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Anti-inflammatory activity of Albizia lebbeck Benth., an ethnomedicinal plant, in acute and chronic animal models of inflammation
Journal of Ethnopharmacology 125 (2009) 356–360
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Anti-inflammatory activity of Albizia lebbeck Benth., an ethnomedicinal plant, in acute and chronic animal models of inflammation N. Prakash Babu, P. Pandikumar, S. Ignacimuthu ∗ Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Nungambakkam, Chennai 600 034, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 19 June 2007 Received in revised form 26 January 2009 Accepted 23 February 2009 Available online 9 March 2009 Keywords: Albizia lebbeck Anti-inflammatory activity Carrageenan Dextran Cotton pellet FCA
a b s t r a c t Aim of the study: Albizia lebbeck Benth. is used both in Indian traditional system and folk medicine to treat several inflammatory pathologies such as asthma, arthritis and burns. The aim of the present study was to evaluate the scientific basis of anti-inflammatory activity of different organic solvent extracts of Albizia lebbeck. Materials and methods: The anti-inflammatory activity of Albizia lebbeck was studied using the carrageenan, dextran, cotton pellet and Freund’s complete adjuvant induced rat models. The extracts obtained using petroleum ether, chloroform and ethanol were administered at the concentrations of 100, 200 and 400 mg/kg body weight. Results: The petroleum ether and ethanol extracts at 400 mg/kg, showed maximum inhibition of inflammation induced by carrageenan (petroleum ether—48.6%; ethanol—59.57%), dextran (petroleum ether—45.99%; ethanol—52.93%), cotton pellet (petroleum ether—34.46%; ethanol—53.57%) and Freund’s adjuvant (petroleum ether—64.97%; ethanol—68.57%). Conclusion: The marked inhibitory effect on paw edema shows that Albizia lebbeck possesses remarkable anti-inflammatory activity, supporting the folkloric usage of the plant to treat various inflammatory diseases. © 2009 Published by Elsevier Ireland Ltd.
1. Introduction Inflammation is a complex pathophysiological process mediated by a variety of signaling molecules produced by leucocytes, macrophages and mast cells as well as by the activation of complement factors that bring about edema formation as a result of extravasation of fluid and proteins and accumulation of leucocytes at the inflammatory site (White, 1999). All the steroidal and nonsteroidal anti-inflammatory drugs (NSAID’s), despite their great number, cause undesired and serious side effects. Therefore, development of new and more powerful drugs is still needed. Research on plants with medicinal properties and identification of the chemical components responsible for their activities have corroborated the traditional uses of ancient healing wisdom and lore and have proven the enduring healing potential of many plant medicines even in today’s hi-tech community. Albizia lebbeck Benth. (Mimosaceae), commonly known as Sirisa in Sanskrit, is a tall, unarmed, and deciduous tree distributed throughout India from the plains up to 900 m in the Himalayas. In Siddha system of medicine the bark and flowers of this plant are used to treat arthritis (Mudaliar, 1936). The tribal people in
∗ Corresponding author. Tel.: +91 44 2817 8348; fax: +91 44 2817 5566. E-mail address: [email protected] (S. Ignacimuthu). 0378-8741/$ – see front matter © 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2009.02.041
Himachal Pradesh and Kashmir use this plant to treat inflammation (Srivastava et al., 1986; Jain, 1991; Kapur, 1993). Balasubramaniam (1992) reported that the tribals of point Calimere wild life sanctuary, Tamilnadu use this plant to treat bone fractures. In ayurvedic system of medicine, the stem bark of this plant is used to treat diarrhoea (Nadkarni, 1954), edema, poisoning, asthma and bronchitis (Gupta, 2004). It was previously reported that the alcoholic extract of Albizia lebbeck protects the guinea pig against the antigen induced challenge (Tripathi et al., 1977; Barua et al., 1997). Further it also reduced the level of histamine and raised the plasma cortisol in antigen challenged guinea pigs (Tripathi and Shukla, 1979) as well as in bronchial asthma patients (Tripathi et al., 1978). Das et al. (2003) and Pramanik et al. (2005) previously reported the anti-inflammatory activity of the methanol extract of Albizia lebbeck bark. Many saponins such as Lebbekanin A–H (Varshney and Khan, 1961; Varshney and Sharma, 1969; Varshney et al., 1973, 1976) and Albizziasaponin A–C (Pal et al., 1995), which contain oleanolic acid, echinocystic acid or acacic acid as sapogenins were reported from various parts of this plant. Further, melanoxetin, okanin, (+) pinitol, (−)-leucopelargonidin (Gupta et al., 1966), friedelan-3-one, ␥-sitosterol and macrocyclic spermine alkaloids namely budmunchiamines L1–L6 (Misra et al., 1995; Dixit and Misra, 1997) were also isolated from this plant. The anti-inflammatory agents exert their effect through a spectrum of different modes of action (Martelli, 1977). Carrageenan
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induced edema in the hind paw (acute inflammation) and Cotton pellet granuloma (chronic inflammation) are widely employed in screening the new anti-inflammatory compound (Billingham and Davis, 1979). Adjuvant induced arthritis is also an often used model of inflammation (Newbold, 1963). This study investigated the anti-inflammatory effect of various solvent extracts of Albizia lebbeck on acute and chronic phases of inflammation. The anti-inflammatory effect of the extracts in adjuvant induced arthritis was also studied. 2. Materials and methods 2.1. Plant material The bark of Albizia lebbeck was collected in August 2005 from Kancheepuram District of Tamil Nadu. The botanical identity of the plant material was confirmed with the help of taxonomist, Department of Botany, Loyola College, Chennai. The voucher specimens were deposited in the herbarium of Entomology Research Institute, Loyola College, Chennai. 2.1.1. Preparation of the extracts The barks were chopped into small pieces, air-dried and powdered. The powdered plant material (1 kg) was sequentially extracted three times with 3 L of petroleum ether, chloroform, and 95% ethanol at room temperature for 48 h. The filtrates were concentrated under reduced pressure at 40 ◦ C and stored in a refrigerator. The percentage of yield of the petroleum ether, chloroform and ethanol extracts was 0.33%, 1.10% and 13.70% respectively. 2.1.2. Preliminary phytochemical screening of the extracts The preliminary phytochemical tests were carried out for the effective extracts using standard phytochemical methods (Harborne, 1998). 2.1.3. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the Albizia lebbeck crude extract was performed using a SHIMADZU GC–MS (GC-2010) equipped with a DB-5 MS fused silica capillary column. The column constant flow rate was maintained at 1.34 mL/min. Injector temperature was set at 200 ◦ C. The oven temperature was programmed from 60 to 150 ◦ C at 3 ◦ C/min, then held isothermal for 10 min and finally raised to 230 ◦ C. The relative percentage of the volatile constituents was expressed as percentages by peak area normalization. Identification of components of the crude extract was based on GC retention time on DB-5 capillary column, computer matching of mass spectra with those of standards (Wiley 6.0 data of GC–MS system) 2.1.4. Drug administration All the extracts were dissolved in 2% Tween-80 in water. The vehicle alone served as control. Indomethacin (10 mg/kg body weight), served as positive control (Arunachalam et al., 2002). 2.2. Animals Female Wistar rats weighing 130–150 g were procured from King Institute, Chennai. All animals were housed in polypropylene cages in a temperature-controlled room at 24 ± 1 ◦ C. The animals were fed with pelleted rat feed manufactured by TANUVAS with free access to water throughout the experiment. The rats were acclimatized at least one week before starting the experiments. In all experimental models of inflammation the studies were carried out using six rats in each group. This study got clearance from the Institutional Animal Ethical Committee (IAEC).
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2.2.1. Carrageenan induced rat paw edema The acute anti-inflammatory effect was evaluated by carrageenan induced rat paw edema according to the method of Winter et al. (1962). Edema was induced by injection of 1% suspension of carrageenan in 0.9% sterile saline solution into the right plantar region of the rat. The plant extract (100, 200 or 400 mg/kg), Indomethacin (10 mg/kg body weight), or vehicle was administrated orally 1 h before injection of carrageenan. Paw volume was measured with Digital vernier caliper 0, 3rd and 5th hour after injection (Ramprasath et al., 2004). The inhibitory activity was calculated using the following formula. Percentage inhibition =
(Ct − Co) control − (Ct − Co) treated × 100 (Ct − Co) control
2.2.2. Dextran induced rat paw edema The paw edema was induced in the right hind paw by subplantar injection of 0.1 mL of freshly prepared 1% dextran solution. Paw thickness was measured at 0, 45, and 90 min after dextran injection. The rats were treated as above. The percentage of inhibition was calculated. 2.2.3. Cotton pellet induced Granuloma Eleven groups of six rats in each group were included in the study. After shaving off fur, the animals were anaesthetized. Sterile pre-weighed cotton pellets (10 mg) were implanted in the axilla region of each rat through a single needle incision. Plant extract (100, 200 or 400 mg/kg body wt), Indomethacin (10 mg/kg body weight) or vehicle alone was administrated orally for seven consecutive days, from the day of cotton pellet implantation. On the eighth day the animals were anaesthetized, the cotton pellets were removed and made free from extraneous tissues. The pellets were dried at 60 ◦ C for 24 h. Mean weight of granuloma tissue formed around each pellet was evaluated (Winter and Porter, 1957). 2.2.4. Adjuvant induced arthritis Chronic arthritis was induced by the injection of 0.1 mL of Freund’s Complete Adjuvant containing 10 mg of heat killed Mycobacterium tuberculosis, in 1 mL of paraffin oil into the right hind paw of the rat intradermally. Treatment was given orally after 14 days from the day of adjuvant injection for 14 days. The volume of the paw was measured before induction, before treatment, and after treatment and the percentage inhibition was determined. 2.3. Statistical analysis The data obtained were analysed using one-way ANOVA followed by Student’s t-test. Differences were considered significant at the 5% level. 3. Results 3.1. Preliminary phytochemical analysis The petroleum ether extract of Albizia lebbeck had shown positive results for steroids, terpenoids and coumarins. The ethanol extract showed the presence of tannins, flavanoids, anthraquinones, and saponins. 3.2. GC–MS analysis of crude extracts The crude extract of Albizia lebbeck was analysed using GC–MS and the petroleum ether extract had the following major composition: 1-tridecene (2.81%), 1-dodecanol (2.94%), 1ethyldibenzothiophene (2.07%), bis-(3, 5, 5-trimethylhexyl) ether
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Table 1 Effect of Albizia lebbeck stem bark on carrageenan induced paw edemaa .
Table 2 Effect of Albizia lebbeck stem bark on dextran induced paw edemaa .
Groups
Groups
Dose (mg/kg body weight)
Thickness of the injected foot (mm) 3rd hour
5th hour
Control
5.16 ± 0.10
5.03 ± 0.08
Petroleum ether
100 200 400
4.70 ± 0.10 (23.70) 4.25 ± 0.11** (46.54) 4.14 ± 0.04** (48.66)
Chloroform
100 200 400
Ethanol
Indomethacin
Dose (mg/kg body weight)
Thickness of the injected foot (mm) 45 min
90 min
Control
6.05 ± 0.21
5.73 ± 0.07
4.52 ± 0.03 (27.54) 4.14 ± 0.08** (47.17) 4.03 ± 0.03** (50.82)
Petroleum ether
100 200 400
4.92 ± 0.17 (36.71) 4.77 ± 0.11** (39.81) 4.76 ± 0.08** (41.08)
4.61 ± 0.14** (40.45) 4.52 ± 0.04** (41.89) 4.43 ± 0.26** (45.99)
5.11 ± 0.04 (05.01) 5.03 ± 0.08 (09.24) 4.77 ± 0.09* (20.82)
4.93 ± 0.03 (07.44) 4.83 ± 0.06 (13.35) 4.58 ± 0.13* (25.05)
Chloroform
100 200 400
5.75 ± 0.12 (09.91) 5.67 ± 0.16 (12.49) 5.49 ± 0.13* (17.77)
5.43 ± 0.14 (10.92) 5.27 ± 0.13* (16.47) 5.10 ± 0.08** (22.28)
100 200 400
5.00 ± 0.04 (10.35) 4.50 ± 0.05** (31.62) 3.90 ± 0.05** (59.57)
4.82 ± 0.06** (13.23) 4.22 ± 0.08** (40.89) 3.83 ± 0.04** (60.04)
Ethanol
100 200 400
5.49 ± 0.06* (17.35) 5.10 ± 0.18** (24.12) 4.59 ± 0.03** (45.97)
5.18 ± 0.12* (18.93) 4.84 ± 0.09** (30.00) 4.22 ± 0.11** (52.93)
10
3.69 ± 0.10** (69.53)
3.62 ± 0.10** (70.87)
Indomethacin
10
4.39 ± 0.03** (53.16)
4.08 ± 0.03** (58.60)
*
**
Values given in parentheses represent percentage of inhibition. a Each value is the mean ± S.E.M. of six rats. * p < 0.05, compared with control rats; Student’s t-test. ** p < 0.005, compared with control rats; Student’s t-test.
(2.49%), n-hexadecanoic acid (24.24%), methyl 2L, 9d-dimethylteracosanoate (5.08%), 9,12-octadecadienoic acid (Z,Z)-(10.04%), oleic acid (19.30%), 1,13-tetradecadiene (6.59%), 3,3-difluro-1tetradecen-4-ol (2.20%), 1-tridecyn-4-ol (6.34%). The retention time of the above compounds was: 10.933, 13.441, 15.035, 15.696, 17.391, 17.737, 19.209, 19.266, 19.661, 21.402, and 27.581, respectively.
3.3. Carrageenan induced inflammation In carrageenan induced animal models, the petroleum ether extract of Albizia lebbeck at concentrations 100, 200, and 400 mg, inhibited the edema formation in third hour by 23.7% (p < 0.05), 46.5%, and 48.6% (p < 0.005) in a dose-dependent manner respectively. This effect also extended and significantly increased up to the fifth hour (p < 0.005). The ethanol extract at 100, 200, and 400 mg showed 10.35%, 31.62%, and 59.57% of inhibition at third hour and 13.23%, 40.89%, and 60.04% (p < 0.005) of inhibition at fifth hour respectively. The chloroform extract showed a mild activity in higher concentration only. The reference drug group significantly inhibited the edema formation by 69.53% and 70.87% at third and fifth hour respectively (p < 0.005). Results are shown in Table 1.
3.4. Dextran induced inflammation In dextran induced inflammatory model, the petroleum ether extract at 45 min inhibited the paw edema by 36.71%, 39.81%, and 41.08% (p < 0.005) of inhibition at the concentration of 100, 200, and 400 mg respectively. At 90 min, there was increase in the percentage of inhibition by 40.44%, 41.89% and 45.99%, respectively. Likewise, 100, 200, and 400 mg of ethanol extract treated groups showed 17.35% (p < 0.05), 24.12%, and 45.97% (p < 0.005) inhibition at 45 min and 18.93%, 30.00%, and 52.93% inhibition at 90 min respectively. Results were shown in Table 2.
3.5. Cotton pellet induced granuloma In cotton pellet granuloma, the petroleum ether extract inhibited the granuloma formation by 23.53% (p < 0.05), 28.26%, and 34.46% (p < 0.005) at 100, 200 and 400 mg, respectively (Table 3). The ethanol extract at 100, 200, 400 mg inhibited the granuloma formation by 27.17% (p < 0.05), 38.68%, and 53.57% (p < 0.005) respectively.
**
Percentage of inhibition is mentioned in parentheses. a Each value is the mean ± S.E.M. of six rats. * p < 0.05, compared with control rats; Student’s t-test. ** p < 0.005, compared with control rats; Student’s t-test.
Table 3 Effect of Albizia lebbeck stem bark on cotton pellet induced granulomaa . Groups
Dose (mg/kg body weight)
Dry weight of granuloma (mg)
Petroleum ether
100 200 400
105.05 ± 2.55* (23.53) 098.55 ± 1.43** (28.26) 090.03 ± 1.18** (34.46)
Chloroform
100 200 400
135.67 ± 4.83 (01.23) 124.75 ± 5.29 (09.19) 104.22 ± 2.96* (24.13)
Ethanol
100 200 400
100.05 ± 3.10* (27.17) 084.22 ± 4.06** (38.68) 63.77 ± 3.73** (53.57)
10
054.65 ± 3.64** (60.21)
137.37 ± 9.47
Control
Indomethacin
Percentage of inhibition is mentioned in parentheses. a Each value is the mean ± S.E.M. of six rats. * p < 0.05, compared with control rats; Student’s t-test. ** p < 0.005, compared with control rats; Student’s t-test.
3.6. Adjuvant induced arthritis In Adjuvant induced animals, a dose-dependent reduction in foot thickness was exhibited in petroleum ether and ethanol extracts of Albizia lebbeck. At a dose of 400 mg, arthritic swelling was inhibited by 64.97% and 68.57% (p < 0.005) for petroleum ether and ethanol extract treated groups compared to the adjuvant control respectively, where as the Indomethacin treated group showed an inhibition of 73.29% (Table 4). 4. Discussion Alternative medicine for the treatment of various diseases is getting more popular. Many medicinal plants provide relief of symptoms comparable to that of conventional medicinal agents (Verpoorte, 1999). Therefore, the present study was aimed at evaluating the scientific basis for the traditional use of Albizia lebbeck using in vivo inflammatory models. Carrageenan induced rat hind paw edema has been widely used for the discovery and evaluation of anti-inflammatory drugs, since the relative potency estimates obtained from most drugs tend to reflect clinical experience (Winter et al., 1962). The carrageenan induced inflammatory process in the rat involves three phases: an initial, second and third phases caused by the release of histamine
N.P. Babu et al. / Journal of Ethnopharmacology 125 (2009) 356–360 Table 4 Effect of Albizia lebbeck stem bark on adjuvant induced arthritisa . Groups
Dose (mg/kg body weight)
Control
Thickness of the injected foot (mm) Before treatment
After treatment
6.16 ± 0.14
5.86 ± 0.10
Petroleum ether
100 200 400
6.07 ± 0.16 6.30 ± 0.23 5.92 ± 0.13
5.01 ± 0.07** (29.94) 4.70 ± 0.21** (39.63) 4.01 ± 0.05** (64.97)
Chloroform
100 200 400
6.09 ± 0.12 5.94 ± 0.15 6.11 ± 0.15
5.59 ± 0.16 (07.20) 5.47 ± 0.31 (10.99) 5.28 ± 0.21 (18.48)
Ethanol
100 200 400
6.05 ± 0.30 5.82 ± 0.22 6.05 ± 0.21
4.321 ± 0.03** (55.45) 4.07 ± 0.03** (63.86) 3.96 ± 0.05** (68.57)
10
5.97 ± 0.05
3.82 ± 0.06** (73.29)
Indomethacin
Percentage of inhibition is mentioned in parentheses. a Each value is the mean ± S.E.M. of six rats. ** p < 0.005, compared with control rats; Student’s t-test.
and serotonin; bradykinin and prostaglandins respectively (DiRosa et al., 1971; Crunkhon and Meacock, 1971). Both histamine and serotonin are characterized by the increase of vascular permeability. Prostaglandins mediate maximum vascular responses during the third phase of inflammation (Vinegar et al., 1969). In carrageenan induced model, the extracts of Albizia lebbeck were able to significantly reduce paw edema. This antiedematous effect was significant during the first phase of inflammation (results not shown), indicating the inhibition of histamine release. Similarly, it was previously reported that the alcoholic extract of Albizia lebbeck had antihistaminic property, by neutralizing the histamine directly or due to corticotrophic action as evidenced by raise in plasma cortisol levels (Tripathi and Shukla, 1979). In addition, in the present study, the antiedematic effect of the extract was also significantly maintained during the second and third phases of edema development. This may be due to the inhibition of cyclooxygenase enzymes that are involved in the formation of prostaglandins. Dextran induced edema is considered as a consequence of histamine and serotonine liberation from the mast cells (Rowley and Benditt, 1956). It was already reported that the water decoction from the bark of Albizia lebbeck inhibits the mast cell degranulation and thereby inhibits the release of histamine (Tripathi and Shukla, 1979; Johri et al., 1985; Barua et al., 1997). In this study, the dextran-mediated inflammation was reduced probably as a result of antihistaminic effect of the extract, which may be due to the inhibition of mast cell degranulation. The cotton pellet granuloma method has been widely employed to assess the transudative, exudative and proliferative components of chronic inflammation (Spector, 1969). The fluid absorbed by the pellet greatly influences the wet weight of the granuloma and the dry weight correlates well with the amount of granulomatous tissue formed (Swingle and Shideman, 1972). Monocyte infilteration and fibroblast proliferation rather than neutrophil infilteration and exudation take place in chronic inflammation (Dunne, 1990). In this study, the petroleum ether and ethanol extracts of Albizia lebbeck extracts decreased both wet and dry weight of the cotton pellets compared to control groups. This may be due to the ability of Albizia lebbeck in reducing the number of fibroblasts and synthesis of collagen and mucopolysaccharide, which are natural proliferative agents of granulation tissue formation. In the investigation of FCA induced arthritis, it was observed that the swelling and redness developed over a twenty-four hour period in the foot injected with adjuvant. This inflammatory reaction subsided slightly during the next 8–10 days and then increased at the time when disseminated arthritis appeared (Ward and Cloud, 1996). The treatment that was begun 14 days after the day of adju-
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vant induction suppressed the secondary increase in swelling of the injected foot, which occurred, with the appearance of polyarthritis. The determination of foot thickness and changes in body weight of the animals has been used in evaluating anti-inflammatory activity and therapeutic effects of the treatment. In the present study, it was found that the extracts of Albizia lebbeck stem bark exhibited antiarthritic effect evidenced by increase in body weight and decreased edema formation in comparison with arthritic control animals. The loss of body weight that was found in the control animals could be due to reduced absorption of glucose and leucine in rat intestine in arthritic condition (Geetha and Varalakshmi, 1998). Prostaglandins greatly potentiate exudates by inducing relaxation of arteriolar smooth muscle and increasing the blood supply to the tissue (Williams, 1979). The ability of the extracts to reduce edema formation may thus be related to its inhibitory action of prostaglandin synthesis. In summary, our results suggest that Albizia lebbeck displays considerable potency in anti-inflammatory action and has prominent effects on adjuvant arthritis by alleviating paw edema. The mechanism of the effect may be due to the presence of flavanoids and saponins, which are present in Albizia lebbeck as evidenced by previous reports. Future studies will provide new insight into the anti-inflammatory activity of Albizia lebbeck, and eventually lead to development of a new class of anti-inflammatory agent.
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Varshney, I.P., Khan, M.S.Y., 1961. Saponins and sapogenins. XI. On the presence of echinocystic acid and quercetin in flowers of the Albizzia lebbek Benth. Canadian Journal of Chemistry 39, 1721–1723. Varshney, I.P., Sharma, S.P., 1969. Chemical investigation of the Albizzia lebbek leaves and Albizzia lucida seeds and bark. Indian Journal of Applied Chemistry 32, 10–11. Varshney, I.P., Geeta, H., Srivastava, H.C., Krishnamurthy, T.N., 1973. Partial structure of Lebbekanin A, a new saponin from the seeds of Albizzia lebbek Benth. Indian Journal of Chemistry 11, 1094–1096. Varshney, I.P., Pal, R., Vyas, P., 1976. Studies on the lebbekanin E, a new saponin from Albizzia lebbek Benth. Journal of Indian Chemical Society LIII, 859–860. Verpoorte, R., 1999. Exploration of nature’s chemodiversity: the role of secondary metabolites as leads in drug development. Drug Discovery Today 3, 232–238. Vinegar, R., Schreiber, W., Hugo, R., 1969. Biphasic development of carrageenan oedema in rats. Journal of Pharmacology and Experimental Therapeutics 166, 96–103. Ward, J.R., Cloud, R.S., 1996. Comparitive effect of antirhematic drugs on adjuvant induced polyarthritis in rats. Journal of Pharmacology and Experimental Therapeutics 152, 116–121. White, M.J., 1999. Mediators of inflammation and inflammatory process. Journal of Allergy and Clinical Immunology 103, S378–S381. Williams, T.J., 1979. Prostaglandin E2, Prostaglandin I2 and the vascular changes of inflammation. British Journal of Pharmacology 65, 517–524. Winter, C.A., Porter, C.A., 1957. Effect of alteration in side chain upon antiinflammatory and liver glycogen activities in hydrocortisone esters. Journal of American Pharmaceutical Association 46, 515–519. Winter, C.A., Risely, E.A., Nuss, G.W., 1962. Carrageenen-induced edema in the hind paw of rat as an assay for antiinflammatory drugs. Proceedings of Society for Experimental Biology and Medicine 111, 544–547.
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Anti-inflammatory activity of Albizia lebbeck Benth., an ethnomedicinal plant, in acute and chronic animal models of inflammation N. Prakash Babu, P. Pandikumar, S. Ignacimuthu ∗ Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Nungambakkam, Chennai 600 034, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 19 June 2007 Received in revised form 26 January 2009 Accepted 23 February 2009 Available online 9 March 2009 Keywords: Albizia lebbeck Anti-inflammatory activity Carrageenan Dextran Cotton pellet FCA
a b s t r a c t Aim of the study: Albizia lebbeck Benth. is used both in Indian traditional system and folk medicine to treat several inflammatory pathologies such as asthma, arthritis and burns. The aim of the present study was to evaluate the scientific basis of anti-inflammatory activity of different organic solvent extracts of Albizia lebbeck. Materials and methods: The anti-inflammatory activity of Albizia lebbeck was studied using the carrageenan, dextran, cotton pellet and Freund’s complete adjuvant induced rat models. The extracts obtained using petroleum ether, chloroform and ethanol were administered at the concentrations of 100, 200 and 400 mg/kg body weight. Results: The petroleum ether and ethanol extracts at 400 mg/kg, showed maximum inhibition of inflammation induced by carrageenan (petroleum ether—48.6%; ethanol—59.57%), dextran (petroleum ether—45.99%; ethanol—52.93%), cotton pellet (petroleum ether—34.46%; ethanol—53.57%) and Freund’s adjuvant (petroleum ether—64.97%; ethanol—68.57%). Conclusion: The marked inhibitory effect on paw edema shows that Albizia lebbeck possesses remarkable anti-inflammatory activity, supporting the folkloric usage of the plant to treat various inflammatory diseases. © 2009 Published by Elsevier Ireland Ltd.
1. Introduction Inflammation is a complex pathophysiological process mediated by a variety of signaling molecules produced by leucocytes, macrophages and mast cells as well as by the activation of complement factors that bring about edema formation as a result of extravasation of fluid and proteins and accumulation of leucocytes at the inflammatory site (White, 1999). All the steroidal and nonsteroidal anti-inflammatory drugs (NSAID’s), despite their great number, cause undesired and serious side effects. Therefore, development of new and more powerful drugs is still needed. Research on plants with medicinal properties and identification of the chemical components responsible for their activities have corroborated the traditional uses of ancient healing wisdom and lore and have proven the enduring healing potential of many plant medicines even in today’s hi-tech community. Albizia lebbeck Benth. (Mimosaceae), commonly known as Sirisa in Sanskrit, is a tall, unarmed, and deciduous tree distributed throughout India from the plains up to 900 m in the Himalayas. In Siddha system of medicine the bark and flowers of this plant are used to treat arthritis (Mudaliar, 1936). The tribal people in
∗ Corresponding author. Tel.: +91 44 2817 8348; fax: +91 44 2817 5566. E-mail address: [email protected] (S. Ignacimuthu). 0378-8741/$ – see front matter © 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2009.02.041
Himachal Pradesh and Kashmir use this plant to treat inflammation (Srivastava et al., 1986; Jain, 1991; Kapur, 1993). Balasubramaniam (1992) reported that the tribals of point Calimere wild life sanctuary, Tamilnadu use this plant to treat bone fractures. In ayurvedic system of medicine, the stem bark of this plant is used to treat diarrhoea (Nadkarni, 1954), edema, poisoning, asthma and bronchitis (Gupta, 2004). It was previously reported that the alcoholic extract of Albizia lebbeck protects the guinea pig against the antigen induced challenge (Tripathi et al., 1977; Barua et al., 1997). Further it also reduced the level of histamine and raised the plasma cortisol in antigen challenged guinea pigs (Tripathi and Shukla, 1979) as well as in bronchial asthma patients (Tripathi et al., 1978). Das et al. (2003) and Pramanik et al. (2005) previously reported the anti-inflammatory activity of the methanol extract of Albizia lebbeck bark. Many saponins such as Lebbekanin A–H (Varshney and Khan, 1961; Varshney and Sharma, 1969; Varshney et al., 1973, 1976) and Albizziasaponin A–C (Pal et al., 1995), which contain oleanolic acid, echinocystic acid or acacic acid as sapogenins were reported from various parts of this plant. Further, melanoxetin, okanin, (+) pinitol, (−)-leucopelargonidin (Gupta et al., 1966), friedelan-3-one, ␥-sitosterol and macrocyclic spermine alkaloids namely budmunchiamines L1–L6 (Misra et al., 1995; Dixit and Misra, 1997) were also isolated from this plant. The anti-inflammatory agents exert their effect through a spectrum of different modes of action (Martelli, 1977). Carrageenan
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induced edema in the hind paw (acute inflammation) and Cotton pellet granuloma (chronic inflammation) are widely employed in screening the new anti-inflammatory compound (Billingham and Davis, 1979). Adjuvant induced arthritis is also an often used model of inflammation (Newbold, 1963). This study investigated the anti-inflammatory effect of various solvent extracts of Albizia lebbeck on acute and chronic phases of inflammation. The anti-inflammatory effect of the extracts in adjuvant induced arthritis was also studied. 2. Materials and methods 2.1. Plant material The bark of Albizia lebbeck was collected in August 2005 from Kancheepuram District of Tamil Nadu. The botanical identity of the plant material was confirmed with the help of taxonomist, Department of Botany, Loyola College, Chennai. The voucher specimens were deposited in the herbarium of Entomology Research Institute, Loyola College, Chennai. 2.1.1. Preparation of the extracts The barks were chopped into small pieces, air-dried and powdered. The powdered plant material (1 kg) was sequentially extracted three times with 3 L of petroleum ether, chloroform, and 95% ethanol at room temperature for 48 h. The filtrates were concentrated under reduced pressure at 40 ◦ C and stored in a refrigerator. The percentage of yield of the petroleum ether, chloroform and ethanol extracts was 0.33%, 1.10% and 13.70% respectively. 2.1.2. Preliminary phytochemical screening of the extracts The preliminary phytochemical tests were carried out for the effective extracts using standard phytochemical methods (Harborne, 1998). 2.1.3. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the Albizia lebbeck crude extract was performed using a SHIMADZU GC–MS (GC-2010) equipped with a DB-5 MS fused silica capillary column. The column constant flow rate was maintained at 1.34 mL/min. Injector temperature was set at 200 ◦ C. The oven temperature was programmed from 60 to 150 ◦ C at 3 ◦ C/min, then held isothermal for 10 min and finally raised to 230 ◦ C. The relative percentage of the volatile constituents was expressed as percentages by peak area normalization. Identification of components of the crude extract was based on GC retention time on DB-5 capillary column, computer matching of mass spectra with those of standards (Wiley 6.0 data of GC–MS system) 2.1.4. Drug administration All the extracts were dissolved in 2% Tween-80 in water. The vehicle alone served as control. Indomethacin (10 mg/kg body weight), served as positive control (Arunachalam et al., 2002). 2.2. Animals Female Wistar rats weighing 130–150 g were procured from King Institute, Chennai. All animals were housed in polypropylene cages in a temperature-controlled room at 24 ± 1 ◦ C. The animals were fed with pelleted rat feed manufactured by TANUVAS with free access to water throughout the experiment. The rats were acclimatized at least one week before starting the experiments. In all experimental models of inflammation the studies were carried out using six rats in each group. This study got clearance from the Institutional Animal Ethical Committee (IAEC).
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2.2.1. Carrageenan induced rat paw edema The acute anti-inflammatory effect was evaluated by carrageenan induced rat paw edema according to the method of Winter et al. (1962). Edema was induced by injection of 1% suspension of carrageenan in 0.9% sterile saline solution into the right plantar region of the rat. The plant extract (100, 200 or 400 mg/kg), Indomethacin (10 mg/kg body weight), or vehicle was administrated orally 1 h before injection of carrageenan. Paw volume was measured with Digital vernier caliper 0, 3rd and 5th hour after injection (Ramprasath et al., 2004). The inhibitory activity was calculated using the following formula. Percentage inhibition =
(Ct − Co) control − (Ct − Co) treated × 100 (Ct − Co) control
2.2.2. Dextran induced rat paw edema The paw edema was induced in the right hind paw by subplantar injection of 0.1 mL of freshly prepared 1% dextran solution. Paw thickness was measured at 0, 45, and 90 min after dextran injection. The rats were treated as above. The percentage of inhibition was calculated. 2.2.3. Cotton pellet induced Granuloma Eleven groups of six rats in each group were included in the study. After shaving off fur, the animals were anaesthetized. Sterile pre-weighed cotton pellets (10 mg) were implanted in the axilla region of each rat through a single needle incision. Plant extract (100, 200 or 400 mg/kg body wt), Indomethacin (10 mg/kg body weight) or vehicle alone was administrated orally for seven consecutive days, from the day of cotton pellet implantation. On the eighth day the animals were anaesthetized, the cotton pellets were removed and made free from extraneous tissues. The pellets were dried at 60 ◦ C for 24 h. Mean weight of granuloma tissue formed around each pellet was evaluated (Winter and Porter, 1957). 2.2.4. Adjuvant induced arthritis Chronic arthritis was induced by the injection of 0.1 mL of Freund’s Complete Adjuvant containing 10 mg of heat killed Mycobacterium tuberculosis, in 1 mL of paraffin oil into the right hind paw of the rat intradermally. Treatment was given orally after 14 days from the day of adjuvant injection for 14 days. The volume of the paw was measured before induction, before treatment, and after treatment and the percentage inhibition was determined. 2.3. Statistical analysis The data obtained were analysed using one-way ANOVA followed by Student’s t-test. Differences were considered significant at the 5% level. 3. Results 3.1. Preliminary phytochemical analysis The petroleum ether extract of Albizia lebbeck had shown positive results for steroids, terpenoids and coumarins. The ethanol extract showed the presence of tannins, flavanoids, anthraquinones, and saponins. 3.2. GC–MS analysis of crude extracts The crude extract of Albizia lebbeck was analysed using GC–MS and the petroleum ether extract had the following major composition: 1-tridecene (2.81%), 1-dodecanol (2.94%), 1ethyldibenzothiophene (2.07%), bis-(3, 5, 5-trimethylhexyl) ether
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Table 1 Effect of Albizia lebbeck stem bark on carrageenan induced paw edemaa .
Table 2 Effect of Albizia lebbeck stem bark on dextran induced paw edemaa .
Groups
Groups
Dose (mg/kg body weight)
Thickness of the injected foot (mm) 3rd hour
5th hour
Control
5.16 ± 0.10
5.03 ± 0.08
Petroleum ether
100 200 400
4.70 ± 0.10 (23.70) 4.25 ± 0.11** (46.54) 4.14 ± 0.04** (48.66)
Chloroform
100 200 400
Ethanol
Indomethacin
Dose (mg/kg body weight)
Thickness of the injected foot (mm) 45 min
90 min
Control
6.05 ± 0.21
5.73 ± 0.07
4.52 ± 0.03 (27.54) 4.14 ± 0.08** (47.17) 4.03 ± 0.03** (50.82)
Petroleum ether
100 200 400
4.92 ± 0.17 (36.71) 4.77 ± 0.11** (39.81) 4.76 ± 0.08** (41.08)
4.61 ± 0.14** (40.45) 4.52 ± 0.04** (41.89) 4.43 ± 0.26** (45.99)
5.11 ± 0.04 (05.01) 5.03 ± 0.08 (09.24) 4.77 ± 0.09* (20.82)
4.93 ± 0.03 (07.44) 4.83 ± 0.06 (13.35) 4.58 ± 0.13* (25.05)
Chloroform
100 200 400
5.75 ± 0.12 (09.91) 5.67 ± 0.16 (12.49) 5.49 ± 0.13* (17.77)
5.43 ± 0.14 (10.92) 5.27 ± 0.13* (16.47) 5.10 ± 0.08** (22.28)
100 200 400
5.00 ± 0.04 (10.35) 4.50 ± 0.05** (31.62) 3.90 ± 0.05** (59.57)
4.82 ± 0.06** (13.23) 4.22 ± 0.08** (40.89) 3.83 ± 0.04** (60.04)
Ethanol
100 200 400
5.49 ± 0.06* (17.35) 5.10 ± 0.18** (24.12) 4.59 ± 0.03** (45.97)
5.18 ± 0.12* (18.93) 4.84 ± 0.09** (30.00) 4.22 ± 0.11** (52.93)
10
3.69 ± 0.10** (69.53)
3.62 ± 0.10** (70.87)
Indomethacin
10
4.39 ± 0.03** (53.16)
4.08 ± 0.03** (58.60)
*
**
Values given in parentheses represent percentage of inhibition. a Each value is the mean ± S.E.M. of six rats. * p < 0.05, compared with control rats; Student’s t-test. ** p < 0.005, compared with control rats; Student’s t-test.
(2.49%), n-hexadecanoic acid (24.24%), methyl 2L, 9d-dimethylteracosanoate (5.08%), 9,12-octadecadienoic acid (Z,Z)-(10.04%), oleic acid (19.30%), 1,13-tetradecadiene (6.59%), 3,3-difluro-1tetradecen-4-ol (2.20%), 1-tridecyn-4-ol (6.34%). The retention time of the above compounds was: 10.933, 13.441, 15.035, 15.696, 17.391, 17.737, 19.209, 19.266, 19.661, 21.402, and 27.581, respectively.
3.3. Carrageenan induced inflammation In carrageenan induced animal models, the petroleum ether extract of Albizia lebbeck at concentrations 100, 200, and 400 mg, inhibited the edema formation in third hour by 23.7% (p < 0.05), 46.5%, and 48.6% (p < 0.005) in a dose-dependent manner respectively. This effect also extended and significantly increased up to the fifth hour (p < 0.005). The ethanol extract at 100, 200, and 400 mg showed 10.35%, 31.62%, and 59.57% of inhibition at third hour and 13.23%, 40.89%, and 60.04% (p < 0.005) of inhibition at fifth hour respectively. The chloroform extract showed a mild activity in higher concentration only. The reference drug group significantly inhibited the edema formation by 69.53% and 70.87% at third and fifth hour respectively (p < 0.005). Results are shown in Table 1.
3.4. Dextran induced inflammation In dextran induced inflammatory model, the petroleum ether extract at 45 min inhibited the paw edema by 36.71%, 39.81%, and 41.08% (p < 0.005) of inhibition at the concentration of 100, 200, and 400 mg respectively. At 90 min, there was increase in the percentage of inhibition by 40.44%, 41.89% and 45.99%, respectively. Likewise, 100, 200, and 400 mg of ethanol extract treated groups showed 17.35% (p < 0.05), 24.12%, and 45.97% (p < 0.005) inhibition at 45 min and 18.93%, 30.00%, and 52.93% inhibition at 90 min respectively. Results were shown in Table 2.
3.5. Cotton pellet induced granuloma In cotton pellet granuloma, the petroleum ether extract inhibited the granuloma formation by 23.53% (p < 0.05), 28.26%, and 34.46% (p < 0.005) at 100, 200 and 400 mg, respectively (Table 3). The ethanol extract at 100, 200, 400 mg inhibited the granuloma formation by 27.17% (p < 0.05), 38.68%, and 53.57% (p < 0.005) respectively.
**
Percentage of inhibition is mentioned in parentheses. a Each value is the mean ± S.E.M. of six rats. * p < 0.05, compared with control rats; Student’s t-test. ** p < 0.005, compared with control rats; Student’s t-test.
Table 3 Effect of Albizia lebbeck stem bark on cotton pellet induced granulomaa . Groups
Dose (mg/kg body weight)
Dry weight of granuloma (mg)
Petroleum ether
100 200 400
105.05 ± 2.55* (23.53) 098.55 ± 1.43** (28.26) 090.03 ± 1.18** (34.46)
Chloroform
100 200 400
135.67 ± 4.83 (01.23) 124.75 ± 5.29 (09.19) 104.22 ± 2.96* (24.13)
Ethanol
100 200 400
100.05 ± 3.10* (27.17) 084.22 ± 4.06** (38.68) 63.77 ± 3.73** (53.57)
10
054.65 ± 3.64** (60.21)
137.37 ± 9.47
Control
Indomethacin
Percentage of inhibition is mentioned in parentheses. a Each value is the mean ± S.E.M. of six rats. * p < 0.05, compared with control rats; Student’s t-test. ** p < 0.005, compared with control rats; Student’s t-test.
3.6. Adjuvant induced arthritis In Adjuvant induced animals, a dose-dependent reduction in foot thickness was exhibited in petroleum ether and ethanol extracts of Albizia lebbeck. At a dose of 400 mg, arthritic swelling was inhibited by 64.97% and 68.57% (p < 0.005) for petroleum ether and ethanol extract treated groups compared to the adjuvant control respectively, where as the Indomethacin treated group showed an inhibition of 73.29% (Table 4). 4. Discussion Alternative medicine for the treatment of various diseases is getting more popular. Many medicinal plants provide relief of symptoms comparable to that of conventional medicinal agents (Verpoorte, 1999). Therefore, the present study was aimed at evaluating the scientific basis for the traditional use of Albizia lebbeck using in vivo inflammatory models. Carrageenan induced rat hind paw edema has been widely used for the discovery and evaluation of anti-inflammatory drugs, since the relative potency estimates obtained from most drugs tend to reflect clinical experience (Winter et al., 1962). The carrageenan induced inflammatory process in the rat involves three phases: an initial, second and third phases caused by the release of histamine
N.P. Babu et al. / Journal of Ethnopharmacology 125 (2009) 356–360 Table 4 Effect of Albizia lebbeck stem bark on adjuvant induced arthritisa . Groups
Dose (mg/kg body weight)
Control
Thickness of the injected foot (mm) Before treatment
After treatment
6.16 ± 0.14
5.86 ± 0.10
Petroleum ether
100 200 400
6.07 ± 0.16 6.30 ± 0.23 5.92 ± 0.13
5.01 ± 0.07** (29.94) 4.70 ± 0.21** (39.63) 4.01 ± 0.05** (64.97)
Chloroform
100 200 400
6.09 ± 0.12 5.94 ± 0.15 6.11 ± 0.15
5.59 ± 0.16 (07.20) 5.47 ± 0.31 (10.99) 5.28 ± 0.21 (18.48)
Ethanol
100 200 400
6.05 ± 0.30 5.82 ± 0.22 6.05 ± 0.21
4.321 ± 0.03** (55.45) 4.07 ± 0.03** (63.86) 3.96 ± 0.05** (68.57)
10
5.97 ± 0.05
3.82 ± 0.06** (73.29)
Indomethacin
Percentage of inhibition is mentioned in parentheses. a Each value is the mean ± S.E.M. of six rats. ** p < 0.005, compared with control rats; Student’s t-test.
and serotonin; bradykinin and prostaglandins respectively (DiRosa et al., 1971; Crunkhon and Meacock, 1971). Both histamine and serotonin are characterized by the increase of vascular permeability. Prostaglandins mediate maximum vascular responses during the third phase of inflammation (Vinegar et al., 1969). In carrageenan induced model, the extracts of Albizia lebbeck were able to significantly reduce paw edema. This antiedematous effect was significant during the first phase of inflammation (results not shown), indicating the inhibition of histamine release. Similarly, it was previously reported that the alcoholic extract of Albizia lebbeck had antihistaminic property, by neutralizing the histamine directly or due to corticotrophic action as evidenced by raise in plasma cortisol levels (Tripathi and Shukla, 1979). In addition, in the present study, the antiedematic effect of the extract was also significantly maintained during the second and third phases of edema development. This may be due to the inhibition of cyclooxygenase enzymes that are involved in the formation of prostaglandins. Dextran induced edema is considered as a consequence of histamine and serotonine liberation from the mast cells (Rowley and Benditt, 1956). It was already reported that the water decoction from the bark of Albizia lebbeck inhibits the mast cell degranulation and thereby inhibits the release of histamine (Tripathi and Shukla, 1979; Johri et al., 1985; Barua et al., 1997). In this study, the dextran-mediated inflammation was reduced probably as a result of antihistaminic effect of the extract, which may be due to the inhibition of mast cell degranulation. The cotton pellet granuloma method has been widely employed to assess the transudative, exudative and proliferative components of chronic inflammation (Spector, 1969). The fluid absorbed by the pellet greatly influences the wet weight of the granuloma and the dry weight correlates well with the amount of granulomatous tissue formed (Swingle and Shideman, 1972). Monocyte infilteration and fibroblast proliferation rather than neutrophil infilteration and exudation take place in chronic inflammation (Dunne, 1990). In this study, the petroleum ether and ethanol extracts of Albizia lebbeck extracts decreased both wet and dry weight of the cotton pellets compared to control groups. This may be due to the ability of Albizia lebbeck in reducing the number of fibroblasts and synthesis of collagen and mucopolysaccharide, which are natural proliferative agents of granulation tissue formation. In the investigation of FCA induced arthritis, it was observed that the swelling and redness developed over a twenty-four hour period in the foot injected with adjuvant. This inflammatory reaction subsided slightly during the next 8–10 days and then increased at the time when disseminated arthritis appeared (Ward and Cloud, 1996). The treatment that was begun 14 days after the day of adju-
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vant induction suppressed the secondary increase in swelling of the injected foot, which occurred, with the appearance of polyarthritis. The determination of foot thickness and changes in body weight of the animals has been used in evaluating anti-inflammatory activity and therapeutic effects of the treatment. In the present study, it was found that the extracts of Albizia lebbeck stem bark exhibited antiarthritic effect evidenced by increase in body weight and decreased edema formation in comparison with arthritic control animals. The loss of body weight that was found in the control animals could be due to reduced absorption of glucose and leucine in rat intestine in arthritic condition (Geetha and Varalakshmi, 1998). Prostaglandins greatly potentiate exudates by inducing relaxation of arteriolar smooth muscle and increasing the blood supply to the tissue (Williams, 1979). The ability of the extracts to reduce edema formation may thus be related to its inhibitory action of prostaglandin synthesis. In summary, our results suggest that Albizia lebbeck displays considerable potency in anti-inflammatory action and has prominent effects on adjuvant arthritis by alleviating paw edema. The mechanism of the effect may be due to the presence of flavanoids and saponins, which are present in Albizia lebbeck as evidenced by previous reports. Future studies will provide new insight into the anti-inflammatory activity of Albizia lebbeck, and eventually lead to development of a new class of anti-inflammatory agent.
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