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Timestamp: 2019-04-21 21:28:16+00:00

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Vernonia amygdalina is a plant that is commonly cultivated in Africa for food as well as for its medicinal properties5; and there have been several reports on the plant’s antioxidant and antimicrobial properties.6,7 Previous studies8,9 have reported DPPH IC50 values for aqueous and ethanolic extracts of V. amygdalina but there have been no reports on the IC50 values for the ABTS free radical scavenging assay using V. amygdalina extracts. Owhe-Uregheet al.10 reported antimicrobial properties of aqueous and ethanolic extracts of V. amygdalina against Y. enterocolitica. At present, there are no reports on the antimicrobial activity of V. amygdalina against E. coli O157:H7. The present study describes antimicrobial properties of ADE, AME, EE and EAE leaf extracts of V. amygdalina against two food borne pathogens; E. coli O157:H7 and Y. enterocolitica. The study also includes the antioxidant properties of the leaf extracts which were reassessed through IC50 studies using the DPPH and ABTS free radical scavenging assay.
Folin-Ciocalteu’s phenol reagent, Sodium carbonate, methanol and ethyl acetate were from Merck (Germany). 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was purchased from Sigma-Aldrich (Canada). Gallic acid, 2,2-diphenyl-1-picryhydrazyl (DPPH) and Butylated hydroxyanisole (BHA) were purchased from Sigma-Aldrich (USA). Ethanol and dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (UK). Vanillin and trolox were purchased from Acros Organics (USA). Sulphuric acid was purchased from Friendemann Schmidt Chemical (Australia). Diosgenin was purchased from ChromaDex (USA).
For antimicrobial studies, Mueller-Hinton agar (MHA), ampicillin (10 µg/disc) and chloramphenicol (30 µg/disc) were purchased from Oxoid (UK). Chloramphenicol powder was purchased from Bio Basic (Canada) and Mueller-Hinton Broth (MHB) was purchased from Difco (USA).
Escherichia coli O157:H7 vegetable and chicken isolates were obtained from the Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM). Yersinia enterocolitica ATCC 23715 was purchased from Thermo Scientific, USA. The test microorganisms were maintained on TSA agar plates and stored at 2-8 ºC.
The V. amygdalina leaves (8.564 kg) were collected from the Yik Poh Ling Tropical Herbal Farm, Negeri Sembilan on the 17th of September 2012 and a sample of the leaves were sent to UPM for identification. Back in the laboratory, the leaves were weighed and then rinsed to remove the surface dirt and contaminants before being air dried in the shade for a week. After drying, the leaves were weighed once again and the moisture content was determined. The dried leaves were then ground into powder using a commercial blender (Waring Products Division, USA). The powder was stored in sealed plastic containers until needed.
The ADE extract of V. amygdalina were prepared according to the method described by Ola et al.11 with slight modifications. The powdered V. amygdalina leaves (10 g) were boiled in 150 ml ultrapure water (ElgaLabwater, UK) for 10 min before being allowed to cool. After cooling, the solution was filtered through a double rings filter paper using a Buchner funnel and rinsed to bring the volume up to 150 ml. After rinsing, the extract was freeze dried (Martin Christ, Germany) and stored in a desiccator for future use.
The AME, EE and EAE extracts were prepared according to the method used by Margeretha et al.12. Briefly, 100 g of powdered V. amygdalina leaves was placed into three separate 1000 ml Erlenmeyer flasks. Then, 900 ml of ethanol was added to the first flask, 900 ml of ethyl acetate was added to the second flask and 600 ml of ultrapure water was added to the third flask. These flasks were then wrapped in aluminum foil and periodically shaken at room temperature. After 24 h, the ethanol and ethyl acetate solutions from these flasks were filtered using a double rings filter paper and the filtrate concentrated using a rotary vacuum evaporator (Tokyo Rikakikai, Japan)at 45-50 ºC. To the marc, a fresh volume of solvent was added and the maceration continues for another 24 h. The maceration process, followed by concentration via rotary vacuum evaporator, was repeated for 14 days until the addition of the solvent to the marc produces a clear solution. The obtained concentrated filtrates were then combined before the final drying in an oven at 40 ºC to produce a constant weight. The extraction yields of the crude EE and EAE extracts were determined and the extracts were stored in a desiccator at room temperature.
The AME extract, after filtering through a double rings filter paper, was freeze dried. Fresh ultrapure water was then added to the marc and the maceration-freeze drying process was repeated for ten days until a clear solution was obtained. The freeze dried crude extracts were then combined and the extraction yield was determined. The AME extract was then stored in a desiccator at room temperature.
The TPC assay was done according to the method described by Atanassova et al.13 with minor modifications. The assay was carried out by mixing 0.2 ml of sample solution (consisting of either plant extract or gallic acid standard) with 1.8 ml of ultrapure water and 0.2 ml of Folin-Ciocalteu reagent (10X dilution). The mixture was then allowed to incubate for 5 min before 2 ml of 7% aqueous Na2CO3 solution and 0.8 ml of ultrapure water was added. After 90 min of incubation, the absorbance was measured at 750 nm using a Lambda 25 UV/Vis Spectrometer (Perkin Elmer; US). For the reagent blank, the plant extract/ gallic acid standard was replaced with 80% aqueous methanol. To prepare the calibration curve, gallic acid was assayed at concentrations 10, 20, 40, 60, 80 and 100 µg/ml. The TPC values of the plant extracts (1 mg/ml) were determined by substituting the absorbance values obtained into the linear equation generated from the standard curve. The TPC assay for each extract was performed in triplicates and expressed as milligrams of gallic acid equivalents (GAE) per g of dry extract.
The total saponin assay was performed according to the method given by Makkaret al.14 The assay was performed by mixing 0.25 ml of sample solution (consisting of either plant extract or diosgenin standard) with 0.25 ml of 8% vanillin reagent and 2.5 ml of 72% (v/v) H2SO4. The solution was mixed thoroughly before being placed into a hot water bath at 60 ºC for 10 min. After heating, the solution was cooled in ice-cold water for 3-4 min before the absorbance was read at 544 nm. For the reagent blank, the sample solution was replaced with 80% aqueous methanol. The calibration curve was prepared by assaying the diosgenin standard at 50, 100, 150, 200, 300, 400 and 500 µg/ml. The total saponins content of the extracts (1 mg/ml) were quantified by substituting the absorbance values of the plant extracts into the linear equation generated by the standard curve. The total saponin assay was done in triplicates for each plant extract and the values were expressed as milligrams of diosgenin equivalents (DE) per g of dry extract.
The DPPH radical scavenging activity was done according to the method used by Sharma and Goyal.15 In each test tubes 1 ml of sample solution (containing antioxidant from either the plant extract or BHA standard) and 6 ml of methanolic DPPH solution (100 µM ) were mixed together and incubated in the dark for 10 min. After incubation the decrease in absorbance was measured at 517 nm using a Lambda 25 UV/Vis Spectrometer and the % DPPH radical scavenging activity was calculated using Equation 1. Methanol was used as the reagent blank.
where Acontrol is the absorbance of the control and Asample is the absorbance of the sample solution.The IC50 values of each sample were determined from the polynomial equation generated from the plotted curve using the Microsoft Office Excel 2007 program. The IC50 is defined as the concentration of the sample that is required to scavenge 50 % of the DPPH radical. DPPH assay was carried out in triplicates. BHA was used as positive control.
The ABTS.+radical scavenging assay was done according to the method employed by Re et al. and Teowet al.16, 17 with slight modifications. The ABTS.+ solution was prepared by mixing 192.2 mg of ABTS salt with 33.2 mg of potassium persulfate in 50 ml of ultrapure water and this will give the solution a final concentration of 7 mM ABTS and 2.456 mM potassium persulfate. The ABTS.+ solution was then stored in the dark at room temperature for 16 h before use. The ABTS.+ radical scavenging assay was performed by first diluting the ABTS.+ solution at room temperature with 95% ethanol for the Trolox standard, EE and EAE extract; and 76% ethanol for the ADE and AME extracts to an absorbance of 0.70 (+ 0.05) at 734 nm (approximately 7 ml ABTS.+ solution in 500 ml 95% ethanol). After dilution, 4 ml of diluted ABTS.+ solution is placed into each test tube and 40 µl of sample solution (containing either Trolox standard or plant extract) is added. The solution is mixed and the decrease in absorbance was measure at 734 nm after 6 min. For the reagent blank, 95% ethanol was used for the Trolox standard, EE and EAE extract while 76% ethanol was used for the ADE and AME extracts. The ABTS radical scavenging activity was determined using Equation 2.
Where Acontrol is the absorbance of the control and Asample is the absorbance of the sample solution. The IC50 values of each sample were determined from the polynomial equation generated from the plotted curve using the Microsoft Office Excel 2007 program. The concentration at which 50% of the ABTS radical is scavenged is known as the IC50. The ABTS IC50 assay was carried out in triplicates.
The EE extract (500 mg/ml) and EAE extract (500 mg/ml) were prepared by dissolving 500 mg of crude extract into 1 ml of 99.99% DMSO. The ADE extract (500 mg/ml) and AME extract (500 mg/ml) were prepared by dissolving 500 mg of crude extract into 1 ml of sterilised ultrapure water. The ADE and AME extracts were then filtered through a 0.45 µm nylon filter under a laminar flow hood for sterilisation before use.
The screening of the antimicrobial properties of V. amygdalina was done using the disk diffusion method according to the protocol given by the Clinical and Laboratory Standards Institute (CLSI).18 The preparation of the inoculum suspension was done using 24-hour-old cultures and adjusted to that of a 0.5 McFarland standard. The cell suspension was then streaked onto Mueller-Hinton agar (MHA) plates. After streaking, 6 mm antibiotic assay discs impregnated with 25 µl of plant extracts (500 mg/ml) were placed onto the agar surface. The MHA was then inverted and incubated at 35 + 2 ºC under ambient air for 16-18 h. After incubation, the inhibition zones were measured to the nearest millimetre with a ruler and recorded. Extracts exhibiting antimicrobial activity were then chosen for the minimum inhibitory concentration (MIC) assay. For negative controls, antibiotic assay discs containing 99.99% DMSO and sterilised ultrapure water was used. For positive controls, ampicillin (10 µg/disc) was used for the E. coli O157:H7 strains while chloramphenicol (30 µg/disc) was used for Y. enterocolitica ATCC 23715. Aseptic conditions were maintained throughout the antimicrobial susceptibility screening and all relevant apparatus were autoclaved prior to use. The antimicrobial screening was done in triplicates.
The MIC determination was done using the broth macrodilution method.19, 20 Chloramphenicol stock solution (positive control) was subjected to two fold dilutions to give concentrations of 128, 64, 32 and 16 µg/ml. From these prepared concentrations, 0.5 ml of the antibiotic was transferred into screw capped test tubes and after the addition 0.5 ml of inoculum into these test tubes (1:2 dilution), the final concentration of the chloramphenicol solutions were 64, 32, 16 and 8 µg/ml, respectively. Likewise, the AME extract was subjected to two fold dilutions to give concentrations of 500, 250, 125, 62.5 and 31.25 mg/ml. Then, 0.5 ml of the plant extract was transferred into screw capped test tubes and after the addition 0.5 ml of inoculum into these test tubes (1:2 dilution), the final concentration of the AME extract were 250, 125, 62.5, 31.25 and 15.625 mg/ml, respectively.
The MIC assay was done using 24-hour-old Y. enterocolitica cultures. The inoculum was prepared by adjusting the Y. enterocolitica suspension in 0.85% NaCl to match that of a 0.5 McFarland standard turbidity. This gave a cell density of about 1 x 108 CFU/ml. Then, the Y. enterocolitica suspension was further diluted (1:100) with CAMBH to give a cell density of 1 x 106 CFU/ml. Within 15 min of preparation, 0.5 ml of the inoculum was added into the test tubes containing the antimicrobial solution. This gave the inoculum a final concentration of 5 x 105 CFU/ml.
For controls, the growth control consisted of 0.5 ml of test inoculum with 0.5 ml of sterile water. For the broth control tube, 0.5 ml of CAMBH with 0.5 ml of sterile water was mixed together. An additional tube containing 0.5 ml of AME extract and 0.5 ml of CAMBH was also included with the assay to test for the sterility of the extract. To check for the purity of the test organism, 10 µl of suspension was removed from the growth control tube and streaked onto a TSA plate.
Incubation of the samples, controls and purity tests were done at 35 + 2 ºC under ambient air for 16- 20 h. After incubation, the MIC was determined by visually checking the test tubes after gentle agitation. The lowest concentration of antimicrobial agent/extract that completely inhibits the growth of the test organism (detected by lack of visual turbidity) is known as MIC and the values were reported in mg/ml. For the plant extract, the MIC assay was done in triplicates. Aseptic conditions were maintained throughout the MIC assay and all relevant apparatus were autoclaved prior to use.
The statistical analysis was done using the one-way analysis of variance (ANOVA) and Tukey’s multiple comparison post-hoc test on the Prism Graph Pad software. The difference between the means at 5% confidence level was considered significant. The linear regression analysis was done using Microsoft Excel 2007 to compare the correlation between the total phenolics and total saponin against the free radical scavenging activities of the V. amygdalina extracts.
Figure 1. The extraction yields of Vernonia amygdalina using different solvent. ADE = aqueous extract obtained by decoction; AME = aqueous extract obtained by maceration; EE = extract obtained by maceration in ethanol; EAE = extract obtained by maceration in ethyl acetate.
The TP values (reported in mg gallic acid equivalents/ g dry extract) are shown in Table 1. The EE extract of V. amygdalina has the highest TP content. The TP contents of the extracts exhibited the descending order: EE > ADE > AME > EAE. The TP content of the EAE extract and the AME extract were not significantly different (p > 0.05) indicating that both the EAE and AME extracts have comparable amounts of phenolic compounds. The TP for EE, EAE and ADE however were significantly different (p < 0.05). Phenolic compounds are polar aromatic compounds that are readily soluble in ethanol23 and the results shown in Table 1 confirms this as the EE extract of V. amygdalina has the highest TP value calculated. However, the presence of phenolic compounds in the ADE, AME and EAE extract could be attributed to the difference in the phenolic compound’s structure and polarity.
Total saponin content of the extracts is shown in Table1. The TS content of EAE extract was found to be the highest. The TS content of the extracts exhibited the descending order: EAE > EE > ADE > AME. The EAE and EE extracts showed significantly (p < 0.05) higher TS content when compared to the AME and ADE extracts. The TS content of ADE and AME were not significantly different (p > 0.05). The TS content between EE, EAE and ADE extract were significantly different ( p<0.05). The higher TS content found in the EE and EAE extracts may be ascribed to the method, 14 since the vanillin reagent, which acts as a chromogen under acidic conditions, reacts with the aglycone portion of the saponin and not its sugar moiety. Therefore, sapogenins, which are the non-polar aglycone portion of the saponin26 will also be quantified by the assay. The high TS content found in the EAE and EE extracts may indicate that most of the saponins found within the V. amygdalina leaves are sapogenin.
Figure 2 shows DPPH radical scavenging activity of V. amygdalina extracts and the reference standard BHA. Table 2 shows the IC50 values of V. amygdalina extracts for DPPH and ABTS radical scavenging activity. The IC50 value of the AME extract is significantly higher than that of the ADE despite the fact that both of these extracts were prepared using water as the extraction solvent. A possible reason for this could be due to the higher TP content of the ADE as compared to the AME. The DPPH IC50 values of ADE, EE and EAE extracts are not significantly different (p > 0.05) despite the significant differences between the extracts’ TP content. This would imply that there may be other compounds involved that could cause the reduction of the DPPH radical and this is supported by the data moderate correlation between the DPPH radical scavenging activity (IC50) and TP contents (Figure 4 a) of V. amygdalina. Venskutonis27 reported that the hydroxyl group found in monosaccharides (which are water soluble) is capable of acting as a hydrogen donor and this causes the reduction of the DPPH radical from dark purple to yellow. This might explain the higher DPPH radical scavenging activity found in the ADE extract despite its lower TP value when compared to the EE extract. Similarly, saponins content which was found in high in the EAE extract (as shown in Table 1) may also contribute to the DPPH radical scavenging activity28 since there was moderate correlation between the DPPH radical scavenging activity (IC50) and TS contents of V. amygdalina (Figure 4 c).
In a study conducted by Ho et al.9 using aqueous spray dried V. amygdalina extracts, the IC50 value of the aqueous extract was reported to be 600 ± 7.4 µg/ml and this IC50 value is comparable to the IC50 value of the ADE extract obtained in this study. However, the IC50 values of the aqueous suspension of V. amygdalina extract reported by Adetutuet al.8 was different. In Adetutuet al.’s study, 4.0 ± 0.1 µg/ml was the IC50 value reported for the DPPH radical scavenging activity. Comparable results were obtained between the study conducted by Ho et al.9 and this study because both the plants used in the studies were obtained from the same geographical region (Malaysia) while the study conducted by Adetutu et al.8 was done in Nigeria which may have a different geographical environment and thus resulting in a variation in chemical composition. For the EE extract, Ayoola et al.29 reported an IC50 value of 2300 µg/ml and this value differs from the IC50 value obtained in this study due to the reasons mentioned above.
Figure 3 shows ABTS radical scavenging activity of V. amygdalina extracts and the reference standard Trolox. The IC50 values of the ABTS assay was different from the DPPH assay with the AME extract having a lower IC50 value when compared to the EE and EAE extracts (Table 2). The difference in radical scavenging activities between the ABTS and DPPH assays could be attributed to the stereo-selectivity of the generated radicals, the chemical structures of the compounds and the solubility of the V. amygdalina extracts in the test system used.30, 31 Correlation of the TP and TS content against the ABTS radical scavenging activity (Figure 4b) revealed that the TP content had weak correlation with the observed ABTS radical scavenging activity. This indicates that the phenolic compounds in V. amygdalina are not responsible for the ABTS radical scavenging activity and that there may be other bioactive compounds that are responsible for the ABTS radical scavenging activity observed. The correlation between the TS and ABTS radical scavenging activity (Figure 4d) showed strong negative correlation as the ABTS radical scavenging activity decreases with increasing TS content. Currently, Li et al.32, reported similar strong negative correlation between the TS and ABTS radical scavenging activity of Rhizomacimicifugae.
Means with the same letter are not significantly different from each other at p< 0.05 (ANOVA, followed by Tukey test). The values within the table were obtained by calculating the average of three replicates ± standard deviation.
Figure 2: DPPH radical scavenging activity of Vernonia amygdalina extracts and the reference standard BHA.
The results of the antimicrobial screening are shown in Table 3. Of the four V. amygdalina extracts screened at 500 mg/ml, only the AME extract showed antimicrobial activity on Y .enterocolitica with an inhibition zone of 11.33 + 0.58 mm. Therefore, the MIC was performed using only the aqueous maceration extract on Y. enterocolitica. The results of the MIC revealed that the AME extract had an MIC of 31.25 mg/ml against Y. enterocolitica while chloramphenicol had an MIC value of < 8.000 x 10-3 mg/ml. Similar literature report by Owhe-Ureghe et al.10 showed that the Y. enterocolitica O:8 strain was resistant to the ethanolic extract but susceptible to the water extract of V. amygdalina.
Figure 3: ABTS radical scavenging activity of Vernonia amygdalina extracts and the reference standard Trolox.
Figure 4: Correlation between (A) DPPH radical scavenging activity (IC50) and total phenolic contents, (B) ABTS radical scavenging activity (IC50) and total phenolic contents, (C) DPPH radical scavenging activity (IC50) and total saponin contents, (D). ABTS radical scavenging activity (IC50) and total saponin contents.
E. coli O157:H7 (v) = vegetable isolate; E. coli O157:H7 (c) = chicken isolate; NI = no inhibition; - = not applicable. The inhibition zone diameters shown were obtained by calculating the average of three replicates ± standard deviation.
The choice of solvents and extraction method plays important role in the preparation of plant extracts. This study shows that the use of solvents of different polarity affects not only the extraction yield but also the content of the TS and TP; the free radical scavenging activities; the bioactive compounds that can be obtained and the antimicrobial activity of the extracts. The more polar solvents gave higher extraction yields than the less polar solvents. The EE extract showed the highest TP content and the EAE extract showed the highest TS content. This indicates that different bioactive compounds can be extracted with different solvents. The effect of temperature on the extraction yield, TP and free radical scavenging activities can also be seen when comparing the ADE and AME extract. The ADE extract exhibited higher extraction yield, TP content and better free radical scavenging activity than the AME extract. All the extracts demonstrated free radical scavenging activities with the ADE extract having the lowest IC50 value for both the DPPH and ABTS radical scavenging assay. This information helps to establish the potential of V. amygdalinaas a source of dietary antioxidants. For the antimicrobial studies, both the E. coli O157: H7 isolates and Y. enterocoiticashowed resistance against the V. amygdalina extracts tested. The antimicrobial studies provide information on the suitability of V. amygdalinaagainst E. coli O157:H7 and Y.enterocolitica.
1. Wallace RJ. Antimicrobial properties of plant secondary metabolites. Proc Nutr Soc. 2004;63(04):621-9.
2. Pesewu GA, Cutler RR, Humber DP. Antibacterial activity of plants used in traditional medicines of Ghana with particular reference to MRSA. J Ethnopharmacol. 2008;116(1):102-11.
3. Igile GO, Oleszek W, Jurzysta M, Burda S, Fafunso M, Fasanmade AA. Flavonoids from Vernonia amygdalina and their antioxidant activities. J Agric Food Chem. 1994;42(11):2445-8.
4. Dragland S, Senoo H, Wake K, Holte K, Blomhoff R. Several culinary and medicinal herbs are important sources of dietary antioxidants. The J Nutr. 2003;133(5):1286-90.
5. Adesanoye OA, Farombi EO. Hepatoprotective effects of Vernonia amygdalina (astereaceae) in rats treated with carbon tetrachloride. Exp Toxicol Pathol. 2010;62(2):197-206.
6. Salawu S, Ogundare A, Ola-Salawu B, Akindahunsi A. Antimicrobial activities of phenolic containing extracts of some tropical vegetables. Afr J Pharmacol Pharm. 2011;5(4):486-92.
7. Erasto P, Grierson D, Afolayan A. Bioactive sesquiterpene lactones from the leaves of Vernonia amygdalina. J Ethnopharmacol. 2006;106(1):117-20.
8. Adetutu A, Oyewo E, Adesokan A. Protective effects of Vernonia amygdalina against sodium arsenite-induced genotoxicity in rat. Pharmacognosy Res 2013;5(3):207.
9. Ho WY, Liang W, Yeap SK, Beh BK, Noaman AH, Alitheen NB. In vitro and in vivo antioxidant activity of Vernonia amygdalina water extract. Afr J Biotechnol. 2012;11(17):4090-4.
10. Owhe-Ureghe U, Ogiehor S, Ehwarieme D. In vitro susceptibility testing of Yersinia species to eight plant extracts and three natural antimicrobial agents. Afr J Biotechnol. 2012;11(42):10008-14.
11. Ola SS, Catia G, Marzia I, Francesco VF, Afolabi AA, Nadia M. HPLC/DAD/MS characterisation and analysis of flavonoids and cynnamoil derivatives in four Nigerian green-leafy vegetables. Food Chem. 2009;115(4):1568-74.
12. Margeretha I, Suniarti DF, Herda E, Mas’ud ZA. Optimization and comparative study of different extraction methods of biologically active components of Indonesian propolis Trigona spp. J Nat Prod. 2012;5.
13. Atanassova M, Georgieva S, Ivancheva K. Total phenolic and total flavonoid contents, antioxidant capacity and biological contaminants in medicinal herbs. J University Chem Technol Metallurgy. 2011;46(1):81-8.
14. Makkar HPS, Siddhuraju S, Becker K. Plant secondary metabolites. New Jersey: Humana Press; 2007.
15. Sharma SK, Goyal N. In vitro antioxidant activity of root extracts of Heliotropium eichwaldi Stued. ex DC. Res J Pharm, Biol Chem Sci. 2012;3(1):444-9.
16. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999;26(9–10):1231-7.
17. Teow CC, Truong V-D, McFeeters RF, Thompson RL, Pecota KV, Yencho GC. Antioxidant activities, phenolic and ß-carotene contents of sweet potato genotypes with varying flesh colours. Food Chem. 2007;103(3):829-38.
18. CLSI. Performace standards for antimicrobial susceptibility testing: twenty first informational supplement. [place unknown]: Clinical and Laboratory Standards Institute; 2011.
19. CLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A7. [place unknown]: Clinical and Laboratory Standards Institute.; 2006. Available from: http://isoforlab.com/phocadownload/csli/M7-A7.pdf.
20. Qaiyumi S. Macro- and microdilution methods of antimicrobial susceptibility testing. In: Schwalbe R, Steele-Moore L, Goodwin AC, editors. Antimicrobial susceptibility testing protocols. Boca Raton: Taylor & Francis; 2007.
21. Sultana B, Anwar F, Ashraf M. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules. 2009;14(6):2167-80.
22. Luthria DL, Vinjamoori D, Noel K, Ezzel J. Accelerated solvent extraction. In: Luthria DL, editor. Oil extraction and analysis: critical issues and comparative studies. USA: AOCS Press; 2004.
23. Saura-Calixto F, Serrano J, Pérez-Jiménez J. What contribution is beer to the intake of antioxidants in the diet? In: Preedy VR, editor. Beer in health and disease prevention. Burlington: Elsevier Science; 2011. p. 441-4.
24. Wijingaard HH, Trifunovic O, Bongers P. Novel extraction techniques for phytochemicals. In: Tiwari BK, Brunton NP, Brennan C, editors. Handbook of plant food phytochemicals: sources, stability and extraction. West Sussex: Wiley; 2012. p. 419.
25. Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 2010;15(10):7313-52.
26. Gupta VK. X-ray structural behaviour of some significant bioactive steroids and their chemistry in the crystal packing and related matters. In: Brahmachari G, editor. Chemistry and pharmacology of naturally occurring bioactive compounds. Boca Raton: Taylor & Francis; 2013. p. 312.
27. Venskutonis PR. Natural antioxidants in food systems. In: Bartosz G, editor. Food oxidants and antioxidants: chemical, biological, and functional properties. Boca Raton: Taylor & Francis; 2013. p. 264.
28. Ye Y, Guo Y, Wang YF. Free radical scavenging activity and anti-inflammatory property of the saponin from seeds of Camellia oleifera abel. Adv Mat Res. 2012;550:1262-5.
29. Ayoola G, Coker H, Adesegun S, Adepoju-Bello A, Obaweya K, Ezennia E, Atangbayila TO. Phytochemical screening and antioxidant activities of some selected medicinal plants used for malaria therapy in Southwestern Nigeria. Trop J Pharm Res. 2008;7(3):1019-24.
30. Yu LL, Cheng ZH. Antioxidant properties of wheat phenolic acids. In: Yu LL, editor. Wheat antioxidants. New Jersey: Wiley; 2008. p. 61-2.
31. Bhoyar MS, Mishra GP, Naik PK, Srivastava R. Estimation of antioxidant activity and total phenolics among natural populations of Caper (Capparis spinosa) leaves collected from cold arid desert of trans-Himalayas. Aust J Crop Sci. 2011;5(7):912.
32. Li X, Lin J, Gao Y, Han W, Chen D. Antioxidant activity and mechanism of Rhizoma Cimicifugae. Chem Cent J. 2012;6(1):1-10.
33. Noumedem JA, Mihasan M, Kuiate JR, Stefan M, Cojocaru D, Dzoyem JP, Kuete V. In vitro antibacterial and antibiotic-potentiation activities of four edible plants against multidrug-resistant gram-negative species. BMC Complement Altern Med. 2013;13(1):190.
34. Talukdar PK, Rahman M, Rahman M, Nabi A, Islam Z, Hoque MM, Endtz HP, Islam MA.
35.	Antimicrobial resistance, virulence factors and genetic diversity of Escherichia coli isolates from household water supply in Dhaka, Bangladesh. PloS one. 2013;8(4):1-8.

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