Materials and Methods
Plant materials and preparation of aqueous, ethanol and n-butanol fractions
Fresh healthy aerial parts of Tragia hispida ,Justicia adathoda and tubers of Cyperus rotundus were collected from Matara District, in the Southern Province of Sri Lanka during July to September 2017.J. adathoda and tubers of C. rotundus were authenticated by Nawinna Ayurveda research institute. The T.hispida plant was authenticated by the National Herbarium, Royal Botanical Garden, Peradeniya, Sri Lanka and a voucher specimen (Th/Matara/01), was deposited in the National Herbarium, Royal botanical garden, Peradeniya, Sri Lanka.
Fresh aerial parts (leaves and stems) of T. hispida , dried tubers of Cyperus rotundus and fresh aerial parts of Justicia adathoda were washed, air dried and subsequently oven dried at 400C for 4 days and ground using a mechanical grinder. Two 300 g samples of T. hispida were extracted separately by refluxing the plant material for 2 hours in either 1.5 L of distilled water and 80 % ethanol to obtain an aqueous extract (THA) and an ethanol extract (THE) respectively. A portion of the THA extract was freeze dried and a portion of it was mixed with n-butanol to to obtain the butanol soluble fraction of the aqueous extract of T. hispida (THB).
In the preparation of C. rotundus plant extract, two 50 g samples of C. rotundus were extracted separately by refluxing the plant material for 2 hours in either 250 mL of distilled water and 80 % ethanol to obtain an aqueous extract (CRA) and an ethanol extract (CRE) respectively. CRE was evaporated under a vacuum to obtain a sticky brown semi-solid compound. The aqueous extract (CRA) was freeze-dried for 5 days to obtain a light brown coloured powder. Justicia adathoda, was prepared similarly with two 50 g samples extracted separately by 250 mL each of distilled water and 80 % ethanol by refluxing for 2 hours. Then 80 % ethanol extract (JAE) was evaporated under a vacuum to obtain brown semi-solid compound. The aqueous extract (JAA) was freeze-dried for 5 days to obtain a yellowish brown coloured powder.