Bioactivities of Sonneratia Caseolaris (Linn) Leaf and Stem Using Different Solvent Systems

This study was aimed to report the antioxidant, cytotoxic and antibacterial potentials of different fractions of crude ethanol extract of leaf and stem of Sonneratia caseolaris Linn. The liquid-liquid fractionation was conducted among Ethyl Acetate, chloroform and carbon tetrachloride and was designated as EAFS, CFS, CTFS for stem and EAFL, CFL, CTFL for leaf respectively. Antioxidant activity of individual fraction was then evaluated by DPPH free radical scavenging assay; whereas cytotoxic activity was investigated by brine shrimp lethality assay and antibacterial activity by disk diffusion method. In antioxidant assay, the EAFL was found to be more potent (IC 50 12.0±0.12µg/ml) whereas in cytotoxicity test both the EAFS and CTFL demonstrated lowest LC 50 (25.0±0.05 and 25.0±0.07 µg/ml, respectively). Among all the fractions, EAFS and CTFS were found to have highest inhibitory effect against salmonella bacterial strains (zone of inhibition 8-11.5 mm). Promising results from this study support the ethnomedical uses of the plant as antioxidant, cytotoxic and antibacterial agents and further exploration for isolating active drug lead. Abbreviations:


Introduction
. Many of these secondary metabolites can be used as it is or modified structurally in managing human ailments. Thus, the diverse chemical compounds resulting from the mangrove plants may be novel alternative for discovering new drug molecules [2]. The red flowers with 6 valvular lobes containing green septal tube have offensive smell and are opened for only one night. The flower has 6 red petals overshadowed by long showy, numerous white to reddish stamens. Its green, star-shaped leathery berry type also in sprains and swellings, hemorrhage, and in the treatment of smallpox, cough, kidney failure, leucorrhoea, urinary tract infection and piles [3,4]. So far from this plant, some bioactive constituents including fatty acids, hydrocarbons, steroids, pectin, sugars, flavone, luteolin and its 7-O-β-glucoside (cynaroside) have been isolated [5][6][7]. Previous researchers have reported antinociceptive and antidiarrhoeal activities of this palnt [8,9]. However, no cytotoxic and antibacterial activity has yet been reported. As novelty, present study was designed to investigate the antioxidant, cytotoxic and antibacterial activity of different fractions S. caseolaris.

Preparation of Crude Extract
The collected stem and leaves were separated from undesirable materials and shade-dried followed by grinding into a coarse powder with the help of a suitable grinder (Capacitor start motor, Wuhu motor factory, China). The powder samples (600g of stem and 500g of leaf) were then kept wetting in 2.0 litres and 1.5 litres ethanol (98%) respectively, for a period of 14 days accompanying periodical shaking or stirring. After filtration using cotton and Whatman filter paper the filtrate was concentrated by evaporation of solvent in a rotary evaporator and the weight of the crude extract obtained from stem was 8.7g and 7.5g for leaf.

Solvent-Solvent Partition of Crude Extract
The crude extracts (Leaf and stem) was then fractionated in three solvents (carbon tetrachloride, chloroform and ethyl acetate) of varying polarities using separating funnel, and the obtained six samples were designated as EAFS, CFS, CTFS for stem and EAFL, CFL, CTFL for leaf respectively. The amount was found to be 0.35g, 0.43g and 1.44g for EAFS, CFS, CTFS respectively and 0.40g,1.88g and 0.30g for EAFL, CFL, CTFL respectively.

Antioxidant Activity
Free radical DPPH scavenging capability was used as indicator of anti-oxidant potential [10]. Different concentrations (400-1.57 μg/ml) of fractionated extracts were prepared and in 2mL of each of the different concentration, 6mL of freshly prepared DPPH solution (0.004%) was added. The reaction was allowed to complete by keeping the tubes in dark place for 30 minutes followed by measuring absorbance in a UV spectrophotometer at 517 nm against suitable blank. Here ascorbic acid was used as standard.
IC 50 value (the concentration of sample required to scavenge 50% of the DPPH free radicals) was determined by using the following formula: of the test samples were also computed from that data. In this assay vincristin sulfate was used as standard.

Antioxidant Activity
In this investigation, EAFL showed excellent free radical scavenging activity (IC 50 =12.0 µg/ml). Between the other two fractions of leaf, the free radical scavenging activity in chloroform extract (IC 50 =19.0 µg/ml) was superior to that of the carbon tetrachloride extract (IC 50 = 49.0 µg/ml). One the other hand, among the fractions of the stem extract, chloroform extract (IC50=69.0 µg/ ml) showed better radical scavenging activity than the rest of the two (Table 1).

Cytotoxic Activity
The standard drug (vincristine sulphate, LC 50 : 0.156 µg/ml) showed significant mortality compared to control to the tested shrimp nauplii. Among the fractions, the EAFS and CTFL were found to be more lethal (LC 50 :25.0 µg/ml) ( Table 2).

Antibacterial Activity
The EAFS and CTFS showed good antibacterial activity against Salmonella strains (zone of inhibition 8-11 mm) at the dose of 500 µg/disc compare to standard drug ciprofloxacin (zone of inhibition 12-15 mm at the dose of 5 µg/disc). Other fractions showed varying degrees of antibacterial activity on different tested strains. Only the CTFL failed to show any inhibition against the tested organisms (Table 3).

Discussion
In Ethnopharmacology, traditionally used medicinal plants are scientifically screened for diverse biological activities. As different plant parts can be used to cure different life threatening diseases, they are confirmed here to be an important source of novel pharmacophore [14]. Antioxidants prevent the degradation of any chemicals including foodstuffs or drugs and that's why they are widely used in those formulations since ages to prolong the shelf life of food or drugs. However, some issues have limited their uses e.g. some synthetic antioxidants butyl hydroxyl anisole and butyl hydroxyl toluene (BHA and BHT, respectively) need to be replaced with natural antioxidants because of their potential health risks and toxicity. Thus, the search for antioxidants from natural resources has received much attention now a days [15]. Phenolic Brine shrimps lethality bioassay has been considered as pre-screening assay for antitumor, antimalarial and insecticidal activities due to its simplicity and low cost. Therefore it is proposed to be a convenient probe to evaluate bioactivities of plant extracts [12,17]. Various fractions of crude ethanol extract of S. caseolaris leaf and stem exerts notable cytotoxic activity in different dose level. The difference in mortality (  [18,19]. Plant containing Quercetagetin-7-arabinosyl-galactoside, a flavonoid has been used extensively to treat infectious disease [20]. The flavone baicalein is reported to be largely responsible for antimicrobial effects [21]. Flavonoid rich plant extracts from species of Hypericum [22] and Chromolaena [23] have been reported to possess antibacterial activity. Many other phytochemical preparations with high flavonoids or saponin content have also been reported to exhibit antibacterial activity [24][25][26].

Funding
Not applicable