are in Research
AND ANTIINFLAMMATORY ACTIVITY OF CONSTITUENTS OF CANNABIS SATIVA L.
Vol 12, No. 4, 1988
Formukong, A.T. Evans, and F.J. Evans
of Pharmacognosy, The School of Pharmacy University of London,
Brunswick Square London, WC11N 1AX, England
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.
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).
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).
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
folowing were used: aspirin (Sigma Chemical Co., Poole, Dorset.), tripotassium
citrate (analytical grade), all cannabinoids except CBG (Sigma), and CBG (Makor
Chemicals, Jerusalem, Israel).
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.
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).
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.
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%.
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).
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.
results were expressed as percentage inhibition, taken to mean the complete suppression
of erythema in the test animals, as described in reference 19.
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.)
was fully effective only at concentrations above 10 mg/kg Figure 2).
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).
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)
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.
substances were more active than trifluoperazine, 1 mg/5ul, a known phorbol ester
antagonist both in vivo (19) and in vitro (20).
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.
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).
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.
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.
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.
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
are grateful to the Medicinal Research Council and the Government of Cameroon
for financial support.
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