Tropical Journal of Pharmaceutical Research Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria ISSN: 1596-5996 EISSN: 1596-9827 Vol. 8, Num. 2, 2009, pp. 133-137

Tropical Journal of Pharmaceutical Research, Vol. 8, No. 2, June 2009, pp. 133-137

Research Article

Arjun Patra^1* , Shivesh Jha², P. Narasimha Murthy³, Aher Vaibhav D.¹, Pronobesh Chattopadhyay¹, Ghanshyam Panigrahi³ & Devdeep Roy⁴

¹College of Pharmacy, IFTM, Moradabad-244 001, U.P.,
²Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra-835 215, Ranchi, Jharkhanda,
³Royal College of Pharmacy & Health Sciences, Berhampur-760 -002, Orissa, India,
⁴ Master of Research Biomedical Sciences, Department of Immunology, University of Strathclyde, Glasgow, Scotland.
*Corresponding author: E-mail: arjun.patra@rediffmail.com; Tel: +919761459749; +919359526128

Received: 19 July 2008 Revised accepted: 23 November 2008

Code Number: pr09018

Abstract

Purpose: Hygrophila spinosa T. Anders (Acanthaceae) is commonly used in the traditional system of medicine for the treatment of inflammation, pain, jaundice, rheumatism, arthritis, anaemia, etc. In the present study, we investigated the anti-inflammatory and antipyretic activities of the petroleum ether, chloroform, alcoholic and aqueous extracts of the leaf of this plant.
Methods: The anti-inflammatory activity of the various extracts was studied based on their effects on carrageenan-induced paw oedema in rats while antipyretic activity was evaluated on the basis of their effect on Brewer’s yeast-induced pyrexia in rats. The extracts were screened for alkaloids, steroids, proteins, flavonoids, saponins, mucilage, carbohydrates, organic acids, fats and oils.
Results: Preliminary phytochemical screening revealed the presence of alkaloids, steroids, proteins, flavonoids, fats and oils, tannins, mucilage and organic acids in the leaves of H. spinosa. Chloroform and alcoholic extracts of leaves of H. spinosa produced significant (p < 0.05 and p < 0.01) anti-inflammatory and antipyretic activities in a dose-dependent manner. On the other hand, petroleum ether and aqueous extracts did not show significant anti-inflammatory and antipyretic activities. The maximum anti-inflammatory activities produced by chloroform and alcoholic extracts (400 mg/kg) were 33.7% and 47.5%, respectively. These two extracts also reduced elevated body temperature in rats at 200 and 400 mg/kg body weight doses throughout the observation period of 6 h.
Conclusion: Chloroform and alcoholic extracts of H. spinosa leaves have anti-inflammatory and antipyretic activities.

Keywords: Hygrophila spinosa, anti-inflammatory activity, antipyretic activity, leaf extracts

Introduction

Hygrophila spinosa T. Anders (Acanthaceae) is commonly found in water-logged areas throughout India¹. The plant is used as a diuretic and for the treatment of rheumatism, jaundice, inflammation, pain, hepatic obstruction, gout, bacterial infection etc^2-6. The aerial parts of the plant are reported to contain lupeol, stigmasterol and butelin while the seeds mainly contain fatty acids⁷. Its root contains an alkaloid named hygrosterol⁸ while its flower contains apigenin 7-o-glucuronide⁹. However, no data were found regarding the pharmacological and phytochemical evaluation of the leaves of the plant. The aim of the present study is to investigate the antiinflammatory and antipyretic properties of the petroleum ether, chloroform, alcoholic and aqueous extracts of the leaves of H. spinosa.

Material and Methods

Tween 80 (Lobachem, India), Indomethacin (MicroLab, India), carrageenan (Sigma), Brewer’s yeast (Tetragon Chemie), and paracetamol (GlaxoSmithKline) were used in the study.

Plant material

The leaves of H. spinosa were collected from Berhampur, Orissa, India. The plant was identified by Dr. N. K. Dhal, Scientist, Regional Research Laboratory, Bhubaneswar, India and a voucher specimen (no. 9999) was preserved for further references.

Preparation of extracts

The leaves were washed thoroughly, dried under a shade and pulverized. The coarse powder was extracted successively with petroleum ether, chloroform and alcohol using a soxhlet apparatus. Finally, the aqueous extract was prepared by decoction. The extracts were dried using a rotary vacuum evaporator and stored in a desiccator until further use.

Animals

Wistar rats of both sexes, weighing 150 – 200 g were used for the study. The animals were kept in polypropylene cages in a room maintained under controlled atmospheric conditions. The animals were fed with standard diet (Hindustan liver, Mumbai, India) and had free access to clean drinking water. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Royal College of Pharmacy and Health Sciences Berhampur, Orissa, India.

Anti-inflammatory activity

The anti-inflammatory activity of the extracts was determined according to the method of Borgi et al¹⁰ and Vogel et al¹¹. The rats were divided into ten groups of six rats each. The control group received 1% (v/v) Tween 80 in water, p.o. at a dose of 10 ml/kg. The positive control group was treated orally with the standard drug, indomethacin (10mg/kg). Different extracts were administered to the other groups in doses of 200 and 400 mg/kg as shown in Table 1. All the suspensions were administered 30 min before the induction of oedema by administering 0.1 ml of 1% w/v carrageenan in saline^12,13. The degree of paw oedema of all the groups was measured using a plethysmometer at 30, 60, 120, 180 and 240 min after the administration of carrageenan to each group.

Antipyretic activity

Animals were selected for the experiment after confirmation of approximate constant rectal temperature for 7 days. The antipyretic activity of the extracts was evaluated based on Brewer’s yeast-induced pyrexia in rats^14,15 . Pyrexia was induced by subcutaneous injection of 10 ml/kg of 15% w/v Brewer’s yeast suspension below the nape of the neck. The rectal temperature of each rat was measured at time, 0 h, using a telethermometer and before injection of the yeast. At 18 h following yeast injection, the different groups were treated with the vehicle, extracts (200 and 400 mg/kg) and standard drug, paracetamol (150 mg/kg). The rectal temperature was then recorded over a period of 6 h.

Statistical analysis

The results were expressed as mean ± S.E.M. Statistical analysis of the data were carried out using Student’s t-test and results were considered significant when p < 0.05.

Results

The chloroform and alcoholic extracts of H. spinosa produced significant (p < 0.05) antiinflammatory activity, while petroleum ether and aqueous extracts did not. Significant reduction of paw oedema was observed 30 min and 3 h after carrageenan injection, for alcoholic and chloroform extracts, respectively. The reduction in carrageenaninduced paw oedema by 400 mg/kg of chloroform and alcoholic extracts after 4 h was 43.7 and 47.5%, respectively, while oedema reduction by the standard drug, indomethacin (10 mg/kg) was 53.7% (see Table 1).

Antipyretic activity

Chloroform and alcoholic extracts produced significant antipyretic activity (p < 0.05), but petroleum ether and aqueous extracts did not. Chloroform extract significantly decreased the elevated rectal temperature 3 h after the administration of a dose of 400 mg/kg only, while the alcoholic extract reduced the hyperthermia at both 200 and 400 mg/kg doses 1 h after administration. The initial and final rectal temperatures in the groups treated with chloroform extract (400 mg/kg), alcoholic extract (400 mg/kg) and paracetamol (150 mg/kg) were 38.03 ± 0.16 and 37.41 ± 0.26, 38.55 ± 0.14 and 37.81 ± 0.19, and 38.70 ± 0.15 and 37.87 ± 0.18 C, respectively. Paracetamol and alcoholic extract showed significant antipyretic activity throughout the test period of 6 h (see Table 2).

Discussion

Carrageenan-induced paw oedema is a commonly used primary test for the screening of new anti-inflammatory agents and is believed to be biphasic¹⁶. The first phase (1-2 hr) is due to the release of histamine or serotonin and the second phase of oedema is due to the release of prostaglandin^17,18 . The results of this study indicate that the chloroform and alcoholic extracts of H. spinosa significantly reduced carrageenaninduced paw oedema in rats. Therefore, the mechanism of action may be by inhibition of histamine, serotonin or prostaglandin synthesis.

Usually most anti-inflammatory and analgesic drugs possess antipyretic activity. In general, non-steroidal anti-inflammatory drugs produce their antipyretic action through the inhibition of prostaglandin synthetase within the hypothalamus¹⁹. Therefore, the antipyretic activity of chloroform and alcoholic extracts of H. spinosa is probably by inhibition of prostaglandin synthesis in hypothalamus.

Preliminary phytochemical screening revealed the presence of alkaloids, flavonoids, steroids in the chloroform extract, as well as alkaloids, flavonoids, tannins and steroids in the alcoholic extract of the leaves of H. spinosa²⁰. The anti-inflammatory and antipyretic potentials of alkaloids, steroids and flavonoids have been reported in various studies^21-23. Therefore, the anti-inflammatory and antipyretic activities of the chloroform and aqueous extracts may be due to the presence of alkaloids, sterols and flavonoids.

Conclusion

The results of the present study indicate the anti-inflammatory and antipyretic activities of the leaves of H. spinosa. However, further investigations are required to isolate the active constituents responsible for these activities and to elucidate the exact mechanisms of action.

Acknowledgment

The authors are thankful to Dr. N. K. Dhal, Regional Research Laboratory, Bhubaneswar, India for the identification of the plant.

References

1. The Ayurvedic Pharmacopoeia of India. Part I, vol. II, 1^st edn., Govt. of India, Ministry of Health & Family Welfare, Department of ISM & H, Delhi, The Controller of Publications, 1999, pp 88-94.
2. Sharma PC, Yelne MB, Dennis TJ. Database on medicinal plants used in ayurveda. vol. 4, New Delhi, Central Council for Research in Ayurveda & Siddha, 2002, pp 320-331.
3. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. New Delhi, CSIR, 1986, pp 29.
4. Nadkarni KM. Indian Materia Medica. Bombay, India, Popular Prakashan, 1978, pp 667-669.
5. Mazumder UK, Gupta M, Maiti S, Mukherjee D. Antitumor activity of Hygrophila spinosa on Ehrlich ascites carcinoma and sarcoma-180 induced mice. Indian J Exp Biol 1997; 35: 473-477.
6. Boily Y, Vanpuyvelde L. Screening of medicinal plants of Rwanda (Central Africa) for antimicrobial activity. J Ethnopharmacol 1986; 16: 1-13.
7. Quasim C, Dutta NL. Chemical investigation of Asteracantha longifolia Nees. J Indian Chem Soc 1967; 44: 82-83.
8. Usha K, Kasturi GM, Hemlatha P. Hepatoprotective effect of Hygrophila spinosa and Cassia occidentalis on carbon tetrachloride induced liver damage in experimental rats. Indian J Cli Biochem 2007; 22: 132-135.
9. Balraj P, Nagaraj S. Apigenin 7-oglucuronide from the flowers of Asteracantha longifolia Nees. Indian Drugs 1982; 19: 150-152.
10. Borgi W, Ghedira K, Chouchane N. Antiinflammatory and analgesic activities of Zizyphus lotus root barks. Fitoterapia 2007; 78: 16-19.
11. Vogel HG, Vogel WH. Drug Discovery and Evaluation. Verlag Berlin Heidelberg, New York, Springer, 1997, pp 390-417.
12. Akindele AJ, Adeyemi OO. Antiinflammatory activity of the aqueous leaf extract of Byrsocarpus coccineus. Fitoterapia 2007; 78: 25-25.
13. Dimo T, Fotio AL, Nguelefack TB, Asongalem EA, Kamtchouing P. Antiinflammatory activity of leaf extracts of Kalanchoe crenata Andr. Indian J Pharmacol 2006; 38: 115-119.
14. Jain BB, Rathi BS, Thakurdesai PA, Bodhankar SL. Antipyretic activity of aqueous extract of leaves of Cocculus hirsutus. Indian J Nat Prod 2007; 23: 26-29.
15. Metowogo K, Agbonon A, Eklu-Gadegbeku K, Aklikokou AK, Gbeassor M. Anti-ulcer and Antiinflammatory Effects of Hydro-alcohol Extracts of Aloe buettneri A. Berger (Lilliaceae). Trop J Pharm Res 2008; 7: 907-912.
16. Vinegar R, Schreiber W, Hugo R. Biphasic development of carrageenan oedema in rats. J Pharmacol Exp Ther 1960; 166: 96-103.
17. Britto ARMS, Antonio MA.Oral anti-inflammatory and anti-ulcerogenic activities of a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia (Turneraceae). J Ethnopharmacol 1998; 61: 215-228.
18. Saha A, Masud MA, Bachar SC, Kundu JK, Datta BK, Nahar L, Sarker SD. The analgesic and antiinflammatory activities of the extracts of Phyllanthus reticulatus. Pharmaceutical Biology 2007; 45: 335-359.
19. Hayare SW, Chandra S, Tandan SK, Sarma J, Lal J, Telang AG. Analgesic and antipyretic activities of Dalbergia sissoo leaves. Indian J Pharmacol 2000; 32: 357-360.
20. Patra A, Jha S, Murthy PN, Roy D, Sahu AN. Analgesic and antimotility activities of Hygrophila spinosa T. Anders. Pharmacologyonline 2008; 2: 821-828.
21. Mossa JS, Tariq M, Mohsin A, Ageel AM, Al-yahya MA, Al-said MS, Rafatullah S. Pharmacological studies on aerial parts of Calotropis procera Am j Chin med 1991; 19: 223-231.
22. Singh RK, Acharya SB, Bhattacharya SK. Pharmacological activity of Elaeocarpus sphaericus. Phytother Res 2000; 14: 36-39.
23. Al-said MS, Tariq M, Al-yahya MA, Rafatullah S, Ginnawi OT, Ageel AM. Studies on Ruta chalepensis, an ancient medicinal herb still used in traditional medicine. J Ethnopharmacol 1990; 28: 305-312.

© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.

The following images related to this document are available:

Photo images