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Screening of Phytochemical and Antibacterial Activities SCREENING OF PHYTOCHEMICAL AND ANTIBACTERIAL ACTIVITIES OF LEAVES OF SELECTED MEDICINAL PLANTS FORM Al-KHUMS, LIBYA *Salem Mohamed Edrah 1, Fouzy Alafid2, 1Chemistry Department, Science College, El- Mergeb University, Al-Khums Libya *Author & Corresponding Author: [email protected], Tel: +218924748909 2Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Czech Republic Abstract: Human beings benefit from medicinal plants to make life healthy and free from disease. Medicinal properties of plants perform them effective to halt or remedy disease. The present study was to evaluate the phytochemical constituents and antibacterial activity assessment of Crude extract (aqueous and ethanolic) leaves of three medicinally significant plants (Zygophyllum, Ammi visnaga and Rhus tripartita). Qualitative screening of phytochemical components such as Tannins, Phlobatanins, Saponins, Flavonoids, Steroids, Alkaloids, Anthraquinones, Coumarins, Terpenoids and Cardiac Glycosides were present in both extracts whilst steroids not present in all aqueous extracts of these plants and Anthraquinones present only in the Rhus tripartita in aqueous and ethanolic extracts. Whereas the Quantitive screening of phytochemical components percentage’s yields of Zygophyllum, Ammi visnaga and Rhus tripartita were 33%, 26% and 29% respectively, while the percentage’s yields of alkaloids were found as 2.4, 8 and 8.5% correspondingly, and the percentage’s yields of saponins were found as 7.6, 6 and 3.2% respectively. And the percentage’s yields of flavonoids were 9.6, 10 and 8.7% individually. Furthermore, the biological importance of these compounds together with the use of these plants in traditional medicine also encouraged us to investigate it for antibacterial activity. For this determination, the crude extracts (Aqueous and Ethanolic) of the leaves were used. The antibacterial activity of the crude extracts of the tree plants leaves was assessed by Disc Diffusion Method, which showed effective activity against Gram-positive bacterial strains (Staphylococcus aureus and Staphylococcus epidermis) and Gram-negative bacterial strains (Klebsiella pneumonia and Escherichia coli). What is more, the crude extracts of leaves exhibited effective inhibition as the comparison with the used standard Antibiotics. Keywords: phytochemicals, “Zygophyllum”, “Ammi visnaga (L.)Lam”, Rhus tripartita, Antibacterial Activity. 1. Introduction: Since a long while of time, various plants were used as a provenance of medicines, plants were used for the healing of illnesses without knowledge about the chemical ingredients that influence and their method of action. The biological activity of extracts traditionally used plants is still proper owing toward the presence of microbial resistance to several of antibiotics and the occurrence of fatal infection. A wide variety of medicinal plants is used by African healers for the preparation of treatments to eliminate gastrointestinal parasites (McGaw et al.; 2000.). “Zygophyllum album L” (Z. album) is common perennial herbaceous plants grown in Libya. It’s used as antiseptic, stimulant, carminative (Meng et al.; 2002). “Ammi visnaga (L) LAM.” (A. visnaga) obligates its ancestries in the warm climate of the Mediterranean, Likewise indigenous plants prevalent in North African countries (Tutin, 1968; Batanouny, 1999; Bueno, et al, 2006, Chevallier, 1996). Moreover, A. visnaga were used in the treatment of respiratory conditions such as bronchitis, asthma, and cough for mild angina treatment (Farnsworth, 2001; Anonymous, 2007). And found typically useful for the recovery of wound healing, psoriasis, poisonous bites and inflammation conditions (Abdelfattah, et al.; 1983, Valkova, et al 2004). “Rhus tripartita” (R. tripartita) plant was used to treat gastrointestinal and inflammatory conditions (El-Mokasabi, 2014). Medicinal plants constituents own a rich source of active phytochemicals for instance Tannins, flavonoids, Glycosides, Anthraquinones and other constituents which are necessary to show specific beneficial properties in several diseases (Murphy, et al, 2003, Caballero-George et al., 2002). 2. Material and Methods: 2.1 Plant Collection: The fresh healthy leaves of Z. Album, A. visnaga, and R. tripartita taxonomically identified by Department of Biology Science College Alkhums El-Mergeb University Alkhums Libya, Samples of plants were collected during April-Maye 2016 from Alkhums Region and then washed by tap water subsequently by distilled water and dried in shadow for three days and finishing drying in an oven at 50 0C. The samples of dried leaves were pulverized into the fine powder using a grinder consequently packed in sterilized dark polyethene bags. 2.2 Preparation of extracts: 20 g of each pulverized wild plant leaves powder was dissolved separately in 500ml of appropriate solvents (Distilled water, Ethyl Alcohol) using Soxhlet apparatuses for 8-10 hours. Subsequently, the extracts were filtered through Whatman filter paper No. 1 and the solvents was evaporated by Rotary Vacuum Evaporator and a green, brown and black sticky substance was obtained that was stored in the refrigerator at 4 0C until further use. 2.3 Phytochemical screening: 2.3.1 Qualitative determination of the chemical constituency: Qualitative phytochemical analyses for each sample alkaloids, saponins, flavonoids and tannins were determined quantitatively as described below. Chemical tests were carried out both on the extracts (aqueous and ethanolic) and on the powdered specimens using standard procedures to identify the constituents (Harbone, 1973.; Trease, et al 1989; Sofowora, 1993; Dietland, 2009). The specific procedure involved for the evaluation of a particular group of chemicals is mention below: 2.3.1.1 Determination of Tannins: 2 ml of water and 2-3 drops of ferric chloride solution were added in 1 ml of crude extracted solution. The blue colour was observed for Gallic tannins and green black for Catecholic tannins (Iyengar, 1995). 2.3.1.2 Determination of Saponins: Foam test: a few amount of crude extracted solution was shaken with little quantity of water. If foam produced persists for ten minutes it indicates the presence of saponins (Roopashree, et al, 2008). 2.3.1.3 Determination of Flavonoids: (Alkaline Reagent Test): Crude extracted solution was treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on the addition of dilute acid, indicates the presence of flavonoids (Roopashree, et al, 2008). 2.3.1.4 Determination of Steroids: 4 ml of acetic anhydride was added to a 1g ethanolic crude extract of each sample with 4 ml H2S04. The colour changed from violet to blue or green in some samples indicating the presence of steroids. 2.3.1.5 Determination of Terpenoids: (Salkowski’s test): 10ml of each crude extracted solution was mixed in 4 ml of chloroform and concentrated H2SO4 (6 ml) was carefully added to form a layer. A reddish-brown colouration of the interface was formed to show the presence of terpenoids. 2.3.1.6 Determination of Cardiac glycosides: (Keller-Killani test): 10ml of each crude extracted solution was treated with 4 ml of glacial acetic acid containing 2 drops of ferric chloride solution. This was under-layer with 2 ml of concentrated Sulphuric acid. A brown ring of the interface indicates a deoxy-sugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout the thin layer. 2.3.1.7 Determination of Alkaloids: Alkaloids are basic nitrogenous compounds with definite physiological and pharmacological activity. Alkaloid solution produces white yellowish precipitate when a few drops of Mayer’s reagents are added (Siddiqui, et al, 1997). 2.3.1.8 Determination of Anthraquinones: Borntrager’s test: 1 g of the plant extract was shaken with benzene layer separated and half of its own volume of 10% ammonia solution added. A pink, red or violet colouration in the ammoniacal phase indicated the presence of Anthraquinone. 2.3.2. Quantitative determination of the chemical constituency: Quantitative phytochemical analyses for each sample alkaloids, saponins and flavonoids were determined quantitatively well-known tests procedure available in the literature as described below (Harbone, 1973; Obdoni, et al, 2001; Boham et al 1994): 2.3.2.1 Alkaloids determination: 10g of the fine powder of dried plants were weighed into 500 ml beakers to which 400ml of 10% acetic acid in ethanol was added, covered and allowed to stand for about 4 hr. The filtered extracts were concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added dropwise to the extracts until complete precipitation. The precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The alkaloid residue was dried to a constant weight and expressed as the percentage (Harbone, 1973). 2.3.2.2 Saponin determination: 10 g of each were put into a conical flask and 50 ml of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4 h with continuous stirring at about 55°C. The mixture was filtered and the residue re-extracted with another 100 ml 20% ethanol. The combined extracts were reduced to 40 ml over a water bath at about 90°C. The concentrate was transferred into a 250 ml separatory funnel and 10 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered
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