Abstract
A petroleum ether extract of Phyllanthus debelis. Klein. ex Willd whole plant was subjected to analgesic and anti-inflammatory screening using various animal models. The extract exhibited significant anti-inflammatory activities in the acute carrageenan-induced rat paw edema and the chronic granuloma pouch models. However, it was devoid of analgesic activity in the tail-flick model.
Introduction
Phyllanthus amarus. Schum and Thonn (Euphorbiaceae) and Phyllanthus debilis. Klein. ex Willd (Euphorbiaceae) are closely related and similar looking species commonly available in India. (Balachandran & Sivarajan, Citation1994). P. amarus. is widely distributed throughout India, whereas P. debilis. has its distribution restricted toward southern India. These are given for stimulating sluggish liver and as tonic. Isolation of a bitter lignan, phyllanthin, and a nonbitter lignan, hypophyllanthin, fromP. niruri. (Phyllanthus niruri. Linn) was reported earlier (Row et al., Citation1970). Isolation of phyllanthin and hypophyllanthin from P. amarus. was also reported (Aimon et al., Citation1993). Phyllanthus.fraternus. Webster is native probably to West Pakistan and Western India and has been introduced into Africa and the West Indies. Its closest relative is P. debilis.. The two species, though appearing distinct, are considered to be two allopatric subspecies of a single species, which interfered when they came together (Anonymous, Citation1950). P. debilis. has been proven to be a better hepatoprotective than P. amarus. at a dose of 0.66 g/kg against carbon tetrachloride (0.7 ml/kg) induced liver dysfunction (Sane et al., Citation1995). The efficacy of the aqueous extract of leaves was compared with roots and stems of P. debilis. against carbon tetrachloride–induced rat liver dysfunction (Shah et al., Citation2002). The potential hepatoprotective action of the extract of P. debilis. whole plant in various solvents on carbon tetrachloride–induced liver damage rat model was investigated (Shah et al., Citation1999). Recently, we have examined the plant and reported (Chandrashekar et al., Citation2004) the isolation of β.-sitosterol, phyllanthin, and hypophyllanthin from a petroleum ether extract. A literature survey indicates lack of analgesic and anti-inflammatory activities of P. debilis.. In the current investigation, a petroleum ether extract of whole plant of P. debilis. was used for analgesic and anti-inflammatory activity studies.
Materials and Methods
The whole plants of Phyllanthus debilis.were collected from Udupi district. They were identified by K. Gopalkrishna Bhat by comparison with standard specimens deposited at the Department of Botany, Poorna Prajna College, Udupi. Voucher specimens are kept at the NGSM Institute of Pharmaceutical Sciences, Nanthoor, Mangalore, India. These shade-dried plants (1200 g) were powdered and extracted with petroleum ether. The crude extract was obtained as a dark-colored viscous residue (40 g, 3.33%). This total extract was subjected to pharmacological screening.
Experimental animals
Swiss albino mice and albino rats (HA strain) of either sex weighing 20–25 g and 100–150 g, respectively, were obtained from an animal colony of NGSM Institute of Pharmaceutical Sciences, Nanthoor, Mangalore, India. They were housed in polypropylene cages in an air-conditioned area at 25 ± 2°C with 10:14 h light and dark cycle and maintained on Amrut brand balanced animal feed and water ad libitum.. In all experimental sets, 6 rats and 10 mice were used for each treatment.
LD50 and behavioral studies
The petroleum ether extract was insoluble in water. Hence, it was suspended using 1% Tween 80 for further studies. The albino mice were treated in graded doses up to 4000 mg/kg p.o. and were observed for any behavioral changes or mortality up to 7 days. There were few changes in the behavioral responses like alertness, touch response, and restlessness. Therefore, of the maximum tolerated dose (i.e., 400 mg/kg body weight) was chosen for the studies.
Analgesic activity
Acetic acid–induced writhings
Male mice were injected intraperitonially with 1ml/kg of 3% aqueous acetic acid 30 min after oral administration of petroleum ether extract (50, 100, 200, 400 mg/kg)or aspirin (50 mg/kg) orally to various groups of mice. The number of writhing episodes of individual mice were recorded for 30 min after acetic acid treatment.
Tail flick
Male mice were trusted orally with 50, 100, 200, or 400 mg/kg doses of extract or aspirin (50 mg/kg ) to various groups of mice. The mouse was held firmly to immerse its tail in a water bath maintained at the constant temperature of 58°C. The time required for the typical reaction, a violent jerk of the tail, was recorded to assess response to noxious stimulus (Turner, Citation1965).
Anti-inflammatory activity
This activity was studied using acute or chronic treatment in rats.
Acute: Carrageenan-induced rat paw edema
Extract was administered in 50, 100, 200, or 400 mg/kg doses or diclofinac sodium (100 mg/kg), 0.5 h prior to subcutaneous (s.c.) carrageenan in the planter region of the rat hind paw to induce inflammation (Winter et al., Citation1962). The paw volume was measured initially and at 1, 2, 3, 4, and 5 h, by the plethysmographic method of Harris and Spencer (Citation1962) after carregeenan was injected.
Chronic: Cotton pellet–induced granuloma in rats
Four sterilized cotton pellets, each weighing 10 mg, were implanted s.c., one in each axilla and groin in an anesthetized rat, using the method of D'Arcy et al. (Citation1960). After treatment with extract at 50 or 100 mg/kg or diclofinac sodium (5 mg/kg) for 7 days, the rats were sacrificed the next day. The pellets were dissected out and granuloma was dried at 60°C overnight to determine the dry weight.
Statistical analysis
The statistical analysis was carried out to calculate mean (SEM). Further analysis was carried out by Student's t.-test to calculate significance of results. p values > 0.05 were considered as non-significant.
Results
Acetic acid–induced writhing
There was significant reduction in acetic acid–induced writhing due to extract treatment at various doses, and with aspirin pretreatment, as shown in .
Table 1.. Effect of Phyllanthus debilis. whole plant onacetic acid-induced writhing in albino mice.
Tail flick
There was no effect on tail-flick response due to the hot water–induced noxious stimuli after extract pretreatment. However, this response was significantly altered due to aspirin pretreatment.
Carrageenan-induced rat paw edema
The extract as well as diclofinac sodium showed antiphleogestic activity. This anti-inflammatory response was dose-dependent and significant at 3 and 4 h after carrageenan injection, as shown in .
Table 2.. Anti-inflammatory activity of petroleum ether extract of Phyllanthus debilis. in carrageenan-induced rat paw edema.
Cotton pellet–induced granuloma in rats
There was statistically significant reduction in weight of granuloma in extract and diclofinac sodium–treated rats as shown in .
Table 3.. Effect of petroleum ether extract of Phyllanthus debilis. whole plant on cotton pellet-induced granuloma in albino rats.
Discussion
The LD50 of the petroleum ether extract was more than 4000 mg/kg body weight. Hence, doses of 50, 100, 200, and 400 mg/kg body weight were chosen for the study.
The extract demonstrated significant reduction in acetic acid–induced writhings. However, there was no effect on tail-flick response due to noxious stimuli. It is worthwhile to note that central nervous depressants, and antihistaminics, are known to reduce the number of writhings, thus indicating the above activity relates to a central nervous system depressant effect of extract. There was significant and dose-dependent anti-inflammatory activity of the extract in the acute carrageenan-induced rat paw edema model. Further, the extract showed significant reduction in weight of granuloma after chronic treatment, indicating activity of extract on granular tissue formation. It is worthwhile to investigate this important lead.
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