Screening of Ficus religiosa leaves fractions for analgesic and anti-inflammatory activities,Vishal Gulecha1, T Sivakumar2, Aman Upaganlawar3, Manoj Mahajan3, Chandrashekhar Upasani3, : Indian Journal of Pharmacology

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Year : 2011 \| Volume : 43 \| Issue : 6 \| Page : 662--666

Screening of Ficus religiosa leaves fractions for analgesic and anti-inflammatory activities

Vishal Gulecha¹, T Sivakumar², Aman Upaganlawar³, Manoj Mahajan³, Chandrashekhar Upasani³,
¹ Department of Pharmacology, SNJB'S SSDJ College of Pharmacy, Neminagar, Chandwad, Nashik;Department of Medicinal Chemistry, Nandha College of Pharmacy, Erode, Tamilnadu, India
² Department of Medicinal Chemistry, Nandha College of Pharmacy, Erode, Tamilnadu, India
³ Department of Pharmacology, SNJB'S SSDJ College of Pharmacy, Neminagar, Chandwad, Nashik, India

Correspondence Address:
Vishal Gulecha
Department of Pharmacology, SNJB«SQ»S SSDJ College of Pharmacy, Neminagar, Chandwad, Nashik;Department of Medicinal Chemistry, Nandha College of Pharmacy, Erode, Tamilnadu
India

Abstract

Objective : To evaluate the different fractions of dried leaves of Ficus religiosa Linn for analgesic and anti-inflammatory activity using different models of pain and inflammation Materials and Methods : The analgesic activity of F. religiosa carried out using acetic acid-induced writhing in mice and tail flick test in rats. The anti-inflammatory activity was evaluated using carrageenan-induced rat paw edema and cotton pellet-granuloma formation in rats. Five different fractions (FRI, FRII, FRIII, FRIV and FRV) of F. religiosa at the dose level of 20 and 40 mg/kg, p.o were tested. Results : The fraction FRI (40 mg/kg, p.o.) and FRIII (40 mg/kg, p.o) were found to be more effective (P<0.01) in preventing carrageenan induced rat paw edema, cotton pellet granuloma formation, and acetic acid induced writhing compared to the other fractions. FRI (20 mg/kg, p.o.) and FRIII (20 mg/kg, p.o.) were also found to be more effective in increasing latency period in tail flick method. Conclusion : Out of five different fractions of F. religiosa leaves tested, FRI and FRIII possess potent analgesic and anti-inflammatory activities against different models of inflammation and pain.

How to cite this article:
Gulecha V, Sivakumar T, Upaganlawar A, Mahajan M, Upasani C. Screening of Ficus religiosa leaves fractions for analgesic and anti-inflammatory activities.Indian J Pharmacol 2011;43:662-666

How to cite this URL:
Gulecha V, Sivakumar T, Upaganlawar A, Mahajan M, Upasani C. Screening of Ficus religiosa leaves fractions for analgesic and anti-inflammatory activities. Indian J Pharmacol [serial online] 2011 [cited 2023 Jun 6 ];43:662-666
Available from: https://www.ijp-online.com/text.asp?2011/43/6/662/89822

Full Text

Introduction

Despite progress within medical research during the past decades, the treatment of many serious diseases remains problematic. [1] Chronic inflammatory diseases remain one of the world's major health problems. [2] Currently, both steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs (NSAIDs) are used in the aid of inflammation. Steroids have an obvious role in the treatment of inflammatory diseases, but due to their toxicity, they can only be used over short periods except in very serious cases where the risks are acceptable. Prolonged use of NSAIDs is also associated with severe side effects, notably gastrointestinal hemorrhage. [3],[4]

Inflammatory diseases are among the most common health problems treated with traditional remedies. Therefore, it is crucial to evaluate the potential of herbal remedies that might serve as leads for the development of potent drugs. A large number of Indian medicinal plants are attributed with various pharmacological activities owing to there different class of phytochemicals.

Ficus religiosa (FR) L. (Moraceae) commonly known as 'Peepal' is a variety of fig and sacred tree native to India. It is reported to have numerous therapeutic uses in folk medicine viz. leaf juice has been used for the treatment of asthma, cough, sexual disorders, diarrhea, hematuria, ear-ache and toothache, migraine, eye troubles, gastric problems and scabies; leaf decoction has been used as an analgesic for toothache; fruits for the treatment of asthma, other respiratory disorders and scabies; stem bark is used in gonorrhea, bleeding, paralysis, diabetes, diarrhea, bone fracture, antiseptic, astringent, and antidote. [5] In Ayurveda, it is claimed that Ficus religiosa possesses anticonvulsant activity. [6] It also showed acetyl cholinesterase inhibitory activity [7] and antianxiety activity. [8] Fruits of this plant contain numerous amino acids whereas the fig of this plant has been reported to contain highest amount of serotonin [5-HT] as compared to figs of other Ficus species. [9]

Although its bark is extensively used in traditional medicine as analgesic and anti-inflammatory, [10] no scientific evaluation of leaves is available. A study has, therefore, been carried out to investigate the analgesic and anti-inflammatory activity of different fractions of extract of F. religiosa leaves.

Material and Methods

Collection and Authentication of Plant Materials

The leaves of F. religiosa were collected from local region of Nashik, India, in the month of July 2008. The plant material was identified and authenticated by Prof. P.G.Diwakar, Botanical survey of India, Pune and the Voucher No. BSI/WC/Tech/08/340.

Preparation of F. religiosa Fractions

The extraction was carried out using petroleum ether followed by alcohol, then it was allowed to evaporate slowly in shallow dish and resinous mass was discarded. For column chromatography, neutral alumina was first activated at 150 0 C for 3 hrs in an oven. After cooling, slurry was prepared in benzene; it was poured in glass column and set aside for 2 hrs. The residue of petroleum ether extract was dissolved in minimum volume of benzene and it was mixed thoroughly with neutral alumina. It was air dried and charged into the column. The elution was carried out first with benzene (FRI) and successively eluted with ethyl acetate (FRII). The dried alcoholic extract was separated into water soluble and water insoluble portion. Water soluble portion was shaken vigorously with methanol yielded a gelatinous precipitate (FRIII). The water insoluble part was dissolved in minimum volume of absolute alcohol and column chromatography was carried out with benzene (FRIV) and ethyl acetate (FRV).

All the fractions were prepared fresh prior to the administration in 0.5% w/v gum acacia.

Experimental Animals

Albino rats of Wistar strain (150-200 g) and Swiss albino mice (25-30 g) of either sex were used in the entire study. They were housed in standard polypropylene cages and kept under controlled room temperature (24 ± 2°C; relative humidity 60-70%) in a 12 h light-dark cycle. The animals were fed with standard laboratory diet and water ad libitum. Food was withdrawn 12 h before and during the experimental hours. The experimental protocol was approved by Institutional Animal Ethics Committee.

Phytochemical Investigation

Preliminary phytochemical tests for fractions were performed using specific reagents through standard procedure. [11]

Tail flick latency period in rats

Male rats of 125-150 g were divided into six groups containing five animals in each group. A tail flick response was evoked by placing each rat tail over the wire heated electrically, using analgesiometer (Space Scientific, Nashik, India). The intensity of heat was adjusted so that baseline tail flick latency averaged 3-4 sec in all animals. Cut off time was 15 sec in order to avoid injury to tail. The fractions FRI, FRIII, and reference standard ibuprofen were administered orally in their respective doses 1 hr prior to the test. [12]

Acetic acid induced writhing in mice

The writhing syndrome was elicited by intraperitoneal injection of acetic acid (0.1ml of 0.6% solution) and numbers of writhes displayed from 5 to 20 min were recorded. [13] The fractions FRI, FRIII, and reference standard ibuprofen were administered orally in their respective doses 30 min prior to the test.

Carrageenan induced rat paw edema

The anti-inflammatory activity of these fractions (FRI, FRII, FRIII, FRIV, and FRV) were assessed using carrageenan induced rat paw edema. The fractions were suspended in 0.5% w/v gum acacia and were administered in doses of 20 and 40 mg/kg, p.o, respectively 1 hr prior to carrageenan injection (0.1 ml of 1% solution). The paw volume was measured at an interval of 0, 1, 2, and 3 hrs using plethysmometer (UGO Basil, Italy, Model no.7130). [14]

Granuloma formation induced by cotton pellet in rats

Male rats of 125-150 g were divided into seven groups containing five animals in each group. The cotton pellet weighing 50±1 mg was sterilized in an autoclave (Lab hosp, Mumbai, India) handled with sterile instrument. The pellet was inserted in each animal on the back. Control group received vehicle. Group II, III, IV and V were treated with FRI (20 and 40 mg/kg, p.o) and FRIII (20 and 40 mg/kg, p.o) whereas group VI and VII were treated with reference standard i.e. hydrocortisone (30 mg/kg, p.o) and ibuprofen (40 mg/kg, p.o) for consecutive six days. [15],[16] The animals were sacrificed on seventh day and cotton pellet along with granuloma mass were collected; it was weighted and dried at 60 0 C. Results of the assay were calculated as % inhibition of dry weight of granuloma formation by using the formula: 100 (A-B)/A, where, A= gain in dry weight of control pellet (mg), B= gain in dry weight of drug treated (mg).

Statistical Analysis

Results have been indicated in terms of mean±SEM. Difference between the groups was statistically determined by analysis of variance (ANOVA) with Dunnett's multiple comparisons test using GraphPad InStat version 5.00, GraphPad Software, CA, USA. The level of significance was set at P < 0.05.

Results

Phytochemical investigation

Preliminary phytochemical analysis revealed the presence of different phytochemicals present in different fractions of F. religiosa plant [Table 1].{Table 1}

Effect of F. religiosa fractions on tail flick latency period and acetic acid induced writhing in mice

Treatment of FRI and FRIII (20 mg/kg, p.o.) significantly inhibited nociception in rats by 19.26% and 20.92%, respectively. Whereas, FR I and FRIII (40 mg/kg, p.o) significantly inhibited pain perception by 17.5% and 15.29%, respectively. Ibuprofen treatment (40 mg/kg, p.o) significantly inhibited pain perception by 27.92 % (P<0.01). [Figure 1]{Figure 1}

[Table 2] shows the effect of different fractions of F. religiosa against acid induced writhing in mice. It was observed that mice treated with FRI 20 (31.89%) and FRIII 20 (36.21%) shows significant (P < 0.01) protection compared to control group, however, FRI 40 (44.18%) and FRIII 40 (46.51%) was found to be more significant (P<0.01) in protecting acetic acid induced writhing compared to control group. Ibuprofen showed 56.14% protection against acetic acid induced writhing in mice.{Table 2}

Effect of F. religiosa fractions on carrageenan induced rat paw edema and cotton pellet granuloma formation in rats

Out of the different fractions (FRI, FRII, FRIII, FRIV and FRV) tested, FRI and FRIII of F. religiosa significantly (P<0.01, P<0.05, respectively) reduced the rat paw edema induced by carrageenan. FRI and FRIII (40 mg/kg, p.o) reduced inflammation by 43.12% and 48.12%, respectively. However, other fractions (FRII, FRIV and FRV) showed mild inhibition of paw edema induced by carrageenan during the three time points from 1 to 3 h [Table 3].{Table 3}

Treatment with FRI and FRIII (40 mg/kg, p.o) to rats showed a significant (P<0.01) inhibition in the weight of cotton pellet compared to control group and the percentage inhibition was found to be 38.75 and 40.31, respectively. Treatment with the reference standard i.e. hydrocortisone (30 mg/kg, p.o) and ibuprofen (40 mg/kg, p.o) also showed significant inhibition in cotton pellets granuloma formation as compared to control group [Table 4].{Table 4}

Discussion

In the present study, analgesic and anti-inflammatory effects of different fractions of F. religiosa were tested in different experimental models of pain and inflammation.

The animal models for assessing analgesic activity viz. non-narcotic model like acetic acid induced writhing and narcotic model like tail flick method were used. In acetic acid induced abdominal constriction acetic acid causes inflammatory pain by inducing capillary permeability and release of arachidonic acid via cyclooxygenase and prostaglandin biosynthesis which plays a role in the nociceptive mechanism. [17-19]

Results from the study revealed that the intensity of antinociception of fractions of F. religiosa treated groups was higher than the control group in acetic acid induced writhing in mice. The mechanism of analgesia by fractions of F. religiosa could probably be due to blockade of the effect or the release of endogenous substances that excite pain nerve endings similar to that of ibuprofen and other NSAIDs.

The most common test to test narcotic drugs is the tail flick method. This test is based on phasic stimulus of high intensity. The nociceptive experience is short lasting and it is well accepted that agonist of μ opoid receptors producing analgesia in acute pain models. [20] Therefore, it is believed that substances effective in tail flick exert their effects predominantly through μ opioid receptors. The fractions of F. religiosa showed increased latency for tail flick which suggest that the analgesic activity may in part be mediated by opoid receptors. Therefore, fractions of F. religiosa exhibited analgesic activity in all the animal models of nociception used in this study possibly exerted their effect through diverse mechanism that may involve both central and peripheral pathways.

It is well known that carrageenan induced paw edema is characterized by biphasic event with involvement of different inflammatory mediators. In the first phase (during the first 2 h after carrageenan injection), chemical mediators such as histamine and serotonin play role, while in second phase (after 3 h carrageenan injection), kinin and prostaglandins are involved. [21] Our results showed that administration of test fractions inhibited the edema starting from the first hour and during all phases of inflammation which is probably inhibition of different steps and chemical mediators of inflammation. In cotton pellet induced granuloma, the test fractions produced significant anti-inflammatory activity at the dose of 20 and 40 mg/kg which might be due to the presence of various active constituents in the leaves of F. religiosa. Phytochemical investigations on F. religiosa have revealed the presence of various phytoactive constituents such as glycosides, sterols, tannins, amino acids, campesterol, isofucosterol, stigmasterol, and lupeol. Leaf extract of F. religiosa had been shown to contain high amounts of tannins, phenols, triterpenoids, glucosides, and sterols. [22],[23],[24] Analgesic and anti-inflammatory effects of flavonoids, steroids, and tannins have been reported earlier. [25],[26] Deleterious effects of excessive releases of nitric oxide (NO) have been implicated in tissue damage and inflammation. Tannic acid and polyphenols have been shown to be potent inhibitors of NO synthetase activity and NO production independent of their antioxidant activity. [27] Therefore, the analgesic and anti-inflammatory properties observed might thus be related in part to the tannin content of this plant. Hence, the analgesic and anti-inflammatory effects produced by these fractions may be attributed individually or collectively to the tannins and steroids.

Conclusion

The results presented here demonstrate that F. religiosa fractions inhibit pain and inflammation with an interesting analgesic and anti-inflammatory activity profile similar to other compounds of this kind previously described. The results confirm that F. religiosa collected at Nashik, India, has great value as a source of tannins and polypenols compounds with analgesic and anti-inflammatory properties.

References

1	Bohlin L. Structure-activity studies of natural products with anti-inflammatory effects. In: Hostettmann K, editor. Phytochemistry of Plants Used in Traditional Medicine. Oxford: Clarendon Press; 1995. p. 137-61.
2	Yesilada E, Ustun O, Sezik E, Takaishi Y, Ono Y, Honda G. Inhibitory effects of Turkish folk remedies on inflammatory cytokines: interleukin-1, interleukin-1 and tumor necrosis factor. J Ethnopharmacol 1997;58:59-73.
3	Robert A, Hanchar AJ, Lancaster C, Nezamis JE. Prostacyclin inhibits enteropooling and diarrhea. In: Vane JR, Bergström S, editors. Prostacyclin. New York: Raven Press; 1979. p. 147-58.
4	Miller TA. Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms. Am J Physiol 1983;245:601-23.
5	Ripu MK, Rainer WB. Ficus (Fig) species in Nepal: A review of diversity and indigenous uses. Lyonia 2006;11:85-7.
6	Vyawahare NS, Khandelwal AR, Batra VR, Nikam AP. Herbal anticonvulsants. J Herbal Med Toxicol 2007;1:9-14.
7	Vinutha B, Prashanth D, Salma K, Sreeja SL, Pratiti D, Padmaja R, et al. Screening of selected Indian medicinal plants for acetylcholinesterase inhibitory activity. J Ethnopharmacol 2007;109:359-63.
8	Ratnasooriya WD, Jayakody JR, Dharmasiri MG. An aqueous extract of trunk bark of Ficus religiosa has anxiolytic activity. Med Sci Res 1998;26:817-9.
9	Bliebtrau JN. The Parable of the Beast. New York: Macmillan Company; 1968. p. 74.
10	The wealth of India: A dictionary of Indian Raw Materials and Industrial Products- Raw Materials series. Publication and Information Directorate. New Delhi: CSIR; 1956. p. 38-9
11	Khandelwal KR, Kokate CK, Pawar AP, Gokhle SR. Practical Pharmacognosy techniques and experiments. Pune: Nirali Prakshan; 1996. p. 9.
12	Davies OL, Raventos J, Walpole AL. Method for evaluation of analgesic activity using rats. British J Pharmacol 1946;1:255-64.
13	Koster R, Anderson M, De Beer EJ. Acetic acid for analgesic screening. Proc Soc Exp Biol 1959;18:412-5.
14	Winter CA, Risley EA, Nuss GW. Carrageenan induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol 1962;111:544-7.
15	WinderCV, Wax J, Scothi T, Scherrer RA, Short FW. Antiinflammatory, Antipyretic and Antiniciceptive properties of N-(2-3 xylyl) anthranilic acid. J Pharmacol Exp Thera 1962;138:405-13.
16	Swingle KF, Shideman FE. Phase of the inflammatory response to subcutaneous implantation of a cotton pellet and their modification by certain anti-inflammatory agents. J Pharmacol Exp Thera 1972;183:226-34.
17	Amico-Roxus M, Caruso A, Trombadore S, Scifo R, Scapagini U. Gangliosides antinoceptive effects in rodents. Arch Int Pharmacodyn Ther 1984;272:117.
18	Raj PP. Pain mechanism in pain medicine: A comprehensive review. In: Raj PP, editor. 1 ^st ed. Missouri: mosby-year book; 1996. p. 23.
19	Franzotti EM, Santos CV, Rodrigues HM, Mourao RH, Andrade MR, Antoniolli AR. Anti-inflammatory, analgesic activity and acute toxicity of Sida cordifolia L. (Malva- branca). J Ethnopharmacol 2000;72:273-7.
20	Shuanglin H, Osamu T, Hiroshi I. Intrathecal endomorphin- I produces antinociceptive modulated by alpha 2 adrenoceptors in rat tail flick, tail pressure and formalin test. Life Sci 2000;6:204.
21	Hernandez-Perez M, Rabanal Gallego R. Evaluation of the antiinflammatoryand analgesic activity of Sideritis canariensis var.pannosa in mice. J Ethnopharmacol 2002;81:43-7.
22	Mehrotra BN, Rastogi RD. Compendium of Indian Medicinal Plants. Central Drug Research Institute, Lucknow and National Institute of Science Communication and Information Resources, New Delhi; 2004. p. 188.
23	Nadkarni KM. Indian Materia Medica. 3 ^rd ed. Bombay: Popular Prakashan Private Limited; 2002. p. 552.
24	Swami KD, Bisht NP. Constituents of Ficus religiosa and F. infectoria and their biological activity. J Indian Chem Soc 1996;73:631.
25	Das PC, Das A, Mandal S. Antimicrobial and anti-inflammatory activities of the seed kernel of Mangifera indica. Fitoterapia 1989;60:235-40.
26	Ahmadiani A, Hosseiny J, Semnanian S, Javan M, Saeedi F, Kamalinejad M, et al. Antinociceptive and anti-inflammatory effects of Elaeagnus angustifolia fruitextract. J Ethnopharmacol 2000;72:287-92.
27	Srivastava RC, Hussain MM, Hassan SK, Athar M. Green tea polyphenols and tannic acid act as potent inhibitors of phorbol esters-induced nitric oxide generation in rat hepatocytes independent of their antioxidant properties. Cancer Lett 2000;153:1-5.