Edewor-Kuponiyi / Journal of Pharmacy Research 2012,5(5),2934-2937 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Phytochemical and antimicrobial analyses of extracts of pellucida (L) Edewor-Kuponiyi Theresa. Ibibia Department of Pure and Applied Chemistry, Ladoke Akntola University of Technology, Ogbomoso, Nigeria Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012

ABSTRACT The leaves of Peperomia pellucida were studied with the aim of investigating the antimicrobial and phytochemical properties of the species that thrives in Ogbomoso, Nigeria. The phytochemical screening of the extracts revealed presence of flavonoids and saponins while alkaloids, steroids and tannins were absent in the methanolic and ethyl acetate extracts. All the phytochemicals screened were absent in the n-hexane extract. The antimicrobial activity of the extracts was tested against seven bacteria and two fungal strains. It showed inhibitory activity against , Salmonella typhii, and Shigella. dysenteriae was inactive against , Klebsiella pneumoniae, Proteus mirabilis and Proteus vulgaris with minimum inhibitory concentration ranging from 1.25 – 2.50 mg/mL. It did not exhibit any inhibitory activity against the screened fungi, candidida albicans and Aspergillus niger. The results from this present study indicate that the observed phytochemicals are responsible for its medicinal properties; which is an affirmation of the use of this in the management of abdominal ailments only. The additive or synergistic action of these phytochemicals at target sites associated with physiological process may be responsible for the beneficial effects exerted by Peperomia pellucida.

KEY WORDS: Peperomia pellucida (L); Phytochemical; Antimicrobial activity; TLC

INTRODUCTION There is a worldwide increase in life threatening infections caused by patho- a medicinal in tropical Africa. In Nigeria (Ogbomoso). It is used mainly for genic microorganisms (Khan et al., 2010). These microorganisms are becom- the treatment of abdominal pains. Literature of many researchers prove that ing resistant to known antimicrobial agents (Ghani et al., 1998). Therefore the plant contain alkaloids, saponins, tannins and cardenolides (Khan et al., there is an increased interest in the search for antimicrobial compounds. This 2010) flavonoids, essential oils and carotol (Khan et al., 2002). This paper has led to the pharmacological and chemical investigations of medicinal . describes the phytochemical and antimicrobial study of the leaves of These investigations have provided important advances in the therapeutic Peperomia pellucida L that thrives in Ogbomoso, Nigeria. approach to several pathogens. MATERIALS AND METHODS Peperomia pellucida is an annual, shallow-rooted herb that belongs to the family (Ghani, 1998). It is commonly known as shiny bush. It is General procedure found in Nigeria, Sierra Leone and Ghana. Other places where it is found Thin layer chromatography was carried out on precoated plates with silica include China, Brazil and DR Congo. Other places where found are southern gel 60 (Merck 0059381). All solvents were routinely distilled prior to use. America and Asian countries (Ghani et al., 1998; Bayma et al., 2000; Santos Other chemicals were of commercial grade and used without further purifica- et al., 2001; Mde et al., 2002; Arrigoni-Blank et al., 2004). It grows in clumps, tion. thrives in loose, humid soils, tropical and subtropical climate. It usually grows to a height of about 15 to 45 cm and is characterized by succulent PREPARATION AND EXTRACTION OF PLANT MATERIAL stems, shiny, heart-shaped, fleshy leaves and tiny, dot-like seeds attached to Fresh plants were collected from its natural habitat in Ogbomoso, Nigeria and several fruiting spikes (Dos-Santos et al., 2001). The ethnomedical proper- taken to the Department of Pure and Applied Biology, Ladoke Akintola ties of P. pellucida are well known. It is used for treating abdominal pain, University of Technology, Ogbomoso, Nigeria for identification. The plant abscesses, acne, boils, colic, fatigue, gout, headache, renal disorders, and was pulverized using a sterilized food grinder. The pulverized plant was rheumatic joint pain (Ragasa et al., 1998; Khan and Omoloso, 2002). In extracted using solvents of different polarities (n-hexane, chloroform, ethyl Bolivia, the whole plant is used to stop hemorrhages by Alteños Indians. The acetate and methanol). 500 g of the plant was soaked in each of the solvents roots are used to treat fevers and the aerial parts are used as dressing for for 24 hours (3X), filtered through Whatman No.1 filter paper into screwed wounds (Munoz et al., 2000). In northeastern Brazil, the plants have been vials and concentrated using a rotary evaporator bath under reduced pres- used as a hypocholesteremic agent (Bayma et al., 2000). It is a popular cough sure. Some of the extracts were diluted with dimethylsulphoxide (DMSO) suppressant, emollient, and diuretic in Guyana and the Amazon as well as and used for antimicrobial screening. effective in the treatment of proteinuria (Arrigoni-Blank et al., 2002; De Fatima Arrigoni-Blank et al., 2004). A decoction of the plant is used in the PHYTOCHEMICAL ANALYSIS Philippines to decrease uric acid levels and to treat renal problems. It is also used topically for skin disorders such as acne and boils. It is widely used as The method used is that described by Harborne (1993).

Test for flavonoids: 1 mL of NaOH was added to 3 mL of each extract. A *Corresponding author. yellow colouration indicates that flavonoids are present. Theresa. Ibibia Department of Pure and Applied Chemistry, Test for steroids: 1 mL of concentrated sulphuric acid was added to 2 mL of Ladoke Akntola University of Technology, each of the extracts. Observation of a red colouration indicates presence of Ogbomoso, Nigeria steroids.

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2934-2937 Edewor-Kuponiyi / Journal of Pharmacy Research 2012,5(5),2934-2937 Test for saponins: 2 mL of distilled water was added to 2 mL of each extract Disc diffusion test and shaken vigorously. A persistent frothing indicates presence of saponins. Disc diffusion method by Bauer et al., 1963 was employed. This involved the use of filter paper disc as carrier for the antibacterial agents. Sterilized Test for tannins: 2 mL of 5% ferric chloride was added to 1 mL of each discs cut from Whatman no. 1 filter paper were impregnated with solutions extract. Observation of a greenish precipitate indicates the presence of tannins. of the antibacterial agents. The solvent was evaporated and the disc dried Test for alkaloids: 1 mL of 1% HCl was added to 3 mL of each extract in a test properly. The nutrient agar medium was inoculated with the test organism tube. The mixture was heated for 20 mins, cooled and filtered. The filtrate and the impregnated disc placed on the surface of the nutrient agar. The was used for the following test: 2 mL of Meyer’s reagent was added to 1 mL antimicrobial agent upon contact with the agar diffused into all directions. of the filtrate. Observation of a creamy precipitate indicates the presence of The ability of the test organism to grow or not in the presence of the test alkaloids. sample was then determined within 24 hours by measuring the zones of inhibition. The plates were incubated upside down at 37oC. All tests were CHROMATOGRAPHY done in quadruplicate and the antimicrobial activity was expressed as a mean of inhibition diameters (mm) produced by the plant extracts and Streptomy- Spotting of samples cin, standard antibiotic. Precoated Merck silica gel plates were used for the qualitative analysis. The plate was marked lightly with two pencil lines 0.5 cm from each end. A micro Minimum inhibitory concentration (MIC) drop of a solution of the extract in redistilled solvent (methanol, chloroform The minimum inhibitory concentration (MIC) of the extracts against the and ethyl acetate) was placed precisely on one of the two lines with the aid sensitive microorganisms was determined using the disc diffusion method. of a micro-capillary tube. Serial dilutions of the isolated compounds were prepared (10.0, 5.0, 2.5, 1.25, 0.625, 0.313 and 0.156 mg/ml). Each of the innocula was poured into Development of plate each Petri-dish and the agar was later poured and allowed to set. Wells were After spotting the sample on the plates, it was left for 5mins for the solvent made using the sterile 3 mm cork borer. Serial dilutions of the isolated com- to evaporate off and was subjected to linear ascending development to a pounds were added into the marked wells. The plates were incubated at 37oC distance of about 90 mm in different solvent systems of ethyl acetate and for 24 h. The growth was observed to determine the sensitivity of the micro- chloroform in the ratio of 9:1 (v/v) and 1:9 (v/v ) using a chromatographic organism using clear zones of no microbial growth. The least concentration of developing tank. The tank was left undisturbed until the rising solvent front the extracts that had inhibitory effect was taken as the minimum inhibitory has reached the upper line. The plate was then removed and the solvent concentration (MIC) of that extract against such microorganism. allowed to evaporate from it. RESULTS AND DISCUSSION Visualization The plate is placed in an iodine tank to visualize the colorless components. Phytochemical analysis The iodine vapor is absorbed on the plate wherever there is concentration of The phytochemical analysis of the leaf extracts showed presence of alkaloids organic material and produces a brown spot. After the colour has developed and flavonoids in the methanol and ethyl acetate extracts only. Tannins, the plate is removed and a circle penciled around each spot. On exposure to saponins and glycosides were observed to be absent in these. All the screened air the brown iodine spots evaporate gradually. phytochemicals were absent in the n-hexane extract (table 1).

Determination of Rf value Table 1: Phytochemical analysis of the leaf extracts of Peperomia pellu- The distance travelled by the solvent (solvent front) and the components cida (L) were measured and used to calculate the Rf values of the components. Extractants Alk Sap Fla Tan Gly Ste

ANTIMICROBIAL SCREENING n- hexane ------Methanol + - + - - - Ethyl acetate - - + - - - Organisms Chloroform - - + - - - The microorganisms (Pseudomonas aeruginosa, Salmonella typhii, Shigella dysenteriae, Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, Key: Alk – Alkaloids, Sap – Saponins, Fla – Flavonoids, Tan - Tannins, Gly – Proteus vulgaris, Aspergillus niger and Candida albacans) used were clini- Glycosides, Ste – Steroids, + - Present, - - Absent. cal isolates collected from Baptist Medical Center, Ogbomoso, Nigeria. Percentage weight of extracts The percentage weights of leaf extract increased as the polarity of the solvent Preparation of the medium increased. The order of increase is n-hexane, chloroform, ethyl acetate and Nutrient agar medium was prepared by dissolving 2.8 g of nutrient agar in methanol (fig. 1). The weight percents varied from 3.76 for n-hexane to 100 mL of distilled water. The medium was sterilized in an autoclave at 12.08% for methanol extracts. 121oC for 15 minutes. It was cooled to 45oC and poured into sterile Petri dishes to solidify.

Preparation of test samples 1.0 mg each of the extracts was dissolved in 1.0 mL each of dimethylsulphoxide (DMSO). The activity of Streptomycin standard antibiotic was also deter- mined and used as the positive control; 1.0 mg was dissolved in 1 mL of DMSO. Fig. 1: Weight percent of extracts (MeOH - methanol, EA - Ethyl acetate, CH – Chloroform, Hex - Hexane) Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2934-2937 Edewor-Kuponiyi / Journal of Pharmacy Research 2012,5(5),2934-2937 extract. The presence of these classes of compounds in the extracts could be Thin layer chromaotography partly responsible for the observed antibacterial effect of the leaves of this The thin layer chromatographic analysis revealed presence of six compo- plant. This result is in contrast to that obtained by Egwuche et al., 2011 who nents in the methanolic extract while the ethyl acetate and chloroform ex- observed an additional presence of saponins, tannins and cardenolides. Thin tracts showed presence of four compounds each. layer chromatographic analysis of the ethyl acetate extract using the solvents ethyl acetate and chloroform in the ratio 9:1 (v/v) revealed four spots with R Table 2: TLC analysis of methanol and ethyl acetate and chloroform f values of 0.78, 0.68, 0.51 and 0.42 while that of the methanolic extract using extracts of Peperomia pellucida (L) leaves the solvents ethyl acetate in the ratio 1:9 (v/v) revealed six spots with Rf

Extracts Number of spots Rf values values 0.8, 0.71, 0.64, 0.52, 0.47 and 0.33. Literature revealed that spots with R values 0.78 and 0.33 are for Kaempferol and quercetin respectively Methanol 6 0.8, 0.78, 0.68, 0.53,0.42, 0.33 f Ethyl acetate 4 0.78, 0.68, 0.51,0.42 (table 2) (Sutthanut et al., (2007); Tewtrakul et al., (2009)). The antibacterial Chloroform 4 0.78, 0.68, 0.51, 0.42 analysis of the crude leaf extracts showed inhibitory activity against the following bacteria: S. typii, P. aeruginosa, and S. dysenteriae but not active Antimicrobial analysis against S. aureus, B. subtili, E. coli, P. mirabilis, K. pneumoniae and P. The results of the antimicrobial activity of the leaf extracts of Peperomia vulgaris (Table 3). From the zones of inhibition obtained for all the extracts, pellucida and the standard antibiotics ciprofloxacin are shown in tables 2 and it was observed that the chloroform extract was the least active and followed 3. The n-hexane was not active against any of the isolates while the methanolic by that of ethyl acetate while methanol had the highest inhibitory activity. extract was the most potent against S. typii, P. aeruginosa, and S. dysenteriae. This effect could be due to the fact that the active ingredients are more The methanolic extract compared favourably with the standard antibiotic soluble in the alcoholic solvent (methanol) than the others. In addition, it had ciprofloxacin. The minimum inhibitory concentration ranged from 1.25 – been observed that natural products are more extractable using alcoholic 2.50 mg/mL. solvents (Odoemena and Essien, 1995; Majekodunni et al., 1996; Martinez et al., 1996; Ahmed et al., 1999). This is evident in the number of spots Table 3: Antimicrobial activity of leaf extracts of Peperomia pellucida revealed in the thin layer chromatography (table 2). The MIC of the extracts (L) at concentration of 1 mg/mL. ranged from 1.25-2.50 mg/mL. The inhibitory activity of the extracts against S. typii, P. aeruginosa, and S. dysenteriae was observed to be comparable to Test Organisms Zones of inhibition (mm) that of the standard antibiotic, streptomycin. The antibacterial effects of the Bacteria Hex MeOH Chl EtOAc Str plant leaf suggest that it may possess remarkable therapeutic action in the Bacillus subtillis - - - - 22.5 treatment of gastrointestinal infections (Olaleye, 2007). This activity was Staphylococcus aureus 9.0 ± 0.20 - 8.0 ± 0.12 21.0 more pronounced against Gram-negative than Gram-positive bacteria. This Kliebsiella pneumonia - - - - - Proteus mirabilis - - - - 19.0 could be as a result of the morphological differences between these micro- Shigella dysenteriae - 22.0 ± 0.03 18.0 ± 0.05 20.0 ± 0.30 22.0 organisms. The extracts did not exhibit any antifungal activity. Comparing - 8.0 ± 0.10 - - - the antimicrobial activity of the methanolic extract to that obtained by Khan Proteus vulgaris - - - - 20.5 et al., 2010, some variations in the activity against the screened microorgan- Pseudomonas - 20.0±0.06 17.0 ± 0.05 18.0 ± 0.40 30.0 aeruginosa isms was observed. Salmonella typhii - 25.0±0.04 20.0 ± 0.20 22.0 ± 0.40 26.0 Fungi REFERENCES Candida albicans - - - 7.0 ± 0.25 - Aspergillus niger - 10.0±0.18 - 6.0 ± 0.12 - 1. Ahmed E-T, Gwiria MHS and Sami AK, Antiplasmodial Activity of Selected Sudanese Medicinal Plants with Emphasis on Acacia Experiments were in triplicate. nilotica, Phytotherapy Research, 13 (6), 1999, 474-478. + = present, - = no activity, hex = hexane; MeOH = methanol; Chl = chloroform; 2. Al-Bari MAA, Khan A, Islam MR, Kudrat-E-Zahan M, Rahman EtOAc = ethyl acetate,Ciprofloxacin (Sigma) was used as positive control. 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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2934-2937