Evans/CBD/Antiinflam/1988



ANALGESIC AND ANTIINFLAMMATORY ACTIVITY OF CONSTITUENTS OF CANNABIS SATIVA L.

INFLAMMATION, Vol 12, No. 4, 1988 pp. 361-371

E.A. Formukong, A.T. Evans, and F.J. Evans

Department of Pharmacognosy, The School of Pharmacy University of London,
29-39 Brunswick Square London, WC11N 1AX, England

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.

INTRODUCTION

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.

RESULTS

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).


DISCUSSION

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.

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