Analgesic and Anti-inflamatory Activity of Constituents of Cannabis Sativa L
Formukong, A.T. Evans, and F.J. Evans
Department of Pharmacognosy, The School of Pharmacy University of London
Abstract---Two extracts of Cannabis sativa herb, one being cannabinoid--free (ethanol) and the other containing the cannabinoids (petroleum), were shown to inhibit PBQ- induced writhing in mouse when given orally and also to antagonize tetradecanoylphorbol acetate (TPA) -induced erythema of mouse skin when applied topically. With the exception of cannabinol (CBN) and delta-1-tetrahydrocannabinol (delta-1-THC), the cannabinoids and olivetol (their biosynthetic precursor) demonstrated activity in the PBQ test exhibiting their maximal effect at doses of about 100 mcg/kg. Delta-1-THC only became maximally effective in doses of 10 mg/kg. This higher dose corresponded to that which induced catalepsy and is indicative of a central action. CBN produce a 40% inhibition of PBQ-induced writhing. Cannabidiol (CBD) was the most effective of the cannabinoids at doses of 100 mcg/kg. Doses of cannabinoids that were effective in the analgesic test orally were used topically to antagonize TPA-induced erythema of skin. The fact that delta-1-THC and CBN were the least effective in this test suggests a structural relationship between analgesic activity and antiinflammatory activity among the cannabinoids related to their peripheral actions and separate from the central effects of delta-1-THC.
Various preparations of Cannabis sativa have been employed for their medicinal effects, including antipyretic, antirheumatic, antiallergic, and analgesic purposes (1). Extracts of Cannabis have been shown to possess analgesic activity (2, 3), and delta-1-tetrahydrocannabinol (delta-1-THC), the psychoactive component of Cannabis has also been shown to possess this activity in various models (4-6). In addition, cannabinol (CBN) but not cannabidiol (CBD) was shown to exhibit analgesic activity in vivo (7).
It is possible that the antiinflammatory and antiasthmatic properties of this herb are mediated through effects on arachidonate metabolism. However, constituents of Cannabis are known to stimulate (8,9) and inhibit (10-12) prostaglandin (PG) release by influencing enzymes of this pathway (13, 14).
A cannabinoid or an extract of Cannabis with little or no central effects could be of use therapeutically. In this paper, we have examined the antiinflammatory potential of two extracts of Cannabis, pure cannabinoids and olivetol (a cannabinoid biosynthetic precursor) in two models of inflammation, in an attempt to separate on a structural basis the peripheral from the central action of these phenolic drugs.
MATERIALS AND METHODS
The folowing were used: aspirin (Sigma Chemical Co., Poole, Dorset.), tripotassium citrate (analytical grade), all cannabinoids except CBG (Sigma), and CBG (Makor Chemicals, Jerusalem, Israel).
Preparation of Drugs: PBQ Test. Cannabinoids and cannabis extracts were suspended in a 1% ethanolic solution containing 2.5% w/v Tween. Aspirin was dissolved in a 40 mg/ml solution of tripotassium citrate.
Phenyl Benzoquinone Writhing (PBQ) and Preparation of PBQ Solution. A 0.04% solution of PBQ was prepared immediately before use by dissolving PBQ in warm ethanol and diluting with water at 40 degrees C ( 15) bringing the ethanolic concentration to 5% (16). The bottle was stoppered, foil paper wrapped around it, and the solution maintained at 34 degrees C. Deterioration of the solution occurs if left exposed to light and air (17).
Administration of Drugs. Male CDI male (Charles River) weighing 18-20 g were starved overnight for the experiment. Animals were placed in a thermostatically controlled environment maintained at 34 degrees C. Mice were orally administered test drug 20 min before the intraperitoneal injection of PBQ (4 mg/kg). Five minutes after injection, a hand tally counter was used to record the number of stretching movements for each mouse in a 5-min period. Control animals were only administered the vehicle. Note less than five animals were used per dose.
Statistical Analysis. Results are expressed as mean percentage inhibition of control (+SEM) in the case of PBQ test. IC-50s were obtained from graphs relating probit percentage inhibition (ordinate) against log dose (abscissa). The IC-50 is that dose of drug which would inhibit PBQ-induced writhing by 50%.
Tetradecanoyl phorbol-acetate-induced (TPA) Erythema of Mouse Ear. In order to exclude the possibility of a central mechanism of action (see Discussion), compounds also were tested for their ability to inhibit TPA-induced erythema on mouse ears in 100% of the animals was chosen as the challenging dose for inhibition studies, measured 4 h after application (18).
Test drugs were dissolved in ethanol and 5 ul applied to the inner ear of the mouse 15 min before the application of 1 mcg TPA in 5 ul acetone. Only one dose of test dug was used for this experiment, 100 mcg/mcl ethanols, except trifluoperazine at 1 mg/5 ul. The other ear acted as a control.
The results were expressed as percentage inhibition, taken to mean the complete suppression of erythema in the test animals, as described in reference 19.
PBQ-Induced Writhing. CBD, CBG, olivetol, ethanolic extract, and petroleum spirit extract produced significant inhibition at doses up to 10 mg/kg (Figures 1-3). CBN was only marginally active (Table 1.)
Delta-1-THC was fully effective only at concentrations above 10 mg/kg Figure 2).
The ethanolic and petroleum extract, CBD, olivetol, CBG, and cannflavon were more potent than aspirin. The petroleum spirit extract was about four times more potent than the ethanolic extract, which was virtually equipotent with CBD. Cannflavon, isolated from the ethanolic extract was 14 times less potent than the ethanolic extract of the dried herb (Table 2).
There was a decline in response following the administration of doses greater than 0.1 mg/kg of some substances. This is most evident in the bell shaped dose-response curve of the petroleum spirit extract (Figure 1). The activity of the ethanolic extract and CBD was also found to decrease slightly at higher dose levels. (Figures 1 and 2)
TPA-Induced Erythema. In general, the ability of compounds to inhibit TPA-induced erythema correlated well with their potency in the PBQ-writhing test. Thus, CBN and delta-1-THC were the least active followed by CBG, CBD, and cannflavon. Again, the extracts were the most active (Table 3). Twenty-four hours after application, the ethanolic extract still produced 16% inhibition of TPA-induced erythema of the animals. All other substances were without activity after 24 h.
All substances were more active than trifluoperazine, 1 mg/5ul, a known phorbol ester antagonist both in vivo (19) and in vitro (20).
The PBQ-induced writhing response is believed to be produced by the liberation of endogenous substance(s), notably metabolites of the arachidonic cascade (21, 22). However, the PBQ test is not specific for weak analgesics such as the nonsteroidal antiinflammatory drugs, as it also detects centrally active analgesics (16, 17). Therefore, in the elucidation of the action of the cannabinoids as inflammatory drugs, it was necessary to perform more than one test. In this case, peripheral rather than central action was confirmed in the mouse ear erythema assay.
TPA-induced erythema was inhibited by the extracts cannflavon, cannabinoids, and olivetol. The activity of TPA has been shown to be dependent upon PG release in mouse epidermis (23) and mouse peritoneal macrophages (24) possibly via the initial stimulation of protein kinase C (for a review see reference 25). It has also been shown that compounds that show moderate to very potent antiinflammatory potential in standard in vivo inflammation models will also inhibit TPA-induced edema of the mouse ear (26), and phorbol-ester-induced erythema (19).
It is possible that the cannabinoids and their extracts are inhibiting both PBQ-induced writhing and TPA-induced erythema by effects on arachidonate release and metabolism. Cannabinoids and olivetol have been shown to inhibit PG mobilization (11, 12) and synthesis (14). The noncannabinoid constituents of Cannabis, for example, cannflavon, have been shown to be mainly cyclooxygenase inhibitors (14). Cannabinoids, however, stimulate and inhibit phospholipase A2 (PLA2) activity (13), as well as inducing an inhibition of cyclooxygenase and lipoxygenase (14). The activity of Cannabis herb or resin is complex, in that activities can be demonstrated on at least three major enzymes of the arachidonate cascade.
The mechanism by which delta-1-THC inhibits PBQ-induced writhing may differ from that of the other substances. At concentrations greater than 10 mg/kg, delta-1-THC may be inhibiting PBQ-induced writhing by acting on central rather than peripheral functions. It is possible that prostaglandins modulate certain inhibitory pathways in the brain, bringing about an increase in the pain threshold. This dose of delta-1-THC is capable of bringing about the cataleptic effect (27), which is a standard test for central involvement. Central analgesics have higher efficacies than peripheral ones, and this may explain the effectiveness of delta-1-THC (Figure 2). The central involvement of delta-1-THC is perhaps the primary reason why delta-1-THC was recognized as an analgesic before other cannabinoids.
Our results suggest that the response of the ethanolic extract cannot be solely due to cannflavon. Other structurally related phenolic substances, known to be present in this complex extract, may account for the higher activity seen either due to cumulative or synergistic effects upon cyclooxygenase. The activity of the petroleum ether extract is likely to be largely due to the presence of CBD and CBN. GLC analysis of the extract has shown that this extract contained 14.13% CBD, 9.08% CBN, and 6.68% delta-1-THC (27). On the basis of our results, it is possible to separate the centrally active cannabinoid delta-1-THC from peripherally active compounds of the herbal extracts. An attempt has been made to differentiate them structurally (Table 3). It can be seen that the olivetolic nucleus together with a free C-5 hydroxyl group are structural requirements for peripheral effects, involving both cyclooxygenase and lipoxygenase inhibition (14). Substances possessing this structure possess antiinflammatory and analgesic activities without central hallucinogenic effects. Delta-1-THC and CBN, which are cyclized derivatives exhibiting no C-5 hydroxyl moiety, have little if any peripheral action.
The traditional use of Cannabis as an analgesic, antiasthmatic, and antirheumatic drug is well established. Our results would suggest that cultivation of Cannabis plants rich in CBD and other phenolic substances would be useful not only as fiber-producing plants but also for medicinal purposes in the treatment of certain inflammatory disorders.
Acknowledgments----We are grateful to the Medicinal Research Council and the Government of Cameroon for financial support.
1. Pars, H.G., R.J. Razdan, and J.F. Howes. 1977. Potential therapeutic agents derived from the cannabinoid nucleus. Adv. Drug. Res. 11.
2. O.L. Davies, J. Raventos, and A.L. Walpole, 1946. A method for evaluation of analgesic activity using rats. Br. J. Pharmacol. 1: 255-264.
3. Gill, E.W., W.D.M. Paton, and R.G. Pertwee, 1970. Preliminary experiments on the chemistry and pharmacology of Cannabis. Nature 228: 134-136.
4. Dewey, W.L., L.S. Harris, and J.S. Kennedy, 1972. Some pharmacological and toxicological effects of 1-trans-delta-8- and 1-trans-delta-9-THC in laboratory rodents. Arch. Int. Pharmacodyn. 196: 133-145.
5. Chesher, G.B., C.J. Dahl, M. Everingham, D.M. Jackson, H. Marchant-Williams, and G.A. Starmer, 1973. The effect of cannabinoids on intestinal mobility and their antinociceptive effect in mice. Br. J. Pharmacol. 49: 588-594.
6. Buxbaum, D., E. Sanders-Bush, and D.H. Efron. 1969. Analgesic activity of tetrahydrocannabinol in the rat and mouse. Fed. Proc. 28: 735.
7. Sanders, J., D.M. Jackson, and G.A. Starmer. 1979. Interactions among the cannabinoids in the antagonism of abdominal constriction response in the mouse. Psychopharmacology 61: 281-285.
8. White, H.L., and R.L. Tansik. 1980. Effects of delta-9-THC and cannabidiol on phospholipase and other enzymes regulating arachidonate metabolism. Prostaglandins Med. 4: 409-411,
9. Burstein, S., and S.A. Hunter. 1978. Prostaglandins and Cannabis VI. Release of arachidonic acid from HeLa cells by delta-1-THC and other cannabinoids. Biochem. Pharmacol. 27: 1275-1280.
10. Burstein, S. and A. Raz. 1972. Inhibition of prostaglandin E2 biosynthesis by delta-1-tetrahydrocannabinol. Prostaglandins 2: 369.
11. Burstein, S.E., Levine, and C. Varanelli. 1973. Prostaglandins and Cannabis II. Inhibition of biosynthesis by the naturally occurring cannabinoids. Biochem. Pharmacol. 22: 2905-2910.
12. Barrett, M.L., D.Gordon, and F.J. Evans. 1985. Isolation from Cannabis sativa L of cannflavin: A novel inhibitor of prostaglandin production. Biochem. Pharmacol. 34: 2019-2024.
13. Evans, A.T., E.A. Formukong, and F.J. Evans. 1987. Activation of phospholipase A2 by cannabinoids. Lack of correlation with CNS effects. FEBS Lett. 211: 119-122.
14. Evans, A.T., E.A. Formukong, and F.J. Evans. 1987. Actions of Cannabis constituents on enzymes of prostaglandin synthesis: Antiinflammatory potential. Biochm. Pharmacol. 36: 2035-2037.
15. Parkes, M.W., and J.T. Pickens. 1965. Conditions influencing the inhibition of analgesic drugs of the response to intraperitoneal injections of phenylbenzoquine in mice. Br. J. Pharmacol. 25: 81-87.
16. Siegmund, E.A., R.A. Cadmus, and G. Lu. 1957. A method for evaluating both nonnarcotic and narcotic analgesics. Proc Soc. Exp. Biol. 95: 729-731.
17. Hendershot, L.C., and J. Forsaith. 1959. Antagonism of the frequency of phenylbenzoquinone induced writhing in the mouse by weak analgesics and nonanalgesics. J. Pharmacol. Exp. Ther. 125: 237-240.
18. Kinghorn, A.D., and F.J. Evns. 1975. A biological screen of selected species of the genus Euphorbia for skin irritant effects. Planta Med. 28: 325.
19. Williamson, E.M., and F.J. Evans. 1981. Inhibition of erythema induced by proinflammatory esters of 12-deoxyphorbol. Acta Pharmacol. Toxicol. 481: 47-52.
20. Williamson, E.M., J. Westwick, V.V. Kakkar, and F.J. Evans. 1981. Studies on the mechanism of action of 12-DOPP, a potent platelet aggregating phorbol ester. Biochem. Pharmacol. 30: 2691-2696.
21. Collier, H.O.J., L.C. Dineen, C.A. Johnson, and C. Schneider. 1968. Abdominal constriction response and its suppression by analgesic drugs in the mouse. Br. J. Pharmacol. Chemother. 32: 295-310. (22)
23. Marks, F., G. Furstenberger, and E. Kownatzki, 1981. Prostaglandin E-mediated mitogenic stimulatin of mouse epidermis in vivo by divalent cation ionophore A23187 and by tumor promoter 12-O-tetradecanoyl phorbol-13-acetate. Cancer Res. 41: 696-702.
24. Humes, J.L., S. Sadowski, M. Galavage, M. Goldenberg, E. Bubers, R.J. Bonney, and F.A. Kuehl, 1982. Evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal macrophages. J. Biol. Chem. 257: 1291-1594.
25. Edwards, M.C., and F.J. Evans. 1987. Activity correlations in the phorbol ester series. Bot. J. Linn. Soc. 94: 231-246.
26. Calson, R.P., L. O'Neill-David, J. Chary, and A.J. Lewis. 1985. Modulation of mouse ear edema by cyclooxygenase and lipoxygenase inhibitors and other pharmacological agents. Agents Actions 17: 197-204.
27. Formukong, E.A., A.T. Evans, F.J. Evans. 1987. Inhibition of the cataleptic effect of delta-1-tetrahydrocannabinol by noncataleptic constituents of Cannabis sativa L. J. Pharm. Pharmacol. (in press).