Abstract
We studied the anti-inflammatory activity of a petroleum ether extract of Phyllanthus singampattiana. Sebastine and A.N. Henry leaves, which was tested in Wistar rats with TPA-induced ear inflammation and carrageenan-induced paw edema. Carrageenan-induced paw edema was significantly reduced at a dose of 100 mg petroleum ether extract (p < 0.05%). From the crude extract, β.-sitosterol was identified and confirmed.
Introduction
Plants are a valuable source of new natural products (Hostetmann, 1999). India has an immense wealth of biodiversity. According to an estimate, there are about 45,000 species of wild plants world wide, and out of this 7500 species are used medicinally in indigenous health practices. In India, a small proportion of wild plants has been investigated both phytochemically and pharmacologically. The genus Phyllanthus. (Euphorbiaceae) is widely distributed in most tropical and subtropical countries. It is a very large genus consisting of approximately 750 species and 10 or 11 subgenera (Unander et al., 1995; Calixto et al., Citation1998). In vivo. and in vitro. studies of anti-inflammatory activity of the genera Phyllanthus emblica. and Phyllanthus fraternus. (Hukeri et al., Citation1985; Asmawi et al., Citation1992; Jantan et al., Citation1996; Ihantola-Vormisto et al., Citation1997) has recently been reported. Phyllanthus singampattiana. Sebastine and A.N. Henry (Euphorbiaceae) is a shrub, endemic to Tirunelveli hills, found in the Courtallam, Kannikatty, Valayar, and Kothaiyar regions of Tirunelveli District, Tamil Nadu, India. The medicinal importance of this species is not yet reported. The current study was undertaken to evaluate the anti-inflammatory activity of this plant extract on TPA-induced rat ear edema and carrageenan-induced rat paw edema in Wistar rats.
Materials and Methods
Animals
Six Wistar rats weighing 25–35 g were used in the current experiment. All animals were maintained in suitable nutritional and environmental conditions throughout the experiments.
Plant material
The leaves of P. singampattiana. were collected from Upper Kothaiyar, Kalakad-Mundanthurai Tiger Reserve (KMTR), Tirunelveli. A voucher specimen (no. XCH. 16711) was deposited in the Centre for Biodiversity and Biotechnology, St. Xavier's College (Autonomous), Palayamkottai, Tamil Nadu, India.
Extraction and isolation
Air-dried and powdered leaves of P. singampattiyana. were exhaustively extracted with petroleum ether (40–60°C) for 8 h. The solvent was removed under reduced pressure, to give finally a brown-colored extract in 2.52% (v/w) yield. The extract was chromatographed over a column of silica gel afforded from benzene-chloroform (8:1) elute on colorless compound, identified by positive sterol color test with Libermann-Burchard reaction and thin-layer chromatography (TLC) on authentic sample.
TPA-induced rat ear edema
Edema was induced in the right ear by topical application of 2.5 µg/ear of TPA in 20 µl of acetone. The left ear (control) received the vehicle (acetone). Selected doses of P. singampattiyana. leaf extract (as shown in ) were applied to the ear, simultaneously with TPA. The thickness of the ears was measured using a micrometer before and 4 h after induction of inflammation. The edema was measured as an increase in the ear thickness due to TPA application as shown in .
Table 1.. Topical anti-inflammatory activity of leaf extract of Phyllanthus singampattiyana. (values are mean±SD, no. animals = 6, significant p < 0.05).
Carrageenan-induced rat paw edema
Edema was induced in the right hind foot of rat by subplantar injection of 0.05 ml of solution of 3% carrageenan in 0.09% saline (w/v). The volumes of the injected and control paws measured using a plethysmometer 1, 2, 3, and 5 h after induction of inflammation, and edema was expressed as an increase in paw volume due to carrageenan injection. Test doses of leaf extract of P. singampattiyana. were administered orally 100 mg/kg (0.5 ml) 1 h before carrageenan injection (Table. 2). The control group received the vehicle only.
Table 2. Acute anti-inflammatory activity of the leaf extract of Phyllanthus singampattiyana. on carrageenan-induced rat paw (values are mean±SD, no. animals = 6, significant p < 0.05).
Statistical analysis
The results of the experiments were analyzed by Student's t.-test using the program Statistica (Win 5.0 version).
Results and Discussion
Leaf extract of P. singampattiyana. was tested in two models of inflammation in Wistar rat: TPA-induced ear edema at a dose of 0.5 mg/ear in topical application and carrageenan induced paw edema at a dose of 100 mg/kg in oral administration. The anti-inflammatory activity of three doses of 25 mg, 50 mg, and 100 mg leaf extract were tested, and the results showed a notable immunogenic activity against TPA-induced edema (). The leaf extract with 100 mg dose induced the most active inhibition of 92.16%. When these doses were administered orally to experimental rats of 100 mg/kg carrageenan-induced edema (), significant anti-inflammatory response was found in the rats treated with 100 mg leaf extract of P. singampattiyana.. Maximum inhibition of paw edema at highest dose of Caesalpina bonducella. and Phyllanthus fraternus. has been reported (Hukeri et al., Citation1985; Gupta et al., Citation2003). After separation of crude extract by column chromatography, the corresponding fraction, which may be active, was collected, and the known compound was identified as β.-sitosterol. The anti-inflammatory activity of β.-sitosterol compounds was recommended as previously reported (Gupta et al., Citation1979).
Conclusions
Based on the results of the current study, it was inferred that the leaf extract of Phyllanthus singampattiyana. exhibited significant anti-inflammatory activity in the Wistar rat.
Acknowledgment
The authors are grateful to Rev. Dr. A. Antonysamy, principal, St. Xavier's College (Autonomous), for providing all laboratory facilities to carry out this research work.
References
- Asmawi MZ, Kankaanranta H, Moilanen E, Vapaatalo H (1992): Anti-inflammatory activities of Emblica officinalis. Gaertn leaf extracts. J Pharm Pharmacol 45: 581–584. [CSA]
- Calixto JB, Santos ARS, Filho VC, Tunes RA (1998): A review of the plants of the genus Phyllanthus.: Their chemistry, pharmacology and therapeutic potential. J Med Biol Res 31: 225–258. [CSA]
- Gupta MB, Nath R, Srinivasa N, et al., (1979): Anti-inflammatory and antipyretic effect of β-sitosterol. Planta Med 3: 157–163. [CSA]
- Gupta M, Mazumder UO, Sambathkumar R Sivakumar T (2003): Studies on anti-inflammatory, analgesic and antipyretic properties of methanol extract of Caesalpinia bonducella. leaves in experimental animals models. Iranian J Pharmacol. Therapeutics 2: 30–34. [CSA]
- Hostetmann K (1997): Strategy for the biological and chemical evaluation of plant extracts. International Conference on Biodiversity and Bioresources: Conservation and utilization, 23–27 November, Phuket, Thailand.
- Hukeri VI, Kalani GA, Kakrani HK, Bagi MK (1985): Preliminary phytochemical and experimental anti-inflammatory evaluation on petroleum ether extract of Phyllanthus fraternus.. In: Sadique, ed., Satellite Symposium on Traditional Medicine as Adjunct to Asian Congress of Pharmacology. Pudukottai, Tamil Nadu, India, Sarma's Santorium Press.
- Ihantola-Vormisto A, Summanen J, Kankaanranta Vulorela H, Moilanen E, Asmawi MZ, Vapaatalo H (1997): Anti-inflammatory activity of extracts from leaves of Phyllanthus emblica.. Planta Med 63: 518–524. [PUBMED], [INFOTRIEVE], [CSA]
- Jantan IB, Kang H, Suh DH, Han BH (1996): Inhibitory effects of Malaysian medicinal plants on the platelet-activating factor (PAF) receptor binding. Nat Prod Sci 2: 86–89. [CSA]
- Unander DW, Webster GL, Blumberg BS (1992): Usage and bioassays in Phyllanthus. (Euphorbiaceae): A compilation III. The subgenera Eriococcus, Conami, Gomphidium, Botryanthus, Xylophylla. and Phyllanthodendron., a complete list of the species cited in the three-parseries. J. Ethnopharmacol 36: 103–112. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]