World Journal of Agricultural Sciences 7 (6): 659-666, 2011 ISSN 1817-3047 IDOSI Publications, 2011

Preliminary Phytochemical and Antimicrobial Activity Studies on the Leaves of the Indian Plant Thevetia neriifolia Juss.
1

K. Buvaneswari, 1D. Ramamoorthy and 2J. Velanganni

Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry-605, 104 India 2 Kanchimamunivar Centre for Post Graduate Studies, Puducherry, India
Abstract: The leaves of Thevetia neriifolia Juss., an important medicinal plant was subjected to phytochemical screening, pharmacognosy and antimicrobial investigations. Preliminary phytochemical screening of leaves extracts revealed the presence of the bioactive compounds, such as alkaloids, anthroquinones, flavonoids, phenolic compounds, saponins, steroids and tannins in the leaves. Pharmacognostical studies such as extractive values and fluorescence analysis brought out the standardized data in the quality control of this drug. Hexane, chloroform, ethanol and aqueous extracts of leaves showed significant antimicrobial activity against the tested human pathogens. Hexane and ethanol extracts exhibited maximum activity against the tested human pathogens, while aqueous extracts showed minimum activity against fungal pathogens only. The bioactive compounds responsible for these antimicrobial activities could be isolated and identified to develop a new drug of pharmaceutical interest. Key words: Thevetia neriifolia INTRODUCTION Pharmacognostical Phytochemical screening and Antimicrobial activity been extensively investigated as a source of medicinal agents [11]. Thus it is anticipated that phytochemicals with adequate antibacterial efficacy will be used for the treatment of many bacterial infections [12]. Several phytochemical surveys have been published, including the random sampling approach which involved some plant accessions collected from all parts of the world. The major chemical substances of interest in these surveys were the alkaloids, steroids and saponins, however other diverse are naturally occurring phytocomponents such as flavonoids, tannins, unsaturated steroids, triterpenoids and essential oils [13]. Phytochemical screening of any plant extract was carried out according to the methods and tests described by Dey and Sitaraman [14] to decipher the presence or absence of various phytocomponents. Thevetia neriifolia is used medicinally in Philippine Islands, Guiana, Brazil and Gold Coast. T. neriifolia is used to treat various inflammatory and cardiovascular diseases, beside the antiviral and antifungal properties. Generally, T. neriifolia leaves are applied in cardiac disorder, fever, ringworms, wasp sting and measlestreatment [15]. Moreover, the use of T. neriifolia

Medicinal plants, used in the traditional treatments of various diseases on an empirical basis [1-5]. Herbal medicines have been widely practiced throughout the world from ancient time. These medicines are safe and environment friendly. Nearly 80% of the world population depends upon traditional systems of health care [2]. In India, 9500 herbal medicinal plants and also 8000 higher plants have been used in the Indian medicinal industries. The potential for developing antimicrobials from higher plants appears rewarding as it might lead to the development of phytomedicine, which would play a prominent role against microbes. Plant-based antimicrobials have enormous therapeutic potential as they can serve purpose with lesser side effects that are often associated with synthetic antimicrobials [7]. Several plants and herb species used traditionally have potential antimicrobial and antiviral properties [8, 9] and this has raised the optimism of scientists about the future phyto-antimicrobial agents [10]. In recent years secondary plant metabolites and phytochemicals, previously with unknown pharmacological activities, have

Corresponding Author: D. Ramamoorthy, Department of Ecology and Environmental Sciences, Pondicherry University, India. Tel: +91-0413-2654327.

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tree in folk medicine is well known [16]. However no report are available on the phtyochemical, pharmacognostical, and antimicrobial activities of the plant. Hence the present study was taken up to bring out the above features. MATERIALS AND METHODS Plant Material and Preparation of the Extracts: The leaves of Thevetia neriifolia Juss. (APOCYNACEAE) were collected from the campus of Pondicherry University, Puducherry and its botanical identity was confirmed at Herbarium, French Institute of Pondicherry. The herbarium specimens were deposited at Department of Ecology and Environmental Sciences, Pondicherry University for further reference (Voucher no. BKRDVJ35). The collected leaves were chopped into small pieces, shade-dried and coarsely powdered with suitable pulverizer. The coarse powder was subjected to successive extraction with warm organic solvents hexane, chloroform and ethanol by soxhlet method [17]. The extracts were collected and distilled off on a water bath at atmospheric pressure and the last trace of any solvent was removed in vacuum (Table 3). The resulted extracts were used for, preliminary phytochemical screening pharmacognostical and antimicrobial studies. Antimicrobial Activities: Hexane, chloroform, ethanol and aqueous extracts of leaves were used to prepare different concentrations (100, 50 and 25 mg/ml). These concentrations were used for antimicrobial studies against specific microorganisms. Test Microorganisms: The following cultures of human pathogens used were procured from IMTECH, Chandigarh,the gram-negative bacteria [Escherichia coli (MTCC 724), Proteus vulgaris (MTCC 426), Pseudomonas aeruginosa and Salmonella typhi (MTCC 733), Vibrio vulnificus (MTCC 1145)],also the gram positive bacteria [Bacillus subtilis (MTCC441), Staphylococcus aureus (MTCC96), Staphylococcus pneumonia (MTCC 655)] and the fungal species [Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus and Candida albicans (MTCC 96)]. Determination of Antimicrobial Activity: Agar welldiffusion method [18] was applied to determine the antimicrobial activity. Nutrient Agar (NA) plates were swabbed with 8 hrs old - broth culture of respective 660

bacteria and potato dextrose Agar plates for fungal organisms. Four wells (8mm diameter) were made in each of these plates using sterile cork borer. About 0.3 ml of different concentrations of plant solvent extracts were added using sterilized dropping pipettes into the wells and allowed to diffuse at room temperature for 2 hours. The plates were incubated at 37C for bacteria and 28C for fungal pathogens for 18-24 hrs. For fungal pathogens respective proper controls of solvent plant extract were also maintained. Diameters of the inhibition zones were recorded. Triplicates were maintained and the experiment was repeated thrice and the average values were recorded for antimicrobial activity. Preliminary Phytochemical Screening: All the extracts were subjected to preliminary phytochemical tests followed by the methods of Harborne [17] and Trease and Evans [19]. Test for Alkaloids: To the test solution in 10 ml methanol, add 1 % (w/v)HCl and any of Mayors reagents, Wagners reagent or Dragendroff reagent (6 drops). A creamish or brownish red or orange precipitate indicated the presence of alkaloids. Test for Anthraquinones: To the test solution add a benzene drop and ammonia drop, a pink colour indicates the presence of anthraquininones. Test for Flavonoids: 5 ml of dilute ammonia solution was added to a portion of the aqueous filtrate of each plant extract followed by addition of concentrated H2SO4. A yellow color in each extract indicated the presence of flavonoids. The yellow color disappeared on standing. Few drops of 1 % aluminum solution were added to portion of each filtrate. A yellow color indicates the presence of flavonoids. A portion of the powdered plant sample was in each case heated with 10 ml of ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. A yellow color indicates opposite test for flavonoids. To the test solution in 10 ml of ethanol add conc. HCl Mg ribbon developing a pink-tomato red color indicates the presence of flavonoids. Test for Coumarins: To the test solution add a drop of sodium sulphate developing yellow colour. Indicates the presence of coumarins.

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Test for Phenols: To the test solution add a drop of ferric chloride. Developing of intense colour develops which indicates the presence of phenols. Test for Saponins: About 2 g of the powdered sample was boiled in 20 ml of distilled water in a water bath and filtered. 10ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth, which indicates the presence of saponins. Test for Steroids (Liebermann-burchard Test): 2 ml of acetic anhydride was added to the test solution along with 2 ml of conc.H2SO4. The colour changed from violet to blue or green in some samples. This indicates the presence of steroid. Test for Terpenoids (Salkowski Test): 5 ml of each extract mixed with 2 ml of chloroform, and 3 ml concentrated H2SO4 was carefully added to form a layer. A reddish brown colour of the inter face was formed to show positive results for the presence of terpenoids. Test for Tannins: About 0.5 g of the leaves was dried and powdered samples was boiled in 20 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blueblack colouration. Test for Amino Acid and Protein (Ninhydrin Reaction): Take 2-3 ml of sample solution in a test tube. Add 3-4 drops of ninhydrin solution and heat. Appearance of purple or violet colour indicates the presence of protein. Test for Carbohydrates (Benedicts Test): Add 1 ml of Benedicts reagent to test tube and heat the mixture to boiling in a water bath for 2 minutes. The formation of an orange red precipitate due to the formation of a copper (I) oxide indicates the presence of reducing sugars. Determination of Extractive Values: Extractive values of crude drugs are useful for their evaluation, especially when the constituents of a drug cannot be readily estimated by any other means, Further; these values indicate the nature of the constituents present in a crude drug. Hexane Soluble Extractive Value: Macerate 5 g of the air dried drug, coarsely powdered, with 100 ml of hexane of the specified strength in a closed flask for 24 h, shake 661

frequently during the first 6 h and allow to stand for 18 h. Therefore, filter rapidly taking precautions against loss of hexane, evaporate 25 ml of the filtrate to dryness in a tarred flat-bottomed shallow dish, dry at 105C and weigh. Fluorescence Analysis: The analysis of extract under daylight is unreliable due to lack of fluorescence. So it was evaluated under daylight and UV light. RESULTS AND DISCUSSION In present era, plant and herb resources are abundant, but these resources are dwindling fast due to the onward march of civilization [20]. Although, a significant number of studies have been carried out to obtain purified plant chemical, very few screening programmes have been initiated on crude plant materials. It also been widely observed and accepted that the medicinal value of plants lies in the bioactive phytocomponents present in the plants [21]. In this investigation, the active phytocomponents of Thevetia neriifolia was studied and further the antimicrobial activity of the plant extracts was assayed in vitro by agar well diffusion method against 8 bacterial and 4 fungal species. Table 4, summarizes the microbial growth inhibition of hexane, chloroform and ethanol extracts of the screened plant species. Successive solvent extraction values in various organic solvents were observed as hexane 1.5%, chloroform 5.2%, ethanol 8.9%, xylene 15.4%, toluene rectified 4.9%, butane 5.8%, ammonia 19.6%, benzene 21.7%, and acetone 5.4% as shown in Table 1. The percentage of benzene, ammonia, and xylene soluble extractive values is best for pharmacognostical work (Table 1) The traditional plants may represent new sources of anti-microbial with stable, biologically active components that can estabilish a scientific base for the use of plants in modern medicine [22]. All the three extracts possessed the phytochemical constituents such as flavonoids, phenolics, steroids, saponins and tannins. Hexane and chloroform extracts showed the presence of flavonoids, phenolics, steroids, saponins and tannins, while alkaloids, amino acids, anthraquinones, coumarins, terpenoids and carbohydrates were absent. On the other hand, ethanol extract showed the presence of alkaloids, anthraquinones, flavonoids, phenolics, steroids, saponins and tannins, while amino acids, coumarins, proteins, terpenoids and carbohydrate were absent.

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Table 1: Extract values of leaves of Thevetia neriifolia in various solvents Sample No 1 2 3 4 5 6 7 8 9 Solvent Hexane Chloroform Ethanol Xylene Toluene rectified Butane Ammonia Benzene Acetone Extracts value (g) 1.5 5.2 8.9 15.4 4.9 5.8 19.6 21.7 5.4

Table 2: Fluorescence analysis of Thevetia neriifolia leaf extracts,acids Benedict's solution Fluorescence colour ---------------------------------------------------------------------------S. No 1 2 3 4 5 6 7 8 9 10 11 12 Acids and Benedict's solution 1 2 3 4 5 Sulphuric acid Perchloric acid Arthophosphoric acid Hydrochloric acid Benedicts Black Black Black Black Green Black Black Black Black Dark green Solvents Hexane Chloroform Ethanol Benzene Butane Acetone Toluene rectified Xylene Sodium Thio-sulphate Acetic anhydride Silver nitrate Ammonia Daylight Light green Dark green Green Brown Green Green Green Yellowish green Pale green Pale green Greenish brown reddish brown UV-light Dark green Black Dark green Dark greenish brown Dark green Dark green Dark green Dark green Green Green Green Dark reddish brown

Table 3: Preliminary phytochemical screening of various extracts of Thevetia neriifolia leaves Solvents ----------------------------------------------------------------------------------------S. No. 1 2 3 4 5 6 7 8 9 10 11 12 Phytoconstituents Alkaloids Amino acid Anthraquinones Coumarins Carbohydrates Flavonoids Phenolics Proteins Steroids Saponins Tannins Terpenoids Hexane + + + + + Chloroform + + + + + Ethanol + + + + + + + -

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Table 4: Antimicrobial activity of Thevetia neriifolia leaf extracts against some microorganisms. Zone of inhibition (mm) -----------------------------------------------------------------------------------------------------------------------------------------Tested microorganism --------------------------SampleNo. 1 2 3 4 5 6 7 8 Fungi 9 10 11 12 Aspergillus niger Aspergillus flavus Aspergillus fumigatus Candida albicans 20 18 24 22 17 16 20 20 12 12 19 19 28 35 29 32 30 32 29 26 25 29 24 25 22 28 22 22 20 25 21 20 34(P) 30(P) 32(P) 36(P) Extract (mg/ml) Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Salmonella typhi Vibrio vulnifigus Bacillus subtilis Staphylococcus aureus Staphylococcus pneumoniae Gram-negative bacteria 28 16 17 22 15 20 17 18 25 15 16 19 13 19 15 15 22 12 14 12 12 17 12 13 37 37 36 36 33 36 32 38 37 37 32 32 32 34 38 38 28 24 25 20 26 27 22 25 22 22 21 18 18 26 19 23 20 19 20 16 16 25 17 22 36(A) 36(CI) 25(Cf) 32(K) 35(CI) 32(A) 30(C) 35(C) Hexane ----------------------------------------100 50 25 Std Chloroform extract ------------------------------------------100 50 25 Std Ethanol extract ----------------------------------------100 50 25 Std

Gram-positive bacteria

Table 5: Antimicrobial activity of aqueous extracts of Thevetia neriifolia leaves against some microorganisms Zone of inhibition(mm) Aqueous extract (mg/ml) ----------------------------------------------------------------------------------------------------------------Sample No. Gram-negative bacteria 1 2 3 4 5 Gram-positive bacteria 6 7 8 Fungi 9 10 11 12 Aspergillus niger Aspergillus flavus Aspergillus fumigatus Candida albicans 16 19 20 22 14 14 17 19 12 12 14 16 30 (P) 37 (P) 32 (P) 35 (P) Bacillus subtilis Staphylococcus aureus Staphylococcus pneumoniae 39 (A) 38 (C) 36 (C) Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Salmonella typhi Vibrio vulnifigus 37 (A) 37 (CI) 36 (Cf) 37 (K) 37 (CI) Tested microorganism 100 50 25 Std

Hexane extract showed maximum activity from 19 to 28mm against Gram negative bacteria such as E.coli at 100 and 50 mg/ml concentrations and against the fungi species, A. fumigatus at 100 mg/ml concentration only. Gram positive bacteria did not show maximum value of inhibition. Moderate zone of inhibition observed was from 19 to 23 mm against Gram positive bacteria such as B. subtilis, Gram negative bacteria such as E. coli and S. typhi and against fungi as A. fumigatus, A. niger and 663

C. albicans at 25, 50 and 100 mg/ml concentrations (Table 2). The minimum inhibition zone observed was from 1 to 21mm against Gram positive bacteria, (B. subtilis, S. aureus and S. pneumonia), Gram negative bacteria (P. aeruginosa, P. vulgaris, S. typhi and V. vulnificus) and fungi (A. flavus and A. niger) at 100, 50 and 25 mg/ml concentrations,respectively. Concerning, the chloroform extract it did not show any activity against all the tested bacteria and fungi (Figure 1).

World J. Agric. Sci., 7 (6): 659-666, 2011

Fig. 1: Plates showing the colonies of E.coli under different extracts.

Antimicrobial activity of aqueous extract of Thevetia neriifolia leaves against fungi
25 20 15 10 5 0 Aspergillus niger Aspergillus flavus Aspergillus fumigatus Candida albicans 100 50 25

Fungal species

Fig. 2: The aqueous extract of Thevitia neriifolia did not show activity against Gram negative and Gram positive bacteria while it showed activity against all fungi. With ethanol extract, the maximum activity was observed from 25 to 29 mm against gram negative bacteria (E. coli, P. aeruginosa and V. vulnifigus), Gram positive bacteria (B. subtilis and S. pneumonia) and fungi (A. flavus, A. niger and C. albicans) at 25 mg/ml, 50 mg/ml 664 and 100 mg/ml concentrations. On the other hand, the moderate zone of inhibition was observed from 20 to 24 mm against gram negative bacteria (E. coli, P. aeruginosa, P. vulgaris, S. typhi and V. vulnificus), gram positive bacteria such as S. aureus and S. pneumoniae, and

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against fungi (A. fumigatus, A. niger and C. albicans) at 25, 50 and 100 mg/ml concentrations,respectively (Figure 2).While, the minimum inhibition zone observed was from 13 to 19 mm against Gram positive bacteria (B. subtilis) only, gram negative bacteria (P. vulgaris, S. typhi and V. vulnificus) at 25 and 50 mg/ml concentrations (Table 5). There was no minimum value of inhibition against tested fungi. CONCLUSION A variety of microorganisms lead food into spoilage that is encountered as one of the most important matter concerning the food industry. So far, many pathogenic microorganisms, such as Escherichia coli, Staphylococcus aureus have been reported as the causal agents of food-borne diseases and/or food spoilage [23]. Raw and/or processed foods are open to contamination during the production, sale and distribution of food [23]. Due to the economical impacts and the consumers concerns over safety, the reliability of synthetic chemicals used for treating foods has been in question. Thus, a lot of attention has been paid to naturally derived components or natural products [24]. The results of the present study indicate that the leaves have a medicinal potential to develop into a new drug of pharmaceutical interest. REFERENCE 1. Hutchings, A. and J. Van Staden, 1994. Plants used for stress-related ailments in traditional Zulu, Xhosa and Sotho medicine: part 1 plants used for headaches. J. Ethnopharmacol., 43: 89-124. Hutchings, A., A.H. Scott, G. Lewis and A. Cunningham, 1996. Zulu Medicinal Plants; An Inventory. University of Natal Press, Scottsville, South Africa, pp: 195-196. Jager, A.K., A. Hutchings and J. Van Staden, 1996. Screening of Zulu medicinal plants for prostaglandin-synthesis inhibitors. J. Ethnopharmacol., 52: 95-100. Salie, F., P.F.K. Eagles and H.M.J. Leng, 1996. Preliminary phytochemical screening of four South African Asteraceae species. J. Ethnopharmacol., 52: 27-33. McGaw, L.J., A.K. Jager and J. Van Staden, 1997. Prostaglandin synthesis inhibitory activity in Zulu Xhosa and Sotho medicine plants. Phytotherapy Res., 11: 113-117. 665

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20. Vogel, H.G., 1991. Similarities between Various system of traditional medicine. Considerations for the future of ethnopharmacology. J. Ethnopharmacocol., 35: 179-90. 21. Veeramuthu, D., A. Muniappan and I. Savarimuthu, 2006. Antimicrobial activity of some ethnomedicinal plants used by paliyar tribe from Tamilnadu, India. BMC Complementory and alternative Medicine, 6(35).

22. Gandhiraja, N., S. Sriram, V. Meenaa, J.S. Kavitha, C. Sasikumar and R. Rajeswari, 2009. Phytochemical screening and antimicrobial activity of the plant extracts of Mimosa pudica L. against selected microbes. J. Ethnobotanical Leaflets, 13: 618-24. 23. Deak, T. and L.R. Beuthat, 1996. Handbook of Food Spoilage. New York, USA: CRS Press. 24. Alzoreky, N.S. and K. Nakahara, 2003. Antimicrobial activity of extracts from some edible plants commonly consumed in Asia. Int. J. Food Mictrobiol., 62(2): 183-193.

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