Evaluation of anti-inflammatory activity of Alstonia macrophylla Wall ex A. DC. leaf extract.
Methanolic extract of dried leaves of Alstonia macrophylla Wall ex A. DC. and its fractions were investigated for its anti-inflammatory activity. The extract at a concentration of 200 mg [kg.sup.-1] and 400 mg [kg.sup.-1], p.o. and its fractions at 25 mg [kg.sup.-1] and 50 mg [kg.sup.-1], p.o. showed the significant dose dependent antiinflammatory activity in carrageenan and dextran-induced rats hind paw edema (acute models) as well as in cotton pellet-induced granuloma (chronic model) in rats. Anti-inflammatory activity of the tested extract and its fractions was comparable with that of the standard drug Indomethacin (10 mg [kg.sup.-1]).
Key words: Alstonia macrophylla, Anti-inflammatory activity, Bay Islands.
Alstonia macrophylla Wall ex A. DC. (Apocynaceae), locally known as Chuharoi by Onge and Tachoroi in Nicobarese is a pantropical tree native to Malaysia and stretching to the sea level at Katchal, Passa, Koshin, Kopenheat; Inland and littoral forests of Great Nicobar, North Nicobar, and Carnicobar; as well as in North, Middle and South Andamans. The plant is a 5-10 m tree with blackish green branch, bearing 3-5 velvety (glossy above and rough below) leaves containing tiny hairs on bare sides, arranged in a whorl, with slightly bending margins and 10-20 pairs of veins (less than 40 in number). Ethnobotanical studies indicate that the decoctions of Alstonia macrophylla leaves and stem bark is widely used among the tribal populations of Bay Islands, India, to treat stomachache (Dagar and Dagar, 1991); skin diseases and urinary infections (Bhargava, 1983; Chakraborty and Vasudeva Rao, 1988). Leaves are also reported to have anticholeretic and vulnerary effect (Asolkar et al., 1992). In some area leaves greased wi th hot coconut oil, have been used as poultice for sprains, bruises and dislocated joints and also used as febrifuge (Wealth of India, 1985). Recent report indicates that the plant has got moderate antibacterial and limited antifungal activity (Chattopadhyay et al., 2001). However, there is no report on the anti-inflammatory activity of this plant though inflammation is a common occurrence in infective conditions. Therefore, the aim of the present work is to evaluate, for the first time, the anti-inflammatory activity of Alstonia macrophylla leaf extract and its fractions.
Materials and Methods
The leaves of Alstonia macrophylla Wall ex A. DC were collected from the rain forests of Middle and South Andamans, India, during April, June and October 1999. The voucher specimens have been identified and deposited at the Herbarium Section of the Botanical Survey of India, Andaman & Nicobar Circle, Port Blair, India.
Extraction and fractionation
Coarsely powdered dry leaves (1 kg) were successively extracted in cold with 95% methanol for 72 h at room temperature. The whole extract was filtered and the solvent were evaporated to dryness under reduced pressure in a Eyela Rotary Evaporator (Japan) at 40 [degrees]C-45 [degrees]C. The w/w yield of the prepared extract was 8.9 [+ or -] 0.21. Further fractionation and purification of the crude extract yielded three major and a group of minor fractions as stated earlier (Chattopadhyny et al., 2001). The Whole extract and three major fractions A, B and C dissolved in propylene glycol for the studies.
Albino Wistar rats of either sex weighing 160-180 g each were housed in standard metal cages at room temperature. They were provided with food and water ad libitum. The rats were allowed a one-week acclimatization period before the experimental sessions.
Carrageenan -induced rat paw edema
The rats were divided into eleven groups, each group consisting of six animals. Edema was induced by subplantar injection of 0.1% freshly prepared carrageenan suspension into the right hind paw of each rat. The paw volume was measured at 0 h and at 3 h after the injection of carrageenan, using a plethysmometer (Winter et al, 1962). The methanol extract of Alstonia macrophylla at 200 and 400 mg [kg.sup.-1] and its fractions at 25 and 50 mg [kg.sup.-1] doses was administered orally to nine groups of rats. The tenth and eleventh groups of rats received 5 ml [kg.sup.-1] propylene glycol orally as vehicle control or 10 mg [kg.sup.-1] Indomethacin as drug control respectively, for assessing comparative pharmacological significance. Drug pretreatment was given 1 h before the injection of carrageenan.
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 (Parmar and Ghosh, 1978). Paw volume were measured 30 mm before and after dextran injection. The rats were treated as described above. The percentage inhibition of edema was calculated for both models as described by Kavimani et al (1996).
Cotton pellet-induced granuloma
The eleven groups of rats, eight in each group was included in this study. After shaving off the fur the animals were anaesthetized. Sterile preweighed cotton pellets (10 mg) were implanted in the axilla region of each rat through a single needle incision (D'Arcy et al, 1960). Methanol extract of Alstonia macrophylla leaves at 200 and 400 mg [kg.sup.-1], and its fractions at 25 and 50 mg [kg.sup.-1], indomethacin at 10 mg [kg.sup.-1] (standard) or propylene glycol at 5 ml [kg.sup.-1] (control) were administered orally to the respective group of animals for seven consecutive days, from the day of cotton-pellet implantation. On the eighth day, the animals were anaesthetized again, the cotton pellets were removed surgically and made free from extraneous tissues. The pellets were incubated at 37 [degrees]C for 24 h and dried at 60 [degrees]C to constant weight. The increase in the dry weight of the pellets was taken as measure of granuloma formation (Winter and Porter, 1957).
The results were expressed as mean [+ or -] S.E and the significance were evaluated by Student's t-test compared with control (Woodson, 1987).
The results presented in Table 1 showed that the methanolic extract at 200 and 400 mg [kg.sup.-1], p.o and its fractions at 25 and 50 mg [kg.sup.-1], p.o. exhibited significant anti-inflammatory activity in all the experimental models. The methanolic extract at 400 mg [kg.sup.-1] exhibited maximum inhibition (65.77%) of paw edema and its fractions at 50 mg [kg.sup.-1] concentrations showed 56.08% (fraction A), 64.25% (fraction B), 66.53% (a combination of fraction A and B) and 60.83% (fraction C) inhibition respectively, in carrageenan-induced rat paw edema; while indomethacin showed 67.77% inhibition of edema volume after 4h of drug treatment (Table 1). The results of the dextran-induced rat paw edema, presented in Table 2 showed that the edema suppression at 400 mg [kg.sup.-1] of methanolic extract was 65.77%. While the fractions at 50 mg [kg.sup.-1] showed 50.48% (fraction A), 50.00% (fraction B), 60.48% (fraction A+B), and 52.92% (fraction C) inhibition of edema respectively whereas indomethacin produced 67.77% inhibition. In Cotton pellet granuloma (chronic inflammation model) test the methanolic extract at 400 mg [kg.sup.-1] exhibited maximum inhibition of 52.96%. The fractions at 50 mg [kg.sup.-1] doses, on the otherhand, exhibited maximum inhibition of 51.00% (fraction A), 53.49% (fraction B), 50.65% (a combination of fraction A and B) and 52.07% (fraction C) respectively in granuloma weight compared with 54.07% for the standard drug indomethacin at a concentration of 10 mg [kg.sup.-1] (Table 3).
The results of the three antiinflammatory models indicated moderate to strong anti-inflammatory activity of the methanolic extract of Alstonia macrophylla leaf and its lipophilic fractions. However, the polar fractions containing two major alkaloid picrinine and picralstonine do not have recognisable antiinflammatory activity. Alstonia macrophylla is reported to contain picrinine, picralstonine, 0-benzoyl vincamajine and quebrachidine having CNS depressant effect (Asolkar et al., 1992). We have for the first time, isolated three major compounds namely, [beta]- sitosterol (fraction A), ursolic acid (fraction B) and [beta]-sitosterol glucoside (fraction C), alongwith a mixture of minor compounds (as detected in TLC only) in the n-butanol fraction from this ethnomedicine (Chattopadhyay et al., 2001). Contemporary research revealed that ursolic acid isolated from herbal sources have several pharmacological actions like anticancerous, metastatic, antiulcer, antihyperlipidemic, antimicrobial, antiinflammatory, anti viral and hepatoprotective (Kim, 1997; Young et al., 1995; Liu, 1995). While [beta]-sitosterol and its glucoside are reported to have antiinflammatory, antipyretic (Gupta et al., 1996), antiulcer, antidiabetic, anticancerous activities, and is helpful in controlling rheumatoid arthritis (Pegel, 1980).
Alstonia macrophylla and its fractions showed significant anti-inflammatory activity comparable to that of indomethacin against carrageenan induced acute pedal edema, dextran induced edema and cotton pellet induced granuloma. The carrageenan induced paw edema is believed to be biphasic, of which the first phase is mediated by early release of histamine and 5HT followed by the release of kinin in later phase (Castro et al., 1968). On the otherhand, dextran mediated inflammation (edema) was reduced probably as a result of antihistamine effects of the extract and its fractions, as dextran is known to cause inflammation through both histamine and serotonin (Ghosh et al., 1963). This may be due to the presence of ursolic acid (fraction B), as ursolic acid isolated from Melaleuca leucadendron is reported to inhibit concavalin A induced histamine release (Tsuruga et al., 1991). The extract and different fractions also exhibited significant anti-inflammatory activity in the cotton-pellet granuloma test. This reflecte d its efficacy to inhibit the increase in the number of fibroblasts and synthesis of collagen and mucopolysaccharides during granuloma tissue formation (Arrigoni-Martellie, 1977). Here both the crude extract and fractions of Alstonia macrophylla leaf showed significant anti-inflammatory effects at dose dependent manner in all the animal models tested. It may be mentioned here that the antiinflammatory activity of Phillyrea latifolia L is found to be due to ursolic acid, which inhibit cyclooxygenase and 5-lipooxygenase enzymes of arachidonate cascade (Diaz et al., 2000). However, the exact mechanism of antiinflammatory action of Alstonia macrophylla leaf extract and it fractions required further studies.
The present investigation for the first time, confirms that there is potential anti-inflammatory activity in the crude extract of Alstonia macrophylla leaves, which appears to be due to the action of either fraction B alone or the combination of fractions A, B and C.
Table 1. Effect of leaves of Alstonia macrophylla methanol extract and its fractions on Carrageenan-induced Rat paw edema. Treatment Dose Paw volume % inhibition (mg/kg, p.o.) Propylene glycol 5 ml/kg 5.26 [+ or -] 0.39 -- Indomethacin 10 1.71 [+ or -] 0.31 * 67.77 Leaf extract 200 2.43 [+ or -] 0.21 * 53.80 400 1.80 [+ or -] 0.29 * 65.77 Fraction A 25 2.74 [+ or -] 0.18 * 47.90 50 2.31 [+ or -] 0.12 * 56.08 Fraction B 25 2.40 [+ or -] 0.22 * 54.37 50 1.88 [+ or -] 0.28 * 64.25 Fraction A+B 50 1.76 [+ or -] 0.14 * 66.53 Fraction C 25 2.62 [+ or -] 0.10 * 50.19 50 2.06 [+ or -] 0.14 * 60.83 Values are mean [+ or -] SE, n = 6, * p < 0.001 compared with control, Student's t-test's. Table 2. Effect of leaves of Alstonia macrophylla methanol extract and its fractions on Dextran-induced Rat paw edema. Treatment Dose Paw volume % inhibition (mg/kg, p.o.) Propylene glycol 5 ml/kg 4.10 [+ or -] 0.17 -- Indomethacin 10 1.60 [+ or -] 0.03 * 67.77 Leaf extract 200 3.20 [+ or -] 0.53 * 53.80 400 2.10 [+ or -] 0.41 * 65.77 Fraction - A 25 2.52 [+ or -] 0.18 * 38.53 50 2.03 [+ or -] 0.14 * 50.48 Fraction - B 25 2.32 [+ or -] 0.23 * 43.41 50 2.05 [+ or -] 0.17 * 50.00 Fraction - A+B 50 1.62 [+ or -] 0.11 * 60.48 Fraction - C 25 2.23 [+ or -] 0.21 * 45.60 50 1.93 [+ or -] 0.11 * 52.92 Values are mean [+ or -] SE, n = 6, * p < 0.001 compared with control, Student's t-test's Table 3. Effect of leaves of Alstonia macrophylla methanol extract and its fractions on Cotton-pellet granuloma in Rats. Treatment Dose Weight of % inhibition (mg/kg, p.o.) granuloma (mg) Propylene glycol 5 ml/kg 70.42 [+ or -] 3.60 -- Indomethacin 10 32.34 [+ or -] 1.71 * 54.07 Leaf extract 200 47.37 [+ or -] 2.00 ** 32.73 400 33.12 [+ or -] 1.52 * 52.96 Fraction - A 25 45.22 [+ or -] 0.56 ** 35.78 50 34.50 [+ or -] 0.64 *** 51.00 Fraction - B 25 46.24 [+ or -] 0.34 ** 34.33 50 34.75 [+ or -] 0.81 *** 50.65 Fraction - A+B 50 32.75 [+ or -] 0.81 *** 53.49 Fraction - C 25 43.82 [+ or -] 0.36 ** 37.77 50 33.75 [+ or -] 0.67 *** 52.07 Values are mean [+ or -] S.E. * p < 0.05, ** p < 0.01, *** p < 0.001 Compared with control, Student's t-test's
The Authors are grateful to the Department of Biotechnology, Government of India, for financial assistance through the project Grant No. BT/PRO 237/OSC 17/005/96. Thanks are due to Dr. Sreekumar, Senior Scientist, and the Officer In-Charge, Botanical Survey of India, Andaman & Nicobar Circle, Port Blair, Andamans for identification of the plant and Shri Aloke Kumar Mallick, Laboratory Technician, ICMR Virus Unit, Kolkata for his kind help in some experiments. We express our gratitude to Dr. S. K. Bhattacharya, Director, National Institute of Cholera & Enteric Diseases, Kolkata and Officer In-Charge, ICMR Virus Unit Kolkata, for his constant encouragement, help and suggestions.
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G. Arunachalam (1), D. Chattopadhyay (1), S. Chatterjee (1), A. B. Mandal (2), T. K. Sur (3), and S.C. Mandal (4)
(1.) ICMR Virus Unit Calcutta, I.D. & B.G. Hospital, Beliaghata, Kolkata
(2.) Biotechnology Laboratory, Central Agricultural Research Institute, Port Blair, Andaman & Nicobar Islands
(3.) Department of Pharmacology, Dr. B. C. Roy P.G. Institute of Basic Medical Sciences, University College of Medicine, Kolkata and
(4.) Division of Pharmacognosy, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India
Dr. D. Chattopadhyay M. Sc., Ph.D. Fellow BSAC (England), Senior Research Officer, ICMR Virus Unit Calcutta,, Infectious Diseases & Beliaghata General Hospital, GB 4, 1st Floor, 57, Dr. S.C. Banerjee Road, Kolkata 700 010, India Tel.: ++91-33-353 7425; Fax: 91-33-353 7424 e-mail: firstname.lastname@example.org