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Volume 4, Issue 3, March 2014, Pages 189–193 ObjectiveTo investigate the antibacterial activity and photochemicals of five green leafy vegetables against a panel of five bacteria strains.MethodsDisc diffusion method was used to determine the antibacterial activity, while kanamycin was used as a reference antibiotic. The phytochemical screening of the extracts was performed using standard methods.ResultsAll methanol extracts were found active against all the test bacterial strains. Overall maximum extracts shows antibacterial activity which range from 6 to 15 mm. Proteins and carbohydrates was found in all the green leaves, whereas alkaloid, steroids, saponins, flavonoids, tannins were found in most of the test samples.ConclusionsThe obtain result suggests that green leafy vegetables have moderate antibacterial activity and contain various pharmacologically active compounds and thus provide the scientific basis for the traditional uses of the studied vegetables in the treatment of bacterial infections.

Green leafy vegetables have been used as medicine since ancient times and have been playing a very important role in our diet and nutrition. They are the most readily available sources of carbohydrates, fats, important proteins, vitamins, minerals, essential amino acids, and fibers[1]. Their bioactive substances have a wide range of biological functions, including antioxidant and antimicrobial activities[2, 3, 4 and 5] and can be helpful in management of oxidative stress and age related human aliments[6]. They are rich source of carotene, ascorbic acid, riboflavin, folic acids and minerals like calcium, iron and phosphorus[7]. Being a photosynthetic tissue, leafy vegetables have higher levels of vitamin K when compared with other fruits and vegetables due to direct involvement of vitamin K (phylloquinone) in photosynthesis process. Vegetables as medicinal plants contain none or less toxic effects[8 and 9], and have the ability to synthesize several secondary metabolites of relatively complex structures possessing antimicrobial activities[10, 11 and 12].

Green leafy vegetables are also rich in compounds having antidiabetic[13], anti-histaminic[14], anti-carcinogenic[15]and hypolipidemic[16] properties and possess preventive or curative properties against cardiovascular disease, ageing, obesity, hypertension, insomnia and ageing[17, 18 and 19].
bamix deluxe m150 black immersion hand blenderLeafy vegetables are natural source of antioxidants and rich in phytochemicals[20 and 21].
margaritaville blender saskatoonThe present work was therefore designed to investigate the antibacterial effects of five leafy vegetables namely Coriandrum sativum (C. sativum), Lactuca sativa (L. sativa), Mentha piperita (M. piperita), Portulaca oleracea (P. oleracea) and Raphanus sativus (R. sativus) against some bacteria strains and their phytochemical screening.
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Fresh leaves of C. sativum, L. sativa, M. piperita, P.oleracea and R. sativus free from disease were purchased from local farms in Al-Qassim.
kitchenaid blender parts model ksb3wh4Samples were labeled and stored at 4 °C in polythene bags till they were processed.
breville blender bbl600xl partsCollected materials were washed thoroughly in running tap water, rinsed in distilled water and shade dried for one week in open air, and then crushed using mortar and pestle, reduced to powder using Waring laboratory blender (MX-7011G) for 5 min at high speed and then stored in airtight closed bottles for two days before used for analysis.
magic bullet blender kijkshopFifty grams of all the fresh samples were stored for juice preparation.
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Bacteria cultures of Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Bacilllus subtillis, and Pseudomonas aeruginosa (clinical isolates) were obtained from Botany Department of King Saud University. The strains were maintained on agar slant at 4 °C and activated at 37 °C for 24 h on nutrient agar (Sigma-Aldrich, Germany) before any susceptibility test.Fifty grams of raw leave samples after washing with water were crushed by grinder without adding any solvent. The residue was removed by filtering through 8 layers of muslin cloth. The filtrate was collected in clean airtight bottle and stored at 4 °C until use for antibacterial activity test.Ten grams of dry powder of samples were dissolved in 30 mL of 0.01 mol/L HCl containing 0.15 mol/L NaCl. (sample:extract solution, 1:3 w/v). The residue was then removed by filtering through cheese cloth. The filtrate was then centrifuged at 8100×g, for 5 min. These leaves and extract were subjected to antibacterial activity experiments and protein determination[ 22].

Ten grams of powdered sample was dissolved in 100 mL of methanol in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 190-220 r/min for 24 h. The supernatant was collected slowly and evaporated in wide mouthed evaporating bowls at room temperature for 2-3 d till the final volume was reduced to one fourth of the original volume of the solvent used, giving the concentration of 400 mg/mL[23] and stored at 4 °C in airtight bottles.Twenty three grams of nutrient agar (Sigma-Aldrich, Germany) were dissolved in 1000 mL of distilled water and bring to boil. Agar was then autoclaved for 15 min at 121 °C and left to cool at room temperature. Once the LB medium was cooled (about 45 °C), it was poured into Petri dishes. Each Petri dish was left on the flat surface for 30-40 min until completely set.Antibacterial activity was assayed by disc diffusion method. For all bacteria strains, overnight culture grown in broth was adjusted to an inoculum's density of 100 μL: 0.1A600 culture containing 3.2×108 colony forming unit.

Further, 20 μL was spread onto 20 mL of sterile agar plates by using a sterile cotton swab. The surface of the medium was allowed to dry for about 3 min. Sterile filter paper discs (5 mm in diameter) impregnated with different test extracts (100 μL disc) were then placed on the surface of inoculated agar plates. Kanamycin (30 μg/disc) was used as positive control. The plates were then incubated at 37 °C for 24 h after which microbial growth was determined by measuring the diameter of the inhibition zone (mm) using a transparent scale. Each extract was analyzed in triplicate, the mean values are presented. Kanamycin disc (30 μg/disc) was used for comparing the bioassay.At first 0.5 g of each powder was dissolved separately in 5 mL of distilled water and filtered. Few drops of Molisch's reagent were added to each solution, this was then followed by addition of 1 mL of concentrated H2SO4 by the side of the test tube. The mixture was then allowed to stand for 2 min and then diluted with 5 mL of distilled water.

Formation of a red or dull violet colour at the interphase of the two layers was taken as positive test[24].A given weight of 0.1 g of each powder was dissolved in 5 mL of methanol separately and then filtered. A volume of 2 mL of each filtrate from each sample were stirred with 5 mL of 1% aqueous HCl on water bath and then filtered. Of the filtrate, 1 mL was taken individually into 2 test tubes. To the first portion (1 mL), few drops of Dragendorff's reagent were added. Occurrence of orange-red precipitate was taken as positive. To the second 1 mL, Mayer's reagent was added and appearance of buff-colored precipitate was taken as positive test for the presence of alkaloids[24].At the beginning 0.2 g of crude powder of each sample was dissolved in 2 mL of acetic acid separately. The solutions were cooled well in ice followed by the addition of concentrated H2SO4 carefully. Color development from violet to blue or bluish-green indicated the presence of a steroidal ring[24].One gram of crude powder of each sample was boiled with 5 mL of distilled water separately and then filtered.