PHYTOCHEMICAL AND PHARMACOLOGICAL STUDIES … · ISSN 0975-6299 Vol.1/Issue-4/Oct-Dec.2010 ... PHYTOCHEMICAL AND PHARMACOLOGICAL STUDIES OF ETHANOL ... vivo …

ISSN 0975-6299 Vol.1/Issue-4/Oct-Dec.2010

www.ijpbs.net Pharmacology

272

International Journal of Pharma and Bio Sciences

PHYTOCHEMICAL AND PHARMACOLOGICAL STUDIES OF ETHANOL

EXTRACT OF DALBERGIA SISSOO SEEDS: AN APPROACH FOR THE IN-VIVO

ANALGESIC AND ANTIPYRETIC ACTIVITIES

MALLINATH H. HUGAR1, KALLAPPA M. HOSAMANI*1 AND MD.

LIYAKATH AHMED2

1P.G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar,Dharwad-580 003, India

2Luqman College of Pharmacy and Research, Gulbarga-585102. Karnataka,India

*Corresponding Author [email protected]

ABSTRACT

In the course of search for the biological active constituents present in the alcoholic extract of Dalbergia sissoo seeds (Shisham or Sisam).The peripheral analgesic activity of Sisam seed extract (SSE) was studied using acetic acid-induced writhing in mice and by Randall-Selitto assay in rats. Further, the central analgesic activity of SSE was studied by tail-clip test and hot plate method in mice. The antipyretic activity of SSE was studied in Brewer’s yeast-induced pyrexia in rats. Results showed significant decreased writhing movements in mice by acetic acid-induced writhing test and significant increased in the pain threshold capacity in rats in Randall-Selitto assay and the reaction time in hot-plate test but not in tail-clip test for analgesic activity. Moreover, it also showed significant antipyretic activity in Brewer’s yeast-induced pyrexia in rats throughout the observation period of 6 h. Thus, SSE has moderate analgesic and remarkable antipyretic activities.

KEYWORDS

Dalbergia sissoo; Analgesic activity; Antipyretic activity; Tail-clip test; Hot plate method; Brewer’syeast-induced pyrexia.

INTRODUCTION

Dalbergia sissoo Roxb. belongs to Leguminoseae plant family which is native to India and had been long cultivated in Egypt has shade tree on the banks of irrigation canals [1]. It is a large deciduous tree, often with crooked trunk and light crown. Under favorable conditions the tree attains a height of about 100 ft and girth up to 8 ft. It grows well on porous soils containing sand, pebbles and boulders. It reaches its finest development in the riverain tracts [2].

Dalbergia sissoo includes many

members which are broadly used in folk medicine for several diseases [3-7]. Current literature [8,9] reveals that the isolation of several compounds of confirmed biological activity such as flavones, isoflavones, quinines and coumarins from Dalbergia sissoo. It also contains tectoridin, caviunin-7-O- glucoside, iso-caviunin, tectorigenin,dalbergin, bio-chanin-A, and 7-hydroxy -4-methylcoumarin. The heartwood gave 3,5-dihydroxy-trans-stibene,



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biochanin A, dalbergichromene, dalbergenone and iso-dalbergin [10-13].

The concentrated extract of heartwood in milk was prescribed in fevers; extract of leaves in jaundice, bark extract is used as anti-inflammatory agent in piles, sciatica, and as blood purifier. The oil was used externally in the skin diseases and infected ulcers [13]. The wood exhibits alterative, stomachic, anthelmintic, antileprotic and cooling properties. It was found useful in scalding of urine. Arial parts showed significant spasmolytic activity. Further more, it is mainly used as aphrodisiac, abortifacient, expectorant, anthelmintic, Dalbergia sissoo leaves extract has marked analgesic and antipyretic activity [7] and also it is used in the treatment of emesis, ulcers, arthritis, leucoderma, dysentery, stomach troubles and skin diseases [3,14]. Many authors have reported the isolation of several compounds of confirmed biological activity for Dalbergia sissoo leaves, stem bark, heart wood and root bark [15-18].

However, there are no reports on the in-vivo biological activities of the ethanolic extract of Dalbergia sissoo seeds. Hence, this work has been launched to study the pharmacological activities of Dalbergia sissoo seeds. Therefore, we have reported for the first time in-vivo analgesic and antipyretic activities of ethanolic extract of Dalbergia sissoo seeds.

2. RESULTS AND DISCUSSION

The peripheral analgesic activity of SSE was investigated by the acetic acid induced writhing test in mice [19] and Randall-Selitto assay in rats [20] (Randall-Selitto apparatus, Ugo Basile, Italy) . The writhing movements were observed and counted for 20 min after acetic acid administration .The central analgesic activities were assessed by hot-plate method [21] and tail-clip test [22] in mice. The antipyretic activity of SSE was evaluated using brewers yeast–induced pyrexia in rats. The antipyretic activity of SSE may be related to the inhibition of prostaglandin synthesis in

hypothalamus. In conclusion, the present study demonstrates that SSE has marked moderate analgesic activities and significant antipyretic activities.

Thus, SSE was used in the doses of

100, 300 and 1000 mg/kg orally in the study test. Aspirin (300 mg/kg, p.o.) and pethidine (5 mg/kg, i.p.) were used as standard drugs for comparing analgesic effects at peripheral and central levels, respectively. Six animals were used in each treatment group. Where as SSE (1000mg/kg) and Aspirin (300 mg/kg) showed significant antipyretic activity throughout the observation period up to 6 h. The analgesic activity of SSE was studied for central (narcotic) in hot plate method in mice and peripheral (non narcotic) activity in acetic acid induced writhing test in mice. The analgesic activity of SSE against acute inflammatory pain was moderate as compared to potent inhibitory activity of aspirin. Aspirin and indomethacin offer relief from inflammatory pain by suppressing the formation of pain substances in the peripheral tissues, where prostaglandins and bradykinin were suggested to play an important role in the pain process.

Therefore, it is likely that SSE might suppress the formation of these substances or antagonize the action of these substances and thus exerts its analgesic activity in acetic acid-induced writhing test in mice and in Randall-Selitto assay in rats. In the present study, SSE (1000 mg/kg) significantly increased the reaction time in hot-plate test, suggesting its central analgesic activity. However, it did not show centrally-mediated analgesic effect in tail-clip test. Presence of flavonoids was reported in Dalbergia species and flavonoids are known to inhibit prostaglandin synthetase [23]. Since prostaglandins are involved in pain perception and are inhibited by flavonoids, it could be suggested that reduced availability of prostaglandins by flavonoids of SSE might be responsible for its analgesic activity. It appears that antipyretic activity of SSE may be related to the inhibition of prostaglandin synthesis in hypothalamus.



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3. CONCLUSION

The present study demonstrates that, SSE significantly decreased the writhing movements in mice in acetic acid-induced writhing test. SSE (1000 mg/kg) significantly increased the pain threshold capacity in rats in Randall-Selitto assay and the reaction time in hot-plate test but not in tail-clip test. SSE also showed significant antipyretic activity in Brewer’s yeast-induced pyrexia in rats throughout the observation period of 6 h. SSE has marked moderate analgesic and significant antipyretic activities.

4. EXPERIMENTAL 4.1 Materials and Methods 4.1.1 Preparation of D. sissoo seed extract (SSE)

Dalbergia sissoo seeds were collected from the forest origin. The air-dried seeds (5 kg) were powdered and extracted with 95 % ethanol by heating under reflux yielded 10 % of ethanol extract (500 g). Then, the ethanol extract was concentrated to dryness in a rotary evaporator under reduced pressure and controlled temperature (40-50 oC). The concentrated ethanol extract was suspended in distilled water and fractionated by the light petroleum ether (40-60 oC), chloroform and acetic acid. The collected fractions were separately concentrated to yield the reddish brown semi solid ethanol extract (400 g). Then, the obtained residue was subsequently washed with chloroform and ethyl acetate to get the separated greenish powder solid material. This was found to be amorphous having light green colour. Thus, the ethanol extract contains flavonoids, isoflavonoids and neoflavonoids as its main constituents which was determined by gas chromatographic analysis. The characterization of constituents of ethanol extract is under progress.

This has generated much interest in the ethanol extraction of Dalbergia sissoo seeds,

their by to explore analgesic and antipyretic activities.

4.1.2 Experimental animals

Albino-Swiss mice weighing (20-25 g) and albino-Wistar rats weighing (150-200 g) were used for studying acute toxicity. Animals were maintained under standard laboratory conditions. Study protocol was approved by the institutional Animal Ethics Committee (IAEC, Reg. No. 346/ CPCSEA: Dated. 03-01-2001) before experiment.

Albino-Swiss mice and albino Wistar rats from Laboratory Animal Resource Section, Dept of pharmacology, Luqman College of pharmacy, Gulbarga were used in the study. The animals were kept in polypropylene cages and maintained on balanced ration with free access to clean drinking water.

4.1.3 Analgesic effect of SSE

The peripheral analgesic effect of SSE was investigated by acetic acid-induced writhing test in mice, and Randall-Selitto assay in rats (Table 1). The writhing movements were observed and counted for 20 min after acetic acid administration .SSE was used in doses 100, 300 and 1000 mg/kg, p.o.in the study. Aspirin (300mg/kg p.o) and Pethidine (5mg/kg i.p) were used as standard drugs for comparing analgesic effect at peripheral and central levels respectively. Six animals were used in each treatment group. In Randall-Selitto assay pain threshold capacity increased significantly (P<0.01) at the dose of 1000 mg/kg at 1 h. Aspirin, significantly increased the pain threshold throughout the observation period of 1 to 3 h (Table -1). The results of hot-plate test indicated a significant increase in reaction time at 2 and 3 h with 1000 mg/kg SSE, whereas reference drug pethidine hydrochloride significantly increased the reaction time at 1 and 2 h (Table 2 & Figure 1,3,4,5). In tail clip test, SSE failed to alter the reaction time significantly even at the highest dose (1000 mg/kg) employed in the present study.



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Fig. 1 Determination of central analgesic activity by hot plate method

4.1.4 Antipyretic activity of SSE

The antipyretic activity of SSE was evaluated using Brewer’s yeast-induced pyrexia in rats [24]. Fever was induced by injecting 20 ml/kg (s.c.) of 20 per cent aqueous suspension of Brewer’s yeast in normal saline below the nape of the neck and rectal temperature was recorded by clinical thermometer immediately before (-18 h) and 18 h after (0 h) Brewer’s yeast injection. Prior to the experiment, the rats were maintained in separate cages for 7 days and the animals with approximately constant rectal temperature

were selected for the study. Aspirin (300 mg/kg, p.o.) was used as standard drug for comparing the antipyretic action of SSE. The experimental rats showed a mean increase of about 0.86 oC in rectal temperature, 18 h after Brewer’s yeast injection. SSE at 100 and 300 mg/kg produced significant (P<0.05 and P<0.01, respectively) antipyretic activity at 1 h after drug administration, whereas SSE (1000 mg/kg) and aspirin (300mg/kg) showed significant anti pyretic activity throughout the observation period up to 6h (Table 3 & Figure 2,6).

Fig. 2 Administration of aqueous suspension of Brewers yeast in normal saline to induce

fever 4.1.5 Statistical analysis



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The results are presented as mean ± SEM. Statistical analysis of data was performed using Student’s‘t’ test to study the differences amongst the means [25]. Aspirin and indomethacin offer relief from inflammatory pain by suppressing the formation of pain substances in the peripheral tissues, where as prostaglandins and bradykinin were suggested to play an important role in the pain process [26]. It is well known that most of the anti-inflammatory analgesic drugs possess antipyretic activity. SSE revealed weak antipyretic effect at low doses (100 and 300 mg/kg) but at higher dose (1000 mg/kg), it produced marked antipyretic activity in Brewer’s yeast induced febrile rats. In general, non-steroidal anti-inflammatory drugs produce their antipyretic action might be responsible for its analgesic effect. However, non-steroidal anti-inflammatory drugs produce their action

through inhibition of prostaglandin synthetase within the hypothalamus [27,28]. Therefore, it appears that antipyretic action of SSE may be related to the inhibition of prostaglandin synthesis in hypothalamus. In conclusion, the present study demonstrates that SSE has marked significant antipyretic and moderate analgesic activities.

ACKNOWLEDGEMENTS This research work is financially

supported by the Council of Scientific & Industrial Research (CSIR), New Delhi - 110 012. (Ref. No. 01(2301)/09/EMR-II dated 19-03-2009). The authors are thankful to Chairman and Principal of Luqman College of pharmacy, Gulbarga, for providing necessary facility to carryout this research work.

writhing

0

10

20

30

40

50

60

0gm 100gm 300gm 1000gm

DOSE

No.of writhing

writhing

Fig. 3

Analgesic effect of SSE on acetic-acid induced writhing in mice per 20 min

pressure

0

20

40

60

80

100

120

0gm 100gm 300gm 1000gm

DOSE

Pressure on paw

pressure

Fig. 4

Analgesic effect of SSE by Randall-Selitto assay in rats. Pressure on paw in gm at 1hr.



277

reacn time

0

2

4

6

8

10

12

14

16

18

20

0gm 100gm 300gm 1000gm 5gm drug

DOSE

Reaction time in sec at 3hr

reacn time

Fig. 5 Effect of SSE on thermic stimulus-induced pain (hot plate test) in mice

rectaltemp

36

36.5

37

37.5

38

38.5

39

0gm

100gm

300gm

1000gm

300gmdrug

DOSE

Rectal temp in degree

celceous

rectaltemp

Fig. 6 Effect of SSE on Brewer’s yeast-induced pyrexia in rats at 6 hrs



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Table 1

Analgesic effect of SSE on acetic-acid induced writhing in mice and Randall-Selitto assay in rats

Randall-Selitto assay

Pressure on paw in gm

Drug Dose

(mg/kg)

Writhing test

No. of

writhings

(per 20 min) 0h 1h 3h

Control - 52.17 ± 3.28 49.17 ± 6.11 55.17 ± 7.63 57.67 ± 6.15

SSE 100 33.33 ± 3.67** 55.17 ± 7.35 71.0 ± 7.26 72.66 ± 6.94

300 32.16 ± 3.75** 56.00 ± 9.83 70.83 ± 12.66 68.50 ± 8.48

1000 27.17 ± 3.98*** 61.67 ± 5.43 109.33 ± 8.33** 72.83 ± 11.92

Aspirin 300 13.17 ± 2.52*** 63.50 ± 4,22 161.83 ±15.15*** 88.50± 10.72*

n: six animals in each group; Values are mean ± SEM.

* P<0.05, ** P<0.01, *** P<0.001 when compared to control

Table 2

Effect of SSE on thermic stimulus-induced pain (hot plate test) in mice

Reaction time in sec at time (h) Drug Dose

(mg/kg) 0h 0.5h 1h 2h 3h

Control - 9.25 ± 1.56 9.69 ± 1.06 9.20 ± 0.78 13.28 ± 1.11 12.85 + 1.46

SSE 100 10.0 ± 1.56 8.22 ± 0.74 10.48 ± 0.74 15.12 ± 1.30 12.69 ± 1.30

300 9.39 ± 1.17 10.16 ± 1.25 12.17 ± 1.81 16.90 ± 1.05 14.86 ± 2.07

1000 9.51 ± 0.82 11.57 ± 1.06 11.32 ± 0.74 18.67 ± 1.65* 17.96 ± 1.65*

Pethidine 5 10.33 ± 1.05 11.64 ± 1.27 17.59 ± 3.02* 19.34 ± 1.51** 15.98 ± 0.94


*P<0.05, ** P<0.01, *** P<0.001 when compared to control



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Table 3

Effect of SSE on Brewer’s yeast-induced pyrexia in rats

Rectal temperature in oC at time (h) Drug Dose

(mg/kg)

-18

a 0

b 1h 3h 5h 6h

Control - 37.73 ±

0.15

38.49 ± 0.08

(+0.76)c

38.71 ± 0.11 38.40 ± 0.08 38.36 ± 0.12 38.46 ± 0.13

SSE 100 37.43 ±

0.11

38.33 ± 0.08

(+0.90)c

38.18 ±

0.15*

38.18 ± 0.21 37.95 ± 0.26 37.97 ± 0.24

300 37.71 ±

0.09

38.55 ± 0.12

(+0.84)c

37.93 ±

0.17**

38.08 ± 0.18 38.06 ± 0.24 37.95 ± 0.24

1000 37.68 ±

0.06

38.63 ± 0.11

(+0.95)c

38.08 ±

0.15**

38.11 ±

0.07*

37.81 ±

0.11**

37.68 ±

0.08***

Aspirin 300 37.75 ±

0.11

38.58± 0.24

(+0.83)c

37.48±

0.14***

37.00±

0.11***

36.93±

0.11***

37.03±

0.11***


* P<0.05, ** P<0.01, *** P<0.001 when compared to control.

a temperature just before yeast injection

b temperature just before drug administration

c change in temperature following yeast injection

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PHYTOCHEMICAL AND PHARMACOLOGICAL STUDIES … · ISSN 0975-6299 Vol.1/Issue-4/Oct-Dec.2010 ... PHYTOCHEMICAL AND PHARMACOLOGICAL STUDIES OF ETHANOL ... vivo …