Microsoft Word - 100-109 100 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 Evaluation of Antibacterial Activity of Ethanolic Extracts for Three Local Plants Kudair A. H. Al-Ameri Depart. of Biology/ College of Education for Pure Science( Ibn Al-Haitham )/ University of Baghdad. Receivd in:7/ September/2014, Accepted in :24/November/2014 Abstract There is an increasing interest in the use of plant extracts as therapeutic agents, particularly their capacity to inhibit the growth of pathogenic microorganisms. In this study antibacterial effect of Malva sylvestris, Anastatica hierochuntica and Vitis vinifera leaves extracts were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and Proteus mirabilis. The in vitro antibacterial activity was performed using agar well diffusion method and the minimum inhibitory concentration (MIC) was determined by microtitration technique. The result indicated that the extract of V. vinifera leaves inhibited with the growth of gram-positive bacteria, as well as gram-negative bacteria while the extract of A. hierochuntica showed inhibitory activity against B. subtilis and S. aureus. Finally the extract of M. sylvestris showed inhibitory activity against E. coli, P. aeruginosa, S. aureus and P. mirabilis. Phytochemical studies showed that the active component in ethanolic extract of these plants have amino acid, glycoside, phenol, tannins and alkaloids. Key words: Malva sylvestris, Anastatica hierochuntica, Vitis vinifera leaves, antibacterial activity. 101 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 Introduction Over the past decade, there has been an explosion of interest in antibacterial and antifungal activity of natural products. Recommendation for the use of various natural products for infectious diseases is widespread and appears in a number of popular and other easily obtainable texts [1]. Today in most countries of the world, the knowledge of plant therapy has become an agenda in scientific researches and the use of herbal medicine is constantly growing. Medicinal plant have many healing properties without showing any significant side effect. Antibacterial activity is one of those properties among many others [2]. Malva sylvestris Linn. (Malvaceae) is an important medical plant whose flowers are used as a remedy for cut wound, eczema, dermal, infected wounds, bronchitis, digestive problems and urinary tract infections [3, 4]. Anastatica hierochuntica is a small gray annual herb that rarely grows above 15 centimeters, bears minute white flowers [5, 6]. Anastatica is found in arid areas in the middle east, including parts of North Africa, regions of Iran, Egypt, Palestine, Jorden, Iraq and Pakistan [7, 8]. It is used to treat gastrointestinal disorders, depression, high blood pressure, indigestion, cold, fever and diabetes [9, 10]. Grape (Vitis vinifera) belongs to family vitaceae [11]. Vitis vinifera is a deciduous woody climber with coiled climbing tendrils and large leaves. It has small, pal, green flowers in the summer followed by bunches of berry fruits that range from green to purple-black [12]. In Iraq, grape leaves are used in traditional food (dolma). Grape leaves with antioxidant activity have been reported to treat chronic venous insufficiency in human and nephrotoxicosis induced by citrine [13]. Theoretical Selected bacteria are common bacteria in hospital infectious disease. P. aeruginosa is one of the important opportunistic pathogens in hospital which is clearly seen in affected to out immune system deficiency and in scaled, respiratory diseases, cancerous patients under chemotherapy, heredity cysticfibrosis bacteremia, septicemia and many other hospitals infections [14]. S. aureus is the main cause of bacteremia, surgical wound infections and the most common cause of skin and soft tissue infections [15]. E. coli is the agent of major urinary tract hospital infection [16]. B. subtilis is only known to cause disease in severely immunocompromised patients [17] Proteus include pathogens responsible for many urinary tract infections [18]. The aim of this study is to evaluate the antibacterial activity of M. sylvestris, A. hierochuntica and V. vinifera leaves against pathogenic Gram positive and Gram negative bacteria. Material and Methods Plant samples: M. sylvestris, A. hierochuntica and V. vinifera leaves were purchased from Baghdad markets, Iraq. The plants were dried at room temperature for fifteen days. Preparation of plant extracts: 25 gm of each plant was extracted in 250 ml of solvent (80% ethanol) by soxhlet extraction techniques for 4 h. The extract kept for the next day at room temperature, then filtered by filter paper No. 42 extracts were concentrated by rotary evaporator [19]. Different concentrations of (25, 50, 75 and 100) mg/ ml of each plant extracts were prepared using dimethyl sulfoxide (DMSO). Bacterial strains: Five bacteria (E. coli, P. aeruginosa, S. aureus, B. subtilis and P. mirabilis) were used to test the antibacterial activity in the plant extract. These strains were collected from center for market research and consumers protection-Baghdad University. 102 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 Culture media and antibacterial assay: Bacterial strains were grown on Muller Hinton agar (MH) and Nutrient agar (NA) at 37° C for 24 h were appropriately diluted using sterile normal saline solution to obtain cell suspension at 1.5 ×108 colony forming units (CFU)/ml. To evaluate inhibiterial assay, an agar well diffusion method was used described by [20]. The organisms were spread on MH :NA agar plates by cotton swab. Well were punched into the agar medium and filled with 50 µL of plants extracts. The plates were incubated for 24 h at 37° C. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organism. Minimum inhibitory concentration (MIC): Determination of MIC of the plant extracts against bacterial strains was preformed according the microtitration technique described by [21]. Determination of phytochemical constituents: The freshly prepared extract was subjected to standard photochemichal analyses for different constituents such as alkaloids, glycosides, phenols, tanines and amino acid as earlier described by [22]. Results Table (1) and Fig. (1) showed the effect of ethanolic extract of A. hierochuntica against the growth of bacteria. A. hierochuntica exhibited the strongest antibacterial activity against S. aureus (19 mm) at (100 mg/ ml). The ranking of antibacterial activity of A. hierochuntica against the five bacterial strains was S. aureus > B. subtilis > P. aeruginosa but it wasn't active against E. coli and P. mirabilis. Table (2) and Fig. (2) showed the effect of ethanolic extract of V. vinifera leaves. V. vinifera leaves exhibited maximum antibacterial activity against E. coli (15 mm) at (100 mg/ ml). The ranking of antibacterial activity of V. vinifera leaves against the five bacterial strains was E. coli > S. aureus > P. mirabilis > P. aeruginosa > B. subtilis. Table (3) and Fig. (3) showed the effect of ethanolic extract of M. sylvestris. M. sylvestris exhibited the strongest antibacterial activity against S. aureus (22 mm) at 100 mg/ ml. The ranking of antibacterial activity of M. sylvestris against the five bacterial strains was S. aureus > P. aeruginosa > P. mirabilis > E. coli and it had negative activity against B. subtilis. The results showed that the ethanolic extract of V. vinifera leaves has the highest effective, the second was the extract of M. sylvestris while the extract of A. hierochuntica was the lowest. Table (4) represented the chemical test for the active compounds like amino acid, glycoside, phenol compounds, tannins and alkaloids which was the active material of the plants extracts. Table (5) showed the minimum inhibitory concentration of those studied local plants. Discussion The reason for the effectiveness of antibacterial to contain plant extracts like alkaloids, tannins, phenol and glycosides. Activity of the studied plant extract alkaloids characterized by their ability to break into the bacterial cell and interfering with DNA. Tannins have the ability to inhibit enzymes and transportation of proteins in the cell membrane. Phenolic in usability the formation of a complex with a soluble extracellular protein and activity was in cell wall lead to the treating of the cell[23, 24]. The common name for M. sylvestris is mallow and its local name is Khabaz. The active ingredients are found in the flowers and leaves, which are rich in mucilage; it is used in treatment for their expectorant properties [26]. This plant was largely used to soothe mucous and membrane in inflammations. M. sylvestris exhibited maximum antibacterial activity against S. aureus. The study was conducted by [27]. M. sylvestris is good for skin disorders, 103 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 as well as having good antibacterial and anti-inflammatory activity [28]. V. vinifera leaves are rich in phenolic compound such as myricetin, ellagic acid, kaempfrol, quercetin and gallic acid all of these compound have antibacterial activity [28, 29]. Also this antibacterial properties could be used to increase the shelf-life of food [30]. All part of A. hierochuntica are rich in minerals, these phenolic, have high antioxidant and free radical scavenging activities. These properties explain the therapeutic activities of these plants [31]. Conclusion This study will help to identify active ingredients for the treatment of bacterial diseases. Additional studies are needed to assess the effect of the selected plants on other pathogenic organisms. Finally we can conclude from this study that V. vinifera leaves extracts is good antibacterial with in little concentration. This study could be done on each active compound (alkaloids, amino acid, phenolic compounds, glycosides and tannins) in the ethanolic extract. References 1-Shahidi Bonjar, G. H.; Aghighi, S. and Karimi, N. K. (2004). Methods for testing the antibacterial activity of extracts. Rev. J. Biol. Sci., 4: 405-412. 2-Zarei, B.; Saifi, T.; Fazeli, A.; Khodadadi, E. and Namavar, A. (2013). Evaluation of antibacterial effects of marshmallow (Althaea officinalis) on four strains of bacteria. Internat. J. Agricult. & Crop Sci., 5: 1-4. 3-Ghorbani, A. (2005). Studies on pharmaceutical ethnobotany in the region of Trkman Sahra, north of Iran. J. Ethnopharm., 102: 100-107. 4-Zargari, A. (1990). Medicinal plants. Tehran University Publishers, Tahran, Iran, 4th edition. 5-Doughari, G. H. (2007). Antifungal activity of extracts of mangiferaindical L. J. Res. Physisc. Sci., 2: 7-8. 6-Ticzon, R. (2001). Ticzon herbal encyclopedia Romeo R. Ticzon publishing Phillipines, 2: 231-232. 7-Friedman, J. and Zipporahstein (2012). The influence-seed-dispersal mechanisms on the dispersion of Anastatica hierochuntica in the Neger desert. Rev. J. Biol. Sci., 4(1). 8-Conway, P. (2006). Tree medicine-a comprehensive guide to the healing power of over 10 trees Judy Piatkus publisher 1 td: 213-217. 9-Balanouny, K. H. (1999). Wild medicinal plants in Egypt. The Palm press, Cairo: 207 pp. 10-Eman, A. S.; Tailang, M. and Benyounes, S. (2011). Antibacterial and hepatoprotective effects of Ertira plants of Anastatica hierochuntica. Int. J. Res. Phytoch. Pharm., 1: 24-27. 11-Jayaprrakasha, G. K.; Selvia, T. and Sakariah, K. K. (2003). Antibacterial and antioxidant activities of grape (Vitis vinfera) seed extracts. Food Res. Internat., 3: 117-122. 12-Davis, P. H. (1997). Flora of Turkey and east Aegean islands. Food Res. Internat., 3(6): 117-122. 13-Kiesewetter, H. 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Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J. Clin. Microbiol., 36(1): 325-326. 18-Guentzel, M. N. (1996). Barron's medical microbiology 4th ed. University of Texas medical branch: 963-964. 19-Harborne, J. B. (1976). Photochemical methods a guide to modern techniques of plant analysis. Halsted press, John Wiley and Sons, Inc. New York: 1-26. 20-Nogponga, K. W.; Aporn, M.; Duangtip and Sukon, T. (2008). Screening and identification of lactic acid bacteria producing antibacterial compound from pig gastrointestinal tracts. Kmitl Sci. Tech. J., 8:1. 21-Eloff, J. N. (1998). A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extract for bacteria. Plant Med., 64: 711-713. 22-Odebiyi, A. and Sofowora, A. E. (1999). Phytochemical screenings of Nigerian medical plants, part II, Lyodia, 41:234-246. 23-Simor, A. E.; Ofner-Agostini, M.; Bryce, E.; McGeer, A.; Paton, S. and Mulvey, M. R. (2001). Canadian hospital epidemiology committee and Canadian nosocomial infection surveillance program. J. Infect. Dis., 186(5): 652-660. 24-Balcht, A. and Raymond, S. R. (1994). Pseudomonas aeruginosa infections and treatment. Marcel Deker, New York. 25-Todar, K. (2007). Pathogenic E. coli. Textbook of bacteriology. University of Wisconsin, Madison. Department of Bacteriology. 26-Yeole, N. B.; Sandhya, P. Chaudhari, P. S. and Bujbal, P. S. (2010). Evaluation of Malva sylvestris and Pedalium murex mucilage as suspending agent. Internat. J. Pharm Tech. Res., 2(1): 385-389. 27-Pirbalouti, A. G.; Yousefi, M.; Nazari, H.; Karimi, I. and Koohpayeh, A. (2009). Evaluation of burn healing properties of Arnebia euchorma and Malva sylvestris. Electronic J. Biol., 5(3): 62-66. 28-Pastrana-Bonilla, E.; Akoh, C. C.; Sellapan, S. and Krewer, G. (2003). Phenolic content and antioxidant capacity of muscadine grapes. J. Aric. Food Chem., 51: 5497-4503. 29-Rodriguez-Naquero, M. J.; Alberto, M. R. and Manca-de-Nadra, M. C. (2007). Antibacterial effect of phenolic compounds from different wines. Food Contr., 18: 93-101. 30-Fred, O. J. O. and Honeybell, I. M. (2009). Nutritional and antibacterial properties of Jatropha tanjorensis leaves. Amer. Euras. J. Sci. Res., 4: 7-10. 31-Ilhsanullah, D. (2012). Chemical properties of the medicinal herb Anastatica hierochuntica and its relation on to folk medicine use. African J. Microbiol. Res., 6(23): 5248-5051. 105 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 Table No. (1): The effect of ethanolic extract of A. hierochuntica against the growth of bacteria strains. Concentration  mg/ml  Inhibitory diameters (mm) mean  E. coli  P. aeruginosa P. mirabilis B. subtilis  S. aureus 25  ‐  ‐ ‐ 4 ‐  50  ‐  ‐  ‐  8  8  75  ‐  ‐  ‐  12  18  100  ‐  15  ‐  16  19  DMSO  ‐  ‐  ‐  ‐  ‐  Note: '-', no inhibitory diameter Table No. (2): The effect of ethanolic extract of V. vinifera against the growth of bacteria strains. Concentration  mg/ml  Inhibitory diameters (mm) mean  E. coli  P. aeruginosa  P. mirabilis  B. subtilis  S. aureus  25  4  2  3  2  4  50  8  5  6  3  7  75  11  7 9 4 10  100  15   10 12 5 13  DMSO  ‐  ‐  ‐  ‐  ‐  Note: '-', no inhibitory diameter Table No. (3): The effect of ethanolic extract of M. sylvestris against the growth of bacteria strains. Concentration  mg/ml  Inhibitory diameters (mm) mean  E. coli  P. aeruginosa P. mirabilis  B. subtilis  S. aureus  25  3  4  5  ‐  5  50  6  8  9  ‐  11  75  9  12  14  ‐  16  100  12  16 14 ‐ 22  DMSO  ‐  ‐ ‐ ‐ ‐  Note: '-', no inhibitory diameter 106 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 Table No. (4): Result of photochemical screening of ethanolic extract of plant Photochemical class  A. hierochuntica V. vinifera  M. sylvestris  Alkaloids  +  +  +  Amino acid  +  +  +  Phenolic content  +  +  +  Glycosides  + + +  Tannins  + + +  Table No. (5): Minimum Inhibitory Concentration (MIC) of the ethanolic extracts of the plants against the test bacterium Bacterial strain test  A. hierochuntica  V. vinfera  M. sylvestris  E. coli  ‐  25  25  P. aeruginosa  100  25  25  B. subtilis  25 25 ‐  S. aureus  50 25 25  P. mirabilis  ‐  25  25  Figure No. (1): The effect of ethanolic extract of A. hierochuntica against the growth of bacteria strains. 0 2 4 6 8 10 12 14 16 18 20 In h ib it o ry d ia m et er s (m m ) Concentration mg/ml E. coli P. aeruginosa P. mirabilis B. subtilis S. aureus 25 50 75 100 107 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 Figure No. (2): The effect of ethanolic extract of V. vinifera against the growth of bacteria strains. Figure No. (3): The effect of ethanolic extract of M. sylvestris against the growth of bacteria strains. 0 2 4 6 8 10 12 14 16 In h ib it o ry d ia m et er s (m m ) Concentration mg/ml E. coli P. aeruginosa P. mirabilis B. subtilis S. aureus 25 50 75 100 0 2 4 6 8 10 12 14 16 In h ib it o ry d ia m et er s (m m ) Concentration mg/ml E. coli P. aeruginosa P. mirabilis B. subtilis S. aureus 25 50 75 100 108 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 Malva sylvestris Green plant Dry plant Anastatica hierochuntica 109 | Biology 2014) عام 3(العدد 27المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (3) 2014 تقييم التأثير التثبيطي لمستخلصات اإليثانول في ثالثة نباتات محلية خضير عباس حسن العامري / جامعة بغداد)ابن الھيثم(-الصرفة قسم علوم الحياة/ كلية التربية للعلوم 2014/تشرين الثاني /24،قبل البحث في 2014/ايلول /7استلم البحث في : الخالصة ھناك اھتمام واسع باستعمال المستخلصات النباتية كعالج بسبب قدرتھا الواسعة على تثبيط اكبر عدد من االحياء المجھرية. أجريت ھذه الدراسة لمعرفة التأثير التثبيطي ضد البكتريا لمستخلصات نباتات الخبّاز، كف مريم، وورق العنب، و Pseudomonas aeruginosaو Escherichia coliتريا وتم تقويم التأثير التثييطي للمستخلصات ضد بك Bacillus subtilis وStaphylococcus aureus و Proteus mirabilis اتبعت طريقة االنتشار بالحفر. وتقدير . اوطأ تركيز مؤثر. أظھرت النتائج بان مستخلص ورق العنب ثبط كل البكتريا الموجبة والسالبة لملون كرام في حين أظھر . واخيراً اظھر مستخلص نبات الخبّاز قدرة تثبيطية B. subtilisو S. aureusمستخلص كف مريم قدره تثبيطية تجاه . E. coli و P. mirabilis ،P. aeruginosa ،S. aureusتجاه فات أجريت فحوصات كيميائية للمستخلصات لمعرفة مكوناتھا الكيميائية. أعطت المستخلصات نتائج إيجابية لكشو االحماض االمينية، والكاليكوسيدات، والفينوالت، والقلويدات والتانينات. ورق العنب، وكف مريم، ونبات الخبّاز، والفعالية التثبيطية للبكتريا. :الكلمات المفتاحية