Antimicrobial Activity and Phytochemical Analysis of Medicinal Plant Cassia tora

Present study was carried out to investigate in vitro anti-bacterial and anti-fungal activity from the seeds of an Indian traditional medicinal plant Cassia tora. Plant material was separated and successively extracted with various organic solvents. Extracts was evaluated for solubility, moisture content, melting point, FTIR and other qualitative analysis for Photo constituents. In vitro antibacterial and antifungal studies were carried out by disc diffusion method. Two test samples were prepared in concentrations of 100mg/ml. Extract was found efficacious against various strains of bacteria and fungal species. 100mg/ml test sample shows better zone of inhibitions in bacterial strains. It was obtained in the range of 16.67 to 23.00 among them maximum inhibition was observed in pseudomonasaeruginosa (gram positive bacteria) and last inhibition was observed in E. coli (gram negative). It has also shown satisfactory inhibition in fungal species like Candida albicans and Candida glabrata. Thus, cassia tora can be considered as a safer and efficacious herbal candidate for antimicrobial formulation.


Introduction
Cassia tora is a small shrub that grows widely as a weed in almost all the Asian countries [1][2][3][4][5]. Theleguminosae [6,7] commonly known as the legume, pea or been family, are a large and economically important family of flowering plants.
Legumes are a type of species in which the seeds grow to develop in to pods [8,9]. Legumes are a good source of starch, dietary fiber, protein, minerals; legumes are a valuable part of a healthy diet [10][11][12][13][14]. As a group, nutrient composition of legumes makes them ideal Animal foods [15] to meet dietary recommendation. Legumes have been recognized as functional food that promote good health and have therapeutic properties [16,17]. Cassia tora is legume in the sub-family Casual pinioideae [18]. Its name has been derived from Sinhala languages, in which it is called Tora s an annual herb, 30-90 cm high which occurs as wasteland rainy season wild plant in India. Cassia tora are wild crop that grows in most parts of India as a weed [19]. The main useful parts of Cassia tora are leaves, roots and seeds. Gaur gum, also called guaran, is a gelling agent. Gaur gum is extracted from a legume, the gaur been. Gaur gum is widely used in food industry-in baked goods to increase dough yield and improve texture and shelf life, in dairy industry as a stabilizer, in meats as a lubricant, in desserts, frozen food items etc. It has also been considered of interest with regards to both weight loss and diabetic diets [20,21]. Cassia tora has been reported to contain many active substances, including Anthraquinone, Quarcetine, chrysophenol, emodin, rhein, etc. Cassia tora has been reported to exhibit significant antimutagenic activity [1,2,22]. Anthraquinone act as a fluorescence sensor [23][24][25][26] or fluorophores therefore this plant also shown sensing properties. It constitutes an Ayurvedic preparation "Dadhughnavati" which is one of the successful antifungal formulations [27].
India has rich heritage of Ayurveda and other alternative systems of medicines. The knowledge is medicinal plant was already other alternative already implied by prehistoric people since times immemorial. Various ancient Indian literature like Ayurveda, Shushrutsamhita and charaksamhita have variety of details regarding herbs, extracts, surgery and transplant. Discovery of various antibiotics like Penicillin, Cafalosporins, Aminoglycosides, Macrolides, quinolones etc has created a revolution in medical science by curing wide range of disease. It is reported that on a revolution in medical science by curing wide range of diseases [28]. It is reported that, on average, two or three antibiotics derived from microorganisms are launched each year. After a downturn in that pace in recent decades, the pace I again quickening as scientists realize that the effective life span of any antibiotic is limited. Worldwide pending on finding new anti-infective agents (including vaccines) is expected to increase 60% from the spending levels in 1993. New sources, especially plant sources, are also being investigated. Second, the public is becoming increasingly aware of problems with the over prescription and misuse of traditional antibiotics. Therefore, there is a need to revive ancient knowledge of herbs for a safer and economics treatment [29].

Collection and Identification
The plant material was collected in the month of September from Surendranagar district. It was identified by department of Pharmacognosy, C. U. Shah University and herbarium specimen was stored. Dirt particles were removed and it was subjected for shade dying for 15-20 days. After drying, legumes and it was washed thoroughly. Seeds were again subjected for drying in tray dryer legumes and it was washed thoroughly. Seeds were again subjected for drying in tray dryer at 60°C for 4-5 hours. After drying, seeds were collected in a tray and it was inspected visibly for solid impurities. After shifting in a sieve, grinding was done and seeds were converted to fine dry powder. The powder was stored in a air tight container for further use.

Qualitative Analysis of Plant Material
Solubility Study: Solubility study was performed using solvent like water, methanol, ethanol, Chloroform, NaOH (0.1), & HCI (0.1). The procedure was followed as per IP (Indian pharmacopeia).
Moisture Content: Take 1 gm extract in a bottle. Keep it for drying in hot air oven at 100°C for 6 hours. Take initial weight and final weight and calculate the Loss on Drying.
Melting point: Melting point of the drug was determined by taking small amount of the drug in a capillary tube closed at one end and was placed in a melting point apparatus and the temperature at which the drug melts was noted. Average of triplicate reading was taken.

Collections and Identifications
The plant material was collected in the month of September from Surendranagar district. It was identified by department of Pharmacognosy, C. U. Shah University and herbarium specimen was stored. Dirt particles were removed and it was subjected for shade dying for 15-20 days. After drying, legumes and it was washed thoroughly. Seeds were again subjected for drying in tray dryer legumes and it was washed thoroughly. Seeds were again subjected for drying in tray dryer at 60°C for 4-5 hours. After drying, seeds were collected in a tray and it was inspected visibly for solid impurities. After shifting in a sieve, grinding was done and seeds were converted to fine dry powder. The powder was stored in a air tight container for further use.

Method for Extraction
There are various methods of extraction for crude drug like influsion, decoction, digestion, percolation, maceration and hot continuous percolation (Soxhlet). Among all these methods, Hot continuous percolation i.e. Soxhlet method is most reliable and it gives maximum yield of extract. Drug is suitably comminuted. 150gm of powdered drug was packed in a thimble which was placed into extractor. In first stage, 1500ml Petroleum ether 60:80 was added into a round bottom flask. By this step, fatty materials and oils were removed. The continuous cycles were carried out for 5 hours a day for 3 days. After that, it was further extracted with methanol until the clear white solutions from the siphon tube were obtained. After the Soxhlet extraction, a thick dark solvent with extracted material is accumulated in the round bottom flask. Extra solvent was removed by suction pump. Extract was collected and dried in a porcelain dish and placed into desiccators. This extract was further used for experimental purpose.

Preparation of test Sample
For antimicrobial studies of the leaves, the concentration in range of 1-4 mg/ml was prepared by dissolving solid extract in suitable solvent.

Dilutions and inoculums preparations
The dried extract was weighed and dissolved in sterile distilled water to prepare appropriate dilution to get required concentration of 50, 100mg/ml. The antibiotic Ciprofloxacin was weighed and dissolved in DMSO to prepare appropriate dilution to get required concentration of 1mg/ml. The inoculums of bacterium were prepared in nutrient broth medium and were incubated at 37°C for 8 hours.
Procedure for performing the disc Diffusion test The required amount of petri plates is prepared and autoclaved at 121°C for minutes. And they were allowed to cool under laminar air flow. Aseptically transfer about 20 ml of media into each sterile Petri dish and allowed to solidity. 1 ml inoculums suspension was spread uniformly over the agar medium using sterile glass rod to get uniform distribution of bacteria. The sterile discs were loaded with different concentrations of about 50, 100mg/ml of plant extract of Cassia tora and antibiotic (ciprofloxacin for antibacterial) into each separate disc of about 100µl. The paper diffuse discs were incubated at 37°C for 24 hours. The antibacterial activity was recorded by measuring the width of the clear inhibition zone around the disc using zone reader (mm) [30,31].
Antifungal activity Dextrose medium was used. The required amount of Petri plates and autoclave at 121°C for 15 minutes and they were allowed to cool under laminar air flow. Aseptically transfer about 20 ml of media into each sterile Petri dish and allowed to solidify. 1 ml inoculums suspension was spread uniformly over the agar medium using sterile glass to get uniform distribution of the fungi. The sterile discs were loaded with different concentrations about 50, 100mgml of plant extract of Cassia tora and antifungal (Fluconazole for antifungal activity) into each separate disc of about 100µl. The paper diffuse discs were placed on the medium suitably apart and the plates were incubated at 37° for 24 hours. The antifungal activity was recorded by measuring the width of the clear inhibition zone around the disc using zone reader (mm) [32][33][34].

Phytochemical Screening
The Phytochemical screening [35,36] of the drug is a very sensitive aspect in the process of standardization and quality control because the constituents vary qualitatively and quantitatively not only from plant to plant but also in different samples of the same species depending upon various atmospheric factors and storage conditions. The results are presents below:    From the above data, it is observed that Cassia tora seed methanolic extract showed significant anti-bacterial and antifungal activity against various negative bacteria like Escheriachia coli, Salmonella typhi, Pseudomonas aeruginosa, Coagulase negative Staphylococci, Klebsiellapeumonia etc. It has also inhibited gram positive bacteria like Staphylococcus aureus and fungal species like Candida albicans and Candida glabrata. Ofloxacin is the fluoroquinolone derivative that is generally used for urinary tract infection, enteric fever and upper respiratory tract infection. Since it is working as a broad spectrum antibiotic, it is used as standard drug. Two test samples of Cassia toramethanolic extract was taken in two various concentrations of 100mg/ml and 50mg/ml. that showed significant activity with 1mg/ml if standard drug Ofloxacin. Among both the test samples, 100mg/ml sample has shown best antifungal and antibacterial activity.

Conclusion
It is strongly believed that above detailed information from extensive literature survey, on various activity of Cassia tora might provide detailed evidence for the varied pharmacological and medicinal spectrum. Toxicity of plant leaves also was investigated so there is need of further research in regard; how to expel the toxicity of plant leaf. Thus, Cassia tora seed extract was proved to be efficacious against various bacteria and fungus species. It can be further formulated into a topical formulation to treat common skin disease like itching, rashes eczema and dermatitis. Further in vivo studies are necessary to corroborate the findings.