American-Eurasian J. Agric. & Environ. Sci., 11 (4): 542-546, 2011 ISSN 1818-6769 © IDOSI Publications, 2011 Chemical Constituents and Biological Screening of Grewia optiva Drummond ex Burret Whole Plant 1 Waliullah, 1Ghias Uddin, 1Abdur Rauf, 2Bina Shaheen Siddiqui, 1 Taj Ur Rehman, 3Sadiq azam, 4Muhammad Qaisar 1 Institute of Chemical Sciences, University of Peshawar, K.P.K, Peshawar, Pakistan 2 International Center for chemical and biological sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi Pakistan 3 Centre of Biotechnology, University of Peshawar, Peshawar, Pakistan 4 Medicinal Botanic Centre, PCSIR Laboratories Complex, Peshawar, K.P.K. Pakistan Abstract: Grewia optiva, a plant of northern area of Pakistan was screened for the natural products, antimicrobial and insecticidal activities. Phytochemical screening, using conventional natural products identification tests indicated the presence of different phytoconstituents. The ethyl acetate and methanolic extracts of the aerial parts of the tile plant revealed the presence of tannins, while terpenoids, flavonoids and steroids were present in n-hexane and chloroform fractions. n-hex.+ethyl acetate soluble fraction indicated the presence of saponins and tannins, while n-hexane and n-hexane+ethyl acetate insoluble fractions contained steroids, terpenoids, flavonoids and tannins. Ethyl acetate and n-hexane extract of aerial part is the most active, showing activity against four selected bacterial stains and thus displayed inhibitory zone of (13:0 mm) at the tested concentration (28 mg/ml). The n-hexane and n-hex.+ethyl acetate soluble extracts showed activity with inhibition zone of (13:0 mm) at the tested concentration (32 mg/ml). The n-hexane fraction and n-hex,+EtOAc soluble fractions showed some activity against selected fungal strains. The n-hex.+ethyl acetate soluble extract showed moderate insecticidal activity against Callosbruchus analis with 40 percent mortality rate. The medicinal values of Grewia optiva may be attributed due to the presence of detected metabolites. Key words: Phytochemical screening %Antimicrobial activity %Insecticidal activity INTRODUCTION Ahmad and his co-workers [6] Up till now, several alkaloids including harman, 6-methoxyharman and 6hydroxyharman [7], lignans like grewin, nitidanin, bilagrewin [3], flavone glucosides including vitaxin ad isovitexin [8], gulonic acid ( -lactone, 3, 21, 24 trimethyl5,7-dihydroxyhentriacontanoic acid ä-lactone [9] have been reported. Grewia optiva Drummond ex Burret belongs to the family Tiliaceae is a small tree distributed in tropical and subtropical regions [1]. Many species of this genus are used in folk medicine for the treatment of diarrhea, malaria, typhoid fever, dysentery, small pox, cough, irritable condition of bladder and intestine, eczema and rheumatism [2]. An anti-malarial [3] and anti-bacterial [4] activities have been reported from this genus. In view of the biological and medicinal importance of this genus, research has been conducted on its chemical constituents by various Phytochemists. The first chemical study on Grewia genus can be traced back to 1965 when Anjaneyulu et al., [5] reported friedelin from G. tiliaefolia [5] and its occurrence was confirmed in G. biloba by MATERIALS AND METHODS Plant Material: Grewia optiva whole plant was collected from Dir, Khyber Pakhtunkhwa (KPK) province of Pakistan in the month of October 2009. The plant was identified by Prof. Dr. Jehandar Shah, Vice Chancellor Benazir University, Sheringl, Upper Dir and a Voucher specimen No Bot 9105 was deposited at the herbarium of the said department. Corresponding Authors: Waliullah, Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan. 542 Am-Euras. J. Agric. & Environ. Sci., 11 (4): 542-546, 2011 Extraction and Fractionation: Whole plant was dried at room temperature for 20 days. The dried plant material was crushed to fine powder. The powdered material was soaked in methanol for 5 days. The extract was concentrated under vacuum at 40°C, using a rotavapor. This crude extract of aerial parts was suspended in water and successively partitioned with hexane, chloroform and ethyl acetate to afford the corresponding fractions while that of stem bark were fractionated to n-hexane, nhex.+ethyl acetate soluble and insoluble fractions and nBuOH fraction. % inhibition of fungal growth = 100 − Linear growth and test (mm) Linear growth in control (mm) Miconazole and amphotericin B were used as standard drugs, while miconazole, amphotericin B and DMSO were used as positive and negative controls [11]. Insecticidal Assay of Different Fractions of Stem Bark of Grewia optiva: Different fractions were tested against different Insects viz, Tribolium castaneum, Rhyzopertha dominica and Callos bruchuanalis. The test samples were prepared by dissolving 1019.10 µg/cm2 of crude fractions in 3 ml acetone and loaded in a Petri dish covered with filter paper. After 24 hours, 10 test insects were placed in each plate and incubated at 27°C for 24 hours with 50% relative humidity in growth chamber. The results were evaluated as percentage mortality, calculated with reference to the negative and positive controls. Permethrin was used as a standard drug, while Permethrin, acetone and test insects were used as positive and negative controls. Micro-Organism Assortment and Preservation: Three strains of Gram-positive bacteria (Proteus merablus, Escherichia coli, Staphylococcus aureus and Bacillus cereus) were obtained from stock culture of Center of Biotechnology, University of Peshawar, Peshawar, Pakistan, five strains of fungi (aspergillus flavus, microsporum canis, Candida albicans, Candida glabrata and fusarium solani) and stored in müller-hinton agar at low temperature (4°C) prior to subculture. Antimicrobial Assay of the Different Fractions Against Particular Bacterial Strains: Modified agar well diffusion method was adopted to test the antibacterial activity of the fractions with the use of Müller-Hinton agar as medium. The cultures were equipped in triplicates at 37°C incubation temperature for a period of 24 to 72 hours. 0.6mL of the broth culture of the test organism was put in a sterile Petri-dish and added 20 ml of the sterile molten MHA. Wells were jaded into the medium using 0.2ml of the fractions using Streptomycin (2 mg/ml) as a standard of the antimicrobial agent. Inoculation was done for 1 h ensure the diffusion of the antimicrobial agent into the medium. The inoculation plates were incubated for 24 h at 37°C and the diameters of the zone of inhibition of microbial growth were measured in millimeters. Phytochemical Screening of Aerial Parts and Stem Bark: Chemical tests were performed on the hexane, chloroform, ethyl acetate and methanolic fractions of Grewia optiva L. followed the standard procedures illustrated by Sofowora [12] Trease and Evans [13] and Harborne [14] for detection of the phytochemicals. Alkaloids: 0.2g of each of the fractions was warmed with 2% H2SO4 for two minutes. The reaction mixtures were filtered and added a few drops of Dragendroff’s reagent to each filtrate. Orange red precipitate indicates the presence of alkaloids moiety. Tannins: A small quantity of each extract was mixed with water and heated on water bath and filtered. A few drops of ferric chloride were added to each of the filtrates. A dark green solution indicates the presence of tannins. Antimicrobial Assay of the Different Fractions Against Particular Fungal Strains: The antifungal activity was determined by the Agar Well Diffusion Method [10] In the described method Griseofulvin was used as the standard drug. The crude extract was dissolved in DMSO (50 mg / 5ml). Sterile Sabouraud’s dextrose agar medium (5ml) was placed in a test tube and inoculated with the sample solution (400 µg /ml) kept in slanting position at room temperature overnight. The fungal culture was then inoculated on the slant. The samples were incubated for 7 days at 29°C and growth inhibition was observed and percentage growth inhibition was calculated with reference to the negative control by applying the formula: Anthraquinones: 0.5 g of each extract was boiled with 10% HCl for few minutes on water bath. The reaction mixtures were filtered and allowed to cool. Equal volume of CHCl3 was added to each filtrate. Few drops of 10% ammonia was added to each mixture and heated. Rosepink colour formation indicates the presence of anthraquinones. Glycosides: Each extract was hydrolyzed with HCl and neutralized with NaOH solution. A few drops of Fehling’s solution A and B were added to each mixture. Formation of red precipitate indicates the presence of glycosides. 543 Am-Euras. J. Agric. & Environ. Sci., 11 (4): 542-546, 2011 Table 1: Weight of crude extract and percentage yield of crude extract of the aerial parts of Grewia optiva Solvent Weight of crude extract Hexane extract (g) Chloroform extract (g) Ethyl acetate extract (g) Methanol extract (g) 2.62 1.362 0.179 17.5 Steroids: 2 ml of acetic anhydride was added to the mixture of 0.5 g of each extract and H2SO4 (2 ml). The colour change from violet to blue or green in some samples indicates the presence of steroids. Percentage yield 15.01 7.78 1.01 13.46 Terpenoids: 0.2 g of the each extract was mixed with 2 ml of chloroform and concentrated H2SO4 (3ml) was carefully added to form a layer. The formation of a reddish brown coloration at the interface indicates positive results for the presence of terpenoids. Table 2: Weight of crude extract and percentage yield of crude extract of the stem bark Grewia optiva Solvent Weight of crude extract n-Hexane fraction (g) n-Hex.+E.A (soluble) (g) n-Hex.+E.A (insoluble) (g) n-BuOH (g) 70 5.0 12.0 40.0 Percentage yield 10 0.7142 1.714 5.714 RESULTS The weight percentage yield of aerial parts and stem bark are shown in Tables 1 and 2. The results of antimicrobial activities of aerial parts and stem bark while insecticidal activity of various fractions of stem bark of Grewia optiva whole plant are shown in Table 3, 4 and 5 respectively. Phytochemical screening of Grewia optiva aerial parts and stem bark is shown in Tables 6 and 7, respectively. DISCUSSION Reducing Sugars: Each extract was shaken with distilled water and filtered. The filtrates were boiled with few drops of Fehling, s solution A and B for few minutes. An orange red precipitate indicates the presence of reducing sugars. Saponins: 0.2 g of each extract was shaken with 5ml of distilled water and heated to boiling. Frothing (appearance of creamy miss of small bubbles) shows the presence of saponins. The result of the whole plant extracts of Grewia optiva showed that n-hexane, methanolic and n-BuOH fractions contain a greater proportion by mass of the polar component compounds. The medicinal value of this plant can be correlated to the presence a variety of natural products present in the title plant. For example all the fractions taken from the crude extract of this plant are positive for terpenoids which shows different activities like antitumor and anticancer, Anti-inflammatory and antiviral/antibacterial [15]. Flavonoids: 0.2 g of each extract was dissolved in diluted NaOH and few drops of HCl were added. A yellow solution that turns colourless indicates the presence of flavonoids. Phlobatanins: 0.5g of each extract was dissolved in distilled water and filtered. The filtrate was boiled with 2% HCl solution. Red precipitate shows the presence of phlobatanins. Table 3: Antibacterial assay of various fractions aerial parts of Grewia optiva Aerial parts Stem bark --------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ fractions P. m. S.a F1 x F2 F3 E.c B.c Fractions x X X 12 x X 11 x X F4 x x DMSO (–) x Imipenem 10µg/Disc (+) 28 P. m. S.a E.c B.c A1 x x 16 16 12 A2 13 x x x 10 A3 x 13 x 11 9 X A4 x x x 12 x x X 23 34 32 Concentration of stock solution 3mg/ml and 100 µL was used for assay Keywords: well size= 6mm MeOH=F1, EtoAc=F2, n-hex.=F3(Aerial parts), CHCl3=F4, n-hex.=A1(stem bark), n-hex.+EtoAc sol.=A2, n-hex.+EtoAc insol.=A3, n-BuOH=A4. P.m= Proteus merablus, S.a= Staph aureus, E.c= Escherichia coli, B.c= Bacillus cereus 544 Am-Euras. J. Agric. & Environ. Sci., 11 (4): 542-546, 2011 Table 4: Antifungal assay of different fractions of stem bark of Grewia optiva Fractions Name of Fungus % inhibition Std. Drug Mic µg/mL n-hexane fraction Candida albicans Aspergillus flavus Microsporum canis Fusarium solani Candida glabrata 0 25 20 30 0 Miconazole (110.8) Amphotericin B(20.20) Miconazole (98.4) Miconazole (73.25) Miconazole (110.8) n-Hex.+EtOAc fraction Candida albicans Aspergillus flavus Microsporum canis Fusarium solani Candida glabrata 0 30 35 10 0 Miconazole (110.8) Amphotericin B(20.20) Miconazole (98.4) Miconazole (73.25) Miconazole (110.8) n-BuOH fraction Candida albicans Aspergillus flavus Microsporum canis Fusarium solani Candida glabrata 0 0 0 10 0 Miconazole (110.8) Amphotericin B(20.20) Miconazole (98.4) Miconazole (73.25) Miconazole (110.8) Table 5: Insecticidal assay of different fractions stem bark of Grewia optiva Fractions Name of Insects % Mortality n-Hexane fraction Tribolium castaneum Rhyzopertha dominica Callosbruchus analis 0 0 20 Pet.ether+EtOAc fraction Tribolium castaneum Rhyzopertha dominica Callosbruchus analis 0 0 40 n-BuOH fraction Tribolium castaneum Rhyzopertha dominica Callosbruchus analis 0 0 20 Table 6: Phytochemical screening of n- hexane, chloroform, ethyl acetate and methanol crude extracts of the aerial parts of Grewia optiva Chemical components n-Hexane extract ~ + – ~ ~ ~ ~ ~ ~ ~ Alkaloids Steroids Terpenoids Flavonoids Anthraquinones Tannins Phlobatanins Saponins Glycoside Reducing sugars Chloroform extract – + + + ~ ~ ~ – ~ ~ Ethyl acetate extract Methanol extract ~ ~ + + ~ + ~ ~ ~ – – + ~ ~ + ~ – ~ – Key: – = absent, + = present, ext = extract Table 7: Phytochemical screening of n- hexane, P.E+E.A (soluble), P.E+E.A (insoluble) n-BuOH fractions of the stem bark of Grewia optiva Chemical components Alkaloids Steroids Terpenoids Flavonoids Anthraquinones Tannins Phlobatanins Saponins Glycoside Reducing sugars n-Hexane extract ~ + – + ~ ~ ~ ~ ~ ~ P.E+E.A(soluble) P.E+E.A(insoluble) n-BuOH – + + + ~ ~ ~ + ~ ~ ~ ~ + ~ ~ + ~ ~ ~ – – ~ ~ ~ ~ + ~ + ~ – Key: – = absent, + = present 545 Am-Euras. J. Agric. & Environ. Sci., 11 (4): 542-546, 2011 The pharmacological activity of this plant can also be confirmed from the antimicrobial and insecticidal activities of various fractions. The ethyl acetate and n-hexane extract of aerial part is the most active, showing activity against four selected bacterial stains and thus displayed high inhibitory zone of (13:0 mm) at the tested concentration (28 mg/ml). n-hexane and n-hexane+ethyl acetate soluble extracts showed high activity with inhibition zone of (13:0 mm) at the tested concentration (32 mg/ml). n-hexane fraction and n-hex,+EtOAc soluble fractions showed some activity against selected fungal strains, aspergillus flavus, microsporum canis and fusarium solani, showing its medicinal importance in the treatment of gastroenteritis and pneumonia. 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