Anti-inflammatory activity of Dalbergia sissoo leaves

Fitoterapia 72 Ž2001. 131᎐139

Anti-inflammatory activity of Dalbergia sissoo leaves S.W. Hajare, S. ChandraU , J. Sharma, S.K. Tandan, J. Lal, A.G. Telang Di¨ ision of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar-243 122 (U.P.), India

Received 17 May 2000; accepted in revised form 9 September 2000

Abstract The possible anti-inflammatory activity of the 90% ethanolic extract of Dalbergia sissoo leaves ŽDSELE. was studied in different models of inflammation in rats after oral administration at doses of 100, 300 and 1000 mgrkg. DSELE significantly inhibited carrageenin, kaolin and nystatin-induced paw oedema, as well as the weight of granuloma induced by a cotton pellet. It also inhibited dye leakage in acetic acid-induced vascular permeability test in mice. DSELE was devoid of ulcerogenic effect on the gastric mucosa of rats in acute and chronic tests. In acute toxicity studies, it was found to be safe up to 10.125 grkg, p.o. in the rat. It was concluded that the D. sissoo leaf extract possessed significant anti-inflammatory activity Žin acute, sub-acute and chronic models of inflammation. without any side effect on gastric mucosa. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Dalbergia sissoo; Anti-inflammatory activity

1. Introduction Plants of the genus Dalbergia are reported to be useful in the treatment of arthritis, gonorrhoea and rheumatic pains w1᎐3x. Phytochemical examination of Dalbergia genus has provided a large number of compounds, which include flavonoids, furans, benzophenones, styrenes, and terpenoids w4,5x. In spite of the U

Corresponding author.

0367-326Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 0 . 0 0 2 7 2 - 0

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evidence for the presence of promising chemical constituents supposed to be responsible for anti-inflammatory activity, no such information is available on the biological activity of D. sissoo Roxb. ŽFabaceae.. The present study was, therefore, conducted to assess the anti-inflammatory activity of ethanolic extract of D. sissoo leaves.

2. Experimental 2.1. Plant material D. sissoo mature green leaves were collected in November᎐December 1996, from the campus of Indian Veterinary Research Institute, Izatnagar ŽIndia.. A voucher specimen, authenticated by Dr B.N. Pandey ŽDepartment of Botany, Bareilly College, Bareilly, India., was deposited in the herbarium of the Division of Pharmacology and Toxicology of the Institute. 2.2. Preparation of extract Shade-dried, powdered leaves were Soxhlet extracted with 90% EtOH. The extract was concentrated under reduced pressure and made free of solvent to afford a semi-solid residue ŽDSELE; yield: 7.66%.. Preliminary phytochemical screening w6,7x of DSELE gave positive tests for flavonoids, terpenoids and tannins, and negative tests for alkaloids and saponins. 2.3. Animals Wistar rats Ž150᎐180 g. and Swiss mice Ž15᎐25 g. of either sex were used. They were kept in standardised environmental conditions and maintained on a standard rodent diet Žprovided by the Feed Technology Unit of the Institute . and water ad libitum. 2.4. Anti-inflammatory acti¨ ity 2.4.1. Carrageenin-induced paw oedema Acute inflammation was induced by injecting 0.1 ml of 1% Žwrv. carrageenin into the plantar aponeurosis of the right hind paw of the rats w8x. DSELE Ž100, 300 and 1000 mgrkg. or phenylbutazone ŽPHB; 100 mgrkg. was administered orally 1 h before carrageenin injection. Paw volume was measured with a plethysmometer ŽUGO Basile, Italy. before and 3 h after the carrageenin injection. 2.4.2. Kaolin᎐carrageenin-induced hind paw oedema Hind paw oedema was induced by the administration of 0.2 ml of 20% kaolin and 1% carrageenin mixture into subplantar region of hind paw of rats w9x. DSELE Ž100, 300 and 1000 mgrkg. or acetylsalicylic acid ŽASA; 300 mgrkg. were given

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133

orally three times Ž10.0 AM, 1.0 PM and 5.0 PM. beginning at 18 h after phlogistic challenge. The hind paw oedema was measured by plethysmometer just before Ž0 h. and at 3, 6 and 24 h after the first dose. 2.4.3. Nystatin-induced hind paw oedema Rats received DSELE Ž100, 300 and 1000 mgrkg, p.o.. or PHB Ž100 mgrkg, p.o.. 1 h before the subplantar injection of 6% nystatin solution Ž0.1 ml. into the right hind paw of each rat w10x. Paw volume was measured with plethysmometer immediately Ž0 h. and at 2, 4, 6, 24, 48 and 72 h after the nystatin injection. 2.4.4. Granuloma formation Autoclaved cotton pellets, weighing 35.0 " 1.0 mg each, were implanted in symmetric positions in the lateral abdomen of anaesthetised rats. DSELE Ž100, 300 and 1000 mgrkg. or PHB Ž100 mgrkg. were administered orally once a day for 7 consecutive days. On the eighth day, rats were sacrificed and the pellets covered with granulation tissue were dissected out and dried in hot air oven at 60⬚C until a constant weight was obtained w11x. 2.5. Acetic acid-induced ¨ ascular permeability Thirty minutes after the oral administration of DSELE Ž100, 300 and 1000 mgrkg. or ASA Ž300 mgrkg., the mice were injected with 0.25-ml of an aqueous solution of 0.6% acetic acid intraperitoneally. Immediately after treatment, 1% Evan’s blue Ž10 mgrkg. was administered intravenously. Thirty minutes later, the mice were killed and leakage of dye into peritoneal cavity was measured at 610 nm w12x. 2.6. Gastric tolerability 2.6.1. Acute effect Rats fasted for 16 h were orally treated with DSELE Ž100, 300 and 1000 mgrkg., ASA Ž300 mgrkg. or vehicle Žnormal saline 1 mlrkg.. The animals were killed 5 h after drug administration. The stomachs were removed and cut along the greater curvature for the evaluation of petechial haemorrhages and ulcers w13x. 2.6.2. Chronic effect Rats housed in groups of six each had free access to food and water. The rats received test drugs suspended in normal saline orally for 14 consecutive days. The animals, fasted for 16 h, were killed 24 h after the last drug administration. The stomachs were removed and examined as in acute experiment. 2.7. Acute toxicity Mice of either sex, weighing 20᎐25 g, were divided into five groups of six mice each. DSELE was administered orally to four groups of mice, at doses of 3.00, 4.50,

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6.75 and 10.125 grkg, respectively. The fifth group served as control. Observations for toxic symptoms and mortality were recorded up to 24 h and LD50 of the extract was estimated w14x. 2.8. Statistical analysis The results are expressed as mean " S.E.M. Student’s t-test was used to evaluate the significance of differences between the treated and control groups w15x.

3. Results 3.1. Anti-inflammatory acti¨ ity 3.1.1. Carrageenin-induced paw oedema Dalbergia sissoo ethanolic leaf extract ŽDSELE. significantly reduced the oedema induced by carrageenin by 29.55, 34.09 and 43.18% with 100, 300 and 1000 mgrkg, p.o., respectively; PHB Ž100 mgrkg, p.o.. inhibited the oedema volume by 50.0% ŽFig. 1..

Fig. 1. Effect of oral administration of DSELE on carrageenin-induced hind paw oedema. Values are mean " S.E.M. Ž n s 6.; U P - 0.05; UU P - 0.01 vs. control; Student’s t-test. DSELE, Dalbergia sissoo ethanolic leaf extract, PHB, phenylbutazone.

3.1.2. Kaolin᎐carrageenin-induced paw oedema DSELE Ž300 and 1000 mgrkg, p.o.. produced significant anti-inflammatory effect 6 h after administration, which persisted up to 24 h of observation; ASA Ž300 mgrkg, p.o.. produced an inhibition in oedema volume which was greater than that produced by the highest dose of DSELE at all time intervals ŽTable 1..

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135

Table 1 Effect of Dalbergia sissoo ethanolic leaf extract ŽDSELE. on kaolin᎐carrageenin-induced hind paw oedema in rats a Drug

Control ŽSaline, 1 mlrkg. DSELE

ASA a

Dose Žmgrkg, p.o..

Oedema volume Žml. 0h

3h

6h

24 h

᎐

108 " 0.13

1.20 " 0.11

1.15 " 0.11

0.61 " 0.07

100 300 1000 300

1.32 " 0.06 1.14 " 0.07 1.16 " 0.11 1.09 " 0.08

1.12 " 0.08 1.07 " 0.08 1.04 " 0.11 UU 0.27 " 0.04

0.90 " 0.09 U 0.83 " 0.09 U 0.79 " 0.08 UU 0.18 " 0.03

0.55 " 0.06 U 0.40 " 0.06 U 0.26 " 0.04 U 0.16 " 0.03

U

Values are mean " S.E.M.; n s 6; P - 0.05;

UU

P - 0.001 vs. control; Student’s t-test.

3.1.3. Nystatin-induced hind paw oedema DSELE at its highest dose Ž1000 mgrkg, p.o.. produced significant inhibition of nystatin-induced oedema from 6 to 72 h of nystatin injection; the inhibition induced by PHB Ž100 mgrkg, p.o.. was greater than that produced by highest dose of DSELE at all time intervals ŽTable 2.. 3.1.4. Cotton-pellet granuloma DSELE at all the doses significantly decreased the granuloma weight, as compared to control animals, the degree of inhibition Žapprox. 30%. being similar to that of the reference drug, PHB ŽTable 3.. 3.2. Acetic-acid-induced ¨ ascular permeability The dye leakage induced by acetic acid was significantly inhibited by DSELE at 300 and 1000 mgrkg, p.o.; the inhibitory activity of ASA Ž300 mgrkg, p.o.. was comparable to that produced by highest dose of DSELE ŽFig. 2.. 3.3. Gastric tolerability 3.3.1. Acute effect Acetylsalicylic acid Ž300 mgrkg, p.o.. produced gastric lesions in 83.33% rats with a mean ulcer index of 2.42 " 0.55. The gastric lesions were associated with accumulation of fluid in stomach, congestion of gastric mucosa, pin-point haemorrhagic spots and a few ulcers. On the contrary, DSELE did not produce any gastric lesion at any of the doses used Ž100, 300 and 1000 mgrkg, p.o... 3.3.2. Chronic effect As in the acute ulcer model, the chronic administration of DSELE Ž100, 300 and 1000 mgrkg, p.o.. did not induce any ulcerogenic effect while ASA Ž300 mgrkg, p.o.. produced ulcers in 100% rats with an ulcer index of 2.33 " 0.25.

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Drug

Control ŽSaline, 1 mlrkg. DSELE

Dose Žmgrkg p.o..

Oedema volume Žml. 2h

4h

6h

24 h

48 h

72 h

᎐

0.51 " 0.08

0.78 " 0.07

0.85 " 0.06

0.83 " 0.07

0.53 " 0.09

0.46 " 0.07

0.46 " 0.03 Ž9.80. 0.40 " 0.06 Ž21.57. 0.43 " 0.06 Ž15.69.

0.68 " 0.08 Ž12.82. 0.71 " 0.09 Ž8.98. 0.63 " 0.05 Ž19.24.

0.82 " 0.12 Ž3.53. 0.73 " 0.07 Ž14.12. 0.58 " 0.04 Ž31.76.UU

0.56 " 0.09 Ž32.53. 0.54 " 0.02 Ž34.94.UU 0.38 " 0.04 Ž54.22.UU

0.35 " 0.05 Ž33.96. 0.33 " 0.04 Ž33.73. 0.22 " 0.04 Ž58.49.UU

0.30 " 0.06 Ž34.78. 0.26 " 0.09 Ž43.48.U 0.23 " 0.05 Ž50.00.U

0.31 " 0.04 Ž39.22.U

0.46 " 0.05 Ž41.03.UU

0.47 " 0.04 Ž44.71.UUU

0.22 " 0.05 Ž73.49.UUU

0.20 " 0.05 Ž62.26.UU

0.19 " 0.05 Ž58.69.U

100 300 1000

PHB

100

a Values are mean " S.E.M. Ž n s 6.; inhibition.

U

P - 0.05,

UU

P - 0.01;

UUU

P - 0.001 vs. control; Student’s t-test. Figures in parentheses indicate percentage

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Table 2 Effect of Dalbergia sissoo ethanolic leaf extract ŽDSELE. on nystatin-induced hind paw oedema in rats a

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137

Table 3 Effect of Dalbergia sissoo ethanolic leaf extract ŽDSELE. on cotton pellet-induced granuloma in rats a Drug

Control ŽSaline, 1 mlrkg. DSELE

PHB a

Dose Žmgrkg, p.o..

Weight of granuloma Žmg.

Cotton pellet granuloma % Inhibition

᎐

79.50 " 7.71

᎐

100 300 1000 100

54.42 " 4.13 U 54.08 " 2.45 U 52.83 " 3.27 U 52.90 " 6.03

U

30.29 31.97 33.57 33.46

U

Values are mean " S.E.M.; n s 6; P - 0.05 vs. control; Student’s t-test.

3.4. Acute toxicity Since DSELE did not show any acute toxic effect or death up to 10.125 grkg, LD50 could not be determined.

4. Discussion The results obtained in the present study provide evidence that the ethanolic extract of D. sissoo leaves ŽDSELE. possesses anti-inflammatory activity. At oral doses of 100, 300 and 1000 mgrkg, DSELE inhibited the oedema induced in rats by carrageenin, kaolin and nystatin. In chronic inflammatory test Žcotton-pellet

Fig. 2. Effect of oral administration of DSELE on acetic acid-induced vascular permeability in mice. Values are mean " S.E.M. Ž n s 6.; U P - 0.05; UU P - 0.01; UUU P - 0.001 vs. control; Student’s t-test. DSELE, D. sissoo ethanolic leaf extract; ASA, acetylsalicyclic acid.

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granuloma., it significantly reduced the weight of granuloma, indicating an effect in the proliferative phase of inflammation. The activity was similar to that of phenylbutazone. Moreover, DSELE reduced the intensity of the peritoneal inflammation produced by acetic acid in mice, indicating its ability to inhibit the permeability of small blood vessels. These results suggest that the anti-inflammatory activity of DSELE may involve an inhibitory effect against autacoids Žhistamine, serotonin, kinins or prostaglandins. or a stabilisation of lysosomal membranes. The inflammatory response induced by carrageenin is due to prostaglandins release through arachidonic acid metabolism via cyclooxygenase pathway w8x. Thus, the antiinflammatory activity of D. sissoo may be due to inhibition of the synthesis and release of prostaglandins. This hypothesis may be supported by the fact that a related species, D. odorifera, significantly inhibited prostaglandin biosynthesis as well as platelet aggregation induced by arachidonic acid w16,17x. It has been suggested that bioactive compounds such as flavonoids act as anti-inflammatory agents w18x and that the anti-inflammatory properties are a consequence of their inhibitory actions on arachidonic acid metabolism, as demonstrated in vitro and in vivo w19,20x. Bioactive flavonoids that have been found in D. sissoo w5x may be responsible for its anti-inflammatory activity. The main side effect of non-steroidal anti-inflammatory drugs is their ability to produce gastric lesions w21x. DSELE, up to 1000 mgrkg, p.o., was very well tolerated both in acute and chronic Ž14-day. oral administration. The very high LD50 , no mortality, and lack of toxic effects in mice are suggestive of its safety even in high doses. The present results show that DSELE has anti-inflammatory effect in acute, sub-acute and chronic models of inflammation without any side effect on gastric mucosa, thus substantiating the potential medicinal value of D. sissoo leaves as anti-inflammatory agent.

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