|
INTRODUCTION
In 1932, Grewal reported that on the isolated ileum the alkaloid from Mitragyna
speciosa reduced the amplitude and tone of the smooth muscle. Perry (1980)
quoted a report by Field and Quisumbing which stated that Mitragynine has a
local anaesthetic effect and that the side-effects include dryness of the mouth,
diuresis, constipation, with stools becoming small and dark, loss of appetite
and subsequently a reduction in body weight. Jansen and Prast (1988) reported
that the alkaloid from M.speciosa had opiate-like effects such as analgesia,
antitussive and also cause hypothermia in animals. On the other hand, its action
was not reversed by Nalorphine though it suppresses the opioid withdrawal
syndrome.
It was thought that this novel analgesic action from the alkaloids of M.
speciosa would be interesting to study since Nalorphine did not seem able to
overcome its action (Idid et al., 1992). The alkaloid may be useful in the
treatment of opiate addiction as a replacement therapy without the attendant
dependence if it should act as an opioid agonist-antagonist.
METHODS
Preparation of Mitragynine
Mitragynine was extracted from fresh leaves of M. speciosa according to the
method described by Ikram and Houghton (1986).
Preparation of Drug Solutions
Morphine sulphate, paracetamol and mitragynine were dissolved or suspended in 4%
acacia gum.
Antinociceptive Assay
Three types of antinociceptive assays were employed in this study; these were i)
Writhings test, ii)
Hot tail-flick and iii) Cold tail-flick test.
(i) Writhing tests The abdominal constriction test described by Collier et al.
(1968) was used to measure the antinociceptive actions of morphine, mitragynine
and paracetamol. Male or female albino mice weighing between 20-25 g were fasted
for 24 hours with water given ad libitum, and were pretreated with oral 4%
acacia gum solution (0. 15 ml/10 g), morphine (5 mg/kg), paracetamol(100 mg/kg)
and mitragynine (200 mg/kg) 30 minutes prior to intraperitoneal injection of 0.
15 ml/l 0 g of 0.6% acetic acid to cause a typical stretching response.
Writhings or stretchings (abdominal constrictions) were coun-ted for a period of
5 minutes under a double blind observation. The antinociceptive effects of drugs
were measured by calculating the mean reduction in the number of abdominal
constrictions for each drug, as compared to gum acacia controls.
(ii) Hot tail-flick Male or female albino mice weighing between 20 - 25 g were
fasted for 24 hours with water given ad libitum, maintained at room temperature
and were divided into 4 groups of six mice. Mice were treated with 4% acacia gum
(0.15 m1/10g), morphine (5 mg/kg), paracetamol (100 mg/kg) and mitragynine (200
mg/kg). Antinociceptive effect of the test substances was determined by the hot
tail-flick method described by Sewell and Spencer (1976). One to two cm of
the tail of mice was immersed in warm water kept constant at 50 °C. The reaction
time was the time taken by the mice to deflect their tails. The first reading is
discarded and the reaction time was taken as a mean of the next two readings.
The latent period of the tail-flick response was taken as the index of
antinociception and was determined before and at 15, 30, 45 and 60 min after the
administration of drugs. The maximum reaction time was fixed at 15 seconds. The
maximum possible analgesia (MPA) was calculated as:
Test reaction time - Saline reaction time
MPA =15- Saline reaction time
(iii) Cold tail-flick Male or female albino mice weighing between 20 - 25 g were
fasted for 24 hours with water given ad libitum, maintained at room temperature
and were divided into 4 groups of six mice. They were then given the same
treatment as in the writhing test. The reaction time of the mice was measured at
15, 30, 45 and 60 min. The antinociceptive activity was determined by the cold
tail-flick test described by Pizziketti et al. (1985). One to two cm of the tail
of mice was immersed in a cold 1: 1 mixture of water and ethylene glycol kept
constant at -10 °C. The reaction time was the time taken by the mice to deflect
their tails. The first reading is discarded and the reaction time was taken as
the mean of the last two readings.
ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) May 1998
The maximum reaction time was fixed at 30 seconds . The maximum possible
analgesia (MPA) was calculated as:
Test reaction time - Saline reaction time
MPA = 30- Saline reaction time
STATISTICAL ANALYSIS
The experimental data were analyzed by two-tailed impaired student's ttest. P
<0.05 was considered significant. Data were presented as mean ±SEM of n
observations.
RESULTS AND DISCUSSION
Acetic Acid-induced Writhings
Intraperitoneally injected acetic acid produced abdominal constrictions which is
characterized by a stretching response. Mean writhings observed in control
treated with vehicle (acacia gum) animals over a period of five minutes was 17.5
+ 2.8 counts. Morphine (5 mg/kg) and mitragynine (200mg/kg) significantly
(p<0.05) reduced writhings to 7.3 + 0.6 and 9.6 + 0.6 counts, respectively
(Figure 1). On the other hand, paracetamnol (100 mg/kg), did not significantly
reduce writhings induced by acetic acid.
This test also showed that 5mg/kg of morphine is about equipotent to 200mg/kg of
the crude extract. Other investigators have shown that the cold tail- flick
method is a selective method able to screen centrally acting opiate-fike
analgesic agents, and is not sensitive to analgesics acting peripherally, such
as aspirin, or non-analgesic drugs acting on the central nervous system, such as
chlorpromazine (Pizziketti et al., 1985).
On the other hand, the hot tail-flick method is shown here as incapable of
differentiating between opiate and non-opiate analgesics, or between
peripherally acting or centrally acting substances. It is possible that the
induction of analgesia by the alkaloid has both a peripheral and a central
component. In the report of Jansen and Prast (1988) they found that nalorphine
did not antagonize the effect caused by alkaloid from M speciosa.
Hot Tail-flick Response
Throughout the 60 min observation, animals pretreated with acacia gum did not
show significant effect on the latent period of tail-flick response. The
antinociceptive effect of morphine was evident within 15 min following oral
administration. The mean possible analgesia (MPA) increased from 3.9 ± 3.7 to
35.1 + 4.8 % which remained elevated above the basal levels throughout the
observation period (Figure 2 and Table 1). Likewise, paracetamol also exhibited
significant antinociception which began at 15 min following oral administration
and the effect remained significant throughout the 60 min observation period
(p<0.05). The MPA calculated for paracetamol increased to 27.8+ 2. 1 %.
Similarly, the antinociceptive effect of mitragynine was also observed at 15
mins following oral administration and the effect remained significant
throughout the 60 min observation period. The MPA calculated for mitragynine
increased to 42.8 + 7.2
Cold Tail-flick Response
While animal groups treated with gum acacia and paracetamol showed no
significant effect on the latent period of tail-flick response, the
antinociceptive effect of morphine was evident only after 30 min, reaching its
peak at 45 min and this effect remained significant until the 60 min (p<0.05)
test period. The peak MPA value calculated at 45 min was 66.2 ± 2.4 % compared
to 0.1 + 7.2 % induced by the control acacia gum (Figure 3, Table 1). On the
other hand, the antinociceptive effect of mitragynine was evident after 15 min
reaching its peak at 30 min and this effect remained significant until the 45
min (p<0.05) test period. The peak MPA calculated at 30 min was 49.0 ± 5.9 %.
Use In Traditional Medicine
Reports of the various uses of Mitraynine in traditional medicine in the
countries of the South East Asia region (Jansen & Prast, 1988; Suwanlert, 1974)
spurred research into the pharmacological aspects of its chemical constituents.
More than twenty-five alkaloids have been identified from this plant (Ikram,
1885) but of these the major ones (in terms of % yield) are three indoles i.e.
mitragynine, speciogynine and paynanthine and two oxindoles i.e. mitraphylline
and speciofoline.
In a previous study (unpublished report) we had detected the analgesic property
of the crude alkaloidal extract using the hot-plate method of Ankier (1974) and
the tail-flick method of Sewell and Spencer (1976). The effect showed
similarities to analgesia caused by morphine but its effect was not antagonized
by Naloxone in the hot-plate test. This is interesting because although
paracetamol also exhibited a significant effect in the hot tail-flick test, our
additional experiment using the cold tail-flick test further separated the
analgesic efficacy of the alkaloidal extract and morphine from that of
paracetamol. The failure of paracetamol to exhibit antinociceptive effect in the
cold tail-flick test suggests that analgesia induced by morphine and the extract
is by a different pathway involving other mediators than that due to paracetamol.
This test also showed that 5 mg/kg of morphine is about equipotent to 200mg/kg
of the crude extract. Other investigators have shown that the cold tail- flick
method is a selective method able to screen centrally acting opiate-fike
analgesic agents, and is not sensitive to analgesics acting peripherally, such
as aspirin, or non-analgesic drugs acting on the central nervous system, such as
chlorpromazine (Pizziketti et al., 1985).
On the other hand, the hot tail-flick method is shown here as incapable of
differentiating between opiate and non-opiate analgesics, or between
peripherally acting or centrally acting substances. It is possible that the
induction of analgesia by the alkaloid has both a peripheral and a central
component. In the report of Jansen and Prast (1988) they found that nalorphine
did not antagonize the effect caused by alkaloid from M speciosa.
Although it is recognized that the acetic acid writhing test is a useful measure
of analgesic activity, the method often shows positive responses even to
non-analgesic compounds such as central nervous system depressants (Hendershot
and Forsaith, 1959). Consistent anti-nociceptive activity of the M.speciosa
extract in all three methods used in the present study is in contrast with the
typical antinocipceptive activity exhibited by paracetamol, which shows
significant activity only in the hot tail-flick test. The failure of paracetamol
to inhibit acetic acid induced writhings may imply that suppression of writhings
may only be achieved by analgesic agents of higher efficacy or at higher doses
of paracetamol. Antinociceptive effects of morphine and the alkaloidal extract
were evident from the fact that both of these substances were markedly effective
in reducing the responses in all three tests viz the writhing and the tail-flick
tests. Thus the alkaloid from M.speciosa has definite analgesic properties but
further study must be carried out to determine in which particular alkaloid this
effect resides.
Our present study indicates that the alkaloidal extract has analgesic properties
similar to opioid and, similar to morphine is effective in the acetic-acid
writhing test, the hot tail-flick and the cold tail-flick tests. We have also
shown that aracetamol is only effective in the hot tail-flick test.
ACKNOWLEDGEMENT
We wish to thank Miss Christina Gertrude Yap from the department of
Pharmacology, UKM and Miss Nancy Yeap from the department of Pharmacology,
Universiti Malaya for their assistance.
REFERENCES
Ankier, S.I. 1974. New hot-plate test to quantify antinociceptive and narcotic
antagonist. Eur. J Pharmacol. 27: 1-4
Collier, H.O.J., L.C. Dinneen, C.A. Johnson and C. Schneider 1968. The abdominal
constriction response and its suppression by analgesic drugs in the mouse. Br. J
Pharmacol. Chemother. 32: 295.
Grewal, K.S. 1932 Observations on the pharmacology of Mitragyna. In:
Pharmacology of mitragynine. J Pharmacol. Exp. Ther. Vol XLVI(3): 251-271.
Hendershot, L.C. and Forsaith, J. 1959 Antagonism of the frequency of
phenylquinone-induced writhing in mouse by peak analgesics and nonanalgesics. J
Pharmacol Exp. Ther., 125: 23 7-24 1.
Houghton, P.J & 1kram M. Said. 1986. 3- Dehydromitragynine: an alkaloid from
Mitragyna speciosa. Phytochemistry 25: 2910-2912.
Idid, S.Z.,, K. Norehan and A. Roslan. 1992. The involvement of the
noradrenergic system in analgesia induced by the alkaloidal extract of Mitragyna
speciosa in the rat. Proc. 3rd Medical Colloquim, UKM. pp 337-340.
Ikram, M.S. 1985. Studies on the components of fresh leaves of Mitragyna
speciosa. In: M.S.
lkram & Z.Zakaria (Eds.) Proceedings of 2nd Meeting of the Natural Products
Research Group, Chemistry Dept., UKMalaysia. pp 123-127.
Jansen K.L.R. and C.J.Prast, 1988. Ethnopharmacology of Kratom and the Mitragyna
alkaloids. J Ethnopharmacology 23: 115-119,
Perry, L.M. 1980. Medicinal plants of East and South East Asia, MIT Press,
U.S.A.
Pizziketti, R.J., N.S. Pressman, E. B. Geller and M.W. Adler 1985. Rat cold
water tail-flick: A novel analgesic test that distinguishes opioid agonist from
mixed agonist-antagonist. Eur J Pharm. 119: 23-29.
Sewell, R.D.E. and P.S.J. Spencer 1976. Antinociceptive activity of narcotic
agonist and partial agonist analgesics and other agents in the tail-immersion
test in mice and rats.
Neuropharmacol. 15, 23-29.
Suwanlert, 1974. A study of kratom eaters in Thailand. Thai Bull. on Narcotics.
26: 21-27.
Specific to Mitragyna speciosa
(Kratom):
|
