991).
Aromatherapy: evidence for sedative effects of the essential oil of
lavender after inhalation. Z Naturforsch [C] 46: 1067–1072.
Buchbauer G, Jirovetz L, Jager W, Plank C, Dietrich H (1993).
Fragrance compounds and essential oils with sedative effects upon
inhalation. J Pharm Sci 82: 660–664.
Calkins A (1871). Opium and the Opium-Appetite: with Notices of
Alcoholic Beverages, Cannabis Indica, Tobacco and Coca, and Tea
and Coffee, in Their Hygienic Aspects and Pathologic Relationships.
J.B. Lippincott: Philadelphia, PA.
Campbell WE, Gammon DW, Smith P, Abrahams M, Purves TD
(1997). Composition and antimalarial activity in vitro of the
essential oil of Tetradenia riparia. Planta Med 63: 270–272.
Carlini EA, Cunha JM (1981). Hypnotic and antiepileptic effects of
cannabidiol. J Clin Pharmacol 21 (Suppl.): 417S–427S.
Carlini EA, Karniol IG, Renault PF, Schuster CR (1974). Effects of
marihuana in laboratory animals and in man. Br J Pharmacol 50:
299–309.
Carrier EJ, Auchampach JA, Hillard CJ (2006). Inhibition of an
equilibrative nucleoside transporter by cannabidiol: a mechanism
of cannabinoid immunosuppression. Proc Natl Acad Sci USA 103:
7895–7900.
Carvalho-Freitas MI, Costa M (2002). Anxiolytic and sedative effects
of extracts and essential oil from Citrus aurantium L. Biol Pharm
Bull 25: 1629–1633.
Cascio MG, Gauson LA, Stevenson LA, Ross RA, Pertwee RG (2010).
Evidence that the plant cannabinoid cannabigerol is a highly
potent alpha2-adrenoceptor agonist and moderately potent 5HT1A
receptor antagonist. Br J Pharmacol 159: 129–141.
Cawthorne MA, Wargent E, Zaibi M, Stott C, Wright S (2007). The
CB1 antagonist, delta-9-tetrahydrocannabivarin (THCV) has antioebesity activity in dietary-induced obese (DIO) mice. Proceedings
17th Annual Symposium on the Cannabinoids. International Cannabinoid Research Society: Saint-Sauveur, QC, p. 141.
Choi HS, Song HS, Ukeda H, Sawamura M (2000).
Radical-scavenging activities of citrus essential oils and their
components: detection using 1,1-diphenyl-2-picrylhydrazyl. J Agric
Food Chem 48: 4156–4161.
Christison R (1848). A Dispensatory Or Commentary on the
Pharmacopoeias of Great Britain and the United States. Lea and
Blanchard: Philadelphia, PA.
Christison A (1851). On the natural history, action, and uses of
Indian hemp. Monthly J Med Sci Edinburgh, Scotland 13: 26–45.
117-121.
Clarke RC (2010). Hashish!, 2nd edn. Red Eye Press: Los Angeles, CA.
Comelli F, Bettoni I, Colleoni M, Giagnoni G, Costa B (2009).
Beneficial effects of a Cannabis sativa extract treatment on
diabetes-induced neuropathy and oxidative stress. Phytother Res 23:
1678–1684.
Cornwell PA, Barry BW (1994). Sesquiterpene components of
volatile oils as skin penetration enhancers for the hydrophilic
permeant 5-fluorouracil. J Pharm Pharmacol 46: 261–269.
Costa B, Trovato AE, Comelli F, Giagnoni G, Colleoni M (2007).
The non-psychoactive cannabis constituent cannabidiol is an orally
effective therapeutic agent in rat chronic inflammatory and
neuropathic pain. Eur J Pharmacol 556: 75–83.
Couladis M, Ozcan M, Tzakou O, Akgul A (2003). Comparative
essential oil compostion of various parts of the turpentine tree
(Pistacia terebinthus) growing wild in Turkey. J Sci Food Agric 83:
136–138.
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1357
Crippa JA, Zuardi AW, Hallak JE (2010). [Therapeutical use of the
cannabinoids in psychiatry]. Rev Bras Psiquiatr 32 (Suppl. 1):
S56–S66.
Crippa JA, Derenusson GN, Ferrari TB, Wichert-Ana L, Duran F,
Marti NSRO et al. (2011). Neural basis of anxiolytic effects of
cannabidiol (CBD) in generalized social anxiety disorder: a
preliminary report. J Psychopharmacol 25: 121–130.
Croteau R (1987). Biosynthesis and catabolism of monoterpenoids.
Chem Rev 87: 929–954.
De Oliveira AC, Ribeiro-Pinto LF, Paumgartten JR (1997). In vitro
inhibition of CYP2B1 monooxygenase by beta-myrcene and other
monoterpenoid compounds. Toxicol Lett 92: 39–46.
D’Souza DC, Perry E, MacDougall L, Ammerman Y, Cooper T,
Wu YT et al. (2004). The psychotomimetic effects of intravenous
delta-9-tetrahydrocannabinol in healthy individuals: implications
for psychosis. Neuropsychopharmacology 29: 1558–1572.
Davalos SD, Fournier G, Boucher F, Paris M (1977). [Contribution
to the study of Mexican marihuana. Preliminary studies:
cannabinoids and essential oil (author’s transl)]. J Pharm Belg 32:
89–99.
Davis WM, Hatoum NS (1983). Neurobehavioral actions of
cannabichromene and interactions with delta
9-tetrahydrocannabinol. Gen Pharmacol 14: 247–252.
De Oliveira AC, Ribeiro-Pinto LF, Paumgartten JR (1997). In vitro
inhibition of CYP2B1 monooxygenase by beta-myrcene and other
monoterpenoid compounds. Toxicol Lett 92: 39–46.
De Petrocellis L, Di Marzo V (2010). Non-CB1, non-CB2 receptors
for endocannabinoids, plant cannabinoids, and synthetic
cannabimimetics: focus on G-protein-coupled receptors and
transient receptor potential channels. J Neuroimmune Pharmacol 5:
103–121.
De Petrocellis L, Vellani V, Schiano-Moriello A, Marini P,
Magherini PC, Orlando P et al. (2008). Plant-derived cannabinoids
modulate the activity of transient receptor potential channels of
ankyrin type-1 and melastatin type-8. J Pharmacol Exp Ther 325:
1007–1015.
De Petrocellis L, Ligresti A, Moriello AS, Allara M, Bisogno T,
Petrosino S et al. (2011). Effects of cannabinoids and
cannabinoid-enriched Cannabis extracts on TRP channels
and endocannabinoid metabolic enzymes. Br J Pharmacol
DOI:10.1111/j.1476-5381.2010.0166.x
Delgado P, Moreno F (1999). Antidepressants and the brain. Int
Clin Psychopharmacol 14 (Suppl. 1): S9–16.
Delong GT, Wolf CE, Poklis A, Lichtman AH (2010).
Pharmacological evaluation of the natural constituent of
Cannabis sativa, cannabichromene and its modulation by
Delta(9)-tetrahydrocannabinol. Drug Alcohol Depend 112: 126–133.
Denson TF, Earleywine M (2006). Decreased depression in
marijuana users. Addict Behav 31: 738–742.
Devane WA, Dysarz FA 3rd, Johnson MR, Melvin LS, Howlett AC
(1988). Determination and characterization of a cannabinoid
receptor in rat brain. Mol Pharmacol 34: 605–613.
Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA,
Griffin G et al. (1992). Isolation and structure of a brain constituent
that binds to the cannabinoid receptor. Science 258: 1946–1949.
Deyo R, Musty R (2003). A cannabichromene (CBC) extract alters
behavioral despair on the mouse tail suspension test of depression.
Proceedings 2003 Symposium on the Cannabinoids. International
Cannabinoid Research Society: Cornwall, ON, p. 146.
Dirikoc S, Priola SA, Marella M, Zsurger N, Chabry J (2007).
Nonpsychoactive cannabidiol prevents prion accumulation and
protects neurons against prion toxicity. J Neurosci 27: 9537–9544.
Dobrosi N, Toth BI, Nagy G, Dozsa A, Geczy T, Nagy G et al. (2008).
Endocannabinoids enhance lipid synthesis and apoptosis of human
sebocytes via cannabinoid receptor-2-mediated signaling. FASEB J
22: 3685–3695.
Douthwaite AH (1947). Choice of drugs in the treatment of
duodenal ulcer. Br Med J 2: 43–47.
Dreher M (2002). Crack heads and roots daughters: the therapeutic
use of cannabis in Jamaica. J Cannabis Therap 2: 121–133.
Duru ME, Cakir A, Kordali S, Zengin H, Harmandar M, Izumi S
et al. (2003). Chemical composition and antifungal properties of
essential oils of three Pistacia species. Fitoterapia 74: 170–176.
Elisabetsky E, Marschner J, Souza DO (1995). Effects of Linalool on
glutamatergic system in the rat cerebral cortex. Neurochem Res 20:
461–465.
ElSohly HN, Turner CE, Clark AM, ElSohly MA (1982). Synthesis
and antimicrobial activities of certain cannabichromene and
cannabigerol related compounds. J Pharm Sci 71: 1319–1323.
Esposito G, De Filippis D, Carnuccio R, Izzo AA, Iuvone T (2006a).
The marijuana component cannabidiol inhibits
beta-amyloid-induced tau protein hyperphosphorylation through
Wnt/beta-catenin pathway rescue in PC12 cells. J Mol Med 84:
253–258.
Esposito G, De Filippis D, Maiuri MC, De Stefano D, Carnuccio R,
Iuvone T (2006b). Cannabidiol inhibits inducible nitric oxide
synthase protein expression and nitric oxide production in
beta-amyloid stimulated PC12 neurons through p38 MAP kinase
and NF-kappaB involvement. Neurosci Lett 399: 91–95.
Eubanks LM, Rogers CJ, Beuscher AE 4th, Koob GF, Olson AJ,
Dickerson TJ et al. (2006). A molecular link between the active
component of marijuana and Alzheimer’s disease pathology. Mol
Pharm 3: 773–777.
Evans FJ (1991). Cannabinoids: the separation of central from
peripheral effects on a structural basis. Planta Med 57: S60–S67.
Fairbairn JW, Pickens JT (1981). Activity of cannabis in relation to
its delta’-trans-tetrahydro-cannabinol content. Br J Pharmacol 72:
401–409.
Falk AA, Hagberg MT, Lof AE, Wigaeus-Hjelm EM, Wang ZP (1990).
Uptake, distribution and elimination of alpha-pinene in man after
exposure by inhalation. Scand J Work Environ Health 16: 372–378.
Falk-Filipsson A, Lof A, Hagberg M, Hjelm EW, Wang Z (1993).
d-limonene exposure to humans by inhalation: uptake,
distribution, elimination, and effects on the pulmonary function. J
Toxicol Environ Health 38: 77–88.
Fan X, Gates RA (2001). Degradation of monoterpenes in orange
juice by gamma radiation. J Agric Food Chem 49: 2422–2426.
Fan X, Sokorai KJ (2002). Changes in volatile compounds of
gamma-irradiated fresh cilantro leaves during cold storage. J Agric
Food Chem 50: 7622–7626.
Farag RS, Shalaby AS, El-Baroty GA, Ibrahim NA, Ali MA,
Hassan EM (2004). Chemical and biological evaluation of the
essential oils of different Melaleuca species. Phytother Res 18:
30–35.
Favrat B, Menetrey A, Augsburger M, Rothuizen L, Appenzeller M,
Buclin T et al. (2005). Two cases of ‘cannabis acute psychosis’
following the administration of oral cannabis. BMC Psychiatry
5: 17.
BJP EB Russo
1358 British Journal of Pharmacology (2011) 163 1344–1364
Fellermeier M, Eisenreich W, Bacher A, Zenk MH (2001).
Biosynthesis of cannabinoids. Incorporation experiments with
(13)C-labeled glucoses. Eur J Biochem 268: 1596–1604.
Fischedick JT, Hazekamp A, Erkelens T, Choi YH, Verpoorte R
(2010). Metabolic fingerprinting of Cannabis sativa L., cannabinoids
and terpenoids for chemotaxonomic and drug standardization
purposes. Phytochem 71: 2058–2073.
Formukong EA, Evans AT, Evans FJ (1988). Analgesic and
antiinflammatory activity of constituents of Cannabis sativa L.
Inflammation 12: 361–371.
Franz C, Novak J (2010). Sources of essential oils. In: Baser KHC,
Buchbauer G (eds). Handbook of Essential Oils: Science,
Technology, and Applications. CRC Press: Boca Raton, FL,
pp. 39–82.
Fride E, Russo EB (2006). Neuropsychiatry: Schizophrenia,
depression, and anxiety. In: Onaivi E, Sugiura T, Di Marzo V (eds).
Endocannabinoids: The Brain and Body’s Marijuana and beyond.
Taylor & Francis: Boca Raton, FL, pp. 371–382.
Friedrich RW (2004). Neurobiology: odorant receptors make scents.
Nature 430: 511–512.
Fukumoto S, Morishita A, Furutachi K, Terashima T, Nakayama T,
Yokogoshi H (2008). Effect of flavour components in lemon
essential oil on physical or psychological stress. Stress Health 24:
3–12.
Fusar-Poli P, Allen P, Bhattacharyya S, Crippa JA, Mechelli A,
Borgwardt S et al. (2010). Modulation of effective connectivity
during emotional processing by Delta9-tetrahydrocannabinol and
cannabidiol. Int J Neuropsychopharmacol 13: 421–432.
Fusar-Poli P, Crippa JA, Bhattacharyya S, Borgwardt SJ, Allen P,
Martin-Santos R et al. (2009). Distinct effects of
{delta}9-tetrahydrocannabinol and cannabidiol on neural activation
during emotional processing. Archiv Gen Psychiatr 66: 95–105.
Gaoni Y, Mechoulam R (1964a). Isolation, structure and partial
synthesis of an active constituent of hashish. J Am Chem Soc 86:
1646–1647.
Gaoni Y, Mechoulam R (1964b). The structure and function of
cannabigerol, a new hashish constituent. Proc Chem Soc 1: 82.
Gaoni Y, Mechoulam R (1966). Cannabichromene, a new active
principle in hashish. Chem Commun 1: 20–21.
Gattefosse R-M (1993). Gatefosse’s Aromatherapy. C.W. Daniel:
Essex, MD.
Gauson LA, Stevenson LA, Thomas A, Baillie GL, Ross RA,
Pertwee RG (2007). Cannabigerol behaves as a partial agonist at
both CB1 and CB2 receptors. Proceedings 17th Annual Symposium
on the Cannabinoids. International Cannabinoid Research Society:
Saint-Sauveur, QC, p. 206.
Gerdeman GL, Lovinger DM (2003). Emerging roles for
endocannabinoids in long-term synaptic plasticity. Br J Pharmacol
140: 781–789.
Gershenzon J (1994). Metabolic costs of terpenoid accumulation in
higher plants. J Chem Ecol 20: 1281–1328.
Gershenzon J, Croteau R (1993). Terepenoid Biosynthesis: the basic
pathway and formation of monoterpenes, sequiterpenes, and
diterpenes. In: Moore TS (ed.). Lipid Metabolism in Plants. CRC
Press: Boca Raton, FL, pp. 339–388.
Gertsch J (2008). Anti-inflammatory cannabinoids in diet: towards
a better understanding of CB(2) receptor action? Commun Integr
Biol 1: 26–28.
Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ et al.
(2008). Beta-caryophyllene is a dietary cannabinoid. Proc Natl Acad
Sci USA 105: 9099–9104.
Ghelardini C, Galeotti N, Salvatore G, Mazzanti G (1999). Local
anaesthetic activity of the essential oil of Lavandula angustifolia.
Planta Med 65: 700–703.
Gil ML, Jimenez J, Ocete MA, Zarzuelo A, Cabo MM (1989).
Comparative study of different essential oils of Bupleurum
gibraltaricum Lamarck. Pharmazie 44: 284–287.
Gill EW, Paton WD, Pertwee RG (1970). Preliminary experiments
on the chemistry and pharmacology of cannabis. Nature 228:
134–136.
Giuffrida A, Leweke FM, Gerth CW, Schreiber D, Koethe D,
Faulhaber J et al. (2004). Cerebrospinal anandamide levels are
elevated in acute schizophrenia and are inversely correlated with
psychotic symptoms. Neuropsychopharmacol 29: 2108–2114.
Gomes-Carneiro MR, Viana ME, Felzenszwalb I, Paumgartten FJ
(2005). Evaluation of beta-myrcene, alpha-terpinene and (+)- and
(-)-alpha-pinene in the Salmonella/microsome assay. Food Chem
Toxicol 43: 247–252.
Guy GW, Stott CG (2005). The development of Sativex- a natural
cannabis-based medicine. In: Mechoulam R (ed.). Cannabinoids As
Therapeutics. Birkhäuser Verlag: Basel, pp. 231–263.
Hampson AJ, Grimaldi M, Axelrod J, Wink D (1998). Cannabidiol
and (-)Delta9-tetrahydrocannabinol are neuroprotective
antioxidants. Proc Natl Acad Sci USA 95: 8268–8273.
Haney M, Hart CL, Vosburg SK, Nasser J, Bennett A, Zubaran C
et al. (2004). Marijuana withdrawal in humans: effects of oral THC
or divalproex. Neuropsychopharmacol 29: 158–170.
Hanus L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D,
Horowitz M et al. (1999). HU-308: a specific agonist for CB(2), a
peripheral cannabinoid receptor. Proc Natl Acad Sci USA 96:
14228–14233.
Harris B (2010). Phytotherapeutic uses of essential oils. In:
Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science,
Technology, and Applications. CRC Press: Boca Raton, FL, pp.
315–352.
Hart CL, Haney M, Ward AS, Fischman MW, Foltin RW (2002).
Effects of oral THC maintenance on smoked marijuana
self-administration. Drug Alcohol Depend 67: 301–309.
Hatoum NS, Davis WM, Elsohly MA, Turner CE (1981).
Cannabichromene and delta 9-tetrahydrocannabinol: interactions
relative to lethality, hypothermia and hexobarbital hypnosis. Gen
Pharmacol 12: 357–362.
Hendriks H, Malingré TM, Batterman S, Bos R (1975). Mono- and
sesqui-terpene hydrocarbons of the eseential oil of Cannabis sativa.
Phytochem 14: 814–815.
Hendriks H, Malingré TM, Batterman S, Bos R (1977). Alkanes of
the essential oil of Cannabis sativa. Phytochem 16: 719–721.
Hill MN, Gorzalka BB (2005a). Is there a role for the
endocannabinoid system in the etiology and treatment of
melancholic depression? Behav Pharmacol 16: 333–352.
Hill MN, Gorzalka BB (2005b). Pharmacological enhancement of
cannabinoid CB1 receptor activity elicits an antidepressant-like
response in the rat forced swim test. Eur Neuropsychopharmacol
15: 593–599.
Hill AJ, Weston SE, Jones NA, Smith I, Bevan SA, Williamson EM
et al. (2010). Delta-Tetrahydrocannabivarin suppresses in vitro
epileptiform and in vivo seizure activity in adult rats. Epilepsia 51:
1522–1532.
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1359
Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D,
Mangieri R et al. (2005). An endocannabinoid mechanism for
stress-induced analgesia. Nature 435: 1108–1112.
Holland ML, Allen JD, Arnold JC (2008). Interaction of plant
cannabinoids with the multidrug transporter ABCC1 (MRP1). Eur J
Pharmacol 591: 128–131.
Hollister LE (1974). Structure-activity relationships in man of
cannabis constituents, and homologs and metabolites of
delta9-tetrahydrocannabinol. Pharmacol 11: 3–11.
Hood LV, Dames ME, Barry GT (1973). Headspace volatiles of
marijuana. Nature 242: 402–403.
Howlett AC (1987). Cannabinoid inhibition of adenylate cyclase:
relative activity of constituents and metabolites of marihuana.
Neuropharmacol 26: 507–512.
Huestis MA (2007). Human Cannabinoid Pharmacokinetics. Chem
Biodivers 4: 1770–1804.
ibn al-Baytar (1291) Kitab al-Yami’ li-mufradat al-adwiya wa-l-agdiya.
Bulaq: Egypt.
ibn Sina (Avicenna) (1294). Al-Qanun fi l-tibb (Canon of medicine).
Bulaq: Egypt.
Ignatowska-Jankowska B, Jankowski M, Glac W, Swiergel AH (2009).
Cannabidiol-induced lymphopenia does not involve NKT and NK
cells. J Physiol Pharmacol 60 (Suppl. 3): 99–103.
Ilan AB, Gevins A, Coleman M, ElSohly MA, de Wit H (2005).
Neurophysiological and subjective profile of marijuana with
varying concentrations of cannabinoids. Behav Pharmacol 16:
487–496.
Ismail M (2006). Central properties and chemcial composition of
Ocimum basilicum essential oil. Pharm Biol 44: 619–626.
Iuvone T, Esposito G, Esposito R, Santamaria R, Di Rosa M, Izzo AA
(2004). Neuroprotective effect of cannabidiol, a non-psychoactive
component from Cannabis sativa, on beta-amyloid-induced toxicity
in PC12 cells. J Neurochem 89: 134–141.
Izzo AA, Borrelli F, Capasso R, Di Marzo V, Mechoulam R
(2009). Non-psychotropic plant cannabinoids: new therapeutic
opportunities from an ancient herb. Trends Pharmacol Sci 30:
515–527.
Jäger W, Buchbauer G, Jirovetz L, Fritzer M (1992). Percutaneous
absorption of lavender oil from a massage oil. J Soc Cosmet Chem
43 (Jan/Feb): 49–54.
Jirovetz L, Buchbauer G, Jager W, Woidich A, Nikiforov A (1992).
Analysis of fragrance compounds in blood samples of mice by gas
chromatography, mass spectrometry, GC/FTIR and GC/AES after
inhalation of sandalwood oil. Biomed Chromatogr 6: 133–134.
Johnson JR, Burnell-Nugent M, Lossignol D, Ganae-Motan ED,
Potts R, Fallon MT (2010). Multicenter, double-blind, randomized,
placebo-controlled, parallel-group study of the efficacy, safety, and
tolerability of THC:CBD extract and THC extract in patients with
intractable cancer-related pain. J Pain Symptom Manage 39:
167–179.
Jones NA, Hill AJ, Smith I, Bevan SA, Williams CM, Whalley BJ
et al. (2010). Cannabidiol displays antiepileptiform and antiseizure
properties in vitro and in vivo. J Pharmacol Exp Ther 332: 569–577.
Karsak M, Gaffal E, Date R, Wang-Eckhardt L, Rehnelt J, Petrosino S
et al. (2007). Attenuation of allergic contact dermatitis through the
endocannabinoid system. Science 316: 1494–1497.
Kavia R, De Ridder D, Constantinescu C, Stott C, Fowler C (2010).
Randomized controlled trial of Sativex to treat detrusor overactivity
in multiple sclerosis. Mult Scler 16: 1349–1359.
Kim JT, Ren CJ, Fielding GA, Pitti A, Kasumi T, Wajda M et al.
(2007). Treatment with lavender aromatherapy in the
post-anesthesia care unit reduces opioid requirements of morbidly
obese patients undergoing laparoscopic adjustable gastric banding.
Obes Surg 17: 920–925.
Kim SS, Baik JS, Oh TH, Yoon WJ, Lee NH, Hyun CG (2008).
Biological activities of Korean Citrus obovoides and Citrus
natsudaidai essential oils against acne-inducing bacteria. Biosci
Biotechnol Biochem 72: 2507–2513.
King LA, Carpentier C, Griffiths P (2005). Cannabis potency in
Europe. Addiction 100: 884–886.
Komiya M, Takeuchi T, Harada E (2006). Lemon oil vapor causes an
anti-stress effect via modulating the 5-HT and DA activities in mice.
Behav Brain Res 172: 240–249.
Komori T, Fujiwara R, Tanida M, Nomura J, Yokoyama MM (1995).
Effects of citrus fragrance on immune function and depressive
states. Neuroimmunomodulation 2: 174–180.
Kose EO, Deniz IG, Sarikurkcu C, Aktas O, Yavuz M (2010).
Chemical composition, antimicrobial and antioxidant activities of
the essential oils of Sideritis erythrantha Boiss. and Heldr. (var.
erythrantha and var. cedretorum P.H. Davis) endemic in Turkey.
Food Chem Toxicol 48: 2960–2965.
Labigalini E Jr, Rodrigues LR, Da Silveira DX (1999). Therapeutic
use of cannabis by crack addicts in Brazil. J Psychoactive Drugs 31:
451–455.
Lad V (1990). Ayurveda: the Science of Self-Healing: A Practical
Guide. Lotus Light Publications: Milwaukee, WI.
Langenheim JH (1994). Higher plant terpenoids: a phytocentric
overview of their ecological roles. J Chem Ecol 20: 1223–1279.
Lapczynski A, Bhatia SP, Letizia CS, Api AM (2008). Fragrance
material review on nerolidol (isomer unspecified). Food Chem
Toxicol 46 (Suppl. 11): S247–S250.
Lawless J (1995). The Illustrated Encyclopedia of Essential Oils :
the Complete Guide to the Use of Oils in Aromatherapy and
Herbalism. Element: Shaftesbury, Dorset, [England]; Rockport, MA.
Ligresti A, Moriello AS, Starowicz K, Matias I, Pisanti S,
De Petrocellis L et al. (2006). Antitumor activity of plant
cannabinoids with emphasis on the effect of cannabidiol on
human breast carcinoma. J Pharmacol Exp Ther 318: 1375–1387.
Lin WY, Kuo YH, Chang YL, Teng CM, Wang EC, Ishikawa T et al.
(2003). Anti-platelet aggregation and chemical constituents from
the rhizome of Gynura japonica. Planta Med 69: 757–764.
Lis-Balchin M (2010). Aromatherapy with essential oils. In:
Baser KHC, Buchbauer G (eds). Handbook of Essential Oils: Science,
Technology, and Applications. CRC Press: Boca Raton, FL,
pp. 549–584.
Lopes NP, Kato MJ, Andrade EH, Maia JG, Yoshida M, Planchart AR
et al. (1999). Antimalarial use of volatile oil from leaves of Virola
surinamensis (Rol.) Warb. by Waiapi Amazon Indians.
J Ethnopharmacol 67: 313–319.
Lorenzetti BB, Souza GE, Sarti SJ, Santos Filho D, Ferreira SH (1991).
Myrcene mimics the peripheral analgesic activity of lemongrass tea.
J Ethnopharmacol 34: 43–48.
Lozano I (1993). Estudios Y Documentos Sobre La Historia Del
Cáñamo Y Del Hachís En El Islam Medievaldoctoral Dissertation.
Universidad de Granada: Granada.
BJP EB Russo
1360 British Journal of Pharmacology (2011) 163 1344–1364
Ludlow FH (1857). The Hasheesh Eater: Being Passages Form the
Life of A Pythagorean. Harper: New York.
McGinty D, Letizia CS, Api AM (2010). Fragrance material review
on phytol. Food Chem Toxicol 48 (Suppl. 3): S59–S63.
McGovern PE, Mirzoian A, Hall GR (2009). Ancient Egyptian herbal
wines. Proc Natl Acad Sci USA 106: 7361–7366.
McHugh D, Hu SS, Rimmerman N, Juknat A, Vogel Z, Walker JM
et al. (2010). N-arachidonoyl glycine, an abundant endogenous
lipid, potently drives directed cellular migration through GPR18,
the putative abnormal cannabidiol receptor. BMC Neurosci 11: 44.
McPartland J (1984). Pathogenicity of Phomopsis ganjae on Cannabis
sativa and the fungistatic effect of cannabinoids produced by the
host. Mycopathologia 87: 149–153.
McPartland JM, Pruitt PL (1999). Side effects of pharmaceuticals
not elicited by comparable herbal medicines: the case of
tetrahydrocannabinol and marijuana. Altern Ther Health Med 5:
57–62.
McPartland JM, Mediavilla V (2001a). Non-cannabinoids in
cannabis. In: Grotenhermen F, Russo EB (eds). Cannabis and
Cannabinoids. NY: Haworth Press: Binghamton, NY, pp. 401–409.
McPartland JM, Russo EB (2001b). Cannabis and cannabis extracts:
greater than the sum of their parts? J Cannabis Therap 1: 103–132.
McPartland JM, Clarke RC, Watson DP (2000). Hemp Diseases and
Pests: Management and Biological Control. CABI: Wallingford.
McPartland JM, Blanchon DJ, Musty RE (2008). Cannabimimetic
effects modulated by cholinergic compounds. Addict Biol 13:
411–415.
Magen I, Avraham Y, Ackerman Z, Vorobiev L, Mechoulam R,
Berry EM (2009). Cannabidiol ameliorates cognitive and motor
impairments in mice with bile duct ligation. J Hepatol 51: 528–534.
Malfait AM, Gallily R, Sumariwalla PF, Malik AS, Andreakos E,
Mechoulam R et al. (2000). The nonpsychoactive cannabis
constituent cannabidiol is an oral anti-arthritic therapeutic in
murine collagen-induced arthritis. Proc Natl Acad Sci USA 97:
9561–9566.
Malingre T, Hendriks H, Batterman S, Bos R, Visser J (1975). The
essential oil of Cannabis sativa. Planta Med 28: 56–61.
Maor Y, Gallily R, Mechoulam R (2006). The relevance of the steric
factor in the biological activity of CBD derivaties-a tool in
identifying novel molecular target for cannabinoids. In: Symposium
on the Cannabinoids. International Cannabinoid Research Society:
Tihany, Hungary, p. 1.
Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G,
Cascio MG et al. (2002). The endogenous cannabinoid system
controls extinction of aversive memories. Nature 418: 530–534.
Matura M, Skold M, Borje A, Andersen KE, Bruze M, Frosch P et al.
(2005). Selected oxidized fragrance terpenes are common contact
allergens. Contact Dermatitis 52: 320–328.
de Meijer E (2004). The breeding of cannabis cultivars for
pharmaceutical end uses. In: Guy GW, Whittle BA, Robson P (eds).
Medicinal Uses of Cannabis and Cannabinoids. Pharmaceutical
Press: London, pp. 55–70.
de Meijer EPM, Hammond KM (2005). The inheritance of chemical
phenotype in Cannabis sativa L. (II): cannabigerol predominant
plants. Euphytica 145: 189–198.
de Meijer EP, Bagatta M, Carboni A, Crucitti P, Moliterni VM,
Ranalli P et al. (2003). The inheritance of chemical phenotype in
Cannabis sativa L. Genetics 163: 335–346.
de Meijer EPM, Hammond KM, Micheler M (2009a). The
inheritance of chemical phenotype in Cannabis sativa L. (III):
variation in cannabichromene proportion. Euphytica 165: 293–311.
de Meijer EPM, Hammond KM, Sutton A (2009b). The inheritance
of chemical phenotype in Cannabis sativa L. (IV): cannabinoid-free
plants. Euphytica 168: 95–112.
Mechoulam R (1986). The pharmacohistory of Cannabis sativa. In:
Mechoulam R (ed.). Cannabinoids As Therapeutic Agents. CRC
Press: Boca Raton, FL, pp. 1–19.
Mechoulam R (2005). Plant cannabinoids: a neglected
pharmacological treasure trove. Br J Pharmacol 146: 913–915.
Mechoulam R, Ben-Shabat S (1999). From gan-zi-gun-nu to
anandamide and 2-arachidonoylglycerol: the ongoing story of
cannabis. Nat Prod Rep 16: 131–143.
Mechoulam R, Shvo Y (1963). Hashish-I. The structure of
cannabidiol. Tetrahedron 19: 2073–2078.
Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE,
Schatz AR et al. (1995). Identification of an endogenous
2-monoglyceride, present in canine gut, that binds to cannabinoid
receptors. Biochem Pharmacol 50: 83–90.
Mechoulam R, Peters M, Murillo-Rodriguez E, Hanus LO (2007).
Cannabidiol – recent advances. Chem Biodivers 4: 1678–1692.
Mediavilla V, Steinemann S (1997). Essential oil of Cannabis sativa
L. strains. J Intl Hemp Assoc 4: 82–84.
Mehmedic Z, Chandra S, Slade D, Denham H, Foster S, Patel AS
et al. (2010). Potency trends of delta(9)-THC and other
cannabinoids in confiscated cannabis preparations from 1993 to
2008. J Forensic Sci 55: 1209–1217.
Mello NK, Mendelson JH (1978). Marihuana, alcohol, and polydrug
use: human self-administration studies. NIDA Res Monogr 20:
93–127.
Merzouki A, Mesa JM (2002). Concerning kif, a Cannabis sativa L.
preparation smoked in the Rif mountains of northern Morocco. J
Ethnopharmacol 81: 403–406.
Mishima K, Hayakawa K, Abe K, Ikeda T, Egashira N, Iwasaki K
et al. (2005). Cannabidiol prevents cerebral infarction via a
serotonergic 5-hydroxytryptamine1A receptor-dependent
mechanism. Stroke 36: 1077–1082.
Miyazawa M, Yamafuji C (2005). Inhibition of acetylcholinesterase
activity by bicyclic monoterpenoids. J Agric Food Chem 53:
1765–1768.
Monti D, Chetoni P, Burgalassi S, Najarro M, Saettone MF,
Boldrini E (2002). Effect of different terpene-containing essential
oils on permeation of estradiol through hairless mouse skin. Int J
Pharm 237: 209–214.
Morgan CJ, Curran HV (2008). Effects of cannabidiol on
schizophrenia-like symptoms in people who use cannabis. Br J
Psychiatry 192: 306–307.
Morgan CJ, Freeman TP, Schafer GL, Curran HV (2010a).
Cannabidiol attenuates the appetitive effects of Delta
9-tetrahydrocannabinol in humans smoking their chosen cannabis.
Neuropsychopharmacology 35: 1879–1885.
Morgan CJ, Schafer G, Freeman TP, Curran HV (2010b). Impact of
cannabidiol on the acute memory and psychotomimetic effects of
smoked cannabis: naturalistic study. Br J Psychiatry 197: 285–290.
Morimoto S, Tanaka Y, Sasaki K, Tanaka H, Fukamizu T, Shoyama Y
et al. (2007). Identification and characterization of cannabinoids
that induce cell death through mitochondrial permeability
transition in Cannabis leaf cells. J Biol Chem 282: 20739–20751.
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1361
Morse K, Mamane D (2001). The Scent of Orange Blossoms :
Sephardic Cuisine from Morocco. Ten Speed Press: Berkeley, CA.
Mukerji G, Yiangou Y, Corcoran SL, Selmer IS, Smith GD,
Benham CD et al. (2006). Cool and menthol receptor TRPM8 in
human urinary bladder disorders and clinical correlations. BMC
Urol 6: 6.
Mukherjee PK, Kumar V, Mal M, Houghton PJ (2007). In vitro
acetylcholinesterase inhibitory activity of the essential oil from
Acorus calamus and its main constituents. Planta Med 73: 283–285.
Murillo-Rodriguez E, Millan-Aldaco D, Palomero-Rivero M,
Mechoulam R, Drucker-Colin R (2006). Cannabidiol, a constituent
of Cannabis sativa, modulates sleep in rats. FEBS Lett 580:
4337–4345.
Musty R, Deyo R (2006). A cannabigerol extract alters behavioral
despair in an animal model of depression. Proceedings June 26;
Symposium on the Cannabinoids. International Cannabinoid
Research Society: Tihany, p. 32.
Musty RE, Karniol IG, Shirikawa I, Takahashi RN, Knobel E (1976).
Interactions of delta-9-tetrahydrocannabinol and cannabinol in
man. In: Braude MC, Szara S (eds). The Pharmacology of
Marihuana, Vol. 2. Raven Press: New York, pp. 559–563.
Naqvi NH, Bechara A (2009). The hidden island of addiction: the
insula. Trends Neurosci 32: 56–67.
Naqvi NH, Bechara A (2010). The insula and drug addiction: an
interoceptive view of pleasure, urges, and decision-making. Brain
Struct Funct 214: 435–450.
Naqvi NH, Rudrauf D, Damasio H, Bechara A (2007). Damage to the
insula disrupts addiction to cigarette smoking. Science 315: 531–534.
Neff GW, O’Brien CB, Reddy KR, Bergasa NV, Regev A, Molina E
et al. (2002). Preliminary observation with dronabinol in patients
with intractable pruritus secondary to cholestatic liver disease. Am J
Gastroenterol 97: 2117–2119.
Nerio LS, Olivero-Verbel J, Stashenko E (2010). Repellent activity of
essential oils: a review. Bioresour Technol 101: 372–378.
Nicholson AN, Turner C, Stone BM, Robson PJ (2004). Effect of
delta-9-tetrahydrocannabinol and cannabidiol on nocturnal sleep
and early-morning behavior in young adults. J Clin
Psychopharmacol 24: 305–313.
Nissen L, Zatta A, Stefanini I, Grandi S, Sgorbati B, Biavati B et al.
(2010). Characterization and antimicrobial activity of essential oils
of industrial hemp varieties (Cannabis sativa L.). Fitoterapia 81:
413–419.
Noma Y, Asakawa Y (2010). Biotransformation of monoterpenoids
by microorganisms, insects, and mammals. In: Baser KHC,
Buchbauer G (eds). Handbook of Essential Oils: Science,
Technology, and Applications. CRC Press: Boca Raton, FL,
pp. 585–736.
Nunes DS, Linck VM, da Silva AL, Figueiro M, Elisabetsky E (2010).
Psychopharmacology of essential oils. In: Baser KHC, Buchbauer G
(eds). Handbook of Essential Oils: Science, Technology, and
Applications. CRC Press: Boca Raton, FL, pp. 297–314.
O’Shaughnessy WB (1843). Indian hemp. Prov Med J Retrosp Med
Sci 5: 397–398.
Oh DY, Yoon JM, Moon MJ, Hwang JI, Choe H, Lee JY et al. (2008).
Identification of farnesyl pyrophosphate and N-arachidonylglycine
as endogenous ligands for GPR92. J Biol Chem 283: 21054–21064.
Opdyke DLJ (1983). Caryophyllene oxide. Food Chem Toxicol 21:
661–662.
Ozek G, Demirci F, Ozek T, Tabanca N, Wedge DE, Khan SI et al.
(2010). Gas chromatographic-mass spectrometric analysis of volatiles
obtained by four different techniques from Salvia rosifolia Sm., and
evaluation for biological activity. J Chromatog 1217: 741–748.
Ozturk A, Ozbek H (2005). The anti-inflammatory activity of
Eugenia caryophyllata essential oil: an animal model of
anti-inflammatory activity. Eur J Gen Med 2: 159–163.
Pacher P, Batkai S, Kunos G (2006). The endocannabinoid system as
an emerging target of pharmacotherapy. Pharmacol Rev 58:
389–462.
Parker LA, Mechoulam R, Schlievert C (2002). Cannabidiol, a
non-psychoactive component of cannabis and its synthetic
dimethylheptyl homolog suppress nausea in an experimental
model with rats. Neuroreport 13: 567–570.
Parker LA, Burton P, Sorge RE, Yakiwchuk C, Mechoulam R (2004).
Effect of low doses of Delta(9)-tetrahydrocannabinol and
cannabidiol on the extinction of cocaine-induced and
amphetamine-induced conditioned place preference learning in
rats. Psychopharmacol (Berl) 175: 360–366.
Parolaro D, Massi P (2008). Cannabinoids as potential new therapy
for the treatment of gliomas. Expert Rev Neurother 8: 37–49.
Pauli A, Schilcher H (2010). In vitro antimicrobial activities of
essential oils monographed in the European Pharmacopoeia 6th
Edition. In: Baser KHC, Buchbauer G (eds). Handbook of Essential
Oils: Science, Technology, and Applications. CRC Press: Boca Raton,
FL, pp. 353–548.
Peana AT, Rubattu P, Piga GG, Fumagalli S, Boatto G, Pippia P et al.
(2006). Involvement of adenosine A1 and A2A receptors in
(-)-linalool-induced antinociception. Life Sci 78: 2471–2474.
Perry NS, Houghton PJ, Theobald A, Jenner P, Perry EK (2000).
In-vitro inhibition of human erythrocyte acetylcholinesterase by
salvia lavandulaefolia essential oil and constituent terpenes.
J Pharm Pharmacol 52: 895–902.
Pertwee RG (2004). The pharmacology and therapeutic potential of
cannabidiol. In: DiMarzo V (ed.). Cannabinoids. Kluwer Academic
Publishers: Dordrecht, pp. 32–83.
Pertwee RG, Thomas A, Stevenson LA, Ross RA, Varvel SA,
Lichtman AH, Martin BR, Razdan RK (2007). The psychoactive
plant cannabinoid, Delta9-tetrahydrocannabinol, is antagonized by
Delta8- and Delta9-tetrahydrocannabivarin in mice in vivo. Br J
Pharmacol 150: 586–594.
Pertwee RG (2008). The diverse CB1 and CB2 receptor pharmacology
of three plant cannabinoids: delta9-tetrahydrocannabinol,
cannabidiol and delta9-tetrahydrocannabivarin. Br J Pharmacol 153:
199–215.
Pliny (1980). Natural History, Books XXIV-XXVII., Vol. 7. Harvard
University Press: Cambridge, MA.
Potter D (2004). Growth and morphology of medicinal cannabis.
In: Guy GW, Whittle BA, Robson P (eds). Medicinal Uses of
Cannabis and Cannabinoids. Pharmaceutical Press: London, pp.
17–54.
Potter DJ (2009). The propagation, characterisation and
optimisation of Cannabis sativa L. as a phytopharmaceutical. PhD,
King’s College, London, 2009.
Potter DJ, Clark P, Brown MB (2008). Potency of delta 9-THC and
other cannabinoids in cannabis in England in 2005: implications
for psychoactivity and pharmacology. J Forensic Sci 53: 90–94.
BJP EB Russo
1362 British Journal of Pharmacology (2011) 163 1344–1364
Pultrini Ade M, Galindo LA, Costa M (2006). Effects of the essential
oil from Citrus aurantium L. in experimental anxiety models in
mice. Life Sci 78: 1720–1725.
Qin N, Neeper MP, Liu Y, Hutchinson TL, Lubin ML, Flores CM
(2008). TRPV2 is activated by cannabidiol and mediates CGRP
release in cultured rat dorsal root ganglion neurons. J Neurosci 28:
6231–6238.
Rahn EJ, Hohmann AG (2009). Cannabinoids as pharmacotherapies
for neuropathic pain: from the bench to the bedside.
Neurotherapeutics 6: 713–737.
Raman A, Weir U, Bloomfield SF (1995). Antimicrobial effects of
tea-tree oil and its major components on Staphylococcus aureus,
Staph. epidermidis and Propionibacterium acnes. Lett Appl
Microbiol 21: 242–245.
Rao VS, Menezes AM, Viana GS (1990). Effect of myrcene on
nociception in mice. J Pharm Pharmacol 42: 877–878.
Re L, Barocci S, Sonnino S, Mencarelli A, Vivani C, Paolucci G
et al. (2000). Linalool modifies the nicotinic receptor-ion channel
kinetics at the mouse neuromuscular junction. Pharmacol Res 42:
177–182.
Ren Y, Whittard J, Higuera-Matas A, Morris CV, Hurd YL (2009).
Cannabidiol, a nonpsychotropic component of cannabis, inhibits
cue-induced heroin seeking and normalizes discrete mesolimbic
neuronal disturbances. J Neurosci 29: 14764–14769.
Resstel LB, Tavares RF, Lisboa SF, Joca SR, Correa FM, Guimaraes FS
(2009). 5-HT1A receptors are involved in the cannabidiol-induced
attenuation of behavioural and cardiovascular responses to acute
restraint stress in rats. Br J Pharmacol 156: 181–188.
Rhee MH, Vogel Z, Barg J, Bayewitch M, Levy R, Hanus L et al.
(1997). Cannabinol derivatives: binding to cannabinoid receptors
and inhibition of adenylylcyclase. J Med Chem 40: 3228–3233.
Riedel G, Fadda P, McKillop-Smith S, Pertwee RG, Platt B,
Robinson L (2009). Synthetic and plant-derived cannabinoid
receptor antagonists show hypophagic properties in fasted and
non-fasted mice. Br J Pharmacol 156: 1154–1166.
Rochefort C, Gheusi G, Vincent JD, Lledo PM (2002). Enriched
odor exposure increases the number of newborn neurons in the
adult olfactory bulb and improves odor memory. J Neurosci 22:
2679–2689.
Rock EM, Limebeer CL, Mechoulam R, Parker LA (2009).
Cannabidiol (the non-psychoactive component of cannabis) may
act as a 5-HT1A auto-receptor agonist to reduce toxin-induced
nausea and vomiting. Proceedings 19th Annual Symposium on the
Cannabinoids. International Cannabinoid Research Society: St.
Charles, IL, p. 29.
Rodrigues Goulart H, Kimura EA, Peres VJ, Couto AS,
Aquino Duarte FA, Katzin AM (2004). Terpenes arrest parasite
development and inhibit biosynthesis of isoprenoids in
Plasmodium falciparum. Antimicrobial Agents Chemother 48:
2502–2509.
Rose JE, Behm FM (1994). Inhalation of vapor from black pepper
extract reduces smoking withdrawal symptoms. Drug Alcohol
Depend 34: 225–229.
Ross SA, ElSohly MA (1996). The volatile oil composition of fresh
and air-dried buds of Cannabis sativa. J Nat Prod 59: 49–51.
Rothschild M, Bergstrom G, Wangberg S-A (2005). Cannabis sativa:
volatile compounds from pollen and entire male and female plants
of two variants, Northern Lights and Hawaian Indica. Bot J Linn
Soc 147: 387–397.
Russo EB (2001). Handbook of Psychotropic Herbs: A Scientific
Analysis of Herbal Remedies for Psychiatric Conditions. Haworth
Press: Binghamton, NY.
Russo EB (2006). The solution to the medicinal cannabis problem.
In: Schatman ME (ed.). Ethical Issues in Chronic Pain Management.
Taylor & Francis: Boca Raton, FL, pp. 165–194.
Russo EB (2007). History of cannabis and its preparations in saga,
science and sobriquet. Chem Biodivers 4: 2624–2648.
Russo EB, Guy GW (2006). A tale of two cannabinoids: the
therapeutic rationale for combining tetrahydrocannabinol and
cannabidiol. Med Hypotheses 66: 234–246.
Russo EB, McPartland JM (2003). Cannabis is more than simply
Delta(9)-tetrahydrocannabinol. Psychopharmacol (Berl) 165:
431–432.
Russo EB, Burnett A, Hall B, Parker KK (2005). Agonistic properties
of cannabidiol at 5-HT-1a receptors. Neurochem Res 30: 1037–1043.
Russo EB, Guy GW, Robson PJ (2007). Cannabis, pain, and sleep:
lessons from therapeutic clinical trials of Sativex, a cannabis-based
medicine. Chem Biodivers 4: 1729–1743.
Russo EB, Jiang HE, Li X, Sutton A, Carboni A, del Bianco F et al.
(2008). Phytochemical and genetic analyses of ancient cannabis
from Central Asia. J Exp Bot 59: 4171–4182.
Ryan D, Drysdale AJ, Pertwee RG, Platt B (2006). Differential effects
of cannabis extracts and pure plant cannabinoids on hippocampal
neurones and glia. Neurosci Lett 408: 236–241.
Salvadeo P, Boggia R, Evangelisti F, Zunin P (2007). Analysis of the
volatile fraction of ‘Pesto Genovese’ by headspace sorptive
extraction (HSSE). Food Chem 105: 1228–1235.
Sanguinetti M, Posteraro B, Romano L, Battaglia F, Lopizzo T,
De Carolis E et al. (2007). In vitro activity of Citrus bergamia
(bergamot) oil against clinical isolates of dermatophytes.
J Antimicrob Chemother 59: 305–308.
Schmidt E (2010). Production of essential oils. In: Baser KHC,
Buchbauer G (eds). Handbook of Essential Oils: Science,
Technology, and Applications. CRC Press: Boca Raton, FL,
pp. 83–120.
Scutt A, Williamson EM (2007). Cannabinoids stimulate fibroblastic
colony formation by bone marrow cells indirectly via CB2
receptors. Calcif Tissue Int 80: 50–59.
Shoyama Y, Sugawa C, Tanaka H, Morimoto S (2008).
Cannabinoids act as necrosis-inducing factors in Cannabis sativa.
Plant Signal Behav 3: 1111–1112.
Silva Brum LF, Emanuelli T, Souza DO, Elisabetsky E (2001). Effects
of linalool on glutamate release and uptake in mouse cortical
synaptosomes. Neurochem Res 26: 191–194.
Singh P, Shukla R, Prakash B, Kumar A, Singh S, Mishra PK
et al. (2010). Chemical profile, antifungal, antiaflatoxigenic and
antioxidant activity of Citrus maxima Burm. and Citrus sinensis
(L.) Osbeck essential oils and their cyclic monoterpene,
DL-limonene. Food Chem Toxicol 48: 1734–1740.
Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y, Morimoto S,
Shoyama Y (2005). Tetrahydrocannabinolic acid synthase, the
enzyme controlling marijuana psychoactivity, is secreted into the
storage cavity of the glandular trichomes. Plant Cell Physiol 46:
1578–1582.
Skold M, Karlberg AT, Matura M, Borje A (2006). The fragrance
chemical beta-caryophyllene-air oxidation and skin sensitization.
Food Chem Toxicol 44: 538–545.
BJP Phytocannabinoid-terpenoid entourage effects
British Journal of Pharmacology (2011) 163 1344–1364 1363
Soares Vde P, Campos AC, Bortoli VC, Zangrossi H Jr,
Guimaraes FS, Zuardi AW (2010). Intra-dorsal periaqueductal
gray administration of cannabidiol blocks panic-like response by
activating 5-HT1A receptors. Behavioural Brain Res 213: 225–229.
do Socorro SRMS, Mendonca-Filho RR, Bizzo HR,
de Almeida Rodrigues I, Soares RM, Souto-Padron T et al. (2003).
Antileishmanial activity of a linalool-rich essential oil from Croton
cajucara. Antimicrob Agents Chemother 47: 1895–1901.
Stahl E, Kunde R (1973). Die Leitsubstanzen der HaschischSuchhunde. Kriminalistik: Z Gesamte Kriminal Wiss Prax 27:
385–389.
Stott CG, Guy GW, Wright S, Whittle BA (2005). The effects of
cannabis extracts Tetranabinex and Nabidiolex on human
cytochrome P450-mediated metabolism. In: Symposium on the
Cannabinoids, June 27. International Cannabinoid Research
Association, Clearwater, FL, p. 163.
Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K et al.
(1995). 2-Arachidonoylglycerol: a possible endogenous cannabinoid
receptor ligand in brain. Biochem Biophys Res Commun 215: 89–97.
Tambe Y, Tsujiuchi H, Honda G, Ikeshiro Y, Tanaka S (1996).
Gastric cytoprotection of the non-steroidal anti-inflammatory
sesquiterpene, beta-caryophyllene. Planta Med 62: 469–470.
Taylor B (1855). The Lands of the Saracens. G.P. Putnam & Sons:
New York.
Thomas A, Stevenson LA, Wease KN, Price MR, Baillie G, Ross RA
et al. (2005). Evidence that the plant cannabinoid delta-9-
tetrahydrocannabivarin is a cannabinoid CB1 and CB2 antagonist.
Br J Pharmacol 146: 917–926.
Thomas A, Baillie GL, Phillips AM, Razdan RK, Ross RA, Pertwee RG
(2007). Cannabidiol displays unexpectedly high potency as an
antagonist of CB1 and CB2 receptor agonists in vitro. Br J
Pharmacol 150: 613–623.
Tisserand R, Balacs T (1995). Essential Oil Safety: A Guide for
Health Care Professionals. Churchill Livingstone: Edinburgh.
Trapp SC, Croteau RB (2001). Genomic organization of plant
terpene synthases and molecular evolutionary implications. Genet
158: 811–832.
Tsokou A, Georgopoulou K, Melliou E, Magiatis P, Tsitsa E (2007).
Composition and enantiomeric analysis of the essential oil of the
fruits and the leaves of Pistacia vera from Greece. Molecules 12:
1233–1239.
Turner CE, Elsohly MA, Boeren EG (1980). Constituents of
Cannabis sativa L. XVII. A review of the natural constituents. J Nat
Prod 43: 169–234.
Turner G, Gershenzon J, Nielson EE, Froehlich JE, Croteau R (1999).
Limonene synthase, the enzyme responsible for monoterpene
biosynthesis in peppermint, is localized to leucoplasts of oil gland
secretory cells. Plant Physiol 120: 879–886.
do Vale TG, Furtado EC, Santos JG Jr, Viana GS (2002). Central
effects of citral, myrcene and limonene, constituents of essential oil
chemotypes from Lippia alba (Mill.) n.e. Brown. Phytomed 9:
709–714.
Varvel SA, Bridgen DT, Tao Q, Thomas BF, Martin BR, Lichtman AH
(2005). Delta9-tetrahydrocannbinol accounts for the
antinociceptive, hypothermic, and cataleptic effects of marijuana in
mice. J Pharmacol Exp Ther 314: 329–337.
Vigushin DM, Poon GK, Boddy A, English J, Halbert GW, Pagonis C
et al. (1998). Phase I and pharmacokinetic study of d-limonene in
patients with advanced cancer. Cancer Research Campaign Phase
I/II Clinical Trials Committee. Cancer Chemother Pharmacol 42:
111–117.
Volicer L, Stelly M, Morris J, McLaughlin J, Volicer BJ (1997). Effects
of dronabinol on anorexia and disturbed behavior in patients with
Alzheimer’s disease. Int J Geriatr Psychiatry 12: 913–919.
Vollner L, Bieniek D, Korte F (1969). [Hashish. XX. Cannabidivarin,
a new hashish constituent]. Tetrahedron Lett 3: 145–147.
Von Burg R (1995). Toxicology update. Limonene. J Appl Toxicol
15: 495–499.
Wachtel SR, ElSohly MA, Ross RA, Ambre J, de Wit H (2002).
Comparison of the subjective effects of delta9-tetrahydrocannabinol
and marijuana in humans. Psychopharmacol 161: 331–339.
Wagner H, Ulrich-Merzenich G (2009). Synergy research:
approaching a new generation of phytopharmaceuticals. Phytomed
16: 97–110.
Walton RP (1938). Marihuana, America’s New Drug Problem. A
Sociologic Question with Its Basic Explanation Dependent on
Biologic and Medical Principles. J.B. Lippincott: Philadelphia, PA.
Wattenberg LW (1991). Inhibition of azoxymethane-induced
neoplasia of the large bowel by 3-hydroxy-3,7,11-trimethyl-1,6,
10-dodecatriene (nerolidol). Carcinogen 12: 151–152.
Wilkinson JD, Williamson EM (2007). Cannabinoids inhibit human
keratinocyte proliferation through a non-CB1/CB2 mechanism and
have a potential therapeutic value in the treatment of psoriasis. J
Dermatol Sci 45: 87–92.
Wilkinson JD, Whalley BJ, Baker D, Pryce G, Constanti A,
Gibbons S et al. (2003). Medicinal cannabis: is
delta9-tetrahydrocannabinol necessary for all its effects? J Pharm
Pharmacol 55: 1687–1694.
Williams SJ, Hartley JP, Graham JD (1976). Bronchodilator effect of
delta1-tetrahydrocannabinol administered by aerosol of asthmatic
patients. Thorax 31: 720–723.
Williamson EM (2001). Synergy and other interactions in
phytomedicines. Phytomed 8: 401–409.
Wirth PW, Watson ES, ElSohly M, Turner CE, Murphy JC (1980).
Anti-inflammatory properties of cannabichromene. Life Sci 26:
1991–1995.
Xi Z-X, Peng X-Q, Li X, Zhang H, Li JG, Gardner EL (2010). Brain
cannabinoid CB2 receptors inhibit cocaine self-administration and
cocaine-enhanced extracellular dopamine in mice. Proceedings 20th
Annual Symposium on the Cannabinoids. International
Cannabinoid Research Society: Lund, p. 32.
Yang D, Michel L, Chaumont JP, Millet-Clerc J (1999). Use of
caryophyllene oxide as an antifungal agent in an in vitro
experimental model of onychomycosis. Mycopathologia 148:
79–82.
Zanelati TV, Biojone C, Moreira FA, Guimaraes FS, Joca SR (2010).
Antidepressant-like effects of cannabidiol in mice: possible
involvement of 5-HT1A receptors. Br J Pharmacol 159: 122–128.
Zuardi AW, Guimaraes FS (1997). Cannabidiol as an anxiolytic and
antipsychotic. In: Mathre ML (ed.). Cannabis in Medical Practice: A
Legal, Historical and Pharmacological Overview of the Therapeutic Use
of Marijuana. McFarland: Jefferson, NC, pp. 133–141.
Zuardi AW, Rodrigues JA, Cunha JM (1991). Effects of cannabidiol
in animal models predictive of antipsychotic activity.
Psychopharmacol 104: 260–264.
Zuardi AW, Crippa JA, Hallak JE, Moreira FA, Guimaraes FS (2006).
Cannabidiol, a Cannabis sativa constituent, as an antipsychotic
drug. Braz J Med Biol Res 39: 421–429.

Phytocannabinoids and terpenoids are synthesized in cannabis, in secretory cells inside glandular trichomes (Figure 1) that are most highly concentrated in unfertilized female flowers prior to senescence (Potter, 2004; Potter, 2009). Geranyl pyrophosphate is formed as a precursor via the deoxyxylulose pathway in cannabis (Fellermeier et al., 2001), and is a parent compound to both phytocannabinoids and terpenoids (Figure 2). After coupling with either olivetolic acid or divarinic acid, pentyl or propyl cannabinoid acids are produced, respectively, via enzymes that accept either substrate (de Meijer et al., 2003), a manifestation of Mechoulam’s postulated ‘Nature’s Law of Stinginess’. Although having important biochemical properties in their own right, acid forms of phytocannabinoids are most commonly decarboxylated via heat to produce the more familiar neutral phytocannabinoids (Table 1). Alternatively, geranyl pyrophosphate may form limonene and other monoterpenoids in secretory cell plastids, or couple with isopentenyl pyrophosphate in the cytoplasm to form farnesyl pyrophosphate, parent compound to the sesquiterpenoids, that co-localizes with transient receptor potential vanilloid receptor (TRPV) 1 in human dorsal root ganglion, suggesting a role in sensory processing of noxious stimuli (Bradshaw et al., 2009), and which is the most potent endogenous ligand to date on the G-protein coupled receptor (GPR) 92 (Oh et al., 2008). An obvious question pertains to the chemical ecology of such syntheses that require obvious metabolic demands on the plant (Gershenzon, 1994), and these will be considered. Is cannabis merely a crude vehicle for delivery of THC? Might it rather display herbal synergy (Williamson, 2001) encompassing potentiation of activity by active or inactive components, antagonism (evidenced by the ability of CBD to reduce side effects of THC; Russo and Guy, 2006), summation, pharmacokinetic and metabolic interactions? Recently, four basic mechanisms of synergy have been proposed (Wagner and Ulrich-Merzenich, 2009): (i) multi-target effects; (ii) pharmacokinetic effects such as improved solubility or bioavailability; (iii) agent interactions affecting bacterial resistance; and (iv) modulation of adverse events. Cannabis was cited as an illustration. Could phytocannabinoids function analogously to the endocannabinoid system (ECS) with its combination of active and ‘inactive’ synergists, first described as an entourage (Ben-Shabat et al., 1998), with subsequent refinement (Mechoulam and Ben-Shabat, 1999) and qualification (p. 136): ‘This type of synergism may play a role in the widely held (but not experimentally based) view that in some cases plants are better drugs than the natural products isolated from them’. Support derives from studies in which cannabis extracts demonstrated effects two to four times greater than THC (Carlini et al., 1974); unidentified THC antagonists and synergists were claimed (Fairbairn and Pickens, 1981), anticonvulsant activity was observed beyond the cannabinoid fraction (Wilkinson et al., 2003), and extracts of THC and CBD modulated effects in hippocampal neurones distinctly from pure compounds (Ryan et al., 2006). Older literature also presented refutations: no observed differences were noted by humans ingesting or smoking pure THC versus herbal cannabis (Wachtel et al., 2002); pure THC seemed to account for all tetrad-type effects in mice (Varvel et al., 2005); and smoked cannabis with varying CBD or CBC content failed to yield subjective differences combined with THC (Ilan et al., 2005). Explanations include that the cannabis employed by Wachtel yielded 2.11% THC, but with only 0.3% cannabinol (CBN) and 0.05% CBD (Russo and McPartland, 2003), and Ilan’s admission that CBN and CBD content might be too low to modulate THC. Another factor is apparent in that terpenoid yields from vaporization of street cannabis were 4.3–8.5 times of those from US National Institute on Drug Abuse cannabis (Bloor et al., 2008). It is undisputed that the black market cannabis in the UK (Potter et al., 2008), Continental Europe (King et al., 2005) and the USA (Mehmedic et al., 2010) has become almost exclusively a high-THC preparation to the almost total exclusion of other phytocannabinoids. If – as many consumers and experts maintain (Clarke, 2010) – there are biochemical, pharmacological and Figure 1 Cannabis capitate glandular (EBR by permission of Bedrocan BV, Netherlands). BJP Phytocannabinoid-terpenoid entourage effects British Journal of Pharmacology (2011) 163 1344–1364 1345 phenomenological distinctions between available cannabis ‘strains’, such phenomena are most likely related to relative terpenoid contents and ratios. This treatise will assess additional evidence for putative synergistic phytocannabinoidterpenoid effects exclusive of THC, to ascertain whether this botanical may fulfil its promise as, ‘a neglected pharmacological treasure trove’ (Mechoulam, 2005). Phytocannabinoids, beyond THC: a brief survey Phytocannabinoids are exclusively produced in cannabis (vide infra for exception), but their evolutionary and ecological raisons d’être were obscure until recently. THC production is maximized with increased light energy (Potter, 2009). It has been known for some time that CBG and CBC are mildly antifungal (ElSohly et al., 1982), as are THC and CBD against a cannabis pathogen (McPartland, 1984). More pertinent, however, is the mechanical stickiness of the trichomes, capable of trapping insects with all six legs (Potter, 2009). Tetrahydrocannabinolic acid (THCA) and cannabichromenic acid (Morimoto et al., 2007), as well as cannabidiolic acid and cannabigerolic acid (CBGA; Shoyama et al., 2008) produce necrosis in plant cells. Normally, the cannabinoid acids are sequestered in trichomes away from the flower tissues. Any trichome breakage at senescence may contribute to natural pruning of lower fan leaves that otherwise utilize energy that the plant preferentially diverts to the flower, in continued efforts to affect fertilization, generally in vain when subject to human horticulture for pharmaceutical production. THCA and CBGA have also proven to be insecticidal in their own right (Sirikantaramas et al., 2005). Over 100 phytocannabinoids have been identified (Brenneisen, 2007; Mehmedic et al., 2010), but many are artefacts of analysis or are produced in trace quantities that have not permitted thorough investigation. The pharmacology of the more accessible phytocannabinoids has received excellent recent reviews (Pertwee et al., 2007; Izzo et al., 2009; De Petrocellis and Di Marzo, 2010; De Petrocellis et al., 2011), and will be summarized here, with emphasis on activities with particular synergistic potential. Geranylphosphate: olivetolate geranyltransferase HO OH COOH cannabigerolic acid O OH COOH delta-9-tetrahydrocannabinolic acid OH OH COOH cannabidiolic acid