Analgesic Activity of Abelmoschus manihot Extracts
S. Jain Pritam,
A. Todarwal Amol,
B. Bari Sanjay
J. Surana Sanjay
The natural products served as important sources of medicines now a day increasing, as they possess the therapeutic activity. Therefore, the present study was carried out to evaluate the analgesic activity of the petroleum ether and methanol extract of Abelmoschus manihot (Malvaceae) leaves using hot plate and tail immersion model. The air-dried, powdered leaves (1000 g) were extracted over Soxhlet with petroleum ether and methanol. The crude dried petroleum ether (10 g) and methanol (25 g) extracts was prepared at the doses of 100, 200 and 400 mg kg-1 and evaluated for analgesic activity using the hot plate and tail immersion test. The results obtained indicate that the extracts possessed significant (p<0.05, p<0.01) analgesic activity, which was found to be dose-dependent. A significant inhibition in pain threshold in hot-plate test was exhibited. However, in flick test, highest analgesic activity was observed only with 400 mg kg-1 dose as compared with the standard drug. This study showed that the petroleum ether and methanol extracts of Abelmoschus manihot leaves possess potential pharmacological active constituents responsible for inhibition of the analgesic effect.
June 13, 2011; Accepted: July 21, 2011;
Published: September 09, 2011
There is an increasing trend of herbal formulation as they have lesser side
effects and are easy to access as well as in some cases these are the only available
treatment against chronic diseases (Karim et al.,
2011). Abelmoschus manihot (Linn.) belongs to family Malvaceae is
a large annual erect hairy plant, 1.2-1.8 m. high, stems with small scattered
prickles. Leaves 15-18 cm, deeply palmately lobed, stipules are 1 cm long, linear-lanceolate
with stiff bristles on the margins. Flowers yellow with a purple centre, capsules
oblong, pointed, hispid, seeds black (Pullaiah, 2002).
The mucilage contains polysaccharides and proteins (Kiritikar
and Basu, 1994). The flower contains quercetin-3-robinoside, quercetin-3-glucoside,
hyperin, myrecetin and anthocyanins (Lai et al.,
2006). The different chromatographic methods have been developed on the
flavones present in the plant (Liang et al., 2007;
Lai et al., 2009; Yi et
al., 2008). The flowers are used in the treatment of chronic bronchitis
and toothache. The ethanol extract of flower was screened for antiviral activity
and it was observed that the hyperoside shown significant anti HBV activity
(Lin-Lin et al., 2007). The phytoconstituents
obtained from the plant possesses different pharmacological activities (Liu
et al., 2009; Wen and Chang, 2007; Xin-Ping
et al., 2006). The leaves were tested on bone loss in ovariectomised
rats and it was observed that it was able to prevent the ovariectomy-induced
femoral osteopenia (Puel et al., 2005).
The phytochemical analysis showed the presence of steroids, triterpenoids and
flavonoids in petroleum ether and methanol extract, respectively which possesses
analgesic (Gupta et al., 2008) antioxidant and
anti-inflammatory activity (Oyedapo et al., 2008;
Raj et al., 2006). Therefore, in this study
the analgesic activity evaluated of the leaves of Abelmoschus manihot.
MATERIALS AND METHODS
Plant material: The leaves of Abelmoschus manihot were collected
in August 2010 from Toranmal Hills of Maharashtra and authentication of the
plant specimen was established by Dr. D.A. Patil was deposited in our departmental
herbarium for future reference (Patil, 2003).
Preparation of extracts: The leaves were chopped into small pieces air-dried and powdered. The powdered plant material was successively soxhlet extracted with petroleum ether and methanol at room temperature for 48 h.
Standard drugs/chemicals solutions: Pentazocine (Neon) (10 mg kg-1), used for the purpose of comparison, was prepared by dissolving, in acacia suspension. Carrageenan (λ4) (C3889-5G) was obtained from Sigma Aldrich, USA.
Animals: Swiss albino mice (25-30 g; 7-10 weeks old) were, obtained from
RC Patel Institute of Pharmaceutical Education and Research, Shirpur and Government
Veterinary College, Mahu, MP (India). The animals were housed in Animal house
of R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, India
in polycarbonate cages, in a room maintained, under controlled room temperature
22±2°C, relative humidity 60-70% and provided with food and water
ad libitum. All the experimental procedures and protocols used in the
study were reviewed by the Institutional Animal Ethics Committee (IAEC) and
the care of laboratory animals was taken as per the guidance of CPCSEA, Ministry
of Forests and Environment, Government of India. The animals were deprived of
food for 24 h before experimentation but allowed free access to water throughout.
All studies were carried out by using six animals in one group for anti-inflammatory
Acute toxicity test: A cute toxicity test was performed according to
OECD (2006) guidelines. Animals were weighed and marked,
a single high dose, 2000 mg kg-1 of petroleum ether extract and
methanol extract as recommended by the OECD guidelines was administered to the
first animal. After a single administration, a sign of toxicity and behavior
was observed each hour up to the 24 h. If this animal was dying, then one lesser
dose of that dose was administered to the next animal. Same procedure was followed
for that animal. If the animal survives, then the same dose was given to the
next 5 animals. All the animals were observed for the signs of toxicity and
mortality for up to the 14 days.
Additional observations like changes in skin, eyes and mucous membranes and also respiratory circulatory, autonomic and Central Nervous system and behavior pattern. Attention was also given to observed precipitation of tremors and convulsions.
Hot plate method: The Swiss albino mice (25-30 g) of either sex were
divided into eight groups containing six animals in each. A control group received
normal saline solution, while three groups received petroleum ether extract
at doses 100, 200 and 400 mg kg-1 p.o, respectively. The remaining
three groups received methanol extract at doses 100, 200 and 400 mg kg-1
p.o. Pentazocine (10 mg kg-1) was administered to eighth group. The
temperature of hot plate was maintained at 55±0.5°C. The rats were
placed individually on hot plate and time between placement and licking of paws,
shaking or jumping off the surface was recorded by using Eddys hot plate apparatus
(Spacelab). As a response latency rat with baseline latencies of less than 5
sec or more than 15 sec were eliminated from the study and cut off latency time
was set at 15 sec to avoid tissue damage. After determination of base line response
latencies, hot plate latencies were re determined at 0, 30, 60, 90 and 180 min
after drug administration (Oloyede et al., 2008).
Tail Immersion method: The Swiss albino mice (25-30 g) of either sex were divided into eight groups containing six animals in each. The rats were fasted for 12 h prior to induction of analgesia. The control group received normal saline solution, while three groups received petroleum ether extract at doses 100, 200 and 400 mg kg-1 p.o., respectively. The remaining three groups received methanol extract at doses 100, 200 and 400 mg kg-1 p.o. Pentazocin (10 mg kg-1) was administered to eighth group. The lower 5 cm portion of the tail was marked. This part of the tail was immersed in a cup of freshly filled water of exactly 55°C. Within a few seconds the rat reacted by withdrawing the tail. The reaction time was recorded by a stopwatch. After each determination the tail was carefully dried by a cloth. The reaction time was determined before and periodically after oral administration of the test substance, e.g., after 0, 30, 60, 90 and 180 min. The cut off time of the immersion was kept 15 sec. The withdrawal time of untreated animals was between 1 and 5.5 s. A withdrawal time of more than 6 sec therefore is regarded as a positive response.
Statistical analysis: The statistical analysis of all the results was carried out using one-way ANOVA followed by Dunnetts multiple comparisons using graph pad in stat 3 Demo and obtained in the study were compared with the vehicle control group. The p values <0.05 were considered statistically significant.
RESULTS AND DISCUSSION
Extraction: The percentage of yield of the petroleum ether and methanol extracts was 0.10 and 0.25% , respectively.
Acute toxicity test: The Abelmoschus manihot leaf extracts did
not produce any mortality even at the dose of 2000 mg kg-1, p.o.,
thus found to be non-toxic.
|| The analgesic activity of petroleum ether and methanol extract
of Abelmoschus manihot using the Hot plate method
|Values represent Mean±SEM, n = 6. One way ANOVA followed
by Dunnetts multiple comparison test *p<0.05, **p<0.01 compare
with control group
|| The analgesic activity of Petroleum ether and methanol extract
of Abelmoschus manihot Assessed using Tail Immersion method
|Values represent Mean±SEM, n = 6. One way ANOVA followed
by Dunnetts multiple comparison test **p<0.01 compare with control
Therefore, doses (100, 200, 400 mg kg-1, p.o.) of A. manihot
three was selected for further pharmacological studies (Table
Hot plate method: The petroleum ether and methanol extracts at the doses of 100, 200 and 400 mg kg-1 p.o. showed significant (p<0.05, p<0.01) and dose dependent increase in the reaction time at 180 min (Table 1). The petroleum ether and methanol extract in the dose of 400 mg kg-1 showed maximum of 14.1±0.21 and minimum of 6.98±0.29 reaction time which showed significant threshold in pain , respectively while the group treated with standard drug Pentazocin (10 mg kg-1) showed significant (p<0.01) increase in the reaction time as compared to control.
Tail immersion method: The petroleum ether and methanol extracts at the doses of 100, 200 and 400 mg kg-1 p.o. showed significant (p<0.05, p<0.01) and dose dependent increase in the reaction time at 180 min (Table 2). The flick test showed the petroleum ether extract showed 6.98±0.21 and 4.83±0.12 reaction time while methanol extract 400 mg kg-1 showed 7.05±0.11 and 4.96±0.21 reaction time, exhibited highest analgesic activity, while the group treated with standard drug Pentazocin (10 mg kg-1) showed significant (p<0.01) increase in the reaction time as compared to control.
The present study indicated that A. manihot has the pharmacological
potential as an analgesic agent when tested on various animal models. The ability
to inhibit/reverse the former and latter tests could also be associated with
the extracts potential to inhibit the induced analgesia (Ballou
et al., 2000). Although the present study did not aim at isolation
and identification of bioactive compounds the phytochemical screening of petroleum
ether and methanol extract demonstrated the presence of flavonoids, steroids,
triterpenoids, which are suggested to act synergistically to exert the observed
pharmacological activity (Maj and Rogoz, 2000). The
presence of steroids and flavonoids in petroleum ether and methanol extract
could possibly lead to the observed activity. The analgesic activity of petroleum
ether and methanol extract could also be linked to the mechanism of action either
on central nervous system or peripheral nervous system. Interestingly, compounds
like flavonoids (Kim et al., 2004) and steroids,
triterpenes in part, have been shown to possess anti-inflammatory, analgesic
activity and the claim made by Attaway and Zaborsky (1993).
Based on the classes of compounds detected in petroleum ether and methanol extract,
several mechanisms of action could be used to explain the observed activities
of the extract. Flavonoids are potent inhibitors of nitric oxide synthase type
2 that are involved in the synthesis of NO (Olszanecki et
al., 2002). The ability of steroids and flavonoids to induce vasodilatation
and the importance of vasodilation in the antinociceptive and anti-inflammatory
mechanisms have also been reported and are worth mentioning (Naseri
et al., 2005). To demonstrate whether petroleum ether and methanol
extract is producing analgesic activity in this model by acting on Pentazocin,
further the effect of petroleum ether and methanol extract was studied on Pentazocin
In hot plate and tail immersion analgesic models petroleum ether and methanol extract and reference drug Pentazocin significantly increased the reaction time at 180 min. In the late phases of this model petroleum ether, methanol extract, Pentazocin showed analgesic activity. The results of present study support the ethno-medical application of A. manihot in the treatment of analgesic diseases. Further experimentation is needed in order to understand the precise mechanism of action in analgesic activities by the extracts.
The present study demonstrated that the petroleum ether and methanol extracts possessed analgesic activity. Thus, the present study confirmed the folklore use of A. manihot fruit for the treatment of various ailments and the plants potential pharmacological activities merit further investigation.
The authors would like to thank Principal, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, M.S., India for the facilities provided.
Attaway, D.H. and O.R. Zaborsky, 1993. Marine Biotechnology: Pharmaceutical and Bioactive Natural Products. Vol. 5, Plenum Press, New York, ISBN-10: 0-306-44174-8, pp: 23.
Ballou, L.R., R.M. Botting, S. Goorha, J. Zhang and J.R. Vane, 2000. Nociception in cyclooxygenase isozyme-deficient mice. Proc. Nat. Acad. Sci. USA., 97: 10272-10276.
Direct Link |
Gupta, M., B.P. Shaw and A. Mukherjee, 2008. Studies on antipyretic-analgesic and ulcerogenic activity of polyherbal preparation in rats and mice. Int. J. Pharmacol., 4: 88-94.
CrossRef | Direct Link |
Karim, A., M.N. Sohail, S. Munir and S. Sattar, 2011. Pharmacology and phytochemistry of Pakistani herbs and herbal drugs used for treatment of diabetes. Int. J. Pharmacol., 7: 419-439.
Kim, H.P., K.H. Son, H.W. Chang and S.S. Kang, 2004. Anti-inflammatory plant flavonoids and cellular action mechanism. J. Pharmacol. Sci., 96: 229-245.
Kiritikar, K.R. and B.D. Basu, 1994. Indian Medicinal Plants. Vol. 3. 2nd Edn., Allahabad, India, ISBN-13: 817089279 pp: 1606-1609, 1783-1792.
Lai, X., H. Liang, Y. Zaho and B. Wang, 2009. Simultaneous determination of seven active flavonols in the flowers of Abelmoschus manihot by HPLC. J. Chromatogr. Sci., 47: 206-210.
Lai, X.Y., Y.Y. Zaho and H. Laing, 2006. Studies on chemical constituents in flower of Abelmoschus manihot. Zhongguo Zhong Yao Za Zhi, 31: 1597-1600.
Liang, H., X. Lai, Y. Zaho, Y. Bai, B. Wang and D. Gao, 2007. SPE-HPLC method for the determination of four flavonols in rat plasma and urine after oral administration of Abelmoschus manihot extract. J. Chromatogr. B Anal. Technol. Biomed. Life Sci., 852: 108-114.
Lin-Lin, W.U., Y. Xin-Bo, H. Zheng-Ming, L. He-Zhi and W.U. Guang-Xia, 2007. In vivo and in vitro antiviral activity of hyperoside extracted from Abelmoschus manihot (L.). Med. Acta Pharmacol. Sin., 28: 404-409.
Liu, M., Q.H. Zaing, J.L. Hao and L.L. Zahou, 2009. Protective effect of total flavones of Abelmoschus manihot L. Medic against poststroke depression injury in mice and its action mechanism. Anat Rec., 292: 412-422.
Maj, J. and Z. Rogoz, 2000. Synergistic effect of amantadine and imipramine in the forced swimming test. Pol. J. Pharmacol, 52: 111-114.
Direct Link |
Naseri, M.K.G., M.N. Hamidi and A. Heidari, 2005. Vasorelaxatory effect of Vitis Vinifera extract on rat aorta. Iran J. Pharmaceut. Sci., 2: 93-99.
Direct Link |
OECD., 2006. Guidelines for testing of chemicals: Acute oral toxicity-up-and-down method. Guidelines 425, OECD.
Oloyede, A.M., J. Okpuzor, O. Omidiji and H.O.C. Mbagwu, 2008. A pharmacological evaluation of a herbal cocktail. Int. J. Pharmacol., 4: 196-201.
CrossRef | Direct Link |
Olszanecki, R., A. Gebska, V.I. Kozlovski and R.J. Gryglewski, 2002. Flavonoids and nitric oxide synthase. J. Physiol. Pharmacol., 53: 571-584.
Direct Link |
Oyedapo, O.A., C.O. Adewunmi, E.O. Iwalewa and V.O. Makanju, 2008. Analgesic, antioxidant and anti-inflammatory related activities of 21-hydroxy-2,41-dimethoxychalcone and 4-hydroxychalcone in mice. J. Boil. Sci., 8: 131-136.
CrossRef | Direct Link |
Patil, D.A., 2003. Flora of Dhule and Nandurbar Districts (Maharashtra). Bishen Singh Mahendra Pal Singh, Dehradun, India, ISBN-13: 81-211-0164-6 pp: 440.
Puel, C., J. Mathey, M.J. Davicco, P. Lebecque, B. Chanteranne, M.N. Horcajada and V. Coxam, 2005. Preventive effect of Abelmoschus manihot (L.) medik on bone loss in the overiectomised rats. J. Ethnopharmacol., 99: 55-60.
Pullaiah, T., 2002. Medicinal Plants in India. Vol. I. Regency Publication, New Delhi, India. pp: 6.
Raj, K.K.V., B. Narayana, B.V. Ashalatha and N.S. Kumari, 2006. New thiazoles containing pyrazolopyrimidine moiety as possible analgesic agents. J. Pharmacol. Toxicol., 1: 559-565.
CrossRef | Direct Link |
Wen, J.Y. and Z.W. Chang, 2007. Protective effect of pharmacological preconditioning of total flavones of abelmoschl manihot on cerebral ischemic reperfusion injury in rats. Am. J. Chin Med., 35: 653-661.
Xin-Ping, C., Q. Song, D. Liu-Yi and Z. Jiang-Ning, 2006. Inhibitory effect of total flavone of Abelmoschus manihot L. Medic on NMDA receptor-mediated current in cultured rat hippocampal neurons. Neurosci. Res., 55: 142-145.
Yi, L., L. Wujie, L.X.L. Xiaomei, L. Yuzhen, Z.Z. Qingying and Z. Yuying, 2008. Simultaneous determination of the active ingredients in Abelmoschus manihot (L.) Medicus by CZE. Chromatographia, 67: 819-823.