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The accumulating healthcare problem of antibiotic resistance and the toxic side effects of synthetic antioxidants have necessitated scientists to discover for more potent and nontoxic alternative of antibacterial and antioxidant agents from dietary plants (Cowan 1999). Several plant-derived compounds or phytochemicals especially from the phenolic group have been reported to share both activities (Basile et al. 2000). An antibacterial agent that also possesses antioxidant activity might provide a better healing efficacy as it is not only inhibiting the germination and/or growth of bacteria, but also reducing the side effect of infection by counteracting and scavenging oxidative stress/pathogenesis-generated free radicals. In addition, a plant extract with this mutual effect could also be a suitable candidate of natural food preservative as it is not only extending shelf life of food product by delaying food contamination and but also retarding the oxidation process of lipid.
The abundance of medicinal plants used to treat bacteria-related diseases and oxidative stress-mediated diseases in the tropical regions has provided ample opportunity for local scientists to explore and develop effective and safe therapeutic interventions against these diseases. The ethnobotanical data approach, one of the numerous approaches used in drug discovery, employs plant selection based on previous information on the folk medicinal usage of the plant. Ethnobotanical data may substantially increase the chances of determining a bioactive compound relative to random approaches (Shelley 2009). Ludwigia octovalvis (Jacq.) P. H. Raven (synonym: Jussiaea suffruticosa L.) was selected for this study because of the lack of scientific evaluation verifying its ethnobotanical uses. This aquatic weedy plant is widely distributed throughout the tropical and sub-tropical regions and belongs to the Onagraceae family (Ipor 2002).
A poultice made of an entire plant is well documented to treat various microbial related diseases and is externally applied to heal skin disorders such as dermatitis, boil, ulcer, impetigo and pimple. Its decoction is drunk as a health drink and to treat gastrointestinal complaints such as diarrhea and flatulence (Chang et al. 2004; Ipor 2002; Burkill 1966). Thus far, a study by Shyur et al. (2005) is the only antioxidant evaluation on L. octovalvis. Among the 26 tested extracts of medicinal plants in Taiwan, its methanol extract has demonstrated the strongest free radical scavenging activity. Moreover, the antibacterial screenings of whole plant extracts of L. octovalvis are limited only to the promising anti-Streptococcus mutans activity of its aqueous extract (Chen et al. 1989), low activity of its 95% ethanol extract against Helicobacter pylori (Wang and Huang 2005), and notable activity of its 95% ethanol extract against several dermatological bacteria (Nanda et al. 2008). The antioxidant and antibacterial activities of different parts of L. octovalvis extracts using lower polarity of solvents have yet to be reported.
Numerous reputed antibacterial and antioxidative phenolic compounds, such as luteolin, quercetin, apigenin, and gallic acid, have been isolated from the plant (Yan and Yang 2005). However, no correlation between total phenolic content (TPC) and antioxidant and antibacterial activities of the extracts has been reported. Therefore, this study was carried out (i) to determine the effect of different polarities of solvent systems and plant parts on the quantitative analyses of TPC, as well as the antioxidant and antibacterial activities of the extracts, and (ii) to correlate the TPC in each extract with its antioxidant and antibacterial activities.
Materials and methods
The whole plant of Ludwigia octovalvis was harvested at its mature stage from the wet area of the Universiti Sains Malaysia main campus, Pulau Pinang. A voucher specimen number (11090) was deposited at the herbarium of the School of Biological Sciences, Universiti Sains Malaysia.
Chemicals and reagents
n-Hexane, chloroform, ethyl acetate, and methanol were purchased from Fisher Scientific (Springfield, NJ). Folin-Ciocalteau's (FC) reagent, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid (98% purity), 2,4,6-tris (2-pyridyl)-1,3,5-triazine (TPTZ), ferric chloride hexahydrate, sodium acetate, acetic acid glacial, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and p-iodonitrotetrazolium chloride (INT) were obtained from Sigma-Aldrich Chemical (St. Louis, MO). Dimethylsulphoxide (DMSO) was purchased from QRec (Germany), nutrient broth and nutrient agar from Oxoid (England), and sodium carbonate anhydrous from Bendosen Laboratory Chemicals (England).
The leaves, stems, and roots of the plant were separated and washed. The samples were then dried at 40 oC and ground into powder. Each powdered plant part (30 g) was sequentially extracted (cold extraction) in a flask with 200 mL of n-hexane by continuous shaking for 8 h. The extract was filtered using a filter paper of 150 mm diameter (Whatman, U.K.). The residue was then dried and successively extracted using chloroform, ethyl acetate, and 80% (v/v) methanol. Each extract was concentrated using a rotary evaporator and stored at -4 oC until further use.
Determination of total phenolic content
The total phenolic content (TPC) in all the extracts was estimated by a colorimetric assay based on the procedure described by Slinkard and Singleton (1977), with slight modifications. The TPC was calculated from the calibration curve using gallic acid as a standard. The results are expressed as milligram of gallic acid equivalents per gram dry weight of extract (mg GAE/g d.w.). DMSO was used to dilute the extract to obtain an initial concentration of 1 mg/mL. Briefly, 0.5 mL of each extract was pipetted in a test tube followed by 1.0 mL of 10% dilution of FC reagent. The contents of the test tube were thoroughly mixed. After 3 min, 3 mL of sodium carbonate (1% w/v) was added. The mixture was kept in the dark for 2 h at 25 oC. The absorbance was measured at 760 nm using a spectrophotometer (Hitachi Model U-1900) with DMSO as blank. The procedure was repeated using different concentrations of standard gallic acid solutions (0.05-0.2 mg/mL). Experiments were carried out in triplicate and the mean value was recorded. The TPC was calculated as gallic acid equivalent (GAE) using the following equation: Y (absorbance) = 8.982 X (µg gallic acid) - 0.01412, r2 = 0.9925 that was obtained from the standard gallic acid graph. The absorbance value was inserted in the abovementioned equation and the total amount of phenolic compound was calculated.
Bacterial strains and growth media
Antibacterial activity was evaluated using the following strains of bacteria, Gram-positive bacteria: Bacillus cereus (ATCC 10876), Bacillus licheniformis (ATCC 12759), Bacillus spizizenii (ATCC 6633), Staphylococcus aureus (ATCC 12600), Staphylococcus epidermidis (ATCC 12228) and Streptococcus mutans (ATCC 25175), Gram-negative bacteria: Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), Pseudomonas stutzeri (ATCC 17588), and Shigella boydii (ATCC 9207). All bacteria strains were cultured on nutrient agar slant at 37 oC for 18 h. The stock cultures were maintained on nutrient slants at 4 oC.
Disc diffusion method
The agar disc diffusion method was used to determine the antibacterial activities of all the extracts (NCCLS 1999). Stock solutions of the extracts were prepared by dissolving 20 mg of each extract in 1 mL DMSO. The stock solutions with a standardized initial concentration of 20 mg/mL were then filtered using 0.2 μm filters. Bacterial inoculums were obtained from the bacterial cultures incubated for 24 h at 37 oC on nutrient agar, and diluted with a sterile physiologic saline solution [0.85% (w/v) sodium chloride] to approximately 108 colony forming units per mL according to the 0.5 McFarland standards. Briefly, 100 µL of suspension of the tested bacteria was spread on the nutrient agar plates (9 cm in diameter) using a sterile swab. Sterile filter paper discs of 6 mm diameter (Whatman 6 mm AA disc, U.K.) were impregnated with 20 µL of extract stock solution to a final concentration 400 µg/mL. For each plate, sterile distilled water and DMSO were used as negative controls. Chloramphenicol and doxycycline were used as positive controls. The discs were placed on the inoculated plates. These plates were incubated overnight at 37 oC, and the diameters of the inhibition zones were measured. All the tests were performed in triplicate.
Broth micro-dilution method
A broth micro-dilution bioassay in 96-well plates was used to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) (Eloff 1998; NCCLS 1999). Stock solutions of the 80% methanol extracts were prepared in 10% DMSO, and filtered using 0.2 μm filters. Serial dilutions of the extracts were made to obtain different concentrations, ranging from 1000-7.80 µg/mL. The bacterial inoculums were prepared using 24-hour cultures, and the suspensions were adjusted to the 0.5 McFarland standard turbidity. The 96-well plates were prepared by dispensing 95 µL of nutrient broth (NB), 5 µL of the bacterial inoculum, and 100 µL of extract (dissolved in DMSO) into each well. The final volume in each well was 200 µL. The growth control (containing 5 µL inoculum and 195 µL NB) and the negative control (containing 100 µL of extract dissolved in DMSO and 100 µL NB without inoculum) were included on each microplate. The plates were covered and incubated overnight at 37 oC. As an indicator of bacterial growth, 40 µL of 0.2 mg/mL INT was added to each well, and the plates were incubated at 37 oC for at least 30 min. Bacterial growth in the wells was indicated by a red-pink color, whereas, clear wells indicated growth inhibition by the tested extracts. The MIC of each extract is defined as the lowest concentration showing clear wells. Each plate included three replicates of each extract at each concentration with two plates per replicate. Average MIC values were obtained.
To determine MBC, broth showing no growth in the MIC assay was sub-cultured on freshly prepared nutrient agar plates. After 24 h of incubation at 37oC, the minimum bactericidal concentration was determined as the lowest concentration of extract that did not allow any bacterial growth on the surface of the nutrient agar plate used.
Determination of antioxidant activity
2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay
The free radical scavenging activity of all the extracts were measured using DPPH scavenging activity as described by Brand-Williams et al. (1995), with slight modifications. Briefly, 150 µL of 300 µM ethanolic DPPH solution was added to 50 µL of 1 mg/mL extracts (diluted in DMSO) in 96-microwell plates. DMSO was used as negative control. The reaction mixture was incubated in the dark at 37 oC for 30 min. The decrease in absorbance value was then measured at 515 nm using a microplate reader (Thermo, Multiskan Ex, Finland). All measurements were carried out in triplicate.
Ferric reducing antioxidant power (FRAP) assay
Reducing power was determined using a FRAP assay as described by Firuzi et al. (2005), with slight modifications. The FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 µM TPTZ solution in 40 mM HCl and 20 mM ferric chloride hexahydrate at a proportion of 10:1:1 (v:v). The FRAP reagent was freshly prepared before analysis and warmed to 37 oC prior to use. The extracts were dissolved in DMSO at a concentration of 1 mg/mL. Extracts (20 µL) were allowed to react with 180 µL of FRAP solution for 5 min in the dark. The absorbance of the reaction mixture was then measured at 593 nm using a microplate reader (Thermo, Multiskan Ex, Finland). All measurements were carried out in triplicate.
Determination of FRAP and DPPH inhibition values
Trolox was used as reference in both assays. Two different standard curves were obtained using Trolox standard solution (in DMSO) at various concentrations. The absorbance of the reaction sample was compared to that of the Trolox standard. The results are expressed in terms of microMolar of Trolox equivalents per milligram dry weight of extract (µM TE/mg d.w.).
All data are reported as the mean ± S.E. of triplicate determinations. The statistical analysis of the data was carried out using one-way ANOVA, followed by Tukey's honestly significant difference (HSD) using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL) at a confidence level higher than 95% (p < 0.05). Pearson's correlation test was conducted to determine the correlation between antioxidant and antibacterial activities and TPC. The test was carried out using the Prism 3.02 statistical software of GraphPadPrism (San Diego, CA).
Results and discussion
Total phenolic content (TPC)
Studies on antioxidant and antimicrobial activities of plant phenolics have been well documented in most literature reviews (Pereira et al. 2007; Almajano et al. 2008; Boussaada et al. 2008; Oliveira et al. 2008; Audipudi and Chakicherla 2010). In this study, three different parts of L. octovalvis were separately and successively extracted by increasing the polarity of solvents (from less polar to polar). The solvents used were n-hexane, followed by chloroform, ethyl acetate, and 80% methanol. The extract obtained using each solvent was quantified for its TPC. As indicated in Table 1, 80% methanol extracts of the leaf and stem showed the highest amount of TPC (264.76 ± 0.23 GAE mg/g d.w. and 239.05 ± 0.29 GAE mg/g d.w., respectively), with significant differences (p < 0.05). The ethyl acetate extract of the root followed with a value of 98.88 ± 0.19 GAE mg/g d.w. A variation in the TPC of L. octovalvis extracts was observed, with the highest content in the most polar extract of the leaf. Ethyl acetate is considered the most efficient solvent for extracting phenolic compounds from the root. However, because of the differences in morphological and anatomical characteristics of the different parts of a plant, different extraction solvent systems may be required to ensure optimum recovery of TPC (Naczk and Shahidi 2006).
The antibacterial activities of the extracts (at a final concentration of 400 µg/mL) were initially estimated by measuring the diameter of the inhibition zone around the discs, followed by the determination of MIC and MBC values. The diameter inhibition zones of bacteria inhibited by the plant extracts and two positive controls, chloramphenicol and doxycycline, are presented in Table 2.
Generally, most of the leaf extracts used in this study inhibited the Gram-positive and Gram-negative bacteria, but only the 80% methanol extracts of the stem and the root generated such finding. Poor antibacterial activity of the n-hexane, chloroform, and ethyl acetate extracts was observed. All of the 80% methanol extracts exhibited a broad spectrum of activity against the bacterial strains used in this study. This finding agrees with the result obtained by Nada et al. (2008), who discovered that the successive extraction of polar extracts (95% ethanol) using Soxhlet apparatus of L. octavalvis exhibited significantly higher antibacterial activity against dermatological bacteria than did the non-polar extracts (petroleum ether, hexane and chloroform). The largest zones of inhibitions were indicated by the 80% methanol extract of the leaf against Staphylococcus epidermidis, Pseudomonas stutzeri, Streptococcus mutans, Echerichia coli and Bacillus spizizenii with values of 17.8 ± 1.2 mm, 15.7 ± 1.1 mm, 15.3 ± 1.6 mm, 14.8 ± 0.8 mm, and 14.0 ± 0.8 mm, respectively. This finding is interesting because conventional antibiotics are often more active against Gram-positive than Gram-negative bacteria. Meanwhile, Staphylococcus aureus and Shigella boydii were found to be resistant against the 80% methanol extract of the leaf with inhibition zones of 7.80 ± 1.70 mm and 8.00 ± 0.80 mm, respectively. The lowest inhibition zone of the 80% methanol extracts of the leaf against S. aureus in this study conform with the reported resistance of this bacteria towards the methanolic extract of Ludwigia adscendens studied by Ahmed et al. (2005).
Moreover, Klebsiella pneumoniae was susceptible to only the 80% methanol extract of the root with a diameter inhibition zone of 8.8 ± 0.5 mm. P. aeruginosa, S. aureus, and S. boydii were also more sensitive to the 80% methanol extract of the root in comparison to other extracts. DMSO did not show an inhibitory effect on any of the bacteria tested. The results also showed that the inhibition zones of the 80% methanol extract of the leaf against S. epidermidis and P. stutzeri were significantly higher (p < 0.05) than that of the positive control, doxycycline. The diameter inhibition zone (15.70 ± 1.05 mm) of P. stutzeri inhibited by the same extract was also larger than that of another positive control, chloramphenicol (12.9 ± 1.5 mm).
Correlation between TPC and inhibitory effect
Different correlations between TPC and diameter inhibition zones of 11 bacteria used in this study were observed (with r values ranging from 0.23-0.85; Table 2). The weak correlation might be associated with the absence of an inhibitory effect of the ethyl acetate extract of the root with higher TPC than its 80% methanol extract. This finding suggests the minor contribution of TPC to the antibacterial activity of the 80% methanol extract of the root. The highest correlation (r = 0.85) was observed between TPC and the diameter inhibition zone of Bacillus licheniformis. The synergistic effect of phenolic compounds in the 80% methanol extracts of the leaf and stem can be considered a main contributor to this antibacterial activity. Research conducted on various isolated phenolic compounds from plant extracts has proved effective bactericidal activities at minimal inhibitory concentrations (Basile et al. 2000). Plant phenolics were found to exert their bactericidal activities by disrupting the membrane fluidity, which results in the efflux of K + ions, a major cytoplasmic action of growing bacterial cells, involved in several key functions of bacterial cells. Potassium leakage is an early indicator of membrane damage (Ultee et al. 1999). By contrast, the lowest correlation (r = 0.23) were determined between the TPC and the diameter inhibition zone of S. boydii. The non-involvement of phenolic compounds in inhibiting this bacterium is suggested. Therefore, the results revealed that the antibacterial activity of phenolic compounds may be influenced by their amounts and types, as well as the species and strains of tested bacteria.
Minimum inhibitory concentration (MIC)
As shown in Table 3, the lowest MIC and MBC of the extracts were 62.50 µg/mL and 125.00 µg/mL, respectively, exhibited by the 80% methanol extract of the leaf against B. spizizenii and E. coli, and the 80% methanol extract of the root against P. aeruginosa. The consistent MIC of the three 80% methanol extracts used in this study against S. mutans (125.00 µg/mL) was much lower than that of the aqueous extract of the entire plant (MIC > 2000 µg/mL) tested by Chen et al. (1989). This finding indicates the higher efficiency of the 80% methanol in extracting anti-S. mutans compounds from different parts of L. octovalvis than water. Although, none of the extracts demonstrated a comparable MIC and MBC values with the positive controls, these findings are of great importance, particularly the higher sensitivity of Gram negative bacteria such as E. coli and P. aeruginosa, which are human pathogens. Comparing the diameter of inhibition zones, MIC and MBC values obtained from this study with that of Aliyu et al. (2008), separate analysis of three different parts of L. octavalvis exhibited higher activity against S. aureus, E. coli and K. pneumoniae. The results obtained in this study indicate that variations in the antioxidant and antibacterial activities of the extracts are dependent upon both the parts of L. octovalvis and the type of extracting solvents. These results agree with the suggestion of Oloke and Kolawole (1998) that bioactive components of different species and parts of plants have different solubility levels in different extracting solvents.
The extracts were also examined for their antioxidant activities using DPPH and FRAP assays. Despite representing different mechanism of actions, these assays have generated a similar finding. The results obtained from both assays are expressed as µM TE/mg d.w. (Table 1), a more meaningful and descriptive expression compared with expressing antioxidant activity as percentage of activity at a specific concentration. Results may provide a direct comparison of the antioxidant activity with that of Trolox (Jaitak et al. 2010). Table 1 shows significant differences (p < 0.05) among the 12 extracts. The 80% methanolic extract of the leaf exhibited the highest antioxidant capacity with DPPH and FRAP values of 1080.84 ± 6.07 µM TE/mg d.w. and 1256.88 ± 5.38 µM TE/mg d.w., respectively. These values were followed by the 80% methanol extract of the stem with DPPH and FRAP values of 905.00 ± 7.37 µM TE/mg d.w. and 912.17 ± 5.06 µM TE/mg d.w., respectively, with significant difference (p < 0.05) from other extracts. The results obtained may assist in providing new scientific evidence of the antioxidant potential of the methanol extract of the entire plant of L. octovalvis, as previously highlighted by Shyur et al. (2005). Confirmation is indicated by the following findings: (i) The leaf has more TPC and a higher antioxidant activity than do the stem and root; (ii) The plant should be extracted using polar solvents to enhance antioxidant activity.
The antioxidant results are complemented by the TPC results as the 80% methanol extracts of the leaf and the stem demonstrated better activity than those of ethyl acetate, chloroform, and n-hexane extracts. Likewise, among the root extracts, ethyl acetate extract showed the highest activity in both assays. Moreover, all n-hexane extracts showed lower DPPH values than did other extracts and the lowest DPPH and FRAP values were respectively detected in n-hexane extracts of the leaf and the stem.
Correlation between TPC and antioxidant activity
Accumulated evidence suggests good correlation between the antioxidant activity of plant extracts and TPC (Ferreira et al. 2007; Yesil-Celiktas et al. 2007). The correlation between TPC and antioxidant activities of the extracts was tested using Pearson's correlation test. The TPC values (mg GAE/g d.w.) of all the extracts were plotted separately against those of DPPH and FRAP. Figures 1 and 2 show significant linear correlations. High correlation coefficients were observed between TPC and DPPH values (r = 0.9926, p < 0.0001), and between TPC and FRAP values (r = 0.9814, p < 0.0001). Hence, TPC played a significant role in altering antioxidant activity. These results also tallied with those of Paixão et al. (2007) and Kubola and Siriamornpun (2008), who also found a significant correlation between TPC and antioxidant activities, evaluated by three different analytical methods.
The results revealed that the extracting solvents and plant parts affect the TPC, antibacterial, and antioxidant activities of the extracts. Despite a moderate to weak correlations between TPC and antibacterial activities, strong correlation between TPC and antioxidant activities were observed. Our findings also revealed the greater effect of the polar extract of the leaf compared with that of the stem and root. The 80% methanol extract of the leaf of L. octovalvis can be potentially used as antibacterial and antioxidant agents. Since this plant is widely distributed in the tropical region, the leaves might be utilized as a cost-effective raw material to produce natural preservative for food industry or as food supplement for human health care.