EVALUATION OF ACUTE TOXICITY AND ANTI-INFLAMMATORY EFFECTS OF BACCHAROIDES SCHIMPERI (DC.) IN EXPERIMENTAL ANIMALS

.


Introduction
Inflammation is a response of living cells and tissues to injury and usually comprises a wide array of enzyme activation, mediator release, extravasations of fluid, cell migration, tissue breakdown and repair (Perianayagam et al., 2006).Pain is frequently associated with inflammation (White et al., 2005;Cunha et al., 2008).Steroidal and non-steroidal anti-inflammatory drugs are the most commonly used to treat inflammation (Hassan et al., 2012), but both are prone to presenting with serious adverse reactions (Dharmasiri et al., 2003;Park et al., 2004).Therefore, the search for new and cost effective anti-inflammatory and analgesic agents with little or no side effects for mankind is necessary.Over the years, medicinal plants have been an important source of new chemical substances with potential therapeutic effects.Hence, the investigation of new plants currently used by folk healers as pain relievers and anti-inflammatory agents should therefore be viewed as a fruitful and logical research strategy (Elisabetsky et al., 1995;Gupta et al., 2006).Baccharoides schimperi DC. (Syn.Vernonia schimperi DC.) belong to the family Astraceae and is largely distributed in the southern part of Saudi Arabia.Literature reports on Baccharoides schimperi DC. do not show any pharmacological work.Previous phytochemical screening on Vernonia.schimperi (DC.) (Syn.Baccharoides schimperi (DC.) exposed the presence of tannins, cardiac glycoside, sterols, alkaloids, phenols, saponins and glycosides (Ahmed et al., 2014).
In view of the above medical properties associated with other species of the genus, Baccharoides schimperi DC. was investigated for in-vivo anti-inflammatory and acute toxicity work.In the present paper we are reporting the anti-inflammatory activity, LD 50 value and acute toxicity of the title plant.

Plant material
The plant material was collected in February, 2007 from Bani Malik, Saudi Arabia.The collected plant material was identified by the taxonomist Dr. M. Yusuf and a voucher specimen of the plant (# 15044) was deposited at the herbarium of the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

Extraction process
The aerial part of the plant was dried under shade and ground.The material (200 g) was extracted in 96% ethanol (1 L × 3) at room temperature for 72 h (24 h × 3).The extract was filtered through filter paper (Whatman No.1) and solvent was evaporated to dryness at 40 o C under reduced pressure using Buchi Rotavapour and yielded green solid mass 9.4 g (4.7%) and labeled as BSE.The ethanol extract (BSE) 4.9 g was further fractionated in sequence at room temperature for 72 hr (24 hr × 3) with n-hexane, chloroform and methanol (100 mL × 3) and the solvent in each case was evaporated as described above, afforded n-hexane fraction (BSH) 0.280 g (5.7%), chloroform fraction (BSC) 1.6 g (32.7%) and methanol fraction (BSM) 2.9 g (59.1%).All samples were kept in air tight bottle at 4 o C in refrigerator for further use.

Animals
Wistar albino rats roughly the same age, weighing 180-200 g b.w. and Swiss albino mice (25-30 g b.w.) of either sex were procured from the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh and used in these studies.The animals were kept at constant temperature (22 ± 2°C), humidity (55%) and light-dark conditions (12/12 hr light/dark).They were provided with Purina chow and free access to drinking water ad libitum.

Acute toxicity study and LD 50
The acute toxicity test was performed on mice using oral route.BSE was dissolved in distilled water and administered at various doses, ranging from (500-10000 mg/kg b.w), to different groups of mice (each group contains six animals).The animals were observed for 24 hr for any behavioral change, symptoms of toxicity and mortality.Finally LD 50 was calculated by Karbar's method.

Carrageenan-induced rat paw edema test
Experimental inflammation was induced according to the method described by Winter et al., 1962.Briefly, 0.05 mL of 1% carrageenan sodium salt was injected into the right hind foot of each rat under the plantar aponeurosis.The test groups (six animals) of rats were treated orally with ethanol extract (BSE) 250 and 500 mg/kg b.w, BSH 400 mg/kg b.w, BSC and BSM, 200 and 400 mg/kg b.w, 1 hr before the carrageenan injection.At the same time, the control group (six animals) was given normal saline 5 mL/kg and the reference group (six animals) was administered an aqueous solution of Phenylbutazone 100 mg/kg b.w orally.The measurements of paw volumes were done by the displacement technique using a plethysmometer (Apelex, France) immediately and 3 hr after the injection of carrageenan.The inhibitory activity was calculated according to the following formula: where 'a' and 'b' is the mean paw volume of treated rats after and before carrageenan injection respectively and whereas, 'c' and 'd' are the mean paw volume of control rats after and before carrageenan injection respectively.

Statistical analysis
Values are given as arithmetic means ± standard error of the mean (S.E.M.).Data was statistically analyzed by using one-way analysis of variance (ANOVA) followed by Student's t-test.

Acute toxicity test
In the acute toxicity assay no deaths were observed during the tested dose of up to 3000 mg/kg b.w of BSE, whereas at the doses of 4000, 8000 and 10000 mg/kg b.w., mean mortality was observed as 0.5, 1.5 and 3 respectively (Table I).The median lethal dose (LD 50 ) of BSE was calculated by Karbar's method and found to be 7.250 g/kg.During this experiment, behavioral changes like writhing, micturition, defecation, sedation and increase respiration rate were observed at higher doses 4000, 8000 and 10000 mg/kg b.w.but at 2000 mg/kg b.w.beside sedation, defecation and writhing were also observed.

Carrageenan-induced rat paw edema
The anti-inflammatory activity of BSE was measured at the dose of 250 and 500 mg/kg b.w. and its fractions BSH was measured at 400 mg/kg b.w.BSC and BSM were measured at the dose of 200 and 400 mg/kg and the results are summarized in Table 2.The BSE showed significant anti-inflammatory activity, 62.91% inhibition at 500 mg/kg b.w. and the results are comparable to that of phenylbutazone standard anti-inflammatory drug.While among the fractions i.e.BSH, BSC and BSM the BSH showed good anti-inflammatory activity, 64.87% inhibition at the dose of 400 mg/kg b.w.Conversely, the BSC did not show any significant activity at both higher and lower doses.The BSM showed negligible activity at lower dose but showed significant activity, 60.24% inhibition at higher dose (400 mg/kg b.w.) and are comparable to that of standard drug phenylbutazone.Abrupt rise in the activity at higher dose of BSM prompted us to determine the activity at 300 mg/kg b.w. and found 41.71 % inhibition which suggested that VSM showing dose-dependent activity.

Discussion
The present study showed the anti-inflammatory activity and toxicity results of Baccharoides schimperi (DC.).BSE showed encouraging results that's why it was further fractionated in sequence to n-hexane, chloroform and methanol soluble fractions and tested for anti-inflammatory activity to get the active fraction.Toxicity is the degree to which a substance is poisonous (Martin, 2002); evaluation of crude plant drugs must be included its possible toxicity and/or safety margin (Ibrahim et al., 2007).In this study the ethanol extract (BSE) was not toxic to animals up to a dose of 3g /kg b.w., presenting LD 50 value 7.250 g/kg b.w.Carrageenan-induced edema is a common and suitable procedure to screen the antiinflammatory activity in experimental animal model for acute inflammation and is believed to be biphasic (Vinegar et al., 1969).The early phase (1-2 hr) of the carrageenan model is mainly mediated by histamine, serotonin and increased synthesis of prostaglandins in the damaged tissue surroundings.The late phase is sustained by prostaglandin release and mediated by radykinin, leukotrienes, polymorphonuclear cells and prostaglandins produced by tissue macrophages (Brito and Antonio, 1998;Gupta et al., 2006).
The inhibitory activity showed by the ethanol extract (BSE) of B. schimperi (500 mg/kg b.w) over a period of 4 hr in carrageenan-induced paw inflammation was quite similar to that of exhibited by the group treated with phenylbutazone.Among the BSE fractions the n-hexane fraction, BSH, (400 mg/kg b.w) showed more inhibition in comparison of BSE itself.The methanol fraction, BSM, (200, 300 and 400 mg/kg b.w) showed the dose-dependent activity and increases as the dose increases and highest activity was recorded at the highest dose used as shown in Table 2.The chloroform fraction does not show any significant activity.The results shown in Table 2 clearly indicated that the anti-inflammatory active compounds in the plant extract distributed among the n-hexane fraction (BSH) and methanol fraction (BSM).
Furthermore, variety of in-vitro and in-vivo experiments have shown that flavonoids, tannins, triterpenoids and other secondary plant metabolites possess analgesic and anti-inflammatory properties (Yuan et al., 2006;Salminen et al., 2008;García-Lafuente et al., 2009).Flavonoids inhibit phosphodiesterases, which are involved in cell activation in the biosynthesis of protein kinases (regulatory enzymes in inflammation) (Manthey et al., 2001).The flavonoid luteolin from vernonia patula (Lin and Wang, 2002) have been reported to inhibit the activity of cyclooxygenase and lipoxygenase, decrease the level of PCE 2 and the release and expression of cyclooxygenase-2 (COX -2) (Shimoi et al., 2000).Tannins are reported as potent inhibitors of cyclooxygenase-1 and also possessed anti-phlogistic activity (Pan et al., 2010).Again the phenolic compounds have also been found to exhibit substantial anti-inflammatory effects against carrageenan-induced paw inflammation by inhibition of leukocyte migration, reduction of serum lysozyme level, NO and PGF 2 (Wu et al., 2006).
The previous phytochemical work done on V. schimperi (Ahmed et al., 2014) revealed that the n-hexane fraction contains glycosides, sterols and phenols on the other hand high polar organic solvent fractions like acetonitrile and methanol fractions contains saponins, tannins, flavonoids, glycosides, cardiac glycosides and phenols.The anti-inflammatory activity of BSH and BSM may be attributed due to the presence of these phytochemicals in n-hexane and methanol fractions.Insignificant inhibitory activity of BSC may be due to presence of some compound having antagonistic effect.

Conclusion
The findings of the present work showed that the plant is safe to the experimental model and recommended as a potential source of antiinflammatory agent.Moreover, further work is required to find out the active anti-inflammatory constituents of the plant.

Table 1 :
LD 50 determination of BSE by Karbar's method