Evaluation of anti-inflammatory activity of Vernonia cinerea Less. extract in rats.Summary
The methanol extract of the whole plant of Vernonia cinerea Less. was evaluated for its anti-inflammatory activity in acute (carrageenin, histamine and serotonin induced rat paw edema) and a chronic model (cotton pouch induced granuloma granuloma /gran·u·lo·ma/ (gran?u-lo´mah) pl. granulomas, granulo´mata an imprecise term for (1) any small nodular delimited aggregation of mononuclear inflammatory cells, or (2) such a collection of modified macrophages ). The methanol extract (250 and 500 mg/[kg.sup.-1] p.o.) exhibited significant activity (p < 0.001) against all phlogistic phlogistic
inflammatory. agents used in a dose dependant manner. In the chronic model (cotton pouch granuloma method) the methanol extract exhibited significant anti-inflammatory activity. All these effects were compared with standard drug phenylbutazone phenylbutazone /phen·yl·bu·ta·zone/ (-bu´tah-zon) a nonsteroidal antiinflammatory drug used in the short-term treatment of severe rheumatoid disorders unresponsive to less toxic agents. (100 mg/[kg.sup.-1] p.o.)
Key words: Vernonia cinerea, paw edema, granuloma, anti-inflammatory
Inflammation is a process, accompanied by local liberation of chemical mediators that include histamine, 5HT, bradykinin bradykinin /brady·ki·nin/ (-ki´nin) a nonapeptide kinin formed from HMW kininogen by the action of kallikrein; it is a very powerful vasodilator and increases capillary permeability; in addition, it constricts smooth muscle and and eicosanoids. The inflammatory response in rheumatoid arthritis is manifested by an acute inflammatory exudate exudate /ex·u·date/ (eks´u-dat) a fluid with a high content of protein and cellular debris which has escaped from blood vessels and has been deposited in tissues or on tissue surfaces, usually as a result of inflammation. of neutrophils, leucocytes, in the synovial synovial /sy·no·vi·al/ (-al)
1. pertaining to a synovial membrane.
2. pertaining to or secreting synovia.
of, pertaining to, or secreting synovia. space and chronic inflammation of the synovial tissues (Lawrence, 1992).
Vernonia cinerea (F. Asteraceae) is a common weed throughout India and it is well known as "Sahadevi" (Sanskrit), Naichette (or) Mukuthipundu (Nadkami, 1976). In traditional system of medicine the whole plant with its small flowers is used medicinally to promote perspiration in febrile conditions (Kirtikar and Basu, 1975). Co-administered with quinine it is beneficial in malarial fevers. Poultice poultice /poul·tice/ (pol´tis) a soft, moist mass about the consistency of cooked cereal, spread between layers of muslin, linen, gauze, or towels and applied hot to a given area in order to create moist local heat or counterirritation. of the leaves is useful against guinea worms. Flowers are administered for conjunctivitis conjunctivitis (kənjəngtəvī`təs), inflammation or infection of the mucosal membrane that covers the eyeball and lines the eyelid, usually acute, caused by a virus or, less often, by a bacillus, an allergic reaction, or an (Nadkarni, 1976). The flower extract of the plant was used in adjuvant-induced arthritis (Latha et al., 1998). In the limelight of the above information the present study was undertaken to evaluate the antiinflammatory potential of Vernonia cinerea Less. extract in several experimental animal models.
Material and Methods
The plant Vernonia cinerea Less. was collected in the month of September 2000 from Chennai, Tamil Nadu, India. The taxonomical identification of the plant was done by the Botanical Survey of India The Botanical Survey of India (BSI) is an institution set up by the Government of India in 1887 to survey the plant resources of the Indian empire. The British East India Company had already established botanical gardens at Sibpur, Poona, Saharanpur and Madras as centres for , Shibpur, Howrah and a voucher specimen GMM-2 is kept in our research laboratory for future reference. The whole plant was dried under shade and then powdered with mechanical grinder. The powder was passed through a sieve no. 40 and stored in an air-tight container.
The powdered plant material was extracted with 80% methanol using Soxhlet extraction apparatus. The solvent was completely removed under reduced pressure and a semisolid sem·i·sol·id
Intermediate in properties, especially in rigidity, between solids and liquids.
A semisolid substance, such as a stiff dough or firm gelatin.
Adj. 1. mass was obtained (yield 8.5% w/w with respect to dried powder). The chemical constituents of the extract were identified by qualitative analysis and confirmed by thin layer chromatography Thin Layer Chromatography (TLC) is a chromatography technique used to separate chemical compounds . It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose immobilised onto a flat, . This indicates the presence of steroids, triterpenoids, flavonoids flavonoids,
n.pl common plant pigment compounds that act as antioxidants, enhance the effects of vitamin C, and strengthen connective tissue around capillaries. and tannins. The extract was suspended in aqueous Tween 80 solution (2%) for further experiments.
Wistar strain rats of either sex (150-180 g: M/S B.N. Ghosh & Co., Calcutta, India) were used. They were kept on standard diet and water ad libitum.
Carrageenin-induced rat paw edema
The rats were divided into 4 groups (n = 6). Acute inflammation was produced by the subplantar administration of 0.1 ml of 1% Carrageenin in normal saline in the right hand paw of the rats. The paw volume was measured at 0 hr and 3 hrs after Carrageenin injection using plethysmometer (Winter et al., 1962, Kavimani et al., 1996). The first group received normal saline (3 ml [kg.sup.-1] p.o.), while the second group received phenylbutazone (100 mg [kg.sup.-1] body wt. p.o). The third and the fourth groups were treated with the methanol extract of Vernonia cinerea (250 and 500 mg [kg.sup.-1] body wt. p.o. respectively). The animals were pretreated with the drug 1 hour before the administration of Carrageenin.
The anti-inflammatory activity of the extract was measured with phlogistic agents (viz. Histamine, 5-HT) which act as mediator of inflammation. The paw edema was induced in rats by subplantar injection of freshly prepared histamine (1 mg [ml.sup.-1]) and serotonin (1 mg [ml.sup.-1]) solutions respectively (Parmar and Ghosh, 1978) and the paw edema was measured as mentioned earlier.
Cotton pouch-induced granuloma
The rats were divided into four groups (n = 6). After shaving the fur, the rats were anaesthetized and 10 mg of sterile cotton pellets were inserted, one in each axilla axilla /ax·il·la/ (ak-sil´ah) pl. axil´lae [L.] the armpit.ax´illary
n. pl. ax·il·lae
See armpit. of the rats. Extract (250 and 500 mg [kg.sup.-1] body wt.) phenylbutazone (100 mg [kg.sup.-1] body wt.) and control vehicle were administered orally for 7 consecutive days from the day of cotton pellet implantation. The animals were anaesthetized on the 8th day and cotton pellets were removed surgically and made free from extraneous tissues. The pellets were incubated at 37 [degrees]C for 24 hrs and dried at 60 [degrees]C to constant weight. Increment in the dry weight of the pellets was taken as measure of granuloma formation (Winter and Porter, 1957).
The results are expressed as mean [+ or -] s.e.m. The significance statistical analysis was performed by Student's t test and the p values p < 0.01, implied significance (Woodson, 1987).
The anti-inflammatory activity of Vernonia cinerea against acute pedal oedema (induced by carrageenin) has been shown in Table 1 and the results were comparable to that of phenylbutazone, a prototype of nonsteroidal anti-inflammatory agent. The methanol extract showed maximum inhibition of 47.9% [+ or -] 2.03 at the dose of 500 mg kg-1 body wt. after 3 hrs of drug treatment in carrageenin induced paw edema (Table 1) whereas the standard drug produced 61.1 [+ or -] 2.3% of inhibition. In case of histamine and serotonin induced paw edema, the methanol extract produced 51.8 + 3.4% and 54.4 [+ or -] 1.35% of inhibition (Table 2 and 3 ) at the dose of 500 mg [kg.sup.-1] whereas phenylbutazone produced 64.6 [+ or -] 2.41 and 59.3 [+ or -] 2.34% of inhibition respectively. The effect of the extract on granuloma pouch in rats is shown in Table 4. The methanol extract of Vernonia cinerea significantly inhibited granuloma formation in rats (p < 0.001) in a dose dependant manner. The methanol extract produced the maximu m inhibition of 47.9 [+ or -] 2.03% at the dose of 500 mg [kg.sup.-1] body wt. when compared with that of the control group.
Since it is evident that carrageenin induced oedema is commonly used as an experimental animal model for acute inflammation and is believed to be biphasic, of which the first phase is mediated by the release of histamine and 5-HT in the early stage followed by kinin kinin /ki·nin/ (ki´nin) any of a group of vasoactive straight-chain polypeptides formed by kallikrein-catalyzed cleavage of kininogens; causing vasodilation and also altering vascular permeability. release and then prostaglandin in the later phase (Castro et al., 1968). So, the effect of the extract against inflammations produced by these individual mediators was studied. The extract effectively suppressed the inflammation produced by histamine and serotonin. So it may be suggested that its anti-inflammatory activity is possibly backed by its anti-5-HT activity which is responsible for the same. The methanol extract was found to possess triterpenes (Misra et al., 1993), sesquiterpenes (Jakupovic et al., 1986), flavonoids and phenyipropanoids (Abeysakera et al., 1999). Many sesquiterpenes were found to possess anti-inflammatory activity (Hall et al., 1979). Thus the anti-inflammatory activity may be due to the terpenoids (Mukherjee et al., 19 97) that are present in the extract. The plant was also found to contain flavonoids which may responsible for its activity. (Alcaraz and Jimenez, 1998, Della Loggia et al., 1986). Further the phenolic constituents of Vernonia cinerea were found to be effective inhibitors of the oxidative burst of activated polymorphonuclear leukocytes and therefore may also contribute to the anti- inflammatory activity (Abeysakera et al., 1999). The extract also showed significant anti-inflammatory activity in cotton-pouch induced granuloma model which reflected its efficacy to inhibit the increase in the number of fibroblasts and synthesis of collagen and mucopolysaccharides during granuloma model tissue formation (Arrigoni-Maretellie, 1977).
Thus the methanol extract of Vernonia cinerea possesses significant anti-inflammatory activity against the tested models.
Table 1 Effect of Vernonia cinerea extract and phenylbutazone in carrageenin induced pedal edema in rats (n = 6) Treatment Dose Paw Percentage * P value (mg/kg) volume of (ml) inhibition Carrageenin 0 0.720 -- -- control Extract 250 0.465 3.44 <0.001 Extract 500 0.37 4.79 <0.001 Phenyl- 100 0.28 6.11 <0.001 butazone * p-value was calculated by comparing with control by Student's t-test. Table 2 Effect of Vernonia cinerea extract and phynylbutazone on histamine induced pedal edema in rats (n = 6). Treatment Dose Paw Percentage * P volume (mg/ volume of kg) (ml) inhibition Histamine 0.565 [+ or -] 0.011 control Extract 250 0.345 [+ or -] 0.006 38.9 [+ or -] 2.61 <0.001 Extract 500 0.272 [+ or -] 0.004 5.18 [+ or -] 3.24 <0.001 Phenyl- 100 0.2 [+ or -] 0.015 64.6 [+ or -] 2.51 <0.001 butazone * p-value was calculated by comparing with control by Student's t-test. Table 3 Effect of Vernonia cinerea extract and phenylbutazone on serotonin induced pedal edema in rats (n = 6). Treatment Dose Paw Percentage (mg/ volume of kg) (ml) inhibition Serotonin Control 0.61 [+ or -] 0.006 Extract 250 0.367 [+ or -] 0.006 40.3 [+ or -] 2.67 Extract 500 0.28 [+ or -] 0.007 54.4 [+ or -] 1.35 Phenylbutazone 100 0.25 [+ or -] 0.01 59.3 [+ or -] 2.34 Treatment * P Value Serotonin Control Extract <0.001 Extract <0.001 Phenylbutazone <0.001 * p-value was calculated by comparing with control by Student's t-test. Table 4 Effect of Vernonia cinerea extract and phenylbutazone on granuloma pouch in rats (n = 6). Treatment Dose Weight of the Percentage * P value (mg/ cotton pellet of kg) (mg) inhibition Control 36.45 [+ or -] 0.35 Extract 250 25.97 [+ or -] 0.34 28.8 [+ or -] 2.2 <0.001 Extract 500 19.0 [+ or -] 0.16 47.9 [+ or -] 1.62 <0.001 Phenylbutazone 100 15.92 [+ or -] 0.25 56.3 [+ or -] 3.27 <0.001 * p-value was calculated by comparing with control by Student's t-test.
The authors are grateful to UGC, New Delhi for financial support for this project.
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U. K. Mazumder, Department of Pharmaceutical Technology, Jadavpur University, Calcutta-700 032, India e-mail: firstname.lastname@example.org