In Vitro antioxidant and antibacterial activity of leaf extracts of Measa lanceolata

ABSTRACT This study investigated total phenolic contents (TPC), total flavonoid contents (TFC), antioxidant activity, and antibacterial activities of different leaf extracts of Measa lanceolata. The TPC and TFC of the extracts were determined by Folin-Ciocalteu and aluminum chloride methods, respectively. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, reducing power, and phosphomolybedinium assays were used to evaluate antioxidant activity. Antibacterial properties were assessed using the disc-diffusion assay based on minimum inhibitory concentration (MIC). Bacteria tested were three strains of gram-negative (Escherichia coli (ATCC-25922), Proteus vulgaris (ATCC-13315), and Pseudomonas aeruginosa (ATCC-43495)), and two strains of gram-positive (Staphylococcus aureus(ATCC-25923), and Enterococcus faecalis(ATCC-29212)). It was found that methanol extract contained the highest TPC (60.6 ± 4.4 mg of gallic acid equivalent/g of dried extract) and TFC (6.4 ± 6.0 mg of catechin equivalent/g of dried extract). Extract showed the strongest DPPH radical scavenging activity (EC50 = 76.7 ± 7.3 µg/mL), iron reducing power (EC50 = 74.0 ± 1.6 µg/mL), and total antioxidant activity (128 ± 4 mg of ascorbic acid equivalent per dried extract). Inhibition zones were observed in water and methanol extracts against bacterial strains, whereas Staphylococcus aureus (ATCC-25923), Escherichia coli(ATCC-25922), Pseudomonas aeruginosa (ATCC-43495), and Proteus vulgaris(ATCC-13315) were resistant against chloroform and ethyl-acetate extracts.


Introduction
Medicinal plants have been identified and used traditionally throughout the world from the beginning of human civilization. [1] Medicinal plants are rich source of novel drugs that form the ingredients in traditional systems of medicine. [2] More than 50,000 plant species are reported as being used across the globe for medicinal purposes. [3] About 80% of the population in Africa primarily depends on traditional medicinal plants for their health care similarly; plants have been used as a source of traditional medicine since ancient times in Ethiopia to combat different ailments and human sufferings. [4] The current account of medicinal plants of Ethiopia, as documented for national bio-diversity strategy and action plan shows that about 887 plant species were reported to be utilized in the traditional medicine. [5] Most of the developing countries depend on traditional medicinal plants for their health care It is therefore, not surprising that some of these plants have chemical compounds of therapeutic value that may be used in the treatment of major diseases such as malaria, cancer and pathogenic microorganisms. [6] As literature showed in Ethiopia more than 80% of the population uses plant-absorbance of the resulting blue color was measured at 765 nm with a UV-visible spectrophotometer (JENWAY, 96500, UK) after incubation for 90 min at room temperature. The total phenolic content was estimated from gallic acid (1-100 μg/mL) calibration curve (y = 0.015 x + 0.081, R 2 = 0.991, p < .001) and results were expressed as mg gallic acid equivalent/g of dry extract (mgGAE/g).

Total flavonoid content (TFC)
The TFC was estimated using the method of Samidha. [16] The extract (1 mL, 1 mg/mL) was diluted with 1.25 mL distilled water and 75 µL 5% NaNO 2 was added to the mixture. After 6 min, 150 µL 10% AlCl 3 was added and then after 5 min, 1 mL 1 M NaOH was added to the reaction mixture. Then, the absorbance (pink in color), was determined at 510 nm versus prepared water blank. A standard curve was prepared using 5-1000 µg/mL of catechin. Results were expressed as milligram of catechin equivalents per milligram of dry extract of the plant extract. All the calculations were done using standard equation (y = 0.002x + 0.35, R 2 = 0.99, p < .001) obtained from standard calibration curve of catechin.

Antioxidant activities
DPPH scavenging activity: DPPH radical scavenging activity of the crude extracts was determined as described by Engeda et al. [17] Different concentrations (50 to1000 μg/mL) of the extracts were taken in different test tubes. Freshly prepared DPPH solution (2 mL) was added in to each of the test tubes containing 1 mL of the extract. The reaction mixture and the reference (BHT) were vortexed and left to stand at room temperature in the dark for 30 min. Absorbance of the resulting solution was taken at 520 nm. Methanol was used as blank. The ability of the scavenge the DPPH radical was calculated using the following equation.
DPPH scavenging = [(Ac-As)/Ac] x100 where Ac is the absorbance of the control and As is the absorbance in presence of the sample of the extracts. The antioxidant activity of each extract was expressed as EC 50 . The EC 50 value is defined as the effective concentration (μg/mL) of extracts that scavenges the DPPH radical by 50%.
Ferric ion reducing power: The presence of antioxidants in the extract causes the reduction of the yellow ferric cyanide complex to the ferrous form which can be monitored by measuring the formation of Perl's Prussian blue at 700 nm. [18] One milliliter of extract solution (final concentration 50-1000 μg/ mL) was mixed with 2.5-mL sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferric cyanide. Then, the mixture was incubated at 50°C for 20 min. Trichloro acetic acid (2.5 mL, 10%) was added to the mixture, which was then centrifuged at 3000 rpm (Centurion, 1000 series, UK) for 5 min. Finally, 2.5 mL of the supernatant solution was mixed with 2.5 mL of distilled water and 0.5 mL FeCl 3 (0.1%) and absorbance was measured at 700 nm. The EC 50 value (µg/mL) is the effective concentration at which the absorbance is 0.5 for reducing capacity and was calculated from the graph of absorbance at 700 nm against the sample concentration.
Total antioxidant activity using phosphomolybdenum method: The method is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds or crude extract and subsequent formation of green Mo (V) complexes with a maximal absorption at 695 nm at acidic medium. [19] Plant extract (0.5 and 1 mg/mL) was mixed with 3 mL of reagent solution mixture containing equal amount of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The samples were incubated at 95°C for 90 min, cooled to room temperature and absorbance was measured at 695 nm and methanol (3 mL) was used as blank. The total antioxidant activity was expressed as milligram ascorbic acid equivalent/gram of dried extract (mgAAE/g) based on the calibration curve; (y = 0.0094x + 0.112, R 2 = 0.99).
Pure cultures of the test organisms were obtained from Ethiopian Biodiversity institute, Addis Ababa, Ethiopia, October, 2016. Bacteria were maintained on nutrient agar medium. Each bacteria culture was further maintained by subculturing regularly on the same medium and stored 4°C before use.
Disc diffusion assay: Antibacterial properties of the extracts were measured using the discdiffusion method. [20] Inoculums (100 μL) were spread evenly onto 20 mL Mueller-Hinton agar set in 90 mm Petri dishes using a sterile cotton swab. Sterilized paper disc (6 mm diameter) was impregnated with extract (20 μL) using a micropipette and firmly placed onto the inoculated agar ensuring even distribution to avoid overlapping of zones. Streptomycin susceptibility discs were used as positive controls. In the assay, each inoculums suspension (20 mL) was spread evenly over the entire nutrient agar (Muller-Hinton Agar) surface by sterile collection swab. Then, discs of diameter 6 mm were sterilized at 121°C for 15 min and loaded with prepared positive control (Gentamicin 10 μg/mL) and extract solutions of Measa lanceolata at various concentrations. The impregnated discs were dried for 3-5 min and dispensed onto the surface of the inoculated plates with flamed forceps. The plates were then labeled and incubated at 37°C for 24 hr. Diameter (millimeter) of zone of inhibition was measured using digital caliber.

Minimum inhibitory concentration (MIC):
The minimum inhibitory concentration (MIC) of the extracts was measured by agar dilution method of Nasro et al [21] with minor modification. Each extract was diluted by twofold. The agar and the plant extracts were mixed thoroughly in sterile container and dispensed to Petri dish labeled with a given concentration of diluted plant extract. The plates were incubated at 37°C for 24 h, after which all plates were observed for growth. The minimum diluted of fraction inhibiting the growth of each organism was taken as the MIC.

Statistical analysis
The data were subjected to analysis of variance (ANOVA), Origen 8 software and Duncan's multiple range tests were used for mean separation at p < .05. Linear regression analysis was used to calculate IC 50 value.

Total phenolic and flavonoid contents
The total phenolic contents in various solvent extracts from the leaf of Measa lanceolata varied widely, ranging from 1.1 to 60.6 mg GAE/g of dried extract (Table 1). It was observed that methanol extract (60.6 ± 4.4 mg GAE/g) showed the highest and chloroform extract (1.1 ± 0.2 mg GAE/g) showed the lowest TPC. The TPC followed the order: methanol > water > ethyl acetate > chloroform extracts. There was no significant difference (p > .05) in TPC between methanol and aqueous extracts but these values were significantly higher (p < .05) than that of ethyl acetate and chloroform extracts (p < .05). TPC of methanol extract obtained from this study was lower than that of reported from South Africa. [22] Similarly, the methanol extract was the richest source of TFC (p < .05) and decreased in the order of methanol > water > ethyl acetate > chloroform extracts (Table 1). There was no significant difference (p > .05) in TPC among water, ethyl acetate, and chloroform extracts but these values were significantly lower (p < .05) than that of methanol extract. Similarly, the TFC of the present study was lower than that of sample collected from South Africa. [22] The lower content of phenolic compounds in chloroform extract compared to methanol may be explained by the low solubility of polyphenols in this solvent. [23] These differences in the amount of TPC may be due to varied efficiency of the extracting solvents to dissolve different compounds. Several studies showed that TPC determined differed with polarity of solvent used in extraction. [24][25][26][27] Results of the present study showed that among all the solvent extracts of Measa lanceolata, methanol extract had the highest TPC and TFC.
Results are expressed as milligram of gallic acid equivalents per gram of dried extract (mg GAE/g) and milligram of catechin equivalents per gram of dried extract (mg CE/g). Values are expressed as mean ± SD (n = 3). Different letters in column after the mean indicate significant differences at p< .05.

Antioxidant activity
DPPH scavenging activity: DPPH radical scavenging assay is the most common method used in the study of antioxidant activity of plant extracts. It results in the formation of stable free radical which can be detected by common spectrophotometric technique. Decrease in absorbance shows the more efficient antioxidant activity of the extract in terms of hydrogen atom donating capacity. This assay is more indirect type as it measures the inhibition of reactive species (free radicals) generated in the reaction mixture and its results depend on the type of reactive species used. [28] The concentration of the plant extracts required to scavenge DPPH showed a dose dependent response. Antioxidant activity of all extracts as measured by ability to scavenge (DPPH) free radicals was compared with the standards butylated hydroxyl toluene (BHT). As the result shows that ( Figure 1) the concentration of sample increased, the percentage inhibition of DPPH radical also increased. It was observed that methanol of leave extract had higher activity than water, ethyl acetate, and chloroform extracts of the leave of Measa lanceolata, at concentration of 1.0 mg/mL. The scavenging activity of methanol extract reached 92.2 ± 2.4%. Water, ethyl acetate, and chloroform extracts reached 88.2 ± 1.4%, 77.0 ± 2.4%, and 69.3 ± 1.8%, respectively.
The EC 50 values of Measa lanceolata extracts were calculated from graph of percentage scavenging activity against concentration of the extracts ( Table 2). The EC 50 was actually used to examine the antioxidant effectiveness of the samples. The lower the EC 50 value, the higher is the scavenging potential. The IC 50 values ranged from 76.7 ± 7.3 μg/mL for methanol extract to 282 ± 50 μg/mL for chloroform extract. The strongest scavenging activity (lower IC 50 value) was recorded for methanol extract which appeared more than three folds stronger than that of chloroform extracts and two times stronger than that of the ethyl acetate extracts and two times stronger than that of the water extracts. The EC 50 values of petroleum ether, aqueous, chloroform and ethyl acetate extracts were significantly different (p < .05), but these values were significantly higher (weaker DPPH scavenging) than (p < .05) the DPPH scavenging activity of methanol extract. Whereas the DPPH scavenging activity of methanol extracts was not significantly different from that of BHT. But this value is weaker than the DPPH scavenging activity of M. Lanceolata reported from South Africa. [22] Ferric ion reducing power: The ferric ion reducing power was used to evaluate the antioxidant properties of the extracts based on their ability to reduce ferricyanide complex to Perl's Prussian blue colored ferrocyanide complex. [29] In this assay Fe 3+ -Fe 2+ transformation in the presence of the extracts was investigated. The reducing power of the extracts may serve as a significant indicator of its potential antioxidant activity. [30] In this assay, the yellow color of the test solution changes to various shades green and blue colors of ferrous ion, depending on the reducing power of test specimen. The presence of reductones, which have been shown to be an impart antioxidant action by breaking the free radical chain by donating a hydrogen atom. The presence of antioxidants in the sample extracts might cause the reduction of Fe 3+ /Ferric cyanide complex to ferrous form which can be monitored by spectrophotometric ally at 700 nm. [31] The trends of reducing potential of different solvent extracts of Measa lanceolata was presented in (Figure 2), the greater the intensity of the blue color, the greater was the absorption; consequently, it has strong antioxidant properties. Similar to DPPH scavenging activity the methanol extract of Measa lanceolata in this assay also, showed the strongest iron reducing power activity. At the concentration of 1 mg/mL, the reducing power of Measa lanceolata leaf extracts decreased in the order of methanol > water > ethyl acetate and chloroform with values of 1.5 ± 0.7 nm, 1.4 ± 0.5 nm, 0.7 ± 0.3 nm, and 0.6 ± 0.3 nm, respectively. The EC 50 values of ferric reducing powers of Measa lanceolata were shown in Table 2. The EC 50 values of water, ethyl acetate, and chloroform extracts were found to be significantly higher (weaker DPPH scavenging) (p < .05) than the IC 50 value of ascorbic acid, while that of the methanol extract was found to be similar (p > .05) to the IC 50 value of ascorbic acid.
Total antioxidant activity using phosphomolybdenum assay: The phosphomolybdenum method is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and the formation of green phosphate/Mo (V) complex with absorption at 695 nm. [32] The total antioxidant capacity of different solvent extracts of Measa lanceolata was also evaluated by the phosphomolybdenum method and expressed as ascorbic acid equivalents (AAE) per gram of dried extract. The total antioxidant activity of Measa lanceolata leaf extract ( Figure 3) For methanol, ethyl acetate, water, chloroform; at 1 mg/mL were found to be (128 ± 4 mg AAE/g), (99 ± 7 mg AAE/g), (57 ± 7 mg AAE/g), and (40.0 ± 3.2 mg AAE/ g) respectively. At 1 mg/mL, the total antioxidant activity of methanol extract was significantly stronger (p < .05) than the total antioxidant activity of water, ethyl acetate and chloroform extracts. Similarly, at 0.5 mg/mL, the total antioxidant activity of methanol extract was the strongest (p < .05) whereas chloroform, and water extracts showed significantly the weakest activity (p < .05). This result is consistent with the strongest total antioxidant effect of methanol extract as determined by the DPPH assay and the weakest total antioxidant activity was found in the water extract. No significant difference (p > .05) was found in the total antioxidant activity of water and chloroform extracts (p > .05). However, these values were significantly lower (p < .05) than the total antioxidant activity of ethyl acetate and methanol extracts.

Antibacterial activity
Evaluation of antibacterial activity of these plant extracts was recorded in Tables 3 and 4. All of the examined extracts showed varied inhibitory activity against all strains, of the tested bacteria.
(Staphylococcus aureus (ATCC-25923) was the most sensitive against water and methanol extracts, but Escherichia coli (ATCC-25922), Pseudomonas aeruginosa (ATCC-43495) Staphylococcus aureus (ATCC-25923), and Proteus vulgaris (ATCC-13315) were the most resistance against chloroform and ethyl acetate. At 100 mg/mL, the highest average inhibition zone (12.8 ± 0.4 mm) was recorded in methanol extract against Proteus vulgaris (ATCC-13315). Whereas the lowest inhibition zone was recorded (6.0 ± 0.5 mm) in the same extract in Escherichia coli (ATCC-25922) strain. At the same concentration, the highest average inhibition zone (10.8 ± 0.1 mm) was recorded for water extract against Staphylococcus aureus (ATCC-25923). In general, water and methanol extracts of Measa lanceolata showed higher antibacterial activity, whereas ethyl acetate and chloroform extracts showed weaker antibacterial activity. The Methanol and aqueous extracts of the present study  showed higher inhibition zone than that of Measa lanceolata studied by Berhan et al [33] and Chemweno et al, [34] but showed lower inhibition zones than Measa lanceolata 80% methanol and acetone leaf extracts. [35,36] The minimum inhibitory concentration values of the Measa lanceolata leaf extract were summarized in Table 4. The MIC values for tested bacterial strains were in the range of 3.1 mg/mL and 12.5 mg/mL. The methanol and water extracts showed the strongest activity against Staphylococcus aureus (ATCC-25923) and Escherichia coli (ATCC-25922) with values of MIC 3.1 mg/mL. Furthermore, Enterococcus faecalis (ATCC-29212) appeared the most sensitive microorganisms tested for chloroform extract with MIC value of 3.1 mg/mL. But, (Staphylococcus aureus (ATCC-25923), Escherichia coli (ATCC-25922), Pseudomonas aeruginosa (ATCC-43495), and Proteus vulgaris (ATCC-13315) were resistant against chloroform and ethyl acetate extracts. Hence, these sensitivity differences between Gram-positive and Gram-negative bacteria to the extract of different extracts might be due to the structural and compositional differences in membranes between the two groups. [37] Indeed, Gram-negative bacteria are more resistant to antibiotics because they possess impermeable outer membrane; consequently, the levels of antibiotics in the cell are reduced. [38,39]

Conclusion
The results obtained showed the in-vitro studies of methanol extract of Measa lanceolata showed a stronger antioxidant activity, which is directly related to the total phenolic and flavonoid contents. Methanol and water extracts showed strongest antimicrobial activity as compared to the chloroform and ethyl acetate extracts. While Staphylococcus aureus (ATCC-25923), Escherichia coli (ATCC-25922), Pseudomonas aeruginosa (ATCC-43495), and Proteus vulgaris (ATCC-13315) showed resistant against chloroform and ethyl acetate extracts. Generally, methanol, water, ethyl acetate, and chloroform extracts exhibited the highest antibacterial activity against the gram-positive bacterial strains. The study revealed that Measa lanceolata contain considerable amount of phenolic compounds and has significant antioxidant and antimicrobial activities, which can be used as easily accessible source of natural antioxidants and in pharmaceutical applications. Further studies are necessary on the isolation and characterization of individual compounds to elucidate their different antioxidant and antimicrobial mechanisms. The isolated compounds need to be evaluated in scientific manner using scientific animal models and clinical mechanisms of action in search of bioactive molecules.