Phytochemical and Antimicrobial Screening of Leaf and Tuber Extracts of Tephrosia calophylla Bedd

Tephrosia calophylla, (Fabaceae) a perennial woody under shrub endemic to south India. It is one of 13 rare or threatened Tephrosia species. Commonly it is known as Adavivempali. The various species of Tephrosia is ascribed to have many medicinal and therapeutic uses. The importance of this study was to preliminary screening of different phytochemical constituents for the detection of various secondary metabolites and evaluation of antibacterial, antifungal activity and Minimum Inhibitory Concentration of the different crude extracts of tuber and leaf. Tuber and leaf both yielded more number of secondary metabolites like alkaloids, phenols, flavonoids tannins, saponins and glycosides with high quantity when compared with the leaf, consisting low quantities of phytoconstituents as steroids and in tuber consisting only tannins. Antibacterial activity of T. calophylla tuber and leaf aqueous and alcohol extracts at 10 mg/well are showing more effective activity on Bacillus subtilis (MTCC-441), Escherichia coli (MTCC-443), Pseudomonas aeruginosa (MTCC741), Klebsealla neumoniae, Proteus vulgaris strains than the control drug Ampicillin 10 mg/well with 30.25-15.00 mm zone of inhibition. The minimum inhibitory concentration (MIC) with leaf and tuber extracts was 0.312 to 2.50 mg/ml compared to that of the 10 mg of Ampicillin. Antifungal screening of aqueous leaf extract was more effective on Candida albicans with 19.25 mm inhibition zone than Aspergillus niger at 10 mg/well compared to Nystatin the control drug at 10 mg/well with 10.2 to 12.1 mm of zone of inhibition. Fungal MIC on both organisms with leaf and tuber extracts ranges from 0.612 mg to 3 mg compared to 10 mg of Nystatin. Original Research Article Kavitha; JPRI, 33(52A): 105-114, 2021; Article no.JPRI.77735 106


INTRODUCTION
The different crude extracts of medicinal plants parts are used with their phytochemical compounds of known antimicrobial activities, can be of great importance in the different therapeutic treatments. In the present years, a number of works have been conducted in various countries to prove such efficiency. Number of species has been used for their antimicrobial traits, which are due to the secondary metabolites synthesized from the plants. These products are known by their active compounds like, phenolic compounds, alkaloids, flavonoids etc. The screening of plant parts for their antimicrobial activity has shown that the most of the plants represents a potential source of novel antibiotic effect. The Fabaceae or Leguminosae commonly known as the pea, legume, or bean family, is a large and economically important family of flowering plants. Tephrosia is a genus of plant which is of Indian origin. The number species of Tephrosia are medicinally proved for their various pharmacological activities [1,2]. As T. calophylla belongs to same genus and also an important in traditional system of medicine like ayurveda and used as antimicrobial [3], heapatoprotective [4], antihyperlipidemic [5], cytotoxic [6], antiprotozoal [7], anticancer and anti -HIV [8], anthelmintic [9] and antiulcer drug [10]. It is also used as alternative cure for diseases of the liver, spleen, heart and blood. In this attempt, study had been conducted to determine phytochemical and anti microbial potentials of tuber and leaf extracts of T. calophylla.

Collection and Identification of Plant Material
The tuber and leaf material of T. callophyla were collected during September -December 2017 from Talakona forest in Tirupati, Andhra Pradesh, India. The taxonomic identification of the plant is confirmed by Prof. N. Yasodamma. The voucher specimen B.K:3 were deposited in the herbarium, (RUK) Department of Botany, Rayalaseema University, Kurnool for future reference as per standard methods [11]. The present work was also carried out in the same Department. Plant material was thoroughly washed and then shade dried for one week. The dried parts were ground in a mixer grinder and sieved. The powder was stored in air tight containers at room temperature for further use.

Preliminary Photochemical Screening
To detect the different classes of secondary metabolites in the crude extracts of tuber and leaf of T. callophylaa preliminary phytochemical analysis was undertaken by adopting standard qualitative methods [12,13,14,15].

Crude drug preparation of aqueous and organic solvent extracts
Dried tuber and leaf powder (50 g in 250 ml) were extracted with aqueous, acetone, alcohol, benzene, chloroform, ethyl acetate, methanol and petroleum ether. The drug was soaked for 72 hrs. and the filtered extract was dried on water bath than stored at 4 0 C in refrigerator. Tirupati. These were further maintained on nutrient agar slants at 4 0 C until further use.

Agar well diffusion method
Antibacterial and antifungal activities of the leaf and tuber extracts were determined by using agar well diffusion method with slight modifications. Nutrient agar was inoculated with the selected microorganisms by spreading the bacterial and fungal inoculums on the media. Four agar wells (9 mm, diameter) were made in each plate equidistantly by cutting out the media using sterile broad end (8.5 mm) of micropipette tip, in order to load test solutions and are filled with 10 mg/well of the extracts in quadruplicates. Control wells containing pure solvents (negative control) or standard antibiotic (positive control) viz., Ampicillin 10 mg/well, Nistatin 10 mg/well. The plates were incubated at 37 0 C for 24 hrs for bacterial and 25 0 C for 48 hours for fungal activity. The antimicrobial activity was assessed by measuring the diameter of the zone of inhibition for the respective drug. The relative antimicrobial activity was calculated by comparing its zone of inhibition with that of the standard drug. The data of crud drug activity is given the mean of quadruplicates along with the standard error [16].

Statistical Analysis
The results were analyzed for statistical significance using One way ANOVA followed by Dunnet t 's test. The p < 0.01 and p < 0.05 was considered significant.

Evaluation of Minimum Inhibitory Concentration (MIC)
Minimum Inhibitory Concentration was determined by broth dilution method. Extracts to be tested were taken ranging from 10 mg/ml. It involves a series of nine tubes for each test extract against each strain. To the first assay tube 4 ml of broth was transferred and then 4 ml of test extracts of 10 mg/4 ml was added and mixed thoroughly. To the remaining nine assay tubes, from the first tube 4 ml of the test solution was transferred into second test tube and this was mixed thoroughly. This twofold serial dilution was repeated up to ninth tube. 0.2 ml of the inoculums was added to all test tubes and also to the control tubes were taken aseptically and incubated for 24 hrs. Next day the absorbance was measured by calorimeter at 620 nm for bacterial and at 530 nm for fungal broth cultures. Bacterial MIC was compared with the control Ampicillin (10 mg/ml) and for fungal MIC was compared with the control Nystatin (10 mg/ml) and minimum inhibitory concentration mg/ml was determined [17,18]. (Table 1) Tuber and leaf both yielded highest quantity and more number of secondary metabolites like alkaloids, phenols, flavonoids tannins, saponins and glycosides, followed by leaf consisting low quantities of phyto-constituents as steroids, whereas tuber consisting low quantities of tannins. Quinones and lignins are totally absence in benzene extract of tuber.    14

Fig. 1. Antibacterial Activity of leaf and tuber extracts of
All the data are expressed as mean ± SEM, n=6 * p< way ANNOVA followed by Dunnett's test

MIC for Antibacterial Activity
Minimum Inhibitory Concentrations with leaf and tuber extracts at 0.312 to 2.50 mg/ml compared to that of the 10 mg of Ampicillin. Fig. 2

MIC for Antifungal Activity
Fungal Minimum Inhibitory Concentrations on both organisms with leaf and tuber extracts ranges from 0.612 to 3 mg compared to 10 mg of Nystatin.

MIC for Antifungal Activity
Fungal Minimum Inhibitory Concentrations on both organisms with leaf and tuber extracts ranges from 0.612 to 3 mg compared to 10

Fig. 2. Antifungal Activity of leaf and tuber extracts of
All the data are expressed as mean ± SEM, n=6 * p< 0.05 and ** p< 0.01 which compared with control group one way ANNOVA followed by

DISCUSSION
Recently, plant based drug development is very essential in primary health care to reduce side effects of synthetic drugs. T. calophylla root different extracts were capable of suppressing the test organisms such as

Antifungal Activity of leaf and tuber extracts of T. calophylla
All the data are expressed as mean ± SEM, n=6 * p< 0.05 and ** p< 0.01 which compared with control group one way ANNOVA followed by Dunnett's test Recently, plant based drug development is very essential in primary health care to reduce side T. calophylla leaf and root different extracts were capable of suppressing the test organisms such as B.
subtilis, E. coli, S. aures, K. pneumonia, aeuriginosa, P. vulgaris, C. albicans This work was also agrees with the work of (06). By conducting qualitative screening proved that alkaloids, phenols, flavonoids, tannins, saponins, steroids and glycosides are present in the different extracts of root and leaf of This work also supports the traditional use of this plant in therapeutic use against microbial infections. The antibiotic principles of plants may be the presence of phytoconstituents like alkaloids, flavanoids and glycosides [19].

CONCLUSION
The results revealed that the methanol, aqueous and ethyal acetate extracts were showing more effective zone of inhibition than the standard drug Ampicillin. Acetone and methanol extracts which were showing 0.312 mg of Minimum Inhibitory Concentration (Bacterial, Fungal) are advised as the drug dosages for the preparation of standard drugs against bacterial pathogens, especially P. vulgaris (causing urinary track infections) and fungal pathogens like C. albicans (causing mucous membrane infections).

RESEARCH SIGNIFICANCE
The study highlights the efficacy of "Ayurved" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.