Pharmacological studies of Stem Bark Extracts of Zanthoxylum tetraspermum Wight and Arn

V.R. Ravikumar¹*, V. Gopal² and T. Sudha³

¹Research Scholar, Sastra University Thanjavur. Tamil Nadu.

Department of Pharmacognosy, The Erode college of Pharmacy. Erode.

²Mothertheresa Post Graduate Research Institute of Health Sciences, Pondicherry.

³Department of Pharmaceutical Analysis, The Erode College of Pharmacy and Research, Erode-638112. Tamil Nadu.

*Corresponding Author E-mail: ravisrkumar@yahoo.com

ABSTRACT:

Present study was undertaken to examine anti inflammatory, analgesic and wound healing activity of ethanolic and aqueous extracts of Zanthoxylum tetraspermum Wight and Arn. Ethanolic and aqueous extracts shows significant anti inflammatory (Carrageenan induced pawn oedema model), analgesic (Hot plate and acetic acid induced model) and wound healing (Excision wound model) activity when compared to the standard like Indomethacin (10mg/kg p.o). Pentazocine, (30mg/kg, i.p.) Indomethacin (5mg/kg), metrozyl gel. respectively. Ethanolic extract shows more significant activities when compared to aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn at different dose level.

KEYWORDS: Zanthoxylum tetraspermum Wight and Arn, analgesic, anti-inflammatory, wound healing, Aqueous and Ethanolic extract.

INTRODUCTION:

Zanthoxylum tetraspermum Wight and Arn (Rutaceae) is a thorny, stout, aromatic, climbing shrub, with brown bark having short recurved prickles, found in the Western Ghats in the Nilgiri and Annaimalai hills and in Kolli hills (Namakal District, TN) at attitudes of 1,200 1,800m and in Kerala and Karnataka. The wood is yellowish and soft. The plant is credited in Sri Lanka with stimulant, astringent and digestive properties and is prescribed in dyspepsia and diarrhoes^1-3. 8-acetonyldihydronitidine, 8- acetonyldihydroavicine, Liriodenine, sesamin, Lichexanthone, (+) - piperitol-gamma- dimethylallayl ether have been reported⁴. The present investigation was undertaken to examine analgesic, anti-inflammatory and wound healing effects of ethanolic and aqueous extract of Zanthoxylum tetraspermum Wight and Arn to validate ethno medical review.

MATERIAL AND METHODS:

Plant material

The stem bark of Zanthoxylum tetraspermum Wight and Arn were collected from Cholakkadu, a village of Kolli hills, 60km away from Namakkal (TN) in the month of August (2007).

The plant was identified by local people of that village and authenticated by G.V.S. Murthy, Join Director, Botanical Survey of India, Southern Circle, Coimbatore, (No. Bsi/sc/5/23/ 07-08/ Tech 715). A herbarium specimen of the plant was preserved in the department of Pharmacognosy of our institute for further references.

Chemicals

All the reagents used were of analytical grade obtained from S.D. Fine Chemicals Ltd, Mumbai.

Preparation of stem bark extracts

The stem barks of Zanthoxylum tetraspermum Wight and Arn were washed with water, air- dried at room temperature and then reduced to coarse powder. The dried powder (250mg) was subjected to soxhelt extraction with petroleum ether (40-60), chloroform, ethyl acetate, ethanol and aqueous (in order of increasing polarity) for continuous hot extraction. The extracts were filtered and the filtrates were concentrated under reduced pressure to obtain the extracts as solid residues. The percentage extraction values (% w/w) were 1.25, 1.37, 1.96, 3.75 and 4.06% respectively. The freshly prepared extracts were chemically tested for the presence of different constituents using standard methods^5-8.

Anti-Inflammatory activity:

Carrageenan-Induced Paw Edema in rats

For this experiment, the male rats (150-200g) were divided into eight groups (n = 6). The first group received 0.5% CMC (10ml/ kg p.o.), while the second group received Indomethacin (10mg/kg p.o). The third, fourth and fifth groups were treated with ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn (100, 200 and 400 mg/ kg p.o.) respectively. The sixth, seventh and eighth groups were treated with aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn (100, 200 and 400 mg/ kg p.o.) respectively. Acute inflammation was produced by the sub plantar administration of 0.1 ml of 1% Carrageenan (in 1% CMC w/v) in the right hind paw of the rats. The paw thickness was measured at 0 hr, 1/2 hr, 1 hr, 2 hr and 4 hr after Carrageenan injection by using Plethysmometer. The animals were pretreated with the test drugs 1 hour before the administration of Carrageenan⁹.

Analgesic Activity:

Eddy’s Hot plate method in mice

The hot plate assay method was employed for the purpose of preferential assessment of possible centrally mediated analgesic effects. The central analgesic drug, Pentazocine, was used for positive control group. In this experiment, eight groups (n=6) of Swiss albino mice (20–25 g) were placed on a hot plate maintained at room temperature for 15 Sec. Food was withdrawn on the preceding night of the experiment. Group-1 served as normal control (0.5% CMC p.o.), and group‑2 received Pentazocine (30mg/kg, i.p.) which was severed as reference standard. Groups 3, 4 and 5 animals were treated with ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn (100, 200 and 400 mg/ kg p.o.) respectively. The groups 6, 7 and 8 animals were treated with aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn (100, 200 and 400 mg/ kg p.o.) respectively. Each animal was then individually placed gently on Eddy’s hot plate at 55^oC. Latency to exhibit nociceptive responses such as licking paws or jumping off the hot plate, were determined one hour after administration of the test drugs or vehicle¹⁰

Acetic acid induced writhing response in mice

This method was used to preferentially evaluate possible peripheral effects of the ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn as analgesic. Groups of 8 Swiss albino male mice (n=6) were fasted overnight prior to the start of the experiment, with free access to water. The peripheral analgesic drug, Indomethacin (5mg/kg), was used as a positive control. Group-1 served as normal control (0.5% CMC p.o), and group‑2 administered with Indomethacin (5mg/kg, p.o.), whereas groups 3, 4 and 5 received ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn (100, 200 and 400 mg/ kg p.o.) respectively. The groups of 6, 7 and 8 animals were treated with aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn (100, 200 and 400 mg/ kg p.o.) respectively. 30 min after treatment, the mice were injected intra peritoneally with 0.1 ml of 1% acetic acid solution to induce the characteristic writhing. 5 min after acetic acid administration, the mice were then placed in an observation box, and the number of writhing was counted in a 5min period. The response of the extract and Indomethacin treated groups were compared with those of animals in the control group¹¹

Wound healing activity:

Excision model

Randomly collected rats of both sexes, weighing between 150-200gm were selected for the study. The animals were divided into six groups of six in each and are placed in different cages. A circular wound of about 500 sq mm was made on depilated dorsal thoracic region of rats under light ether anesthesia in aseptic condition and observed throughout the study. Animals were housed individually. Group-I animals are applied with ointment base. Group II applied with metrozyl gel. Group III and IV applied with 200 and 400 mg of ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn incorporated in ointment base. Group V and VI applied with 200 and 400 mg of aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn incorporated in ointment base. All the test drugs were applied on the wounded area twice daily. Wound area was measured on 1^st 2^nd, 4^th, 8^th and 16^th post wounding days. % of wound contraction was calculated from the day of measurement of wound area.¹²

Statistical Analysis:

All the results were expressed as mean ± standard error mean (S.E.M.). Data were analyzed using one-way ANOVA followed by Dunnett’s t-test. The analysis was carried out using Graph pad software of version 4. p<0.05 was considered as statistically significant.

Table 1. Effect of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn on Carageenan induced paw edema in rats

Drug Treatment	Paw Thickness(mm)
Drug Treatment	0 min	30 min	60 min	120 min	240 min
Control CMC 0.5% W/V	0.29±0.02	0.41±0.01	0.66±0.01	0.49±0.02	0.45±0.04
Indomethacin(10mg/kg)	0.22±0.03	0.29±0.02**	0.41±0.01**	0.37±0.01**	0.12±0.03**
Et Ext(100mg/kg)	0.24±0.03	0.32±0.01*	0.55±0.02**	0.41±0.02**	0.32±0.02**
Et Ext (200mg/kg)	0.23±0.02	0.31±0.01*	0.43±0.02**	0.39±0.01**	0.18±0.01**
Et Ext (400mg/kg)	0.22±0.03	0.30±0.01**	0.42±0.02**	0.38±0.02**	0.13±0.02**
Aq Ext (100mg/kg)	0.25±0.02	0.37±0.01^ns	0.59±0.03^ns	0.43±0.01*	0.39±0.03*
Aq Ext (200mg/kg)	0.24±0.01	0.36±0.01^ns	0.56±0.01*	0.43±0.02*	0.33±0.02**
Aq Ext (400mg/kg)	0.23±0.01	0.32±0.01*	0.50±0.01**	0.40±0.02**	0.32±0.02**

Values are in Mean ± SEM (n=6), *P<0.05 and **P<0.01 Vs Control Group

Figure 1. The Anti inflammatory activity of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn by Carageenan induced paw edema in rats

Figure 2: Effect of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn Hot plate model in mice

RESULTS AND DISCUSSION:

Anti –Inflammatory Activity:

Carageenan-Induced Paw Edema in rats

The anti-inflammatory effect of the ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn were studied on the Carrageenan induced hind paw edema in rats and the results were shown on the table. -1 and figure-1

The ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn at 100 and 200mg/kg showed less significant (P<0.05) anti inflammatory activity only at 30^th minute, but 400mg/kg showed more significant (P<0.01) anti inflammatory activity only at 30^th minute. All the doses of ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn showed more significant anti-inflammatory activity (P<0.01), at 60, 120 and 240^th minutes. Aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn at 100 and 200mg/kg did not showed significant anti inflammatory activity but 400 mg/kg showed significant (P<0.01) anti inflammatory activity until 240 minutes. The reference drug Indomethacin showed significant (P<0.01) anti inflammatory activity. The anti inflammatory effects produced by the ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn at 200, 400mg/kg and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn at 400mg/kg were comparable with that of Indomethacin.

Analgesic Activity:

Hot Plate model

The analgesic effect of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn on hot plate in mice was recorded and the results were shown in table no. 2. and Figure-2.

Table 2. Effect of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn on Hot plate model in mice

S.No.	Drug Treatment	Hot plate reaction time (sec)
1	Control CMC 0.5% W/V	5.00±0.37
2	Pentazocine (30mg/kg)	14.17±0.30^**
3	EtExt (100mg/kg)	10.17±0.30^**
4	EtExt (200mg/kg)	12.00±0.58^**
5	EtExt (400mg/kg)	13.67±0.49^**
6	AqExt (100mg/kg)	6.11±0.49^*
7	AqExt (200mg/kg)	7.11±0.49^*
8	AqExt (400mg/kg)	10.67±0.49^**

Values are in Mean ± SEM (n=6),*P<0.05 and **P<0.01 Vs Control Group

Pentazocine opioid analgesic significantly (P<0.01) increased the pain threshold, when compared to control in thermal pain exhibited by the hot plate in mice. The ethanolic extract at the doses of 100, 200 and 400mg/kg and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn at 400 mg/kg showed on significant (P<0.01) analgesic activity and aqueous of extract of bark of Zanthoxylum tetraspermum Wight and Arn at 100 and 200 mg/kg showed less significant (P<0.05) analgesic activity when compared to ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn and the analgesic activity of both the extracts of bark of Zanthoxylum tetraspermum Wight and Arn were comparable as that of reference drug.

Acetic acid induced writhing response in mice

The results of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn on acetic acid induced writhing in mice and the results were shown on table 3 and Figure-4.

Table 3. Effect of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn acetic acid induced writhing in mice.

S.No.	Drug Treatment	Writhing Response (no/ 5minutes)
1	Control CMC 0.5% W/V	52.00±0.97
2	Pentazocine (30mg/kg)	5.17±0.48^**
3	EtExt (100mg/kg)	6.00±0.57^**
4	EtExt (200mg/kg)	5.19±0.45^**
5	EtExt (400mg/kg)	5.16±0.40^**
6	AqExt (100mg/kg)	21.22±1.78^**
7	AqExt (200mg/kg)	13.46±1.22^**
8	AqExt (400mg/kg)	6.32±0.55^**

Values are in Mean ± SEM (n=6),*P<0.05 and **P<0.01 Vs Control Group

Figure 3. Effect of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn on acetic acid induced writhing in mice.

Table 4. The table shows the wound healing activity and % wound contraction of ethanolic and aqueous extract bark of Zanthoxylumtetraspermum Wight and Arn on excision wound model in rats.

Drug Treatment

Wound Contraction (days)

Control

(Ointment Base)

12.36 ±0.82

(2.47)

50.59±3.26

(10.12)

72.62±5.69

(14.54)

108.81±8.32

(21.76)

192.65±14.45

(38.53)

Metrozyl Gel

26.72±1.16

(5.34)

75.88±5.51*

(15.18)

204.15±10.26**

(40.83)

398.22±18.92**

(79.64)

455.56±24.78**

(91.12)

EtExt (200mg/kg)

19.56±1.97

(3.91)

40.12±2.66

(8.02)

98.56±8.97

(19.71)

256.22±1026

(51.24)**

372.23±22.15

(74.45)**

EtExt(400mg/kg)

24.22±2.13

(4.84)

58.79±4.96

(11.76)

179.86±10.62

(35.97)**

352.45±21.47

(70.49)**

426.31±24.71

(85.26)**

AqExt(200mg/kg)

15.58±0.83

(3.12)

36.59±2.24

(7.32)

89.42±6.78

(17.88)

231.96±17.64

(46.39)*

389.18±19.69

(77.83**

AqExt(400mg/kg)

21.23±1.15

(4.25)

49.81±3.66

(9.96)

168.61±12.45

(33.72)*

315.79±21.21

(63.16)**

400.12±20.14

(80.02)**

Figure 4. The wound healing activity and % wound contraction of ethanolic and aqueous extract of bark Zanthoxylum tetraspermum Wight and Arn on excision wound model in rats.

The control animal showed 52.00 writhing in 5 minutes after acetic acid administration in mice. Indomethacin reference drug reduced the number of writhing induced by acetic acid to 5.17. Both the extract of bark of Zanthoxylum tetraspermum Wight and Arn (100 and 200 and 400 mg/kg) significantly (P<0.01) reduced the acetic acid induced writhing. The effects were equipotent as that of standard drug of Indomethacin.

Wound healing Activity

The wound healing activity of ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn was studied on excision wound model in rats and the results were shown in table-4 and Figure-4.

Metrozyl gel was used as reference standard. The wound contraction was observed for 16 days. There was significant (P<0.001) wound contraction was observed at 400mg of both ethanolic and aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn from 4^th day of treatment. The aqueous extract of bark of Zanthoxylum tetraspermum Wight and Arn showed less wound healing activity when compared to ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn at both the dose level. The ethanolic extract of bark of Zanthoxylum tetraspermum Wight and Arn showed moderate significant wound healing activity and aqueous extract showed significant activity when compared to the standard drug of Metrozyl gel.

CONCLUSION:

This study has revealed that both aqueous and ethanolic extract of bark of Zanthoxylum Tetraspermum Wight and Arn shows significant anti inflammatory, analgesic and wound healing activities. It has further confirmed that the bark extract of Zanthoxylum Tetraspermum Wight and Arn could be used for the treatment of various dental infections. Further research going on our institution to isolate the individual compounds from the same extracts

REFERENCES:

1. Ambasta SP. The Wealth of India, Raw materials, CSIR, New Delhi, Vol XI, X-Z, 1998; pp. 17-25.

2. Chandra YR. The useful plants of India, Publication and Information Directorate, CSIR, New Delhi, 1994; pp.697-699.

3. Hajra PK, Nair VJ and Daniel P. Flora of India. Botanical Survey of India, BSI, Calcutta, Vol 4, 1997; pp. 389-390.

4. Harbone JB Phytochemicals Methods, A guide to modern Techniques of plant analysis, Chapman Hall, London, 1973; pp. 182-189.

5. Nissanka APK, Karunaratathe V and Rathayake Bandara. BM. Antimicrobial alkaloids from Zanthoxylum tetraspermum and caudatum. Phytochemistry 56(8); 2001: 857 – 861.

6. Trease GE and Evans WC Pharmacognosy. ELBS, Publications, London, 12^th, Edn, 1985; pp.126-137.

7. Tyler VE, Brady LR and Robber JE. Pharmacognosy. Lea and Febiger Publication, Philadelphia, 1988; 9th ed: pp. 77-79.

8. Kokate CK. Practical Pharmacognosy, Vallabh Prakashan, New Delhi, 1991; 3rd ed: pp.101-111.

9. Perez S, Meckes M, Perez A and Susunaga MA. Zavala; Anti-inflammatory activity of Lippie dulcis. Journal of Ethno Pharmacology. 102(1); 2005: 1-4.

10. Jeane Silva, Worku Abebeb SM, Sousa VG, Duarte MIL and Machadoc FJA Matos Analgesic and anti inflammatory effects of essential oils of Eucalyptus. Journal of Ethnopharmacology. 89; 2003: 277–283.

11. Mate GS, Naikwade NS, Magdum CS, Chowki AA and Patil SB. Evaluation of anti-nociceptive activity of Cissus quadrangularies on albino mice. International Journal of Green Pharmacy. 2(2); 2008:118-121.

12. Kiranmai M, Kazim SM and Ibrahim M. Combined wound healing activity of Gymnema sylvestere and Tagetes erecta Linn. International Journal of Pharmaceutical Applications. 2(2); 2011: 135-140

Received on 25.09.2012 Modified on 09.10.2012

Research J. Pharm. and Tech. 5(11): Nov. 2012; Page 1396-1401