International Journal of Drug Development and Research

References

1. 1) Mitchell RN and Cotran RS. Robinsons Basic Pathology. ed7. Harcourt Pvt. Ltd: New Delhi, India, 2000.
2. 2) Park JH, Son KH, Kim SW, Chang HW, Bae K, Kang SS, et al. Anti-inflammatory activity of Synurus deltoids. Phytother Res 2004; 18: 930–3.
3. 3) Rang HP, Dale MM, Ritter JM and Flower RJ. Antiinflammatory and immunosuppressant drugs.Textbook of Pharmacology.ed6 ed. Elsevier publications, 2008. pp. 226-45.
4. 4) Li RW, Mayers SP, Leach DN, Lin GD, Leach G. A cross cultural study: Anti-inflammatory activity of Australian and Chinese plants. J Ethanopharmacol 2003; 85:25–32.
5. 5) Dharmasiri JR, Jayakody AC, Galhena G, Liyanage SSP, Rathasooriya WD. Anti-inflammatory and analgesic activity of mature fresh leaves of Vitexnegundo. J of Ethanopharmacol 2003; 87:199-206.
6. 6) Kumara NKVMR. Identification of strategies to improve research on medicinal plants used in Srilanka. 2001, pp 12-14.
7. 7) Duffy JC, Dearden JC, Rostron C. Design, synthesis and biological testing of a novel series of antiinflammatory drugs. J Pharm Pharmacol 2001;53: 1505–14.
8. 8) Nishanta R, Cory SH, Towers GHN. Antimicrobial Activity of Plants Collected from Serpentine Outcrops in Sri Lanka, Pharm Biol 2002;40: 235– 44.
9. 9) J. Raamachandran. HERBS OF SIDDHA MEDICINES-The First 3D Book on Herbs, 2002, pp.4.
10. 10) Parekh J, Karathia N, Chanda S. Screening of some traditionally used medicinal plants for potential antibacterial activity. Indian J of PharmaSci 2006; 68:832-34.
11. 11) Abdul Rahuman A, Gopalakrishnan G, Venkatesan P and Geetha K. Isolation and identification of mosquito larvicidal compound from Abutilon indicum (Linn.) Sweet. Parasitology Res 2008; 102: 867-73.
12. 12) Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non inflammatory pain. Pain1987; 30 (1): 103-14.
13. 13) Turner RA. Screening methods in pharmacology. New York, Academic Press, 1965, pp. 100.
14. 14) Winter CA, Risley EA, Nuss GW. Carrageenan induced edema in hind paw of the rats as an assay for ant-inflammatory drugs. ProcSocExpboil Med 1962; 111: 544-47.
15. 15) Bhutia DY, Vijayraghavan R, Pathak U. Analgesic and anti-inflammatory activity of amifostine, DRDE-07 and their analogs in mice. Indian J pharmacol 2010; 42:17-20.
16. 16) Sawadogo WR, Boly R, Lompo M, Some N, Lamien CE, Guissou IP, et al. Anti-inflammatory, analgesic and antipyretic activities of Diclipteraverticillata. Int J Pharmacol 2006; 2:435–8.
17. 17) Vinegar R, Schreiber WR. Biphasic development of carrageenan edema in rats. J PharmacolExpTher 1969;166: 96–103.
18. 18) Abbott FV, Franklin KB, Westbrook RF. The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 1995; 60(1):91–102.
19. 19) Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain1992; 51: 5–17.
20. 20) Shibate M, Ohkubo T, Takashi H, Inoki R. Modified formalin test. Pain 1989; 38:345-52.
21. 21) Correa CR, Calixto JB. Evidence for participation of B1 and B2 kinin receptors in formalin induced nociceptive response in the mouse. Br J Pharmacol 1993; 110:193-8.
22. 22) Coderre TJ, Melzack R. The contribution of excitatory amino acids to central sensitization and persistent nociception after formaln induced tissue injury. J Neuroscie 1992; 12: 3665-70.
23. 23) Damas J, Liegeois JF. The inflammatory reaction induced by formalin in the rat paw. NaunynSchneidebergs Arch Pharmacol 1999; 359:200.

Key words

Abutilon indicum, pain, inflammation, Paw edema

Introduction

Inflammation is a local response of living mammalian tissues to injury. It is a body defence reaction in order to eliminate or limit the spread of injurious agent. There are various components of an inflammatory reaction that can contribute to the associated symptoms and tissue injury. Edema formation, leukocyte infiltration and granuloma formation represent such components of inflammation [1]. The drugs which are used presently for the management of pain and inflammatory conditions are either narcotics or non narcotics (NSAIDS), and have known toxic and lethal effects [2]. About 34-46% of the users of NSAIDs usually sustain some gastrointestinal damage due to the inhibition of the protective cyclo-oxygenase enzyme in gastric mucosa [3]. On the contrary, herbal medicines with good absorption, less toxicity, and easy availability have been used since ancient times [4]. According to WHO about 80% of the world population still rely mainly on herbal remedies [5-6]. The plant kingdom provides enormous source of pharmacologically active molecules for new drug discovery. Many medicines of plant origin have been used since long time to treat various disorders. It is therefore, essential that efforts be made to introduce new medicinal plants, to develop cheaper, effective and safe analgesic and anti-inflammatory drugs [7].

Abutilon indicum (AI), commonly known as Atibala is a small shrub in the Malvaceae family. In traditional medicine, A. indicum is reported to be used to treat ulcers, headaches, gonorrhea, bladder infection, inflammation and hepatic and pulmonary disorders. It is also used as demulcent, aphrodisiac, laxative, diuretic and sedative (leaves). The bark is astringent and diuretic; laxative, expectorant and demulcent (seeds); anti-inflammatory and anthelmintic (plant); analgesic (fixed oil); diuretic and for leprosy (roots) [8]. The leaves can also be used to treat ulcers, headaches, gonorrhea & bladder infection [9].The plant is very much used in Siddha medicines. In fact the root, bark, flowers, leaves and seeds are all used for medicinal purposes by Tamils. The leaves are used as adjunct to medicines used for pile complaints. The flowers are used to increase semen in men Dr.J.Raamachandran, “Herbs of Siddha Medicines” [10].

A methanol extract of A. indicum had some antimicrobial properties. A chemical compound, β- sitosterol, which has been identified as the active ingredient in many medicinal plants, is present in A. indicum and a petroleum ether extract provided larvicidal properties against the mosquito larvae Culex quinquefasciatus [11].Hence the present study was undertaken to evaluate the therapeutic efficacy of Abutilon indicum in the treatment of peptic ulcer.

Materials and methods

Animals

Male / Female Albino rats of Wistar strain weighing 120–200gm obtained from the Department of Pharmacy, MJRP, were used for the experiment. The animals were housed in cages under standard lab conditions (12:12 hr light / dark cycles at 25±2?C, RH55±10%). They had free access to standard pellet diet and water ad libitum. The animals were acclimatized at least one week prior to experiment. All experiments were approved by Institutional Animal Ethics Committee (1422/PO/A/11/CPCSEA/PN01).

Plant Materials and Extracts

The whole plant of Abutilon indicum was obtained from Morena, MP. The authenticated plant materials were shade dried and powered coarsely. The coarsely powdered drugs were extracted separately in soxhlet apparatus in sufficient volume of redistilled water and ethanol for 18 hrs. The filtrates were collected and evaporated to dryness on rotary evaporator. The solid masses were collected carefully and weighed. Their yields were calculated and then stored in sealed (air tight) glass bottles at 4?C for further experimental work.

Treatment protocol

The animals were divided into four groups of six animals each. Group I served as control and received the suspension of 1% CMC in distilled water. Group II received the reference drug diclofenac sodium 5mg/kg. Group III and IV received 200 mg/kg of ethanolic and aqueous extracts of AI by oral route (p.o.). All the test drugs were suspended in1% CMC and were administered in a volume of 5 ml/kg body weight.

Formalin induced paw licking model

The formalin test comprises the early and late phase assessment of the analgesic effect as described by Hunskaar & Hole [12]. One hour after drug administration by the oral route, 20 μl of 1% formalin was injected subcutaneously sub-plantar in the right hind paw. Then the duration of paw licking as an index of nociception was recorded in periods of 0 to 5 minutes (early phase) and 15 to 30 minutes (late phase) after formalin injection.

Tail flick model

In this model Nichrome wire analgesiometer was used [13]. Individually the tail of each rat was placed over the radiant heat source of the apparatus and the tail withdrawal from the heat (flicking response) was taken as the end point. Analgesic activity was assessed by observing the reaction time in the treated groups. Following the administration of drugs, the reaction time was noted at 0, 30, 60, 90 and 120 min. A cut of time of 15 seconds was considered to avoid tissue injury.

Carrageenan induced paw edema

This test was performed as per the method described by Winter et al [14]. The animals were fasted for 16 hours but water was allowed ad libitum [15]. Acute edema in left hind paws of the rats was induced by the sub plantar injection of 0.1 ml of freshly prepared (1% w/v) carrageenan suspension in normal saline 1h after the drug administration. The paw volume was measured before and 1, 2, 3 and 4 h after the carrageenan injection. The results were expressed as mean ± SEM. The statistical analysis of data was done by the Student's t-test and one-way Analysis of Variance (ANOVA), followed by Dunnet’s test. The results were considered statistically significant at P < 0.05.

Results

Formalin induced paw licking model

The formalin induced paw licking model was used to study the analgesic effects during early and late phase (Table 1). The administration of standard drug diclofenac sodium significantly (P<0.01) inhibited the licking response. The ethanolic extract of Abutilon indicum significantly (P<0.01) inhibited the licking response during the early phase. In the late phase of formalin test, the administration of standard drug highly significantly (P<0.001) inhibited the paw licking response as compared to the control group. The ethanolic and aqueous extracts of Abutilon indicum significantly (P<0.01 and P<0.05 respectively) suppressed the paw licking in the late phase as compared to control group.

Tail flick model

In the tail flick test the ethanolic extract of Abutilon indicum produced a reaction time of 4.89±0.29, 6.65±0.50, 6.96±0.55, 6.96±0.45 seconds after 30, 60, 90 and 120 minutes respectively (Table 2). The reaction time for the same period with control group was found to be 4.04±0.26, 4.02±0.34, 4.42±0.32 and 3.89±0.34 seconds respectively. The reaction time for diclofenac sodium treated group was found to be 4.97±0.35, 8.11±0.65, 7.19±0.50, 9.33±0.58 seconds after 30, 60, 90 and 120 minutes respectively. The reaction time with aqueous extract of Abutilon indicum for the same period was found to be 4.66±0.36, 6.25±0.52, 6.26±0.44 and 6.49±0.44 seconds respectively. The above results show that the increase in reaction time in ethanolic and aqueous extracts treated group was found to be significant after 60, 90 and 120 min ( P <0.01, P <0.01 and P <0.001 respectively ) as compared to control group. The increase in reaction time in standard drug treated group was found to be highly significant (P <0.001) after 60, 90 and 120 minutes. The increase in reaction time was highest in standard drug treated group and was seen until the last phase observation (120 minutes).

Carrageenan induced paw edema

Development of paw edema was observed in both control and treated groups after carrageenan injection. Thickness of the paw was found to be increased initially upon injection of carrageenan due to volume effect. Difference in the thickness of the rat paw edema was further increased during the time interval of 1h – 4h in control group. When compared with control, the treatment with standard drug significantly suppressed the edema at 2h (P<0.01); and at 3 and 4h (P<0.001). When compared with ethanolic and aqueous extract of AI, the anti- inflammatory activity with the standard drug treatment was more powerful and pronounced. When compared with aqueous extract, the ethanolic extract produced more powerful and pronounced anti-inflammatory activity. The ethanolic extract of AI, significantly reduced the paw edema at 2h (P<0.05), 3h (P<0.01) and 4h (P<0.05). The aqueous extract of AI produced significant suppression (P<0.05) of edema at 3h.

Discussion

In the present investigation anti-inflammatory activity of ethanolic and aqueous extracts of Abutilon Indicum was studied by using inhibition of carrageenan induced inflammation which is one of the most feasible methods to screen antiinflammatory agents. Carrageenan-induced inflammation is a useful experimental model of acute inflammation for detecting orally active antiinflammatory agents [16]. Edema formation in the rat paw is a biphasic response. The first phase is mediated through the release of histamine, serotonin, and kinins, whereas the second phase is due to the release of prostaglandin and slow reacting substances [17].The ethanolic and aqueous extract of Abutilon indicum significantly reduced the paw edema. In this experiment the suppression of inflammation may be due to PG and kinin synthesis/release inhibition and antihistamine activities. The maximum inflammation is seen approximately three hours post the carrageenan injection, after which it begins to decline.

The formalin model was developed >30 years ago to assess pain and evaluate analgesic drugs in laboratory animals. In this test, a dilute (0.5–5%) formalin solution (in which formaldehyde is the active ingredient) is injected into the paw of a rodent, and pain-related behaviors are assessed over two temporally distinct phases [18-19]. In the present study the analgesic activity of ethanolic and aqueous extracts of Abutilon Indicum was evaluated by formalin induced paw licking and the tail flick model. The persistent pain model of formalin induced hind paw licking was used in the study. The first phase of pain is attributed to the direct activation of nociceptors and primary afferent fibers by formalin, causing the release of bradykinin and trachykinins [20-21]. This phase is inhibited by opioid analgesics. The second phase is due to an inflammatory reaction caused by tissue injury leading to the release of histamine, serotonin, prostaglandin and excitatory amino acids [22-23]. This late phase is inhibited by non-steroidal anti-inflammatory drugs and opioid analgesics

The tail flick model is used to evaluate the analgesic agents acting through central nervous system. In this method a nichrome wire is used to produce pain. In the present study ethanolic and aqueous extracts of Abutilon indicum significantly inhibited the paw licking in the late phase. In the tail flick test the ethanolic extract of AI significantly increased the reaction time. The results of analgesic and antiinflammatory activities of ethanolic extracts of Abutilon indicum are comparable with standard drug diclofenac sodium.

From the above findings it can be concluded that the ethanolic extract of Abutilon indicum possesses promising analgesic and anti-inflammatory activities. The presence of glycosides, phenolic compounds, carbohydrates, sterols and flavonoids in the ethanolic extract of the plant under study may be responsible for these activities. Further pharmacodynamic investigations are required to understand the precise mechanism of action of Abutilon indicum

Acknowledgement

The authors are grateful to MJRP University, Jaipur and Sunder Deep Pharmacy College, Ghaziabad for the facilities provided to conduct the present study.

Tables at a glance

Table 1 Table 2 Table 3

References

1. 1) Mitchell RN and Cotran RS. Robinsons Basic Pathology. ed7. Harcourt Pvt. Ltd: New Delhi, India, 2000.
2. 2) Park JH, Son KH, Kim SW, Chang HW, Bae K, Kang SS, et al. Anti-inflammatory activity of Synurus deltoids. Phytother Res 2004; 18: 930–3.
3. 3) Rang HP, Dale MM, Ritter JM and Flower RJ. Antiinflammatory and immunosuppressant drugs.Textbook of Pharmacology.ed6 ed. Elsevier publications, 2008. pp. 226-45.
4. 4) Li RW, Mayers SP, Leach DN, Lin GD, Leach G. A cross cultural study: Anti-inflammatory activity of Australian and Chinese plants. J Ethanopharmacol 2003; 85:25–32.
5. 5) Dharmasiri JR, Jayakody AC, Galhena G, Liyanage SSP, Rathasooriya WD. Anti-inflammatory and analgesic activity of mature fresh leaves of Vitexnegundo. J of Ethanopharmacol 2003; 87:199-206.
6. 6) Kumara NKVMR. Identification of strategies to improve research on medicinal plants used in Srilanka. 2001, pp 12-14.
7. 7) Duffy JC, Dearden JC, Rostron C. Design, synthesis and biological testing of a novel series of antiinflammatory drugs. J Pharm Pharmacol 2001;53: 1505–14.
8. 8) Nishanta R, Cory SH, Towers GHN. Antimicrobial Activity of Plants Collected from Serpentine Outcrops in Sri Lanka, Pharm Biol 2002;40: 235– 44.
9. 9) J. Raamachandran. HERBS OF SIDDHA MEDICINES-The First 3D Book on Herbs, 2002, pp.4.
10. 10) Parekh J, Karathia N, Chanda S. Screening of some traditionally used medicinal plants for potential antibacterial activity. Indian J of PharmaSci 2006; 68:832-34.
11. 11) Abdul Rahuman A, Gopalakrishnan G, Venkatesan P and Geetha K. Isolation and identification of mosquito larvicidal compound from Abutilon indicum (Linn.) Sweet. Parasitology Res 2008; 102: 867-73.
12. 12) Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non inflammatory pain. Pain1987; 30 (1): 103-14.
13. 13) Turner RA. Screening methods in pharmacology. New York, Academic Press, 1965, pp. 100.
14. 14) Winter CA, Risley EA, Nuss GW. Carrageenan induced edema in hind paw of the rats as an assay for ant-inflammatory drugs. ProcSocExpboil Med 1962; 111: 544-47.
15. 15) Bhutia DY, Vijayraghavan R, Pathak U. Analgesic and anti-inflammatory activity of amifostine, DRDE-07 and their analogs in mice. Indian J pharmacol 2010; 42:17-20.
16. 16) Sawadogo WR, Boly R, Lompo M, Some N, Lamien CE, Guissou IP, et al. Anti-inflammatory, analgesic and antipyretic activities of Diclipteraverticillata. Int J Pharmacol 2006; 2:435–8.
17. 17) Vinegar R, Schreiber WR. Biphasic development of carrageenan edema in rats. J PharmacolExpTher 1969;166: 96–103.
18. 18) Abbott FV, Franklin KB, Westbrook RF. The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 1995; 60(1):91–102.
19. 19) Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain1992; 51: 5–17.
20. 20) Shibate M, Ohkubo T, Takashi H, Inoki R. Modified formalin test. Pain 1989; 38:345-52.
21. 21) Correa CR, Calixto JB. Evidence for participation of B1 and B2 kinin receptors in formalin induced nociceptive response in the mouse. Br J Pharmacol 1993; 110:193-8.
22. 22) Coderre TJ, Melzack R. The contribution of excitatory amino acids to central sensitization and persistent nociception after formaln induced tissue injury. J Neuroscie 1992; 12: 3665-70.
23. 23) Damas J, Liegeois JF. The inflammatory reaction induced by formalin in the rat paw. NaunynSchneidebergs Arch Pharmacol 1999; 359:200.