Phytochemical Analysis and Antimicrobial Activities of Carissa carandas L.

Universities Research Journal 2011, Vol. 4, No. 1 Phytochemical Analysis and Antimicrobial Activities of Carissa carandas L. Aye Aye Naing Abstract The plant Carissa carandas L. locally known as Khan-pin, belongs to the family Apocynaceae. It is found mostly on sandy and rocky soil in a wild state. It is sometimes cultivated for its fruits. In this research, preliminary phytochemical test, physicochemical properties and antimicrobial activities have been carried out. In phytochemical test, alkaloid, phenolic compound, starch and tannin were prominently observed. Flavonoid, glycoside, saponin, terpenoid, steroid and reducing sugar were also observed, and α-amino acid was absent. According to physicochemical properties examination, the raw material and ash of roots were mostly soluble in water. In elemental analysis, calcium, potassium and Sulphur are principal elements and zinc, iron and strontium are found as trace elements. In antimicrobial tests, the extracts of methanol showed the efficacy on Bacillus subtilis and Candida albicans. Key words: Apocynaceae, Carissa carandas, EDXRF, Bacillus subtilis, Candida albicans. Introduction Phytochemistry or plant chemistry has developed in recent years as a distinct, somewhere in between natural product organic chemistry and plant biochemistry and is closely related to both. It is concerned with the enormous variety of organic substances that are elaborated and accumulated by plants and deals with the chemical structures and their biological functions. The chemical constituents of plants can be classified in a number of different ways. Classification of chemical constituents is based on biosynthetic origin, solubility properties and the presence of certain key function group. The largest and structurally the most several of naturally secondary plant metabolism includes the terpene. The terpene have been isolated from plants and used for a variety of human purposes (Geissman, 1969). In this research, the plant Carissa carandas L. contains alkaloids, cyanogenic glycosides, saponins, tannins and triterpenoids (Trease and Assistant Lecturer, Department of Botany, Yadanabon University

294 Universities Research Journal 2011, Vol. 4, No. 1 Evans, 2002). Roots of this plant also contain fixed oil, volatile oil, dark yellow resin and alkaloid (Nadkarni, 1954). The preliminary phytochemical tests were investigated to determine the presence of various compounds and physicochemical tests were done to obtain important information concerning the moisture content and solubility. The antimicrobial activity was also tested to know whether it possess many medicinal values. Materials and Methods Preliminary phytochemical and physicochemical characterization on roots of Carissa carandas L. Preliminary phytochemical test In this study, only the qualitative determination of organic constituents in extract of the roots was investigated. It was carried out to know the presence or absence of alkaloids, carbohydrates, flavonoids, glycosides, phenolic Compound, saponins, starches, terpenoids, steroids, reducing sugar, tannins and α - amino acid. The experimental procedure was prepared by the methods of British Pharmacopeia, 1968; Harbone, 1973; Trease and Evans, 2002; Marini Bettolo et al. 1981 and Central Council for Research in Unani Medicine, 1987). Physicochemical characters of powdered roots from Carissa carandas L. The physicochemical characters such as moisture contents, total ash, acid insoluble ash, water soluble ash, water, ethanol, methanol, acetone, ethyl acetate, chloroform and pet-ether soluble matter were analyzed according of the method of British Pharmacopoeia (1968), and WHO (1998). Elemental analysis from roots of Carissa carandas L. by using EDXRF The relative concentration of elements was analyzed by using Energy Dispersive X-ray Fluorescence (EDXRF) spectrometer technique at the Universities Research Center, Yangon. The parameters of each part of the spectrometer are given below. Detector type : Si (Li) detector

Universities Research Journal 2011, Vol. 4, No. 1 295 LN 2 supply : Only during measurement Dewar capacity : 3 Liters LN 2 consumption : Less than 1 liter per day Detection area : 10 mm 2 Resolution : Less than 155 ev (Mn K a, 1500 H 2 ) The EDX-700 Shimadzu spectrometer can detect a wide range of the elements form sodium (Na) to uranium (U). The required data can be produced in a few minutes and it has a high degree of resolution for the spectrum evaluation. Due to its high sensitivity the spectrometer can detect the concentrations of elements in the parts per million (ppm) ranges. Antimicrobial activity of crude extracts from roots of Carissa carandas L. Antimicrobial activity of crude extracts For the determination of antimicrobial activity of crude extract agarwell diffusion method was used because of its simplicity, speed of performance, economy and reproducibility (Cruickshank, 1975). Extraction and test organisms The dried powder sample was extracted with petroleum ether (60-80) C, chloroform, acetone, methanol, ethyl acetate, ethanol and water by percolation method. Crude extracts of various solvents of roots were tested on six microorganisms such as Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumalis, Candida albicans, Escherichia coli. The test organisms used in this study were obtained from the Development Centre for Pharmaceutical Technology for determination of the antimicrobial activities. Preparation of sample for the test of antimicrobial activity Nutrient agar was prepared by method of Cruickshank (1975). Nutrient agar was used as basal cultural medium for the bacteria test organisms. The broth culture 20 ml was poured into the sterile petridish. Then, 0.2 ml of test organisms was added into the dishes.

296 Universities Research Journal 2011, Vol. 4, No. 1 The nutrients agar was allowed to set for 2-3 hours. After that, agarwell in the diameter of 7mm was punched with the help of sterilized cork porker (7mm). Then about 0.2 ml of the sample was introduced into the agar-well and incubated at 37 C for 24 to 48 hrs. The inhibition (clear) zone appeared around the agar-well was measured. Results Scientific name - Carissa carandas L. Family - Apocynaceae Myanmar name - Khan English name - Karaunda Outstanding characters Perennial small trees, latex present. Leaves simple, opposite and decussate, coriaceous. Inflorescence terminal dichasial cymes. Flowers white or pale-pink coloured, pentamerous, hypogynous. Stamen 5, epipetalous, anther dithecous, dorsifixed. Ovary 2, 2 ovules in each locule. Fruits ovoid, red to purplish black. Seeds 4, compressed, brown (Figure 1). Fig. 1. Flowers and Fruits of Carissa carandas L.

Universities Research Journal 2011, Vol. 4, No. 1 297 Preliminary phytochemical investigation and physicochemical properties from roots of Carissa carandas L. Preliminary phytochemical investigation The preliminary phytochemical investigation from roots of Carissa carandas L. indicated the presence of alkaloid, carbohydrate, flavonoid, glycoside, phenolic compound, saponin, starch, terpenoid, steroid, reducing sugar and tannin. The α-amino acid was absent. The results of preliminary phytochemical tests were shown in Table (1). Table 1. Preliminary Phytochemical investigation of Carissa carandas L. No. Test Extract Test reagent Observation Results 1. Alkaloids 10% Acetic acid EtOH 2. Carbohydrates H 2 O extract Mayer's reagent white ppt + Wagner's reagent orange ppt + 10% α-naphthol + conc:h 2 SO 4 red ring + 3. Flavonoid Methanol HCl/Mg Pink + 4. Glycoside H 2 O extract 10% lead acetate white ppt + 5. Phenolic Compound H 2 O extract K 3 Fe(CN) 6 and FeCl 3 deep blue + 6. Saponin H 2 O extract Distilled water frothing + 7. Starch H 2 O extract I 2 solution blue black + 8. Terpenoid 9. Steroid Pet-Ether extract Pet-Ether extract 10. Reducing Sugar dil H 2SO 4 + SN NaOH Acetic anhydride + conc: H 2 SO 4 Acetic anhydride + conc: H 2 SO 4 Benedict's solution deep red + deep green + yellow ppt + 11. Tannins H 2 O extract FeCl 3 green +

298 Universities Research Journal 2011, Vol. 4, No. 1 Physicochemical properties From the results of physicochemical properties, the moisture content, total ash, acid-insoluble ash, water-soluble ash content were determined and recorded. All the results were useful for the quality control system regarding the physiological ash and impurities whenever it was used for medicinal purposes. The solubility of root powdered was investigated to find the amount of total solids soluble in solvent. The different soluble matter contents were shown in Table (2). Table (2) Result of physicochemical properties of Carissa carandas L. No. Physicochemical characters Quantity determined Present (%) 1. Moisture content 6.89 2. Total ash 8.00 3. Acid-insoluble ash 10.50 4. Water soluble ash 32.50 5. Water soluble matter 12 6. Ethanol soluble matter 8.00 7. Methanol soluble matter 7.60 8. Acetone soluble matter 4.00 9. Ethyl acetate soluble matter 3.60 10. Chloroform soluble matter 3.20 11. Petroleum ether soluble matter 4.00 Elemental analysis from roots of Carissa carandas L. The elements present in powdered roots were quantitatively determined by EDXRF method, it was found that Calcium (Ca), Potassium (K), Sulphur (S) and Iron (Fe) are found as principal elements and Zinc (Zn) and strontium (Sr) are found as trace elements. The results are shown in Table (3) and Figure (2).

Universities Research Journal 2011, Vol. 4, No. 1 299 Table (3) Relative concentration of elements in Carissa carandas L. Element Content (%) Ca 0.541 K 0.313 S 0.193 Zn 0.020 Fe 0.013 Sr 0.005 CH 98.914 Fig. 2. EDXRF spectrum from roots of Carandas L.

300 Universities Research Journal 2011, Vol. 4, No. 1 Antimicrobial activity of different solvent extracts from roots of Carissa carandas L. by using agar-well diffusion method Different solvent extracts Screening of antimicrobial activities of various extracts were carried out by using six solvents such as petroleum ether (60-80) C, chloroform, acetone, methanol, ethyl acetate, ethanol and water. The results were shown in Table (4) and Fig. (3). Table 4 Inhibition zone exhibited by different extracts from roots of Carissa carandas L. against six microorganisms Solvent Bacillus subtilis Staphylococcus aureus Organisms Pseudomonas aeruginosa Bacillus pumalis Candida albicans Escherichia coli Pet-ether - - - - - - Chloroform 8mm 12mm 9mm 10mm 10mm 10mm Acetone 13mm 13mm 15mm 12mm 15mm 10mm Methanol 18mm 15mm 15mm 15mm 18mm 15mm Ethyl acetate - - - - - - Ethanol 15mm 15mm 15mm 14mm 15mm 14mm Water - - - - - - Agar well - 7mm According to this experiment, the root extracts with chloroform, acetone, methanol, and ethanol showed an effective antimicrobial activity on six different microorganisms but petroleum ether, ethyl acetate and water extract did not show antimicrobial activity on six different microorganisms. Among them methanol extract showed the highest activity especially Bacillus subtilis and Candida albicans.

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302 Universities Research Journal 2011, Vol. 4, No. 1 Discussion and Conclusion In this research, the preliminary phytochemical detection, the physico-chemical characters, elemental analysis of Carissa carandas L. root had been studied. According to the phytochemical detection, alkaloid, carbohydrate, flavonoid, glycoside, phenol, saponin, starch, terpenoid, steroid, reducing sugar, and tannin are tested. Among these findings, the presence of alkaloid, terpenoid, saponin and tannin in this plant which are agreed with Trease and Evans (2002). In the physico-chemical properties, a powdered sample of roots from Carissa carandas L. was mostly soluble in water and ethanol. Elemental analysis (EDXRF) reveals Calcium (Ca), Potassium (K), Sulphur (S) and Iron (Fe) are principal elements and Zinc (Zn) and Strontium (Sr) are found as trace elements. Antimicrobial activity of various crude extracts of petroleum ether, chloroform, acetone, methanol, ethyl acetate, ethanol and water and isolated compounds A and B were tested on six different microorganisms by using agar-well diffusion method. In this result, various crude extracts showed effective antimicrobial activity on six different microorganisms except petroleum ether, ethyl acetate and water extracts. Among them, methanol extract showed the highest activity especially more sensitive against Bacillus subtilis and Candida albicans. From this finding, it can be inferred that Carissa carandas L. can be effective in the formulation of medicine for the treatment of diseases, such as pathogenesis, alimentary tract, gastrointestinal tract, skin infection, sores, Candida sis and intense itching. Acknowledgements I would like to express my sincere thanks to Dr. Khin Maung Oo, Rector of Yadanabon University for his permission to submit this article. I am also indebted to Professor and Head, U Nay Win and Professor Dr. Cho Cho, Department of Botany, Yadanabon University for their invaluable sugessions and guidance. I am grateful to Dr. Nu Nu Swe, Professor, Department of Botany, Meiktila University for providing all kindly necessary instruction.

Universities Research Journal 2011, Vol. 4, No. 1 303 References British Pharmacopoeia (1968). Published under the direction of the General Medical Council. Medical Act 1956. London: William Cloves and Sons, Limited. Central Council for Research in Unani Medicine. (1987). Phytochemical Standard of Unani Formulation. New Delhi Cruickshank, R. J. P. (1975). Medicinal Microbiology. London: Livingstone Ltd. Geissman, T. A. (1995). In modern methods of plant. Analysis. Harborne, J.B. (1973). Phytochemical methods. London : Academic press Inc. Marini-Bettalo, G. B. (1981). Plant screening by chemical and chromatographic procedure under field condition. Journal of Chromatography. Nadkarni, K. M. (1954). Indian materia medica. (Vol. 1). (3th ed.). Bombay. Trease, G. E & Evans, W. C. (2002). Pharmacognosy. (15 th ed.). London : Harcourt Publishers Limited. World Health Organization (1998). Quality control methods for medicinal plant materials. Geneva.