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). The methanol extract (250 and 500 mg/[kg.sup.-1] p.o.) exhibited significant activity (p < 0.001) against all phlogistic 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 (100 mg/[kg.sup.-1] p.o.)

Key words: Vernonia cinerea, paw edema, granuloma, anti-inflammatory

Introduction

Inflammation is a process, accompanied by local liberation of chemical mediators that include histamine, 5HT, bradykinin and eicosanoids. The inflammatory response in rheumatoid arthritis is manifested by an acute inflammatory exudate of neutrophils, leucocytes, in the synovial 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 of the leaves is useful against guinea worms. Flowers are administered for conjunctivitis (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

Plant material

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, 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.

Extraction procedure

The powdered plant material was extracted with 80% methanol using Soxhlet extraction apparatus. The solvent was completely removed under reduced pressure and a semisolid 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. This indicates the presence of steroids, triterpenoids, flavonoids and tannins. The extract was suspended in aqueous Tween 80 solution (2%) for further experiments.

Animals used

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.

Mediator-induced inflammation

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 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).

Statistical analysis

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).

Results

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.

Discussion

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 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.

Acknowledgement

The authors are grateful to UGC, New Delhi for financial support for this project.

References

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Address

U. K. Mazumder, Department of Pharmaceutical Technology, Jadavpur University, Calcutta-700 032, India e-mail: [e-mail]