IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504

INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES

Available online at www.ijpcbs.com Research Article

SCREENING OF PHYTO-CHEMICAL CONSTITUENTS, TRACE METAL CONCENTRATIONS AND ANTIMICROBIAL EFFICIENCY OF TETRAPHYLLA VS. Nandhini and G. Vijistella Bai* Post graduate and Research Department of Botany, Kunthavai Naacchiyar Govt. Arts College for Women (Autonomous), Thanjavur, - 613 007, Tamil Nadu, .

ABSTRACT Phytochemical constituents and trace metals concentrations, and antimicrobial activity of ethanolic extract of leaves were investigated. The phytochemical screening of the crude extract revealed the presence of steroids, reducing sugars, sugars, alkaloids, flavonoids, saponins, tannins and amino acids. The leave powder was subjected to analyze the trace metals using atomic absorption spectroscopy (AAS). The average concentrations of Cd, Cr, Cu, Fe, Ni, Pb and Zn were BDL, 0.03, 0.2, 0.5, BDL, 0.06 and 0.5 mg kg-1, respectively. And also the crude ethanol extract of Rauvolfia tetraphylla were analyzed for in-vitro antimicrobial activity against certain pathogens and the zone of inhibition were compared with control drug Methicillin and Itraconazole. The Salmonella typhimurium is the most sensitive strains (17mm) to test solution while smallest inhibition were recorded in Micrococcus luteus and Cryptococcus sp. (11 mm).

Keywords: Rauwolfia tetraphylla, Antimicrobial activity, Phytochemistry, Trace metals.

INTRODUCTION in the family. Indian medicinal are well-known Approximately 85 species in the genus can universally for its main role in the primary mainly be found in tropical regions. Rauvolfia health care systems. Most of the rural people tetraphylla and is the South still rely largely on traditional system to cure African quinine species. Rauvolfia their ailments. Medicinal plant in their tetraphylla contains a number of bioactive surrounding environments are collected and phytochemicals and is mainly known for its used largely. As they provide low cost or no cost phytochemical reserpine, which was widely for practicing knowledge on such medicinal used as an antihypertensive drug. But very least systems have been in the process of research has been published on the leaves of transmittance from one generation to the other. plant Rauvolfia tetraphylla. It has increased the efficacy and reliability of the Many plants throughout the world, including medicinal uses as it has been in continuous some with documented medicinal properties testing among the population over the years. In contain chemicals that are toxic to recent years, secondary plant metabolites or microorganisms (Hudson, 1987). The phytochemicals, previously with unknown acceptance of traditional medicine as an pharmacological activities, have been alternative form of health care and the extensively investigated as a source of medicinal development of microbial resistance to the agents (Krishnaraju et al. 2005). There were available antibiotics has led researchers to many reports on the antimicrobial activity of investigate the antimicrobial activity of plant extracts against human pathogenic medicinal plants. The use of plant extracts and bacteria (White et al. 2002; Mishra et al. 2009; phytochemical, with established antimicrobial Haris Kumar et al. 2010). properties, could be of great significance in Rauvolfia is a genus of evergreen and preventive and therapeutic approaches. The

47 IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504

most important antimicrobial compounds of heated. Formation of green or purple color plants are alkaloids, flavonoids, tannins and indicates the presence of sugars. phenolic compounds. Although synthetic and semi-synthetic antimicrobial drugs abound in Alkaloids: A 3 ml of test solution was taken various markets today, there is a need for with 2N HCl. Aqueous layer formed was continuous search for new ones to cope with the decanted and then added with one or a few increased evolution of multiple antimicrobial drops of Mayer’s reagent. Formation of white resistant strains of organisms (Hart et al. 1998). precipitate or turbidity indicates the presence of The increasing prevalence of multi-drug alkaloids. resistant strains of bacteria and the recent appearance of strains with reduced Phenols: A 3 ml of test solution in alcohol was susceptibility to antibiotics raised the specter of added with one drop of neutral ferric chloride “untreatable” bacterial infections and adds (5%) solution. Formation of intense blue color urgency to the search for new infection-fighting indicates the presence of phenols. strategies. In contrast to synthetic drugs, antimicrobial of plant origin usually are not Flavonoids: A 3 ml of test solution in alcohol associated with many side effects and have an was added with a bit of magnesium and one (or) enormous anti-infective potential in numerous two drops of concentrated HCl and heated. infectious diseases. In present study aims to Formation of red or orange color indicates the analyze the phytochemical constituents, trace presence of flavonoids. metals and antimicrobial activity of the Rauvolfia tetraphylla. Saponins: A 3 ml of test solution was added with water and shacked. Formation of foamy MATERIALS AND METHODS lather indicates the presence of Saponins. Plant Material Tannins: A 3 ml of test solution was added with The plant materials were collected from water and lead acetate. Formation of white Thanjavur District of Tamil Nadu in India during precipitate indicates the presence of tannins. the period of January to February 2013. The shade dried plant powders (100 g) were Anthroquinones: A 3 ml of test solution was successively extracted with ethanol by soxhlet added with magnesium acetate. Formation of apparatus and is used as test sample. pink color indicates the presence of anthroquinones. Phytochemical screening Qualitative analysis Amino Acids: A 3 ml of test solution was added The solvent extracts were subjected to routine with 1% ninhydrin in alcohol. Formation of blue qualitative chemical analysis to identify the or violet color indicates the presence of amino nature of phytochemical constituents present in acids. sample (Koperuncholan et al. 2011). Catechins: A 3 ml of test solution in alcohol was Steroids: Three ml of test solution and added with Ehrlich reagent and a few drops of minimum quantity of chloroform was added concentrated HCl. Formation of pink color with 3-4 drops of acetic anhydride and one drop indicate the presence of catechins. of concentrated H2SO4. Purple color thus formed changes into blue or green color indicating the Quantitative analysis of phytoconstituents presence of steroids. The chlorophyll pigments in the leaves were estimated following the method of Arnon Triterpenoids: A 3 ml of test solution was (1949). After pre-cleaning, weighted fresh leaf added with a piece of tin and 2 drops of thionyl material was homogenized and extracted thrice chloride. Formation of violet or purple colour in chilled 80% acetone (v/v). The volume of the indicates the presence of triterpenoids. acetone extract was made up to a known one and the optical density was read at 645nm and Reducing Sugars: A 3 ml of test solution was 663nm wavelengths on a spectrophotometer. added with a 2 ml of Fehling’s reagent and 2 ml The concentration of the chlorophyll pigments of water. Formation of reddish orange color was calculated and is expressed in mg/g fresh indicates the presence of reducing sugar. weight. Amino acids were estimated by Ninhydrin method which is calorimetrically Sugars: A 3 ml of the test solution was added measured at 570nm (Hwang, 1975). Proteins with very small quantity of anthrone reagent were estimated by Bradford method and the and a few drops of concentrated H2SO4 and absorbance was measured at 595 nm against

48 IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504

blank/ sample (Bradford, 1976). Carbohydrates were performed in triplicate and the average were estimated by anthrone method which can values are presented. Methicillin – 10mcg (for be measured by using colorimetrically at 620nm bacteria) and Itraconazole – 10mcg (for fungus) (or) by using a red filter (Morris 1948). All the was used as positive control. All the media, trials were performed thrice and the mean standard discs and sterile disc were purchased values were presented. from Hi-Media (Mumbai, India).

Trace metal analysis RESULTS AND DISCUSSION The Rauvolfia tetraphylla plant sample was Phytochemical constituents of secondary collected from the Thanjavur district, Tamil metabolites Nadu. The leaves were carefully removed and The present study revealed that the medicinal washed with sterile distilled water. The cleaned plant such as Rauvolfia tetraphylla contains leaves were dried in shadow area and were bioactive compounds. The phytochemical grinned with mortar and pestle. After drying, 1 g constituents were screened by qualitative and of plant samples was treated with aqua-regia quantitatively methods and the results are mixture in Teflon bomb and was incubated at presented in Table 1 and 2. In Tannin analysis, 140 °C for 2-3 days. After incubation, the the brownish green colour formation indicates reaction mixture was filtered with Whatman the presence of Tannin. Similarly, based on the No.1 filter paper. Then the extraction was test presence or absence of colour change indicate for trace metals (Fe, Cu, Zn, Pd, Cd, Cr and Ni) positive and negative results are indicate. In analysis. The extraction of the studied metals in these screening, the positive results were the solutions was determined by the 797 VA obtained from steroids, reducing sugars, sugars, Computrace voltametry, Metrohm. To avoid the alkaloids, flavonoids, saponins, tannins and contamination, the devices were rinsed with amino acids while triterpenes, phenols, acidified water (10% HNO3) and weighted to catechins, and anthroquinones gave negative dissolve metals before analysis. And all the results. equipments and containers were soaked in 10% This contains a number of bioactive NHO3 for 24 h then rinsed thoroughly in de- phytochemicals and is mainly known for its ionized water before use. Also find the below phytochemical reserpine, which was widely detectable limit of the instruments. used as an antihypertensive drug. It is anticipated that phytochemicals with adequate Determination of Antimicrobial Activity antibacterial efficacy will be used for the Testing of antimicrobial activity treatment of bacterial infections (Balandrin et al. The test strains were: Aeromonas liquefaciens 1985). Tannins are dietary anti-nutrients that MTCC 2645 (B1), Enterococcus faecalis MTCC are responsible for the astringent taste of foods 439 (B2), Klebsiella pneumonia NCIM 2883 (B3), and drinks (Chikezie et al. 2008). Tannins bind Micrococcus luteus NCIM 2871 (B4), Salmonella to both proteins and carbohydrates which has typhimurium NCIM 2501 (B5), Vibrio cholerae several implications for commodities containing MTCC 3906 (B6), Candida albicans MTCC 1637 tannins. Their presence can cause browning or (F1), Cryptococcus sp. MTCC 7076 (F2), other pigmentation problems in both fresh foods Microsporum canis MTCC 3270 (F3), and processed products. The presence of tannin Trichophyton rubrum MTCC 3272 (F4). The in the plants implies they may have astringent cultures were obtained from MTCC, Chandigarh properties and in addition, could quicken the and NCIM, Pune, India. Microbial strains were healing of wounds and burns (Farquar, 1996). tested for antimicrobial sensitivity using the disc This justifies their usage in herbal medicine. diffusion method (Koperuncholan et al, 2010). The antibacterial and antifungal activity of test Trace metals analysis samples was analyzed against certain Heavy metals such as Cu, Zn, Mn and Fe are microorganisms on muller hinton agar (MHA) essential for plant growth; many of them do not and potato dextrose agar (PDA), respectively. A have any significant role in the plant physiology sterile cotton swab was used to inoculate the (Balandrin, et al 1985). Sometimes, it also affects bacterial suspension on surface of agar plate. the photosynthetic ability of leaves, closure of The 15 and 30 μL of sample coated disc were leaf stomata, plant growth, physiology and placed in agar plates, separately. For negative productivity of plants (Hart et al. 1998). Metal control study, the sterile triple distilled water contents of plant samples, Cd, Cr, Cu, Fe, Ni, Pb was used. The plates were incubated at 37±1°C and Zn concentrations are BDL, 0.03, 0.2, 0.5, for 24–48 h (for bacteria) and 25 ±1°C for 48-72 BDL, 0.06 and 0.5 mg kg-1, respectively. Heavy h (for fungus). After incubation, the zone of metals such as Cu, Zn, Mn and Fe are essential inhibition was measured with ruler. The assays for plant growth, many of them do not have any

49 IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504

significant role in the plant physiology (Shaw, found to be more sensitive to crude extract 1990). Contaminated sites often support some when compared to others. Several plant species, which are able to accumulate or phytoconstituents such as terpenoids tolerate high concentrations of metals such as (Scortichini and Rossi, 1991), flavonoids Pb and Zn ( Kumar et al.1995). A small number (Tsuchiya et al. 1966) and tannins (Ya et al. of species are capable of growing on soils 1988) are effective against certain containing high levels of metals, and also microorganisms. The results of the present accumulate these pollutants in high investigation clearly demonstrate the concentrations in the parts above ground. These antibacterial and antifungal activities of the plants are known as hyper accumulators ethanol extracts of the leaves. (Brooks et al. 1977). The uptake of these heavy metals by plants is an avenue of their entry into CONCLUSION the human food chain with harmful effects on This study concluded that the presence of health (Ihekoronye and Ngoddy, 1985). steroids, reducing sugars, sugars, alkaloids, flavonoids, saponins, tannins and amino Antibacterial and Antifungal screening acids are helps to the antimicrobial activity and The antimicrobial activity of Rauvolfia is effective than positive control. The presence tetraphylla was examined with various of heavy metals indicated that the plant was microorganisms using the disc diffusion test. resistant to the trace metal and their secondary The results of the antimicrobial activities are metabolites were not seriously affected by these summarized in Table 4. The two tested metals and it may enter into the human food concentrations such as 15 and 30 μL /disc chain. In this endeavor, traditional herbal produce zone of inhibition on MHA and PDA medicines must perforce be granted the benefits plates for bacteria and fungi, respectively. In the of modern science and technology to serves present study, higher (30 μL/disc) further global needs. The Rauvolfia tetraphylla concentration of sample got greater sensitivity may act as an alternative antibiotic in near than (15 μL/disc) lower concentration in most future. of the microorganisms. In bacteria, the test sample was most effective against Salmonella Table 1: Quantitative phytochemical typhimurium NCIM 2501 (B5) while smaller constituent of Rauwolfia tetraphylla Rauvolfia tetraphylla effect was noticed from Micrococcus luteus NCIM Biochemical constituents 2871 (B4). In fungi, which was effective against (mg/g) Trichophyton rubrum MTCC 3272 (F4) whereas Chlorophyll A 0.117 Chlorophyll B 0.852 smaller effect was observed in Cryptococcus sp. Total Chlorophyll 0.969 MTCC 7076 (F2). All the microbial strains depict Amino acid 150 higher sensitivity to the higher concentration Protein 2.120 (30 μL) for the test sample when compared to Carbohydrate 1.000 the positive control except B3, B4 and B6. There Phenol 0.025 is no antimicrobial activity in solution devoid of sample used as a vehicle control (sterile triple Table 2: Quantitative phytochemical distilled water), reflecting that antimicrobial constituent of Rauwolfia tetraphylla Phytochemical Phytochemical activity was directly related to the sample. Result Result Constituents Constituents Suresh et al. (2008) reported the best Steroids + Catechins - antimicrobial activity of Rauvolfia tetraphylla, Triterpenes - Flavonoids + which showed maximum activity against E. coli Reducing sugars + Saponins + and Enterobacter aerogenes, and various tested Sugars + Tannins + fungi such as A. niger and Penicillium sp, were Alkaloids + Anthraquinones - Phenolics - Amino acids +

Table 3: Concentration of trace metals in Rauvolfia tetraphylla

Sampling Site Name Sample S. Code Name/ Family Cd Cr Cu Fe Ni Pb Zn No. Sample Site No. Site Sampling Thanjavur, Rauvolfia tetraphylla P1 TP1 BDL 0.03 0.2 0.5 BDL 0.06 0.5 Tamil Nadu S1

------BDL – Below detectable limit

50 IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504

Table 4: Antimicrobial activity of the ethanolic extracts of leaves in Rauvolfia tetraphylla Test Zone of inhibition (mm) Diseases Route of Transmission S.No Microorganisms Sample (15 & 30) µL / disc Bacteria 15 30 µL PC Remarks µL 1. Aeromonas Wound Infections / Water / Food liquefaciens 13 16 14 > PC Gastroenteritis B1 2. Enterococcus fecalis Endocarditis / Bladder, Water / Food B2 Prostate, and Epididymal 12 14 8 > PC Infections / Nervous system Infections 3. Klebsiella Acute diarrhoea / Dysentery Water / Food pneumoniae 13 15 28 < PC B3 4. Micrococcus luteus Skin & Pulmonary infections / Soil / Dust / Water / Airways B4 11 12 38 < PC Septic shock / Pneumonia / Food endocarditis 5. Salmonella Typhoid Water / Food typhimurium 14 17 0 > PC B5 6. Vibrio cholarae Cholera Water / Food 11 13 16 < PC B6 Fungi 7. Candida albicans Skin (Integument) Infections / Airways / Wound / 12 15 10 > PC F1 Gastrointestinal tract Infection Soil / Water 8. Cryptococcus sp. Cryptococcal disease / Airways / Wound / F2 11 13 9 > PC Bronchiectasis / Soil / Water Endophthalmitis. 9. Microsporum canis Tinea capitis /Ringworm Airways / Wound / F3 12 14 9 > PC Soil / Water

10. Trichophyton rubrum Tinea corporis / Tinea cruris / Airways / Wound / 13 16 7 > PC Tinea pedis / Onychomycosis Soil / Water F4

PC -Positive Control (Using antibiotic disc; Bacteria – Methicillin (10mcg/disc) ; Fungi – Itraconazole (10mcg/disc) Samples - 15 µL / disc & 30 µL / disc; > PC – greater than positive control; < PC – less than positive control

ACKNOWLEDGEMENT indicator plants. Journal of Geochemical The authors thank the Biospark Exploration. 1977;7:49–58. Biotechnological Research Center (BBRC), 5. Chikezie PC, Agomuo EN and Amadi BA. Tiruchirapalli, Tamil Nadu, India for Trace metal Biochemistry, Practical/Research analysis. Method, A Fundamental Approach. Vol 2, Mega soft publishers. 2008:51-53. REFERENCES 6. Farquar JN. Plant Sterols their 1. Arnon DI. Copper enzymes in isolated biological effects in human. Handbook chloroplasts, polyphenoxidase in beta of Lipids in Nutrition BOCA Rotan HL vulgaris. Plant physiology. 1949;24:1- CRC Press. 1996:101-105. 15. 7. Haris Kumar K, Kullatti KK, Sharanappa 2. Balandrin MF, Kjocke AJ and Wurtele E. P and Sharma P. Comparative Natural plants chemical, sources of antimicrobial activity and TLC - industrial and mechanical materials. bioautographic analysis of root and Science. 1985;228:1154-60. aerial parts of Andrographis 3. Bradford MM. Rapid and sensitive serphyllifolia. International Journal of method for the quantitation of Pharmacy and Pharmaceutical Science. microgram quantities of protein 2010;2:52-54. utilizing the principle of protein-dye 8. Hart CA and Kariuki S. Antimicrobial binding. Anal Biochem. resistance in developing countries. Br 1976;72(1):248–254. Med J. 1998;317(1):647-50. 4. Brooks RR, Lee J, Reeves RD and JaGre 9. Hwang M and Ederer GM. Rapid T. Detection of nickeliferous rocks hippurate hydrolysis method for byanalysis of herbarium specimens of presumptive identification of group B

51 IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504

streptococci. J Clin Anthrone Reagent. Science. Microbiol. 1975;1:114. 1948;107(2775):254-255. 10. Ihekoronye AI and Ngoddy PO. 18. Perez C, Paul M and Bazerque P. Integrated Food Science and Antibiotic assay by agar-well diffusion Technology for the Tropics. Macmillan method. Acta Biologiae Ethnomedicine Publishers. 1985:75 – 77. Experimentalist. 1990;15:113–115. 11. Hudson JB. Plant photosensitizers with 19. Scortichini M and Pia Rossi M. antiviral properties. Journal of Preliminary in vitro evaluation of the Ethnopharmacology. 1987;12(2):55-74. antimicrobial ctivity of terpenes and 12. Koperuncholan M and Ahmed John S. terpenoids towards Erwinia amylovora Antimicrobial and Phytochemical (Burrill). J Appl Bacteriol. 1991;71:109- Screening in Myristica dactyloides 112. Gaertn. Journal of Pharmacy Research. 20. Shaw AJ. Heavy Metal Tolerance in 2011;4:398-400 Plants: Evolutionary Aspects. CRC Press, 13. Koperuncholan M, Sathish Kumar P, Boca Raton, FL. 1990. Sathiyanarayanan G and Vivek G. 21. Suresh K, Saravana Baby S and Phytochemical Screening and Harisaranraj R. Studies on In Vitro Antimicrobial Studies of Some Ethno antimicrobial activity of ethanol medicinal Plants in South-Eastern Slope extracts of Rauvolfia tetraphylla. of Western Ghats. International Journal Ethnobotanical Leaflets. 2008;12:586- of Medicobiological Research. 590. 2010;1:48-59. 22. Tsuchiya H, Satho M, Miyazaki T, 14. Krishnaraj AV, Rao TVN and Fujiwara S, Tanigaki S, Ohyama M, Sundararaju D. Assessment of Tanaka T and Iinuma M. Comparative bioactivity of Indian medicinal plants study on the antibacterial activity of using Brine shrimp (Artemia salina) phytochemical flavonols against lethality assay. Int J Appl Sci Eng. methicillin resistant Staphylococcus 2005;2:125-34. aureus. J Ethnopharmacol. 1996;50:27- 15. Kumar PBAN, Dushenkov V, Motto H 34. and Raskin I. Phytoextraction: the use 23. White DG, Zhao S, Simjees S, Wagner DD ofplants to remove heavy metals from and McDermott PF. Antimicrobial soils. Environmental Science and resistance of food borne pathogens. Technology. 1995;29:1232–1238. Microbes and Infection. 2002;4:405- 16. Mishra US, Mishra A, Kumari R, Murthy 412. PN and Naik BS. Antibacterial activity of 24. Ya C, Gaffney SH, Lilley TH and Haslam ethanol extract of Andrographis E. Carbohydrate polyphenol paniculata. Indian Journal of complexation. P.553. In: Hemingway Pharmaceutical Sciences. 2009;71:436- RW, Karchey JJ (Eds). Chemistry and 438. significance of condensed tannins. 17. Morris DL. Quantitative Determination Plenum press, New York. 1988:553. of Carbohydrates with Dreywood's

52