International Journal of Natural Products Research

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1 Available online at International Journal of Natural Products Research Universal Research Publications. All rights reserved ISSN: Original Article PHYTOCHEMICAL TESTS, ANTIOXIDANT POTENTIAL AND TLC ANALYSIS OF IPOMOEA PES CAPRAE AND CATHARANTHUS ROSEUS A. MALAR RETNA* 1 AND P. ETHALSHA 1 1 Department of Chemistry, ManonmaniumSundaranar University, Scott Christian College (Autonomous), Tamil Nadu, India *Corresponding author malarrobin2012@gmail.com Telephone , Received 03 June 2014; Accepted 20 June 2014 Abstract In this study both the medicinal plants were compared using phytochemical tests, antioxidant potential and TLC analysis. The methanol extracts of the aerial parts (fresh leaves and stem) of both the plants were taken in this investigation. Preliminary phytochemical screening of the crude extracts revealed the presence of alkaloids, flavonoids, tannins, saponins, Quinones and phenolics. Ipomoea pes caprae explores marginally better antioxidant activities in comparison to Catharanthus roseus were done by DPPH assay. IC 50 values were also done by Broth dilution assay to indicate shifts in the susceptibility of bacterial populations to antibiotics. Thin Layer Chromatography had been carried out on those plants, which shows better Rf values. The solvent system selected for the results of TLC was chloroform and methanol. The present study reveals the several phytochemical present in these plants so as to supply evidence its antioxidant property and also shows the many phytoconstituents that contribute to its different kinds of medicinal properties Universal Research Publications. All rights reserved Key words:- Antioxidant, DPPH, TLC, I.pescaprae, C.roseus, Phytochemical 1. Introduction Antioxidant research is an essential topic in the medical field as well as in the food industry. Recent research with important bioactive compounds in many plants and food sources has received much attention [1]. Free radicals and reactive oxygen species are byproducts in aerobic organism and have aroused significant interest among scientists in the past decade. It has been proposed that they could induce cellular damage and might be involved in several human diseases, including cancer, arteriosclerosis, diabetes mellitus, hypertension and AIDS and in aging processes [2]. Of various kinds of natural antioxidants, flavonoids and phenolic compounds have received much attention. Therefore, antioxidants with free radical scavenging activities may have great relevance in the prevention and therapeutics of diseases in which oxidants or free radicals are implicated [3]. In this respect, polyphenolic compounds, like flavonoids and phenolic acids, commonly found in plants have been reported to have multiple biological effects, including antioxidant activity [4]. In the recent years interest in the study of antioxidant activity of plant extracts and isolation from plants has grown due to the fact that the free radicals have been related to degenerative diseases [5]. In this present study was to evaluate the antioxidant and antibacterial activity of two medicinal plants. Ipomoea pes caprae belongs to Convolvulaceae family and is widely distributed in tropical and subtropical countries. This plant is used in traditional medicine as powerful cathartics, diuretic, aphrodisiac and in the treatment of skin diseases, ulcers, bronchitis, inflammation, diabetes, fever and general weakness [6]. Their biological activities mainly include anticancer, antioxidant, anti-diabetic, anti-inflammatory etc. Another one plant of Catharanthus roseus L (apocyanaceae) also known as Vincarosea, is native to the Caribbean Basin and has historically been used to treat a wide assortment of diseases. Vincarosea has a variety of medicinal properties such as antibacterial [7], antifungal [8], antiviral [9], and anticancer [10]. It has more than 400 known alkaloids, some of which are approved as antineoplastic agents to treat leukemia, Hodgkin's disease, malignant lymphomas, neuroblastoma, rhabdomyosarcoma, Wilms' tumor, and other cancers [11]. Its vasodilating and memory-enhancing properties have been shown to alleviate vascular dementia and Alzheimer's diseases [12], hence the present investigation was undertaken to compare and conclude the antioxidant potential of these selected plants. 58

2 2. Material and Methods The aerial parts (fresh leaves and stem) of Ipomoea pes caprae, Catharanthus roseus were collected from the sandy beaches of Kanyakumari, Tamilnadu, India, in the month of March These plants were identified and authenticated by Dr. S. Jeeva, Department of Botany, Scott Christian College (Autonomous), Kanyakumari, Tamilnadu, India. Voucher specimen of this plant was deposited at herbarium of this institute (voucher no.sccn: 3352 and 3353). All chemicals and solvents were of analytical grade (RANKEM). They were washed and air dried over a period of one month. The dried samples were milled into a fine powder by pounding manually with a clean, sterile mortar, stored in sterile cellophane bags in a cool dry place till further use. 100gram of aerial parts of selected plants extracted in a soxhlet sequentially with 1000ml hexane, chloroform, ethyl acetate, methanol and water (2 5=10 Extracts). The process was run for 24h after which the sample was concentrated using reduced pressure distillation under vacuum pump and freeze dried to powdered form. The dried extracts were weighed and kept in labeled sterile specimen bottles. A plant powder was extracted by increasing order of their solvent polarity (figure 1). The selected 10 extracts were primarily tested with eight different bacterial strains in different concentrations. Bacterial strains were obtained from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, and Chandigarh, India.Four Gram negative strains MTCC 443 (Escherichia coli), MTCC 109 (Klebsiellapneumoniae), MTCC 450 (Schigellaflexneri), MTCC 441 (Proteus vulgaris), Four Gram positive strains MTCC 441 (Bacillus subtilis), MTCC 96 (Staphylococcus aureus), MTCC 1457 (Clostridium perfringens) MTCC 1538 (Micrococcus luteus) was used in the present study as test organisms for investigating antimicrobial activity by the Well diffusion assay [13]. 2.1 Preliminary phytochemical investigations The major secondary metabolites of tannins, saponins, terpenoids, flavonoids, alkaloids, phenol, quinine, terpenoids, steroids and glycosides were screened according to the common phytochemical methods [14]. 2.2 Antioxidant activity assays DPPH assay: (2, 2-diphenyl-1-picrylhydrazyl) [15] The Radical Scavenging Activity of different extracts was determined by using DPPH assay. The decrease of the absorption at 517nm of the DPPH solution after the addition of the antioxidant was measured in a cuvette containing μl of 0.1mm ethanol DPPH solution mixed with 20 to 200μg/ml of plant extract and vortexes thoroughly. The setup was left in dark at room temperature and the absorption was monitored after 20 minutes. Ascorbic acid was used as references. The ability of the plant extract to scavenge DPPH radical was calculated by the following equation: % of DPPH Radical Scavenging Activity (% RSA) = Abs. Control Abs. sample * 100 Abs. control Abs. control is the absorbance of DPPH radical + ethanol; Abs. sample is the absorbance of DPPH radical + plant extract. Measurements were performed in triplicates. Absorbance values were corrected for radicals decay using blank solutions. 2.3 Thin Layer Chromatography [16]. Methanol extracts of Ipomoea pes caprae, Catharanthus roseus were subjected to thin layer chromatography studies, to find out the probable number of compounds present in them. Used solvent system - Methanol: Chloroform Preparation of the plate: The TLC plate is placed in the chamber that the spot(s) of the sample do not touch the surface of the eluent in the chamber, and the lid is closed. The solvent moves up the plate by capillary action, meets the sample mixture and carries it up the plate (elutes the sample). When the solvent front reaches no higher than the filter paper should be removed (continuation of the elution will give a misleading result) and dried Visualization of the TLC plate The dry plate is placed in a chamber containing a few crystals of iodine. The iodine vapor in the chamber oxidizes the substances in the various spots, making them visible to the eye. Once the spots are visible, they may be outlined with a pencil before the iodine coloration fades. 59

3 Ipomoea pes caprae Catharanthus roseus Chloroform: methanol (9:1) Chloroform: methanol (9.5:0.5) Figure 2: TLC studies of selected plants Table 1a: Antibacterial activity of crude aerial part of I.pes-caprae Name of the extract Conc.of extract (µg) Zone of Inhibition (mm) Micro organisms E.c K.p P.v S.f B.s M.l S.a C.p Hexane Chloroform Ethylacetate Methanol Water

4 Table 1b: Antibacterial activity of crude aerial part of C.roseus Name of the extract Conc.of extract (µg) Zone of Inhibition (mm) Micro organisms E.c K.p P.v B.s M.l S.a Hexane Chloroform Ethylacetate Methanol Water Table 2: IC 50 Determination broth dilution Assay Concentration (µg) Inhibition (%) I.pescaprae C.roseus Table 3: Phytochemical screening of crude methanol extract of aerial parts of I.pescaprae and C.roseus Phytochemical compounds I.pescaprae C.roseus Alkaloids + + Flavonoids ++ + Phenols Tannins Glycosides - + Reducing sugars ++ + Proteins + - Saponins - + Quinones ++ - Steroids ++ - Amino acids + - Terpenoids Present in minor amounts ++ Present in moderate amounts - not detected +++ Present in higher amounts 61

5 4: Radical Scavenging Activity of each plant Table 5: TLC studies of methanol extract of selected plants Methanol extract of plants Solvent system Ratio Number of spots Rf values 0.94 I.pescaprae chloroform: methanol 9: , 0.44, C.roseus chloroform: methanol 9.5: , 0.36, 0.30, Results 3.1 Antibacterial activity Each plant has five extracts. From all the extracts; the methanol extracts of both the plants could be more active against Bacillus subtillis, Micrococcus luteus and Staphylococcus aureus with the zone of inhibition 17 and 18mm respectively (Table 1a & 1b). 3.2 IC50 Determination IC 50 values were also done by the Broth dilution assay [17] against the Gram positive organism Staphylococcus aureus in different concentrations to indicate shifts in the susceptibility of bacterial populations to antibiotics (Table2). So, further works concentrate only in most active methanol extracts of each plant. Inhibitory concentration 50 was done by broth dilution assay to determine the amount of antibiotic that the patient will receive to treat diseases. IC50 values of Ipomoea pes caprae was (500µg) and (1000µg) in C.roseus. The inhibitory concentrations are determined to use not only to determine the amount of antibiotic that the patient will receive but also the type of antibiotic used, which in turn lowers the opportunity for microbial resistance to specific antimicrobial agents. 3.3 Phytochemical Tests The preliminary phytochemical screening tests for the crude methanol extract of C.roseus and Ipomoea pes caprae were revealed the presence of tannins, saponins, flavonoids, alkaloids, glycosides and phenolics (Table 3). Tannins were present in predominantly and flavonoids, quinones, phenols were present in moderate in I,pescaprae. Phenols were present highly than other tested phytochemicals in C.roseus. 3.4 Antioxidant Activity The results of the antioxidant activity of methanolic extract of aerial parts of C.roseus and I.pes caprae determined by DPPH assays at different concentrations are given in graph 1. It was evident that aerial part of I.pescaprae and 62

6 C.roseus showed moderate antioxidant activity when compared to with standard antioxidant L-ascorbic acid whose antioxidant activity at different concentrations like 100 to 200μg were 80%, 82%, 85%, 90% and 92%. I.pescaprae possessed considerably better antioxidant activities in comparison to C. roseus. 3.5 Thin layer Chromatography The TLC analysis also suggests the presences of different kinds of medicinally important phytochemical in the methanol extract of selected plants are given in Table 5. It reports TLC of C.roseus shows six spots with better Rf values and I.pescaprae possesses four spots with good values. Figure 3 also gives the possible reports of TLC. 4. Discussion In the present study is used to evaluate the presence preliminary phytochemical is can be attributed to its antioxidant activity of these plants and also compare the plants depends upon their activity of all testing methods. They belong to different families. But traditionally they are used to better drugs in a good manner to some common diseases. Such that, rheumatism, diuretic, diabetes and cancer. The presence of tannins, saponins, flavonoids, alkaloids, glycosides and phenolics has potentially significant application against human pathogens, including those that cause enteric infections. The presences of alkaloids interesting as significant quantities are used as antimalarials, analgesics and stimulants [18]. The presence of glycosides moieties like saponins, glycosides and flavonoids, which are known to inhibit tumor growth and also serve to protect against gastrointestinal infections are one of the pharmacoqnostic importances and give evidence for the use of the plant in ethno medicine [19]. Tannins are widely used in traditional medicine in treating wounds and arrest bleeding [20]. Some of these bioactive compounds which are synthesized as secondary metabolites as the plant grows also serve to protect the plant against microbial attacks and predation by animals. The tested phytochemical is widely present in I.pes caprae than C.roseus. Any of these secondary metabolites, singly or in combination with others could be responsible for the anti-oxidant activity of these plants. The DPPH is a relatively stable free radical which, when encounters proton donors such as antioxidants, it gets quenched and the absorbance decreases [21].In the present investigation, the obtained data show that the methanol extract of each plant is a free radical scavenger and may act as primary antioxidants which can react with free radicals by donating hydrogen. The percentage of inhibition is increases with the increased concentration of standard. The final result exhibits I. pescaprae exhibits marginally better antioxidant potential than C.roseus. The TLC experiment was successful with the mobile phase of chloroform: methanol. The 9:1 ratio of used mobile phase is predominantly suitable for C.roseus to determine the number of phytochemical and the ratio of 9.5:0.5 also better matches to find the phytochemicals in I.pescaprae. 5. Conclusion In this present study evaluated antioxidant activity of crude methanol extract of I.pes caprae and C.roseus. The antioxidant activity of the extracts was found to be more significant than one another. These findings enrich our knowledge of the chemical constituents that are responsible for the medicinal uses of the plant and the antioxidant potential of selected plants. Radical scavenging effect of I. pescaprae increases with increasing concentration and maximum better antioxidant activity than C.roseus in all concentrations with the presence of maximum phytochemical. In this study, the most suitable solvent system for TLC analysis is shown to be chloroform: methanol. 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