Phytochemical screening and antibacterial activities of Vernonia ...

Phytochemical screening and antibacterial activities of Veronia ambigua... 69Test for free anthraquinones (Bontragerís test)Small portion of the extract was shaken with10 mL of benzene and filtered. Five milliliters of10% NH 3 solution was added to the filtrate andstirred. The production of a pink-red or violet colorindicates the presence of free anthraquinones.Test for saponins (Frothing test)Small quantity of the extract was dissolved in10 mL of distilled water. This was then shaken vigorouslyfor 30 s and was allowed to stand for 30min. A honey comb formed for more than 30 minindicates saponins.Test for steroids and triterpenes (Lieberman-Burchardís test)Equal volume of acetic anhydride was added tothe extract. One milliliter of conc. H 2 SO 4 was addeddownside the tube and the color change wasobserved immediately and later. Red, pink or purplecolor indicates the presence of triterpenes while blueor blue-green indicates steroids.Test for flavonoids (Shinoda test)About 0.5 g of the extract was dissolved in 1.5mL of 50% methanol and warmed on steam bath.Metallic magnesium and 5 drops of concentratedhydrochloric acid were added. A red or orange colorindicates the presence of flavonoids aglycone.Test for tannins (Ferric chloride test)About 0.5 mL of extract was dissolved in 10mL of distilled water and then filtered. Few drops offerric chloride solution were added to the filtrate.Formation of a blue-black precipitate indicateshydrolyzable tannins and green precipitate indicatesthe presence of condensed tannins.Test for alkaloids (Dragendoffís test)To about 0.5 g of each extract, 1% diluted HCl(20 mL) was added in a conical flask, heated on asteam bath and then filtered. The filtrate was madealkaline with 28% NH 3 solution and then extractedwith chloroform (3 ◊ 5 cm 3 ). The combined CHCl 3extracts were concentrated and treated with equalvolume of 1% HCl. Dragendorffís reagents (2 mL)were added and occurence of orange-red precipitateindicated the presence of alkaloids.Test organismsTen clinical bacterial strains: Klebsiella pneumoniae,Streptococcus pyogenes, Staphylococcusaureus, Corynbacterium ulcerans, methicillin resistantStaphylococcus aureus (MRSA), Salmonellatyphi, Pseudomonas aeruginosa, Shigella dysentriae,Proteus mirabilis and Pseudomonas fluorescencewere obtained from the Department ofMicrobiology, Ahmadu Bello University TeachingHospital (ABUTH) Shika. The isolates were purifiedon nutrient agar (OXOID) plates and characterizedusing standard microbiological and biochemicalprocedures (26, 27). The MRSA strains used inthis study were clinical isolates from urethral swab,seminal fluid, urine, high virginal swab, blood, skinand sputum of patients with symptoms of S. aureusassociateddiseases. The isolates were identified bystandard method (26). The disc diffusion methodoutlined by the NCCLS (28) was used with 1 µgoxacillin disk (Oxoid). The zones of inhibition weremeasured after incubation at 35 O C for 24 h. Isolateswith zones diameter = 10 mm were consideredmethicillin resistant. The organisms were maintainedon agar slope at 4 O C and subcultured for 24 hbefore use.Determination of antibacterial activityThe disc diffusion method was used (29).Stock solution (100 mg/mL) of each extract andfractions were prepared using the extractants. Discs(6 mm diameter) were prepared using Whatman filterpaper and sterilized by autoclaving. The blanksterile discs were placed on the inoculated MuellerHinton Agar (MHA) surface and impregnated with15 µL of stock solutions (1500 µg/dics). Antibioticdiscs of ampiclox (75 µg/dics) and streptomycin (30µg/dics) were used as positive control, whereas discsof the extracting solvents were used as negative control.The plates were incubated at 37 O C for 24 h. Alltests were performed in triplicate and the antibacterialactivities were expressed as the mean diameter ofinhibition zones (mm) produced by the plantextracts.Minimum inhibitory concentration (MIC)The minimum inhibitory concentration (MIC)was determined using micro-broth dilution methodsas outlined by NCCLS (30). Dilutions (1ñ9mg/mL) of concentrations of extract and fractionsthat exhibited sensitivity against the test organismswere prepared using test tubes containing 9 mL ofdouble strength broth. The test tubes were inoculatedwith the suspension of the standardized inocula.These were incubated at 37 O C for 24 h andobserved for growth. The minimum inhibitory concentrations(MICs) of the extracts/fractions foreach test organism were regarded as the lowestconcentration that inhibited visible growth of thetest organisms.