Phytochemical Screening, Antimicrobial Activity and Cytotoxicity of Nepalese Medicinal Plants Swertia chirayita and Dendrobium amoenum

Research on medicinal plants are important to Nepal because most of its rural population relies on it as mode of medicine. Medicinal plants namely Swertia chirayita and Dendrobium amoenum were collected from mid hills of Nepal. The present study was undertaken to find the antimicrobial activity, phytochemical presence and their cytotoxicity in different extraction medium. The percentage yield from the plants were highest in warm methanol extraction with 12.6%, followed by ethyl acetate and lowest was for cold methanol. Plant extract showed the presence of antioxidants like alkaloid, terpenoids, flavonoids, tannin, glycosides. The Brine Shrimp Bioassay of methanol and ethyl acetate extract showed cytotoxicity. Chiraito extract showed LC50 of 199 ppm for Dhunche sample, 128.82 ppm for Daman sample and 131.82 ppm of Illam sample. The antibacterial activity of methanol extract of Chiraito and Dendrobium amoenum showed significant bioactivity by inhibiting growth of microbial species selected for the test. The zone of inhibition shown by the extracts was comparable to the standard antibiotics. Similarly, methanol extract of Chiraito also showed significant antifungal activity with the zone of inhibition comparable to amphotericin.


Introduction
For centuries, medicinal plants are being used as Ayurveda or traditional medicine in Nepal by local tribes and also in most of the rural areas of the Indian subcontinent.Medicinal plants and Ayurveda practice in the sub continent are intertwined.Different parts of plant are used in different herbal medicine as one of the constituents of final composition.Some phytochemicals present in these medicinal plants, have medicinal values and are expected to yield positive biological activities.Nepal, in the middle of the Himalayan belt which extends from Myanmar in the East to Karakorum in the West, possess vast diversity of plants because of its geographical distribution [1,2,3,4].Some of these Medicinal plants are used in indigenous rural remedies, homoeopathic medicines, and allopathic pharmacopeia [5,6,7,8].The exploitation of locally available medicinal plants in health care and economic advancement is a necessity of Nepal.Among all those plants Swertia chirayita and Dendrobium amoenum is the focus of this research.Among 100 species of Swertia genus, 27 species are found in Nepal.Among them around nine species are reported to be traded for medicinal purpose.Swertia chirayita is biennial erect herb which is approximately 50 to 125 cm tall.The plant is a native of temperate Himalayas, found at an altitude of 1200-3000 m.Its stem is robust, branched and cylindrical below, four angled upward and containing large pith, broadly lanceolate leaves with 5-nerve and sub-sessile.It has lurid greenish yellow flowers tinged with purple in large panicles, with egg-shaped capsules and minute seed which are smooth and many angled [13].Chiraito grows mainly in temperate Himalayas and is reported from 40 districts of Nepal.Chiraito grows between open forest and margin of cultivated land, it is predominant in Dolakha and spreads mainly in altitude of 1500 m to 3000 m of Eastern and Central region of Nepal [2,9].Dendrobium contains about 1,200 species and is also known as orchid [10].This genus also occurs in diverse habitats throughout much of South and East Asia, stretching to Oceania and some of Pacific islands.This plant is commonly distributed between 660 m to 2000 m temperate forest of Himalayan region and grow in little light exposure [11,12].The orchids are mostly found in terrestrial, epiphytic and saprophytic habitat.Dendrobium amoenum grows in clustered pendulous five to six slender stems.Morphologically, this species is highly evolved, tall, straight with elongated pseudobulbs covered by modest sized leaves.It has unique floral pattern which is fairly constant varying in size from very small to large, the shape and form of the stems and leaves are divergent.This traditional medicinal species is also ornamental plant and have been cultivated for decorative purpose.
The flowers flourish on an older stem in cluster of two to three per node and the flower is amusingly perfumed [13,14,15].

Sample preparation
Swertia chirayita plants were collected from or near from Dhunche, Daman and Illam and Dendrobium amoenum from Pokhara, during their flowering season.The plants were air dried under room temperature.The dried plant samples were cut and grinded to make it in powder form and kept for storage at room temperature.

Extraction
Three types of extraction method were carried out, viz.warm methanol extraction, cold methanol extraction and ethyl acetate extraction.For warm extraction, Soxhlet apparatus was used.10 g of the crushed sample along with 200 ml of methanol was put into the soxhlet apparatus.The soxhlet was run for 28 hours at 65⁰C.The methanol extract was taken out from the soxhlet apparatus.The pigment was removed using Hexane in the separating funnel.The methanol fraction was then dried using water bath.For cold extraction methanol at room temperature was used.100 g of powdered form of sample was taken and 450 ml methanol, that was just enough to cover the upper layer of sample, was poured on it and was shaken regularly.After 48 hrs in room temperature, filtration was done and the filtrate was stored at room temperature.For ethyl acetate extraction, 10 g of powdered material was dissolved in 25 ml ammonium hydroxide.300 ml of ethyl acetate was then added and left for 72 hrs at room temperature.Extract was then filtered.Ethyl acetate was then left to dry in water bath [16,17,18].

Preparation of Test sample
Stock solution was prepared by dissolving 100 mg of the extract in little amount of dimethyl sulfoxide (DMSO) for initial solublization and then addition of water to final volume of 25 ml to make a stock of concentration of 4000 ppm.The stock solution was further diluted to 1000, 100, 10 ppm concentration.For Hatching of Brine shrimp, 50 mg of Brine shrimp eggs were sprinkled in a beaker with 300 ml of sea water.The transferred sample as then allowed incubating at 32 -35 o C for 24 hrs.

Bioassay
Cleaned test tubes were divided into four groups each group consisting of five test tubes.After 24 hrs of incubation, the nauplii were recovered with a pipette and 10 nauplii were transferred in each test tube.The groups were then treated with different dilutions of sample.The test tubes were then incubated at 32-35 o C overnight.The incubated tubes were observed for the number of survived nauplii and graph was plotted for death percentage versus Log of concentration of the extract.This gives linear equation in the form of y = mx + c.

Calculation of LC50
The death of nauplii was calculated as death percentage as, % death = deaths/initial x 100.The % death was corrected for any control deaths by subtracting the -%death control from % death test.The Lethal concentration 50 (LD50) was derived from the graph from the equation of the straight line [19].

Antimicrobial Screening
Micro-organisms used Six clinical samples of bacteria were collected from Kathmandu University Teaching Hospital (KUTH), Dhulikhel namely Staphylococcus aureus, Escherichia coli, Kleibsella pneumonia, Salmonella paratyphi, Salmonella typhi, Pseudomonas aerugenosa.The Yeast Saccharomyces cerevisiae (YCS2) were taken from microbiology lab of Department of Biotechnology, Kathmandu University.

Preparation of Sample
The different extraction of plant was dissolved in DMSO and water, which was made into different concentrations.

Inoculation
Sterilized petri dishes were filled to a uniform depth, with respective sterilized medium (Nutrient agar for bacteria and Muller Hinton agar for fungus respectively).The solidified media was then inoculated with respective organisms.Holes were cut into the medium using a sterile borer.Solution of various concentrations of the extracts under test was poured into the bored holes with the help of a micropipette and the standard antibiotic discs or solutions were gently placed at the different sites of the petri-dish containing the inoculated medium in the case of cup plate method.In case of filter disc method, filter paper discs prepared from Watman's filter paper were soaked with the test solution and the filter discs are then gently placed into the inoculated medium with the help of a sterile forceps.The plates were then maintained at room temperature for few minutes to allow the antibiotic and the extract solution to diffuse into the medium.In the uniform media, diffusion of the extract solution and the antibiotic discs will occur uniformly around the cup and the concentration gradient will be established around it and at a certain distance from the cup.The plates were then incubated at a suitable temperature (usually 37-39⁰C) for 24-48 hours.After 48 hours, the zones of inhibition were measured with the help of a measuring ruler or antibiotic zone reader [22].

Conclusion:
Researchers have identified compounds used in mainstream medicine derived from plant resources.
Similarly, this research has assessed the phytochemical, biological screening of Chiraito and Dendrobium amoenum.This natural product can bring new and effective antimicrobial agents and serve as alternate source of combating infections in human beings.Hence this research can have a promising potential in various traditional, complementary and alternate systems of treatment of human diseases.Further work on isolation and characterization of these plants and their pharmacodynamics study can be a great contribution.The research also proved to be beneficial in exploiting medicinal plants having biological activities namely cancer.

Figure 1 :
Figure 1: Yield percentage of different plant from various solvents.[A] representsyield percentage from cold extract (Methanol), [B] represents yield percentage from cold extract (Ethyl Acetate) and [C] represents yield percentage from warm extract (Methanol).

Figure 2 :
Figure 2: Zone of Inhibition (ZOI) of different plant samples from cold methanol extract of 2.5 % and 7.5 % against different bacteria.A, B, C, D, E and F represents ZOI of different plant extract against S. paratyphi, E. coli, K. pneumoniae, S. typhi, P. aerugenosa and S. aureus respectively.

Figure 3 : 3
Figure 3: Zone of Inhibition (ZOI) of different Chiraito samples.A represents ZOI of Chiraito (Dhunche) prepared from ethyl acetate against 5 microbes and B represents ZOI of Chiraito (Daman) against Saccharomyces cerevisiae.ZOI (in mm) of plant extract against following fungus and comparison with fungicide solution Figure 3 Brine Shrimp Bioassay

Table 1 ,
indicating that there are high value natural compounds in the plants.Since there are presence of alkaloids, terpenoids, coumarins, flavanoids and sterols these plants could have anti cancer activity.be biologically active.Chirato extract showed LC50 of 199 ppm for Dhunche sample, 128.82 ppm for Daman sample and 131.82 ppm for Illam sample.Since cytotoxicity value given by Brine Shrimp assay directly correlates with its cytotoxicity ability, toxicity at low concentration of Chiraito extracts can have toxicity against cancer cell lines and has the potential to be developed as drugs for cancer.