INTRODUCTION:

The plant Flemingia strobilifera linn. (FS) belongs to the family Fabaceae which is a medicinally important one. It is commonly known as “Kusrunt” and “Wild hops” in English. It is frequently found in forests, particularly in dry places and flowers in cold season¹. It is traditionally used in disease conditions like epilepsy, hysteria, rheumatism, and as a vermifuge for children, seeds of the plant are restorative, astringent, decoction or infusion of the leaves and the flowers is also prescribed by “curanderos” for tuberculosis and a decoction of the leaves is given as a bath after childbirth ².

Linking to the chain of research work done on anti-inflammatory activity of the other plants of same genera and family (Fabaceae), FS which is traditionally used in rheumatism and other ailments were considered to evaluate the anti-inflammatory potential of methanol and hydro-alcoholic extracts of Flemingia strobilifera linn. (MEFS and HAFS respectively) in animal models of inflammation.

MATERIALS:

Plant material:

The aerial parts of the plant FS was procured from the forest of Nagzira, District-Gondia, Maharashtra, India. The plant material was taxonomically identified and authenticated from the Department of Botany, R.T.M., Nagpur University, Nagpur. The voucher specimen of the plant (9187) has been preserved in our laboratory.

Chemicals and drugs:

Carrageenan (Himedia), diclofenac sodium (McNeil Pharma), ketamine and xylazine were used for this study. All other reagents and chemicals used were of analytical grade.

Animals:

Male Swiss albino mice (20–25 g) from inbred colony of Department of Pharmaceutical Sciences, Nagpur were used. The animals were housed in polypropylene cages with not more than six animals per cage and maintained under standard laboratory conditions (temperature 25±2 ^◦C, relative humidity of 45–55 % under 12-h light: 12-h dark cycles). They were fed with standard laboratory animal feed (Katariya Agro. Industries Ltd., Nagpur) and water ad libitum. All the pharmacological experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC, vide sanction no. 92/1999/CPCSEA dated 28/04/1999).

METHODS:

Preparation of extracts:

The aerial parts of the plant was procured; dried and coarse powder was prepared. The dried coarse powder of aerial parts was defatted with petroleum ether (60-80 ^oC).The marc was extracted with methanol (MEFS), in a soxhlet apparatus and then macerated with (1:1) hydro-alcoholic solvent (HAFS). Both extracts were subjected to preliminary phytochemical screening and revealed the presence of tannins, flavonoids, carbohydrates, proteins and amino acids ^3-4.

Table 1: Anti-inflammatory activity of methanol and hydro-alcoholic extracts of Flemingia strobilifera in carrageenan-induced paw oedema model.

Group (n=6)	Dose mg/kg	Swelling thickness (mm) ±S.E.M				% Inhibition
Group (n=6)	Dose mg/kg	0 hr	1 hr	3 hr	5 hr	1 hr	3 hr	5 hr
Control	-	2.786 ±0.23	2.898 ±0.23	3.058 ±0.27	3.02 ±0.39
MEFS	200	2.505 ±0.13	2.13 ±0.15*	2.305 ±0.18*	2.295 ±0.23*	27.64*	25.30*	27.17*
MEFS	400	2.665 ±0.05	2.425 ±0.10	2.0176 ±0.17**	2.5025 ±0.12	15.60	32.27**	19.67
HAFS	200	2.48 ±0.05	2.06 ±0.13**	1.9625 ±0.15**	1.8975 ±0.05**	34.82**	34.98**	35.06**
HAFS	400	2.51 ±0.13	2.1675 ±0.07**	1.9575 ±0.11**	2.01 ±0.14**	26**	37.06**	36.40**
Std.	10	2.4502 ±0.06	2.2507 ±0.09*	2.1609 ±0.07**	2.373 ±0.16*	21.58*	27.42**	20.34*

Data represent mean ± S.E.M. % Inhibition (n = 6), statistically significant from control.* p < 0.05 and ** p < 0.01. p value calculated by comparing with control by ANOVA followed by Dunnett`s test. (The number in the parentheses indicates the percentage of anti-inflammatory activity), FS – Flemingia strobilifera, SEM: standard error mean, MEFS – methanolic extract of FS, HAFS – hydro-alcoholic extract of FS and Std. – Diclofenac sodium.

Table 2: Anti-inflammatory activity of methanol and hydro-alcoholic extracts of Flemingia strobilifera in cotton pellet induced granuloma model.

Group (n=6)	Dose (mg/kg)	Cotton pellet induced granuloma weight of the cotton pellet (mg) (mean±S.E.M )	% Inhibition
Control	--	36.3975±0.47	--
MEFS	200	29.9252±0.24**	18.552±1.79**
MEFS	400	25.6295±0.29**	30.2476±1.57**
HAFS	200	24.7544±0.43**	32.9023±3.35**
HAFS	400	21.6098±0.35**	41.1704±1.69**
Std.	10	17.06±0.35**	53.5479±1.3**

Data represent mean ± S.E.M. % Inhibition (n = 6), statistically significant from control. ** p < 0.01. p value calculated by comparing with control by ANOVA followed by Dunnett`s test. (The number in the parentheses indicates the percentage of anti-inflammatory activity), FS – Flemingia strobilifera, SEM: standard error mean, MEFS – methanolic extract of FS, HAFS – hydro-alcoholic extract of FS and Std. – Diclofenac sodium.

Anti-inflammatory activity:

Carrageenan induced hind paw oedema model:^{5 - 6}

The mice were divided into six groups (n = 6). The different groups were treated with MEFS and HAFS (200 and 400 mg/kg), diclofenac sodium (10 mg/kg) and vehicle control (distill water) and after 1 hour 25 µl of 2 % carrageenan solution in normal saline solution was injected into the sub-plantar tissue of the right hind paw and immediately the paw thickness is recorded. The paw thickness is again recorded after 1^st, 3^rd and 5^th hour. The reduction in the paw thickness was compared to the paw thickness of mice receiving only carrageenan (control) indicated the anti-inflammatory activity. The difference in paw thickness was measured by a digital verneir caliper.

The results are shown below in table no. 1. The activity of the drug is expressed as percent inhibition of oedema.

Cotton pellet induced granuloma in mice:^{7 - 8}

This method is widely used for the study of exudative and proliferative phases of inflammation. Male Swiss albino mice (20–25 g) were divided into six groups (n=6). A sterile cotton pellet (10 mg) was subcutaneously introduced in the dorsum of mice anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg).

The treatment with test drug, std. and control saline was initiated on the next day. I, II, III and IV groups of mice received daily 200 and 400 mg/kg oral dose of MEFS, HAFS extracts, V group received 10 mg/kg of diclofenac and VI group received the distil water for 6 days. On the 7th day, the animals were anaesthetized and the pellets together with the surrounding fibro vascular tissue were removed, dried at 37 °C for 24 h and their mass was determined. The results were expressed as the difference between the initial (10 mg) and the final dry mass was compared with the control. The results are shown below: (Kindly refer Table no. 2)

Statistical analysis:

Values were expressed as mean ± S.E.M. Statistical significance was determined by ANOVA, followed by Dunnett`s test and the p values P < 0.01, implied significance.

RESULTS:

Phytochemical study:

Preliminary phytochemical screening of extracts of Flemingia strobilifera linn. was carried out to reveal the presence of different primary and secondary metabolites. The extracts showed the presence of tannins, flavonoids, carbohydrates, proteins and amino acids.

Anti-inflammatory studies:

The anti-inflammatory activity of MEFS and HAFS was measured at the dose of 200 and 400 mg/kg against acute phase of inflammation by carrageenan induced paw oedema model, and is summarized in Table 1. Both the extracts produced significant (P < 0.05, P < 0.01) anti-inflammatory activity and the results were comparable to that of diclofenac sodium as a standard anti-inflammatory drug.

The MEFS showed an inhibition of 32.27 % (P < 0.01) at the dose of 400 mg/kg (at 3 ^rdh) p.o. and the HAFS showed an inhibition of 35.06 % (P < 0.01) at the dose of 200 mg/kg (at 5^th h) p.o. and the maximum inhibition of 37.06, 36.40 % (P < 0.01) was produced at the dose of 400 mg/kg (at 3 ^rdand 5^th h) p.o. that were found greater and comparable to diclofenac (27.42, 20.34 % at 10mg/kg at 3 ^rdand 5^th h respectively) (P < 0.01) which shows its effectiveness against the second phase of inflammation which is related to the release of prostaglandins.

The effects of MEFS, HAFS and diclofenac sodium on the proliferative phase of inflammation are summarized in Table 2. It was seen that only HAFS showed comparative results to that of standard and the maximum antiproliferative effect was observed by HAFS at inhibition of 41.17 % (P < 0.01) at the dose of 400 mg/kg comparative to diclofenac sodium at inhibition of 53.54 % (P < 0.01) at the dose of 10 mg/kg. The anti-inflammatory activity exhibited in sub chronic model may be attributed to the presence of flavonoids where they are found to act by reducing the release of inflammatory substances thereby reducing tissue exaggeration.

DISCUSSION:

The work presented here deals with the anti-inflammatory effect of MEFS and HAFS in animal models of inflammation. To study the acute phase of inflammation in rodents (rats and mice), carrageenan is widely used irritant or inflammogen or a phlogestic agent. Chemically, it is a sulphated polysaccharide from seaweeds. The experimental tissue injury caused by this irritant initiates a cascade of events leading to formation of exudates. The inflammation induced by it is biphasic in nature. The first phase is attributed to the release of histamine, serotonin and kinins while the second phase is related to the release of prostaglandins. The present investigation revealed that MEFS (200, 400 mg/kg) and HAFS 200 mg/kg showed moderate anti-inflammatory activity and HAFS 400 mg/kg exhibited better anti-inflammatory activity in acute model of inflammation. Similarly in cotton pellet induced granuloma model only HAFS inhibited granuloma formation significantly (P < 0.01) as compared to standard. It reveals that HAFS have potent anti-inflammatory activity ⁹.

The activity exhibited in acute and sub acute model may be attributed due to the presence of polyphenols and flavonoids where they are found to act by reducing the release of inflammatory substances thereby reducing tissue exaggeration ¹⁰.

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