Studies on antioxidant and antibacterial activities of crude extracts of Plantago lanceolata leaves

ISSN: 2410-9649 ABC et al / Chemistry International 3(3) (2017) 277-287 iscientic.org. 278 www.bosaljournals/chemint/ [email protected] A free radical is any species capable of independent existence that contains one or more unpaired electrons (Halliwell, 2000). Free radicals are well documented for playing a dual role in our body as both deleterious and beneficial species. In low/moderate concentrations free radicals are involved in normal physiological functions but excess production of free radicals is a harmful process that causes oxidative stress (Golden et al., 2002; Lee et al., 2003). Oxidative stress can damage the cell structures, including lipids, proteins, RNA and DNA which leads to number of diseases (Sen et al., 2010). The damaging effect of free radical can prevented by a group of substance called antioxidant. An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. A great number of natural medicinal herbs and plants have been tested as naturally occurring antioxidant (Hamid et al., 2010). According to the World Health Organization more than 80% of the world’s population of the developing countries relies mainly on the traditional medicines, mostly plant derived. Among the plants that have been used as traditional medicines, the genus Plantago is known for their medicinal properties. Plantago lanceolata known by a common name ribwort plantain is one of the species of genus plantago. The Leaves of this plant have been used for traditional medicinal activities in various countries (Miser-Salihoglu et al., 2013; Taskova et al., 2002). In most part of the world including Ethiopia, the plant has not been cultivated on piece of land or large farms since it is considered as weed. However, the data generated from leaf extracts of the P. lanceolata plant have demonstrated strong antioxidant and antimicrobial properties. Material and Methods Sample collection and preservation The leaves of the P. lanceolata were washed with tape water to remove dust particles and other waste material from the surface of leaves followed by drying in open air at room temperature. The air-dried leaves of P. lanceolata chopped in to small pieces and milled in to a uniform powder. The bacterial species used to test antimicrobial activities (Staphylococcus aureus (ATCC2923), streptococcus pneumoniae (ATCC49619), Escherichia coli (ATCC2592) and shigella boydii (ATCC9289)), were obtained from Biotechnology Laboratory, Biology Department, Gondar University, Ethiopia. Extraction and Defatting procedure Five different flasks each containing 20 g non-defatted P. lanceolata leaf powder were used for extraction using five different solvents (80% and 90% aqu. methanol, pure methanol, pure chloroform, and pure petroleum ether) at room temperature. After filtration and concentration at 35 oC, crude extracts were labeled as PLS1, PLS2, PLS3, PLS4 and PLS5 for 90% methanol, 100% methanol, chloroform, petroleum ether and 80% methanol solvents respectively. For all analyses we used analytical grade chemicals/reagents and solvents. In addition, 20 g of powdered leaf sample each was first defatted using soxhlet extractor with petroleum ether and chloroform solvents for 4-5h above 70 0C. After defatting, only 90% methanol was used for further extraction. Finally petroleum ether and chloroform defatted leaf samples which were extracted using 90% methanol were labeled as PDBPE and PDBCM. Determination of total polyphenol content, TPC Total phenolic content of P. lanceolata leaf extracts were determined using the Folin ciocalteu method with slight modification (Amin et al., 2006). 5 mL of herb extract was diluted with 45 mL of distilled water followed by addition of 0.25 mL Folin-Ciocalteu reagent and 0.5 mL of 7% sodium carbonate solution. The mixture were diluted to 100 ml and left for 30 min in dark. Finally absorbencies were measured at 750 nm using UV-vis spectrometer. Determination of total flavonoids The total flavonoid content of P. lanceolata crude leaf extracts were determined using aluminum chloride assay (Chang et al., 2002). 2.00 mL of the extract was mixed with 4 mL distilled water with 10 mL volumetric flask followed by an immediate addition of 0.30 ml of 5% NaNO2. 5 min latter, 0.30 mL of 10% AlCl3 solution was added. After 6 min, 2.00 mL of 1.0 M NaOH solution was added. The absorbance at 510 nm using Uv-vis spectrophotometer was taken after 10 min. Antioxidant Activity Determination Reducing Power Assay: The reducing power of crude extracts was determined according to the method developed by Oyaizu (Oyaizu, 1986). From each sample (PLS1, PLS2, PLS3, PLS4, PLS5, PDBPE and PDBCM), different concentrations (12%, 24%, 36%, 48%, (v/v)) of P. lanceolata leaf extracts were prepared. 2.5 mL extract was taken and mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH= 6.6) and 2.5 mL of potassium ferricyanide solution (1%). The ISSN: 2410-9649 ABC et al / Chemistry International 3(3) (2017) 277-287 iscientic.org. 279 www.bosaljournals/chemint/ [email protected] mixture was incubated at 50 oC for 20 min. Finally, 2.5 mL of trichloroacetic acid solution (10% w/v) was added and the resulting mixture was centrifuged at 3000 rpm for 10 min. At last, 2.5 mL of the filtrate (upper layer) was mixed with 2.5 mL distilled water and 0.5 mL ferric chloride solution (0.1%. w/v). Finally, absorbance of the mixture was measured at 700 nm. DPPH radical-scavenging capacity: The antioxidant activities of P. lanceolata leaves were assessed according to the method developed by Brand Manzorro using 2,2 diphenyl-1-picrylhydrazyl (DPPH) (Louli et al., 2004). 90% and 100% methanol, chloroform, and petroleum ether extracts of P. lanceolata leaves with different concentration (48%,36%, 24%, 12%(v/v)) were prepared. 5 mL of 0.004% of DPPH solution was mixed with 1 mL of each extract. Solutions were kept in dark for 30 min; absorbencies were measured at 517 nm using Uv-vis spectrophotometer. Peroxide value determination For peroxide value determination Niger seed oil was used because it is rich in linolic acid which is important component for antioxidant activity research (Gutteridge, Fig. 1: DPPH Hydroxyl radical scavenging activity of aqueous (Aq) and ethanol/water (hydro eth) extracts of Hibiscus Sabdariffa Linn and control (Ascorbic acid). Fig. 2: DPPH radical scavenging activity of P.lanceolata leaf extracts at different concentration ISSN: 2410-9649 ABC et al / Chemistry International 3(3) (2017) 277-287 iscientic.org. 280 www.bosaljournals/chemint/ [email protected] 1995). Twelve different samples that contain Niger seed oil and pure Niger seed oil were prepared for the determination of peroxide value. Six of them were placed at room temperature and the other six samples were placed at 70 oC. From each sample, 5 g were taken and transferred to 12 different 250 mL conical flasks. 30 mL of mixture of glacial acetic acid and chloroform (3:2) were added to each sample. The mixtures were shaken to dissolve and 0.5 mL of saturated KI solution was added to all samples kept both at room temperature and 75 oC. Finally 30 mL of water was added to each sample and titrated with 0.01 N sodium thiosulfate solution. After a yellow color disappears, 5 ml of starch solutions were added to check completion of titration. The titrant was added slowly with continuous shaking until the blue color was disappeared. A blank determination was performed under the same condition. Antibacterial Activity Determination Preparation of Inoculum: The microbial stock cultures were maintained at 4 °C on slopes of Muller-Hinton Agar, MHA. Active cultures for experiments were prepared by transferring a loopful of cells from the stock culture to the test tubes containing Muller-Hinton broth and incubated without agitation for 24 h at 37 °C. To 5 ml of Muller-Hinton Broth, 100μ of culture was inoculated and kept till it reached the turbidity equal to 0.5 MacFarland standard solutions which is equivalent to 1.5×10 CFU/ml (13---.13 Disk Diffusion Methods Disk diffusion assay with Muller Hinton agar (MHA) medium was used to analyze antimicrobial activities of different crude extracts of P. lanceolata leaves. The MHA was melted and then cooled and finally poured into sterile petri dishes to get a solid plate. Then standardized inoculums (0.5 McFarland) were added and streaked on the agar plate surface. Wells were prepared in the seeded agar plates with sterile cork borer (6 mm diameter). The test compound or crude extract (100 μl) were carefully dispensed into the wells. This was done in triplicate parallel with different control antibiotics. Extracts were allowed to diffuse for about 2 h at 37 °C. After overnight incubation, the zone of inhibition was observed and the diameter of inhibition zone was measured. Determination of MIC and MBC Fig. 3: Change in peroxide value (meq/kg) during storage at 70OC (upper) and room temperature lower) with different treatments ISSN: 2410-9649 ABC et al / Chemistry International 3(3) (2017) 277-287 iscientic.org. 281 www.bosaljournals/chemint/ [email protected] MIC and MBC extracts of the plant were determined according to methods described by Shahidi (Cushnie and Lamb, 2005). P. lanceolata leaf extracts were diluted to different concentration (1/4, 1/8) of the original solution. To each diluted crude extracts, nutrient broth tubes were seeded both with 100 μL of the pathogenic standard and resistant clinical bacteria. Negative control tubes with no bacterial inoculation were simultaneously maintained. Tubes were Table 1: Result of phytochemical screening of different solvent extracts of P. lanceolata leaves Phytochemicals Solvents 90% MeOH 100% MeOH Chloroform Petroleum ether