|Year : 2014 | Volume
| Issue : 2 | Page : 89-94
Analgesic effect of ethanolic leaf extract of moringa oleifera on albino mice
Ayon Bhattacharya1, Divya Agrawal2, Pratap Kumar Sahu3, Sanjay Kumar1, Sudhanshu Sekhar Mishra1, Shantilata Patnaik4
1 Department of Pharmacology, Institute of Medical Sciences and SUM Hospital, Siksha O Anusandhan University, Bhubaneswar, Odisha, India
2 Department of Anatomy, Institute of Medical Sciences and SUM Hospital, Siksha O Anusandhan University, Bhubaneswar, Odisha, India
3 Department of Pharmacology, School of Pharmaceutical Sciences, Siksha O Anusandhan University, Bhubaneswar, Odisha, India
4 Department of Pharmacology, Institute of Dental Sciences, Siksha O Anusandhan University, Bhubaneswar, Odisha, India
|Date of Web Publication||20-May-2014|
Department of Pharmacology, Institute of Medical Sciences and SUM Hospital K-8, Kalinga Nagar, B.O. Ghatikia, Bhubaneswar - 751 003, Odisha
Source of Support: None, Conflict of Interest: None
Objectives: Moringa oleifera is a highly valued plant distributed in many countries of the tropic and subtropics. Moringa oleifera leaves are a potential source of phytochemical ingredients claimed to have analgesic property. Pain is an unpleasant sensation, which in many cases represents the only symptom for the diagnosis of several diseases. Therefore analgesic drugs lacking the side effect as alternative to nonsteroidal anti-inflammatory drugs (NSAIDs) and opiates are in demand by the society. The present study is undertaken to evaluate the analgesic activity of Moringa oleifera using acetic acid induced writhing test and Eddy's hot plate test. Materials and Methods: It is a randomized control study. The present study was done using two experimental models. The albino mice were divided into six groups, each group consisting of 6 mice. A total of 36 mice were used in each of the two experimental models. Group I: Control (normal saline given orally at 2 ml/kg body weight); Group II: Standard (diclofenac 10 mg/kg i.p/ morphine 1 mg/kg i.p); Group III, IV, V, VI (ethanolic extract of Moringa oleifera (EMO) 50, 100, 200, 400 mg/kg, respectively). The EMO leaves were administered at 50, 100, 200, 400 mg/kg doses orally 1 hour before the experiments. For peripheral analgesic effect, acetic acid induced writhing test was used. The central analgesic effect was screened using Eddy's hot plate method. The standard drug used in acetic acid induced writhing test was diclofenac and in Eddy's hot plate test was morphine. Results: The EMO leaf showed significant (P < 0.01) analgesic activity at 100, 200, 400 mg/kg in the acetic acid induced writhing test showing 32.21%, 59.71% and 78.61% inhibition of writhes, respectively in comparison with the control. In the Eddy's hot plate test EMO at 400 mg/kg showed significant (P < 0.01) analgesic activity from 15 min to 90 min with a mean rank ranging from 28.92 to 26.00, second mean rank following morphine in comparison with control. In both the tests, EMO showed significant (P < 0.01) analgesic activity in a dose-dependent manner. Conclusion: The ethanolic leaf extract of Moringa oleifera exhibited analgesic activity in both models showing its both central and peripheral analgesic actions.
Keywords: Acetic acid induced writhing test, analgesic, Eddy′s hot plate method, ethanolic extract of Moringa oleifera
|How to cite this article:|
Bhattacharya A, Agrawal D, Sahu PK, Kumar S, Mishra SS, Patnaik S. Analgesic effect of ethanolic leaf extract of moringa oleifera on albino mice. Indian J Pain 2014;28:89-94
|How to cite this URL:|
Bhattacharya A, Agrawal D, Sahu PK, Kumar S, Mishra SS, Patnaik S. Analgesic effect of ethanolic leaf extract of moringa oleifera on albino mice. Indian J Pain [serial online] 2014 [cited 2020 Apr 1];28:89-94. Available from: http://www.indianjpain.org/text.asp?2014/28/2/89/132846
| Introduction|| |
Pain is an unpleasant sensation but a protective mechanism of our body. Analgesics are defined as substances, which decrease pain sensation by increasing pain threshold to external stimuli without altering consciousness. Therefore, analgesic drugs lacking the side effect as alternative to nonsteroidal anti-inflammatory drugs (NSAIDs) and opiates are in demand for the society.  Moringa oleifera is one of the 14 species belonging to the family Moringaceae,  easy to cultivate, fast growing, multipurpose plant. Known as Shigru (Sanskrit); Sahijan (Hindi); drumstick tree (English) is naturalized in the tropical and subtropical regions worldwide. , In folk medicine the African bushmen used it to relieve their rheumatic joint pains.  Moringa oleifera leaves are also a folk remedy for stomach aches,  and headaches.  Leaves are tripinnate, compound bearing several small leaf legs, rachis slender, thickened and articulated at base,  highly nutritious and rich in amino acids, vitamins, potassium, calcium, methionine, cysteine and zinc and natural antioxidants.  The methanolic and ethanol extractions from the leaves have shown to be the best source of antioxidants compounds.  The phytochemical ingredients in leaves showed presence of flavonoids, tannins, saponins, terpenoids, proanthocyanadins, cardiac glycosides, which have analgesic property.  The leaves have a wide array of central nervous system (CNS) activities like analgesic, anti-inflammatory, antipyretic, anxiolytic actions.  All the currently available analgesic drugs such as NSAIDs and opioid analgesics are subject to their own side effects such as gastric erosions with NSAIDs and tolerance, dependence with opioid analgesics. Therefore, man has been on hunt since ages for suitable alternatives to NSAIDs and opioid analgesics. Taking this background into consideration the present study was taken up to evaluate the analgesic activity of this plant and also scientifically validate the folklore claims of this plant.
| Material and Methods|| |
Collection of plant material
The leaves were collected for the local areas of Syampur, Bhubaneswar, Odisha, 751003 and its identity was confirmed by taxonomist of Regional Plant Research Centre (RPRC), Bhubaneswar.
Preparation of extract
Fresh leaves were collected dried in shade and powdered. The powder was extracted with 90% ethanol using continuous hot air percolation method in a Soxhlet apparatus for 18 hrs. Extract filtered using Whitman filter paper no 1 and concentrated in rotary evaporator to yield a semi solid mass of 42 g (yield 8.4% w/w). Extract stored in refrigerator at 4 0 C and used for oral administration. 
Morphine (Morphitroy, Troikaa Pharmaceuticals Ltd, Gujrat, India), acetic acid (Fischer inorganic and Aromatic Ltd, Chennai, India), diclofenac (Voveran, Novartis, Worli Mumbai), and other solvent chemicals used were of analytical grade.
Wistar Albino mice of either sex (20-30 g) were randomly selected from the central animal facility. The animals kept at ambient temperature of 22 ± 1 o C, 12-hr light and dark cycle allowed. Food, water given ad libitum . Animals were acclimatized to laboratory conditions for 7 days prior to taking them for experimentation. The study was approved by the Institutional Animal Ethical Committee (IAEC) of Siksha O Anusandhan University, Bhubaneswar under the approval number 22/12/IAEC/SPS/SOA. All experiments and animal care were according to the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) and Good Laboratory Practice (GLP) Guidelines. No animals were sacrificed at the end of the study.
It is a randomized control study. The animals were randomly divided into 6 groups with 6 mice each; Group I: Control (normal saline given orally at 2ml/kg body weight); Group II: Standard (diclofenac 10 mg/kg i.p / morphine 1 mg/kg i.p ); Group III, IV, V, VI (EMO 50, 100, 200, 400 mg/kg, respectively). The total number of mice used in each experiment was 36, so a total of 72 mice were used in this study [Figure 1].
Acetic acid-induced writhing method
Initially test drug EMO was given orally 1 hr before, and the standard drug diclofenac (10 mg/kg) was given intraperitoneally half-an-hour before the administration of acetic acid. , Next acetic acid 1%, 10 ml/kg was injected intraperitoneally in albino mice and the writhing movements were recorded after 5 min for a total of 10 min. For scoring purposes a writhe is characterized by stretching of the abdomen with simultaneous stretching of at least one hind limb.  The results were permitted to express the percentage of protection of writhes compared with the control.
Eddy's hot plate method: Albino mice were placed on heated plates and the time taken for either paw licking or jumping was taken as the reaction time or latency time.  Albino mice were immediately removed from the hot plate on paw licking or jumping to prevent damage to their paws. The test drug (EMO) was given orally 1 hour before and the standard drug morphine (1 mg/kg) was given intraperitoneally half-an-hour prior to the experiment. The temperature of the hot plate was set at 55 ± 1 o C. The cutoff period was taken at 15 seconds. The latency is recorded before and after 15, 30, 45, 60, 90, 120, 180 min of administration of standard and test drugs and the results were compared with the control.
The Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test for acetic acid-induced writhing test. In Eddy's hot plate test Kruskal Walli's test was used followed by Mann Whitney's test. Level of significance was taken at P < 0.01.
| Results|| |
The ethanolic leaf extract of Moringa oleifera showed significant (P < 0.01) analgesic activity at 100, 200, 400 mg/kg in both the acetic acid-induced writhing test and the Eddy's hot plate test in comparison with control. The results of the acetic acid-induced writhing test along with the percentage inhibition of writhes are given in [Table 1]. Dose-dependent increase in the percentage inhibition of writhes was noted with EMO 100, 200, 400 mg/kg showing 32.21%, 59.71% and 78.61%, respectively. The EMO at 400 mg/kg showed 78.61% inhibition of writhes comparable with the standard diclofenac, which showed 79.71% [Figure 2]. The multiple comparison following Bonferroni test revealed that the effect of diclofenac is significantly better than EMO up to 200 mg/kg, but the effect of diclofenac and EMO up to 400 mg/kg are comparable (P > 0.05) [Table 2].
|Figure 2: Effects of EMO and diclofenac on acetic acid-induced writhing method|
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|Table 1: Analgesic effects of EMO and diclofenac on acetic acid-induced writhing test |
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|Table 2: Multiple comparison of different treatments in acetic acid-induced writhing test using post hoc bonferroni test |
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The result of the hot plate method is given in [Table 3]. From 15 min to 120 min it was found that the group ranks were significantly (P < 0.01) different. At each time interval from 15 min onward the standard drug morphine showed the highest mean rank ranging from 30.75 to 32.83, whereas EMO at 400 mg/kg was the second mean rank ranging from 28.92 to 26.00 following morphine. Further Mann-Whitney U test was conducted to see the difference between morphine and EMO 400 mg/kg [Table 4]. The results showed that there is no significant (P > 0.05) difference between the two from 15 min to 90 min. In Eddy's hot plate test the predicted onset time of EMO was found to be at 75 min.
|Table 3: Analgesic effects of EMO and morphine on Eddy's hot plate test and comparison of their mean rank using Kruskal Wallis test |
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|Table 4: Mann Whitney's U test for comparison between Morphine and EMO 400 mg/kg |
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| Discussion|| |
The EMO showed significant (P < 0.01) analgesic activity in both the central and the peripheral experimental models in a dose-dependent manner.
Analgesic activity using petroleum ether and methanolic extract of Moringa oleifera stem bark,  and the hydro ethanol extract of pods have already been done.  In these studies, flavonoids, tannin, saponins, and alkaloids like ingredients were claimed to possess analgesic activity. The mechanism of action postulated in these studies was due to prostaglandin inhibition and the action on the central pain receptors.
Ethanolic leaf extract of Moringa oleifera has anti-inflammatory,  antioxidant,  anti-cataleptic,  and anti-microbial  actions, as well.
Acetic acid writhing method is used to screen the peripheral analgesic effect. The intraperitoneal injection of acetic acid produced an abdominal writhing response due to sensitization of chemosensitive nociceptors by prostaglandins. Increased level of prostanoids, particularly prostaglandin E2 (PGE 2 ) and prostaglandin F2 (PGF 2 ), as well as lipoxygenase products have been found in the peritoneal injections of acetic acid. , Acetic acid-induced writhing method also liberates endogenous substances like serotonin, bradykinin, histamine, which stimulate the sensory nerve endings.  Recently, it was found that nociceptive activity of acetic acid may be due to release of cytokines like tumor necrosis factor alpha (TNFα), interleukin 1β, interleukin-8 by resident macrophages and mast cells.  Anti-nociceptive action of EMO leaves could be due to inhibition of these mediators. However, this needs to be confirmed by further studies. The anti-nociceptive action of EMO here could be due to its action on the visceral receptors sensitive to acetic acid, to the inhibition of algogenic substances.  (prostaglandin synthesis).
In the hot plate method, phasic stimuli of high intensity are given.  Pain induced by thermal stimulus of hot plate is specific for centrally mediated activity.  Opioid agents (morphine) exert their analgesic action via supraspinal (μ1 , κ3 , σ2 , γ1 ) and spinal (μ2 , κ1 , γ2 ) receptors.  The EMO showed anti-nociceptive activity by increasing the latency to discomfort in hot plate test. This action could be by activating the periaqueductal gray matter to release endogenous peptides (endorphins or encephalins).  This endogenous peptides descend the spinal cord and function as inhibitors of pain impulse transmission at the synapse in the dorsal horn. , The possible mechanism of EMO leaves could be due to its action on the central opioid receptors or promoted release of endogenous opioid peptides.  The present study, thus demonstrates the potential anti-nociceptive effect of EMO in chemical (acetic acid-induced writhing method) and thermal (Eddy's hot plate method) models of nociception, thus postulating that EMO possesses both central and peripheral nociceptive actions.
The phytochemical ingredients found in leaf extract of EMO like flavonoids, ,, which potently inhibit prostaglandins, especially the endoperoxidase, tannins, , alkaloids,  could also contribute to the anti-nociceptive action of EMO.
| Conclusion|| |
In this study, the analgesic effect of EMO is exhibited in a dose-dependent manner and thus scientifically validating the folklore claims about the plant. However, further studies are needed to isolate the active constituents responsible for the observed effect and to reveal the possible mechanism of action responsible for its anti-nociceptive activity.
| Acknowledgment|| |
I would like to extend my utmost gratitude to Dr Manas Naik, Tutor cum Resident, dept of Pharmacology and Dr. B. B Nanda, M.Sc (Stat), Ph.d (Stat), former biostatistician, SCB Medical College, Cuttack.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]