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Investigation on Phytochemical and antimicrobial properties of tuber extracts of Pueraria tuberosa Linn.

In the current scenario, development of drug resistance (Senthilkumar and Prabakaran, 2005; Balaji and Senthilkumar, 2011; Senthilkumar et al, 2014; Behailu etal, 2016) among the disease causing microbes to the commonly used antibiotics has provoked the research for discovery of new antimicrobial agents. Plants are considered as primary choice for dietary supplements and also for exploring for new medicines (Abdallah, 2011; Murugan et al, 2014). Medicinal values of many plants are still remaining unexplored for its enumerable activity of compounds responsible for their activity. Besides, plant materials remain important resources of remedies to combat against serious diseases around the world and plants are known to contain enormous biologically active compounds which possess antibacterial properties (Ahmad and Wajid, 2013). Hence, it is essential to carryout pharmogonostic investigations of plants to find novel drugs or templates for the development of new therapeutic agents (Nasir et al, 2015).

P. tuberosa is a perennial woody climber with large tuberous roots belongs to the family Fabaceae. It grows up to 6 m tall, it has opposite trifoliate compound leaves and the flowers are white. Pods are flat, constricted between seeds (Theng and Korpenwar, 2014). P. tuberosa is an important and potential medicinal plant in Indian traditional system of medicine that is in Siddha and Ayurveda. The Ayurvedic Pharmacopoeia of India is calling P. tuberosa under the name of Indian Kudzu (or) Vidari. The flowers of P. tuberosa are used as cooling agent and as aphrodisiac (Mishra, 2012). The root tuber is sweet, oily and it has cooling effect and used in the form of tonic, demulcent and refrigerant for fever, inflammation, etc. Besides, it is effectively used in aphrodisiac, galacatagogue and diuretic, and used to cure leprosy, blood and urinary related disorders (Verma et al., 2012). In folk medicine, the P. tuberosa root tubers had been used for purification of blood and improve the sperm production. The consumption dried raw root powder had been used control the worm growth in intestine and used to keep clean the stomach (Vijay et al., 2013).

The medicinal value of P. tuberosa is extended as fertility controlling agents such as aphrodisiac, cardiotonic, diuretic and galactogogue and the experimental evidence exhibits antihyperglycemic, anti-hyperlipidemic, anti-fertility in male rats, hepatoprotective, and anti-implantation activities (Hsu et al., 2003; Tanwar et al., 2008). It is used in food supplements as nutritive, diuretic, expectorants and for the management of rheumatism, fever, and bronchitis (Pandey et al., 2013). P. tuberosa tubers have rich in extended vast level of bioactive phytoconstituents like isoflavonoids, puerarin, daidzein, genistein, puetuberosanol and tuberosin (Amal et al., 2014). During the past decade, interest in these isoflavonoids has increased considerably because of the beneficial effects proposed by epidemiologists, nutritionists, and food manufacturers since isoflavonoids could interact with milk proteins, namely, bovine serum albumin, casein micelle, [beta]-lactoglobulin have been reported in case of certain food and antimicrobial drug preparation (Shilpashree et al., 2015). Against these backdrops, in the present study, the exploration of bioactive compounds of P. tuberosa was made using various polar and non-polar solvents, and the evaluation of antimicrobial properties of bioactive compound was made against common human pathogens.

MATERIALS AND METHODS

Collection and preparation of material

The fresh tubers of Pueraria tuberosa were collected from Yercaud hills, India and authenticated at Centre for Advanced Studies in Botany, University of Madras, India. The tubers were washed with mild detergents to remove the adhered dust and soils. The tubers were rinsed several times in tap water till the detergent was completely removed and were shade dried at room temperature till it is completely dried. Dried tubers were powdered with the help of a blender and fine powder was separated by sieving. The powder was tightly packed in sterile polythene bags for further experimental purpose.

Extraction of Bioactive compounds

The extraction of P. tuberosa tuber was made by sequential cold extraction method using the different polar and non-polar solvents such as hexane, benzene, chloroform, ethyl acetate, acetone and methanol (Sadguna et al., 2015). Fifty grams of P. tuberosa tuber powder was mixed with 4 volumes of solvent in a glass stopper flask and kept overnight at room temperature for complete extraction of phytocompounds. The mixture was agitated well and filtered through Whatman No. 1 filter paper. Both filtrate and debris was air dried by evaporating the solvent in open air at room temperature till they attain complete powder form. The filtrated powder was stored in a sterile vial at 4 [degrees]C for further use and the dried debris was subjected to sequential extraction using remaining solvents. The similar extraction method was continued using other solvents and the extracted tuber debris was used as source material for other solvents.

Estimation of bioactive compounds

The different solvent extracts of P. tuberosa root tubers were analyzed for the phytochemicals like total antioxidants, phenolic compounds and flavonoids by using modified methods of Tripathi and Kohli (2013). The experiments for the estimation of bioactive compounds were repeated three times and the average data expressed as mean[+ or -] median.

Total antioxidants

The total antioxidants of P. tuberosa tuber extract were estimated by ABTS (2, 2'-azino-bis 3ethylbenzenethiazoline-6-sulphonic acid) method. The reaction mixture was prepared by adding 25 ml of tuber extract, 1 ml of diluted ABTS solution and 5 ml of distilled water and the mixture was allowed to stand for 5 min. The absorbance of the mixture was read at 734 nm using spectrophotometer against reagent blank. The total antioxidant value was estimated by comparing the absorbance with standard chart prepared using p-carotene.

Total phenolic acids

The total phenolic content of P. tuberosa tuber extract was determined using folin ciocalteau reagent method. The reaction mixture was prepared by adding of 1 ml of tuber extract, 3 ml of distilled water and 0.5 ml of folin ciocalteau reagent and 2 ml of [Na.sub.2]C[O.sub.3] (20%). The reaction mixture was mixed well and warmed for 1 minute for the development of blue colour. The absorbance of the mixture was read at 650 nm against the reagent used as a blank. The concentrations of phenols in the test samples were calculated from the calibration plot and expressed as mg catechol equivalent of phenol/g of sample.

Total flavonoids

The estimation of total flavanoid was done by methanolic aluminium chloride reagent. One ml of tuber extract was added with 1 ml of 2 % of aluminium chloride reagent prepared in methanol. The mixture was agitated well for 2 minutes and absorbance was read at 430 nm against the reagent blank. Total flavonoid content of the tuber extract was expressed by comparing the absorbance within equivalent of Gallic acid standard.

Evaluation of antimicrobial activity Microbial Strains

The microbial strains were selected accordingly, which are human pathogens that commonly cause various diseases. The pure strains were obtained from Microbial type culture collection (MTCC), Chandigarh, India. The test was performed against different strains of bacteria such as Escherichia coli (MTCC 590), Klebsiella pneumonia (MTCC 9509), Shigella boydii (MTCC 11947), Salmonella typhi (MTCC 3224), Staphylococcus aureus (MTCC 1430), Pseudomonas aeruginosa (MTCC 4673), Proteus vulgaris (MTCC 658),Micrococcus luteus (ATCC 106), Vibrio cholera (MTCC 3906) and fungi such as, Candida albicans (MTCC 183), Candida parapsilosis (MTCC 2509) and Candida tropicalis (MTCC 184).

Disc diffusion assay

The antimicrobial activity was carried out by Kirby Bauer method using Muller Hinton Agar (Senthilkumar et al., 2014; Sadguna et al., 2015). The overnight microbial cultures were adjusted to the approximately 1.5 x [10.sup.8] cells/ml according to the McFarland standard. The cultures were swabbed to the sterile dry Mueller Hinton agar (Hi-media, India). Meanwhile, the tuber extract was dissolved in 40% Dimethyl sulphoxide (DMSO) and incorporated to the sterile paper discs for the concentration of 50 mg/ disc. The loaded disks were dried in sterile condition and placed on the surface of the appropriate inoculated Mueller Hinton agar medium. The sterile disc loaded with 10pl of DMSO was maintained as control. The plates were incubated at 37 [degrees]C for 24 h. After the incubation, the zone of inhibition was recorded in millimetres. The experiments for the antimicrobial activity were repeated three times and the average data expressed as mean[+ or -] median.

RESULTS AND DISCUSSION

Experimental plant

As a primary part of this work, the P. tuberosa plant was identified and the tubers were collected. P. tuberosa was a climbing shrub with tuberous roots with trifoliate leaves. The inflorescence is axillary racemes with bluish-purple colour. The tubers of P. tuberosa exhibited different shapes like spherical, semi spherical and cuboidal with fibrous root. The surface has brown thick skin and inner part was whitish brown in colour. The inner flush was annulated with exudates. Bioactive compounds

The extracts from each solvent were subjected to total antioxidant estimation. The results showed that almost all the solvent extracts showed the presence of antioxidants (Table 1). Among the solvents, ethyl acetate has extracted maximum quantity (8.12 mg/g) of antioxidants followed by chloroform extract (7.75 mg/g). The least quantity of antioxidant extraction was observed in almost all remaining solvents. The total flavonoid extraction results revealed the highest yield of 870 [micro]g/g and 580 [micro]g/g of flavonoids by methanol and acetone, respectively whereas, least flavonoids was extracted by hexane. In the case of total phenolic acid extraction, methanol and acetone has extracted more amount of phenolic acid as 905 [micro]g/g and 600 [micro]g/g, respectively. In contrast, Hexane and choloroform extract yielded least quantity of total phenolic acid (100 [micro]g/g) whereas, other solvents extracted moderate quantity. The P. tuberosa contains isoflavonoids in the stem and root, the tuberosin is an active flavonoid which has versatile potentials (Pandey and Tripathi, 2010). Roots of P. tuberosa contain p-sitosterol, stigmasterol, daidzein, puerarin and a new isoflavone C-glycoside - 4', 6' '-diacetyl puerarin. An isoflavone glycoside has also been isolated from roots and tubers of Pueraria alopecuroides (Yang et al., 2016). It is significant to note that P tuberosa which seems almost extinct in vast tracts of the Eastern and Western Ghats of India. The phytochemical analyses have reported the presence of various isoflavonoids of high antioxidant properties including daidzin, genistin, tectoridin, and puerarin in P. tuberosa (Sawale et al, 2013).

Antimicrobial activity

The antimicrobial susceptibility test of P tuberosa revealed that the extracts of ethyl acetate, hexane and acetone showed broad spectrum of inhibitory activity among the tested bacteria (Fig. 1). The bacterial strains showed higher susceptible range over than fungal isolates. Among the fungal strains, C. albicans showed higher susceptible rate than other two fungal strains (Table 2). Among the bacterial strains, M. luteus and S. boydii showed more sensitive to the compounds extracted by almost all the solvents. E. coli and P. aeroginosa showed sensitivity towards most of solvent extracts whereas, S. typhi showed resistance except ethyl acetate and acetone extracts. Among the different solvents used for extraction, the ethyl acetate was found to be an effective solvent that extracts the antimicrobial compounds from P. tuberosa. The previous literature evidences reported that that the ethyl acetate extract of P. tuberosa exhibited broad spectrum of antibacterial activity against K. pneumoniae, M. luteus, S. typhimurium, B. cereus, S. aureus and P. aeruginosa. The ethanol extract showed feeble activity against the test organisms (Ratnam and Raju, 2009). The presence of biologically active compounds such as phenols, polyphenols, tannins, alkaloids flavanoids and terpenoids in the plants are known to possess antibacterial activity. P. tuberosa is an under utilised species and it can be a valuable alternative to much sought after other species. Puerarin, highly abundant in P. tuberosa, has hypothermic, spasmolytic, hypotensive and antiarrhymatic activities (Vidhya and Nishteswar, 2015).

CONCLUSION

The present investigation revealed that the ethanolic tuber extract of the selected P. tuberosa were shown active against all the selected pathogens. The ethyl acetate tuber extracts were found to show a moderate antibacterial activity and the P. tuberosa in particular exhibited greater prosperity to inhibit fungal growth even at low concentration. Further studies on P. tuberosa will certainly aid the isolation of bioactive compounds for their pharmacological importance to check the growth of harmful microbes.

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(14.) Sawale, P.D., Singh, R.R.B., Kapila, S., Arora, S., Rastogi, S., Rawat, A.K.S. Immunomodulatory and antioxidative potential of herb (Pueraria tuberosa) in mice using milk as the carrier. Int. J. Dairy Technol., 2013; 66(2): 202-206.

(15.) Senthilkumar, B., Anbarasu, K., Senbagam, D., Rajasekarapandian, M. Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development. AsianPac. J. Trop. Med., 2014; 7 (12): 933-939.

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(21.) Tripathi, A.K., Kohli, S. Anti-Diabetic Activity and Phytochemical Screening of Crude Extracts of Pueraria Tuberosa DC. (Fabaceae) Grown in India on STZ -Induced Diabetic Rats. Asian J. Med. Pharm. Res., 2013; 3(3): 66-73.

(22.) Verma S.K., Vartika, J., Singh, D.P. Effect of Pueraria tuberosa DC. (Indian Kudzu) on Blood Pressure, Fibrinolysis and Oxidative Stress in Patients with Stage 1 Hypertension. Pak. J. Biol. Sci., 2012; 15: 742-747.

(23.) Vidhya, U., Nishteswar, K. Antiplatelet ayurvedic herbs in the management of cardiovascular disease - A review. Int. Ayurvedic Medical J, 2015; 3(5): 1462-1473.

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M.R. Aruna [1], D.J. Mukesh Kumar [2], D. Senbagam [3] and B. Senthilkumar [1]*

[1]* Department of Biotechnology, Muthayammal College of Arts and Science, Rasipuram, TN, India.

[2] CAS in Botany, University of Madras, Guindy Campus, Chennai, TN, India.

[3] Department of Microbiology, Vivekananda College of Arts and Science for Women, Tiruchengode, TN, India.

(Received:10 December 2015; accepted: 25 February 2016)

* To whom all correspondence should be addressed. Mob.: +91-9443286292; E-mail: senthilmicro@yahoo.co.in; nevithasenthil@gmail.com

Caption: Fig. 1. Antibacterial activity of P. tuberosa tuber extract a) E. faecalis b) E. coli c) P. vulgaris d) P. aeruginosa e) S. typhi f) S. boydii (C - Control; H - Hexane; B - Benzene; CH - Chloroform; E - Ethyl acetate; A - Acetone; M - Methanol)
Table 1. Phytochemical analysis of P. tuberosa tuber extracts

                Bioactive compounds

Solvents            Total       Total flavonoid    Total phenolic
                 antioxidant      ([micro]g/g)        compound
                   (mg/g)                           ([micro]g/g)

Hexane          1.93 [+ or -]   105.66 [+ or -]    103.67 [+ or -]
                    0.153            5.132              4.042

Benzene         2.47 [+ or -]     123 [+ or -]     253.33 [+ or -]
                    0.153            3.606              3.512

Chloroform      7.77 [+ or -]   272.333 [+ or -]   102.67 [+ or -]
                    0.025            2.517              3.055

Ethyl acetate   8.14 [+ or -]   372.33 [+ or -]    600.33 [+ or -]
                    0.025            2.517              0.577

Acetone         0.95 [+ or -]   580.33 [+ or -]    350.33 [+ or -]
                    0.035            2.517              0.577

Methanol        1.33 [+ or -]   873.33 [+ or -]    906.67 [+ or -]
                    0.306            4.163              2.082

Table 2. Antimicrobial properties of P. tuberosa tuber extracts


                   Solvent extracts/ Zone of inhibition (mm)

Test organisms         Control Hexane             Benzene

C. parapsilosis              -                       -
C. albicans        - 8.27 [+ or -] 0.252             -
C. tropicalis                -                       -
E. coli             9.17 [+ or -] 0.208              -
K. pneumoniae       - 10.3 [+ or -] 0.3        10 [+ or -] 0
M. luteus          - 11.3 [+ or -] 0.265    11.17 [+ or -] 0.208
P. vulgaris        - 10.1 [+ or -] 0.581             -
P. aeruginosa        - 8.3 [+ or -] 0.3     11.27 [+ or -] 0.306
S. typhi                     -                       -
S. boydii          - 11.23 [+ or -] 0.208   9.23 [+ or -] 0.252
S. aureus            - 10 [+ or -] 0.0         9 [+ or -] 0.0
V. cholerae                  -                9.3 [+ or -] 0.3

                   Solvent extracts/ Zone of inhibition (mm)

Test organisms          Chloroform            Ethylacetate

C. parapsilosis             -                      -
C. albicans                 -                      -
C. tropicalis      8.33 [+ or -] 0.252             -
E. coli              9.3 [+ or -] 0.2      11.3 [+ or -] 0.2
K. pneumoniae               -             9.17 [+ or -] 0.208
M. luteus          10.43 [+ or -] 0.153   13.3 [+ or -] 0.252
P. vulgaris                 -             11.37 [+ or -] 0.321
P. aeruginosa                             9.46 [+ or -] 2.347
S. typhi                    -             8.45 [+ or -] 0.351
S. boydii          9.23 [+ or -] 0.245    10.46 [+ or -] 2.347
S. aureus                   -             8.37 [+ or -] 0.321
V. cholerae        8.56 [+ or -] 0.343    11.17 [+ or -] 0.208

                   Solvent extracts/ Zone of inhibition (mm)

Test organisms           Acetone                Methanol

C. parapsilosis      9.0 [+ or -] 0.0              -
C. albicans        7.17 [+ or -] 0.208    8.43 [+ or -] 0.153
C. tropicalis               -                      -
E. coli             10.3 [+ or -] 0.2      10.0 [+ or -] 0.0
K. pneumoniae      11.43 [+ or -] 0.153            -
M. luteus           9.1 [+ or -] 0.58     11.27 [+ or -] 0.252
P. vulgaris         10.0 [+ or -] 0.0     9.45 [+ or -] 0.351
P. aeruginosa      13.45 [+ or -] 0.351    9.1 [+ or -] 0.581
S. typhi           9.46 [+ or -] 0.437             -
S. boydii          11.13 [+ or -] 0.367   10.37 [+ or -] 0.321
S. aureus          12.62 [+ or -] 0.141            -
V. cholerae        10.25 [+ or -] 0.345   9.25 [+ or -] 0.351
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Author:Aruna, M.R.; Kumar, D.J. Mukesh; Senbagam, D.; Senthilkumar, B.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Jun 1, 2016
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