Twelve infusions of Ludwigia octovalvis were examined to find their entire phenolic content ( TPC ) , every bit good as antioxidant and antibacterial activities. The highest TPC and antioxidant activities ( evaluated by 2, 2-diphenyl-1-picrylhydrazyl and ferrous cut downing antioxidant power checks ) were detected in 80 % methanol infusion of the foliage ; these are 264.76 ± 0.23 GAE mg/g d.w. , 1080.84±6.07 µM TE/mg d.w. , and 1256.88±5.38 µM TE/mg d.w. A strong correlativity between the TPC and antioxidant activities of both checks was observed ( R & A ; gt ; 0.98 ) . The largest zone of suppression ( 17.8±1.2 millimeter ) was obtained against Staphylococcus epidermidis utilizing the same infusion. The lowest minimum inhibitory concentration ( 62.5 µg/mL ) and minimum bactericidal concentration ( 125.0 µg/mL ) were observed in 80 % methanol infusion of the foliage against Bacillus spizizenii and Escherichia coli, and in 80 % methanol infusion of the root against Pseudomonas aeruginosa. Differences in correlativities between TPC and diameter suppression zones were dependent on bacterium species. TPC yielded the highest rectification to the suppression of Bacillus licheniformis ( r=0.848 ) .
Keywords: Entire phenolic content ; antioxidant ; antibacterial ; Ludwigia octovalvis.
The development of drug opposition of human pathogens against commercial antibiotics and the toxic side effects of man-made antioxidants have necessitated a hunt for new antibacterial and antioxidant agents from workss ( Cowan, 1999 ) . Based on the considerable array of constructions and activities of medicative workss, farther probe into their reputed bioactivities is required. Several plant-derived compounds including phenoplasts, terpenoids, indispensable oils, alkaloids, lectin, polypeptides, and polyacetylenes possess effectual antimicrobic belongingss against a broad scope of antibiotic-resistant micro-organisms ( Al-Fatimi et al. , 2007 ) .
The ethnobotanical informations attack, one of the legion attacks used in drug find, employs works choice based on old information on the common people medicative use of the works. Ethnobotanical information may well increase the opportunities of finding a bioactive compound relation to random attacks ( Shelley, 2009 ) . The copiousness of medicative workss used to handle bacteria-related diseases in the tropical parts has provided ample chance for local scientists to research and develop effectual curative intercessions against these diseases. Ludwigia octovalvis ( Jacq. ) P. H. Raven was selected for this survey because of the deficiency of scientific rating verifying its ethnobotanical utilizations. This aquatic weedy works is widely distributed throughout the tropical and sub-tropical parts and belongs to the Onagraceae household ( Burkill, 1966 ) .
A cataplasm made of an full works is externally applied to handle assorted microbic diseases, and its extract is internally consumed as a wellness drink ( Chang et al. , 2004 ; Burkill, 1966 ) . Therefore far, nevertheless, Shyur et Al. ( 2005 ) is the lone survey that has scientifically validated the antioxidant activity of the methanol infusion of L. octovalvis. Among the 26 tried infusions of medicative workss in Taiwan, this infusion has demonstrated the strongest free extremist scavenging activity. Furthermore, the antibacterial showings of whole works infusions of L. octovalvis are limited merely to the promising anti-Streptococcus mutans activity of its aqueous infusion ( Chen et al. , 1989 ) , low activity of its 95 % ethanol infusion against Helicobacter pylori ( Wang and Huang, 2005 ) , and noteworthy activity of its 95 % ethanol infusion against several dermatological bacteriums ( Nanda et al. , 2008 ) . The antioxidant and antibacterial activities of different parts of L. octovalvis infusions utilizing lower mutual opposition of dissolvers have yet to be reported.
Numerous reputed antibacterial and antioxidative phenoplast compounds, such as luteolin, quercetin, apigenin, and Gallic acid, have been isolated from the works ( Yan and Yang, 2005 ) . However, no correlativity between entire phenolic content ( TPC ) and antioxidant and antibacterial activities of the infusions has been reported. Therefore, this survey was carried out ( I ) to find the consequence of different mutual oppositions of solvent systems and works parts on the quantitative analyses of TPC, every bit good as the antioxidant and antibacterial activities of the infusions, and ( two ) to correlate the TPC in each infusion with its antioxidant and antibacterial activities.
2. Materials and methods
2.1. Plant stuff
The whole works of Ludwigia octovalvis was collected at adulthood from the wet country of the Universiti Sains Malaysia chief campus, Pulau Pinang. A voucher specimen figure ( 11090 ) was deposited at the herbarium of the School of Biological Sciences, Universiti Sains Malaysia.
2.2. Chemicals and reagents
n-Hexane, trichloromethane, ethyl ethanoate, and methyl alcohols were purchased from Fisher Scientific ( Springfield, NJ ) . Folin-Ciocalteau ‘s ( FC ) reagent, 2, 2-diphenyl-1-picrylhydrazyl ( DPPH ) , Gallic acid ( 98 % pureness ) , 2,4,6-tris ( 2-pyridyl ) -1,3,5-triazine ( TPTZ ) , ferrous chloride hexahydrate, Na ethanoate, 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 ) , alimentary stock and alimentary agar from Oxoid ( England ) , and Na carbonate anhydrous from Bendosen Laboratory Chemicals ( England ) .
2.3. Plant extraction
The foliages, stems, and roots of the works were separated and washed. The samples were so dried at 40 oC and land into pulverization. Each powdered works portion ( 30 g ) was consecutive extracted ( cold extraction ) in a flask with 200 milliliters of n-hexane by uninterrupted agitating for 8 h. The infusion was filtered utilizing a filter paper of 150 millimeters diameter ( Whatman, U.K. ) . The residue was so dried and in turn extracted utilizing trichloromethane, ethyl ethanoate, and 80 % ( v/v ) methyl alcohol. Each infusion was concentrated utilizing a rotary evaporator and stored at -4 oC until farther usage.
2.4. Determination of TPC
The TPC in all the infusions was estimated by a colorimetric check based on the process described by Slinkard and Singleton ( 1977 ) , with little alterations. The TPC was calculated from the standardization curve utilizing Gallic acid as a criterion. The consequences are expressed as mg of Gallic acid equivalents per gm dry weight of infusion ( mg GAE/g d.w. ) . DMSO was used to thin the infusion to obtain an initial concentration of 1 mg/mL. Briefly, 0.5 milliliter of each infusion was pipetted in a trial tubing followed by 1.0 milliliters of 10 % dilution of FC reagent. The contents of the trial tubing were exhaustively assorted. After 3 min, 3 milliliter of Na carbonate ( 1 % w/v ) was added. The mixture was kept in the dark for 2 H at 25 oC. The optical density was measured at 760 nanometers utilizing a spectrophotometer ( Model U-1900 Spectrophotometer Hitachi High Technology Corporation 2006 ) with DMSO as space. The process was repeated utilizing different concentrations of standard Gallic acid solutions ( 0.05-0.2 mg/mL ) . Experiments were carried out in triplicate and the average value was recorded. The TPC was calculated as Gallic acid equivalent ( GAE ) utilizing the undermentioned equation: Yttrium ( optical density ) = 8.982 Ten ( µg Gallic acid ) – 0.01412, r2 = 0.9925 that was obtained from the standard Gallic acid graph. The optical density value was inserted in the abovementioned equation and the entire sum of phenolic compound was calculated.
2.5. Determination of antioxidant activity
2.5.1. 2, 2-diphenyl-1-picrylhydrazyl ( DPPH ) assay
The free extremist scavenging activity of all the infusions were measured utilizing DPPH scavenging activity as described by Brand-Williams et Al. ( 1995 ) , with little alterations. Briefly, 150 µL of 300 µM ethanolic DPPH solution was added to 50 µL of 1 mg/mL infusions ( diluted in DMSO ) in 96-microwell home bases. DMSO was used as negative control. The reaction mixture was incubated in the dark at 37 oC for 30 min. The lessening in optical density value was so measured at 515 nanometers utilizing a microplate reader ( Thermo, Multiskan Ex, Finland ) . All measurings were carried out in triplicate.
2.5.2. Ferric cut downing antioxidant power ( FRAP ) assay
Reducing power was determined utilizing a FRAP check described by Firuzi et Al. ( 2005 ) , with little alterations. The FRAP reagent was prepared by blending 300 mM ethanoate buffer ( pH 3.6 ) , 10 µM TPTZ solution in 40 millimeter HCl and 20 millimeter ferrous chloride hexahydrate at a proportion of 10:1:1 ( V: V ) . The FRAP reagent was newly prepared before analysis and warmed to 37 oC prior to utilize. The infusions were dissolved in DMSO at a concentration of 1 mg/mL. Infusions ( 20 µL ) were allowed to respond with 180 µL of FRAP solution for 5 min in the dark. The optical density of the reaction mixture was so measured at 593 nanometers utilizing a microplate reader ( Thermo, Multiskan Ex, Finland ) . All measurings were carried out in triplicate.
2.5.3. Determination of FRAP and DPPH suppression values
Trolox was used as mention in both checks. Two different criterion curves were obtained utilizing Trolox standard solution ( in DMSO ) at assorted concentrations. The optical density of the reaction sample was compared to that of the Trolox criterion. The consequences are expressed in footings of microMolar of Trolox equivalents per mg dry weight of infusion ( µM TE/mg d.w. ) .
2.6. Antibacterial activity
2.6.1. Bacterial strains and growing media
Antibacterial activity was evaluated utilizing the undermentioned strains of bacteriums, Gram-positive bacteriums: Bacillus Cereuss ( 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 bacteriums: Escherichia coli ( ATCC 25922 ) , Klebsiella pneumoniae ( ATCC 13883 ) , Pseudomonas aeruginosa ( ATCC 27853 ) , Pseudomonas stutzeri ( ATCC 17588 ) , and Shigella boydii ( ATCC 9207 ) . All bacteriums strains were cultured on alimentary agar angle at 37 oC for 18 h. The stock civilizations were maintained on alimentary angles at 4 oC.
2.6.2. Disc diffusion method
The agar phonograph record diffusion method was used to find the antibacterial activities of all the infusions ( NCCLS, 1999 ) . Stock solutions of the infusions were prepared by fade outing 20 milligram of each infusion in 1 milliliter DMSO. The stock solutions with a standardised initial concentration of 20 mg/mL were so filtered utilizing 0.2 ?m filters. Bacterial inoculants were obtained from the bacterial civilizations incubated for 24 H at 37 oC on alimentary agar, and diluted with a unfertile physiologic saline solution [ 0.85 % ( w/v ) Na chloride ] to about 108 settlement organizing units per milliliter harmonizing to the 0.5 McFarland criterions. Briefly, 100 µL of suspension of the tried bacterium was spread on the alimentary agar home bases ( 9 centimeter in diameter ) utilizing a unfertile swab. Sterile filter paper phonograph record of 6 millimeters diameter ( Whatman 6 millimeter AA phonograph record, U.K. ) were impregnated with 20 µL of extract stock solution to a concluding concentration 400 µg/mL. For each home base, sterile distilled H2O and DMSO were used as negative controls. Chloramphenicol and Vibramycin were used as positive controls. The phonograph record were placed on the inoculated home bases. These home bases were incubated overnight at 37 oC, and the diameters of the suppression zones were measured. All the trials were performed in triplicate.
2.6.3. Broth micro-dilution method
A broth micro-dilution bio-assay in 96-well home bases was used to find minimum repressive concentration ( MIC ) and minimum bactericidal concentration ( MBC ) ( Eloff, 1998 ; NCCLS, 1999 ) . Stock solutions of the 80 % methyl alcohol infusions were prepared in 10 % DMSO, and filtered utilizing 0.2 ?m filters. Consecutive dilutions of the infusions were made to obtain different concentrations, runing from 1000-7.80 µg/mL. The bacterial inoculants were prepared utilizing 24-hour civilizations, and the suspensions were adjusted to the 0.5 McFarland standard turbidness. The 96-well home bases were prepared by adding 95 µL of alimentary stock ( NB ) , 5 µL of the bacterial inoculant, and 100 µL of infusion ( dissolved in DMSO ) into each well. The concluding volume in each well was 200 µL. The growing control ( incorporating 5 µL inoculant and 195 µL NB ) and the negative control ( incorporating 100 µL of infusion dissolved in DMSO and 100 µL NB without inoculant ) were included on each microplate. The home bases were covered and incubated overnight at 37 oC. As an index of bacterial growing, 40 µL of 0.2 mg/mL INT was added to each well, and the home bases were incubated at 37 oC for at least 30 min. Bacterial growing in the Wellss was indicated by a red-pink colour, whereas, clear Wellss indicated growing suppression by the tried infusions. The MIC of each infusion is defined as the lowest concentration demoing clear Wellss. Each home base included three replicates of each infusion at each concentration with two home bases per replicate. Average MIC values were obtained.
To find MBC, broth demoing no growing in the MIC check was sub-cultured on newly prepared alimentary agar home bases. After 24 H of incubation at 37 oC, the minimal disinfectant concentration was taken as the lowest concentration of infusion that did non let any bacterial growing on the surface of the alimentary agar home base used.
2.6.4. Statistical analysis
All informations are reported as the mean ± S.E. of three findings. The statistical analysis of the information was carried out utilizing one-way ANOVA, followed by Tukey ‘s candidly important difference ( HSD ) utilizing SPSS 16.0 for Windows ( SPSS Inc. , Chicago, IL ) at a assurance degree higher than 95 % ( p & A ; lt ; 0.05 ) . Pearson ‘s correlativity trial was conducted to find the correlativity between antioxidant and antibacterial activities and TPC. The trial was carried out utilizing the Prism 3.02 statistical package of GraphPadPrism ( San Diego, CA ) .
3. Consequences and treatment
Surveies on antioxidant and antimicrobic activities of works phenoplasts have been good documented in most literature reappraisals ( Pereira et al. , 2007 ; Almajano et al. , 2008 ; Boussaada et al. , 2008 ; Oliveira et al. , 2008 ; Audipudi and Chakicherla, 2010 ) . In this survey, three different parts of L. octovalvis were individually and in turn extracted by increasing the mutual opposition of dissolvers ( from less polar to polar ) . The dissolvers used were n-hexane, followed by trichloromethane, ethyl ethanoate, and 80 % methyl alcohol. The infusion obtained utilizing each dissolver was quantified for its TPC. As indicated in Table 1, 80 % methanol infusions of the foliage and root showed the highest sum of TPC ( 264.76 ± 0.23 GAE mg/g d.w. and 239.05 ± 0.29 GAE mg/g d.w. , severally ) , with important differences ( P & A ; lt ; 0.05 ) . The ethyl acetate infusion of the root followed with a value of 98.88 ± 0.19 GAE mg/g d.w. A fluctuation in the TPC of L. octovalvis infusions was observed, with the highest content in the most polar infusion of the foliage. Ethyl ethanoate is considered the most efficient dissolver for pull outing phenolic compounds from the root. However, because of the differences in morphological and anatomical features of the different parts of a works, different extraction dissolver systems may be required to guarantee optimal recovery of TPC ( Naczk and Shahidi, 2006 ) .
3.2. Antioxidant activity
The infusions were besides examined for their antioxidant activities utilizing DPPH and FRAP checks. Despite stand foring different mechanism of actions, these checks have generated a similar determination. The consequences obtained from both checks are expressed as µM TE/mg d.w. ( Table 1 ) , a more meaningful and descriptive look compared with showing antioxidant activity as per centum of activity at a specific concentration. Consequences may supply a direct comparing of the antioxidant activity with that of Trolox ( Jaitak et al. , 2010 ) . Table 1 shows important differences ( P & A ; lt ; 0.05 ) among the 12 infusions. The 80 % methanolic infusion of the foliage 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. , severally. These values were followed by the 80 % methanol infusion of the root 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. , severally, with important difference ( p & A ; lt ; 0.05 ) from other infusions. The consequences obtained may help in supplying new scientific grounds of the antioxidant potency of the methanol infusion of the full works of L. octovalvis, as antecedently highlighted by Shyur et Al. ( 2007 ) . Confirmation is indicated by the undermentioned findings: ( I ) The foliage has more TPC and a higher antioxidant activity than do the root and root ; ( two ) In obtaining the infusions, the usage of polar dissolvers enhanced antioxidant activity.
The antioxidant consequences are complemented by the TPC consequences as the 80 % methanol infusions of the foliage and the root demonstrated better activity than those of ethyl ethanoate, trichloromethane, and n-hexane infusions. Likewise, among the root extracts, ethyl ethanoate infusion showed the highest activity in both checks. Furthermore, all n-hexane infusions showed lower DPPH values than did other infusions, and the lowest DPPH and FRAP values were severally detected in n-hexane infusions of the foliage and the root.
3.3. Correlation between TPC and antioxidant activity
Accrued grounds suggests good correlativity between the antioxidant activity of works infusions and TPC ( Ferreira et al. , 2007 ; Yesil-Celiktas et al. , 2007 ) . The correlativity between TPC and antioxidant activities of the infusions was tested utilizing Pearson ‘s correlativity trial. The TPC values ( mg GAE/g d.w. ) of all the infusions were plotted individually against those of DPPH and FRAP. Figures 1 and 2 show important additive correlativities. High correlativity coefficients were observed between TPC and DPPH values ( r = 0.9926, P & A ; lt ; 0.0001 ) , and between TPC and FRAP values ( r = 0.9814, P & A ; lt ; 0.0001 ) . Hence, TPC played a important function in changing antioxidant activity. These consequences besides tallied with those of Paix & A ; atilde ; o et al. ( 2007 ) and Kubola and Siriamornpun ( 2008 ) , who besides found a important correlativity between TPC and antioxidant activities, evaluated by three different analytical methods.
3.4. Antibacterial activity
The antibacterial activities of the infusions ( at a concluding concentration of 400 µg/mL ) were ab initio estimated by mensurating the diameter of the suppression zone around the phonograph record, followed by the finding of MIC and MBC values. The diameter suppression zones of bacteriums inhibited by the works infusions and two positive controls, Chloromycetin and Vibramycin, are presented in Table 2.
By and large, most of the foliage infusions used in this survey inhibited the Gram-positive and Gram-negative bacteriums, but merely the 80 % methanol infusions of the root and the root generated such determination. Poor antibacterial activity of the n-hexane, trichloromethane, and ethyl ethanoate infusions was observed. All of the 80 % methyl alcohol extracts exhibited a wide spectrum of activity against the bacterial strains used in this survey. This determination agrees with the consequence obtained by Nada et Al. ( 2008 ) , who discovered that polar infusions ( utilizing 95 % ethyl alcohol ) of L. octavalvis exhibited significantly higher antibacterial activity against dermatological bacteriums than did the non-polar infusions. The largest zones of suppressions were indicated by the 80 % methanol infusion of the foliage against Staph. epidermidis, Ps. stutzeri, Strep. mutans, E. coli, and B. spizizenii with values of 17.8 ± 1.2 millimeter, 15.7 ± 1.1 millimeter, 15.3 ± 1.6 millimeter, 14.8 ± 0.8 millimeter, and 14.0 ± 0.8 millimeter, severally. Meanwhile, Staph. aureus and Sh. boydii were found to be immune against the 80 % methanol infusion of the foliage with suppression zones of 7.80 ± 1.70 millimeter and 8.00 ± 0.80 millimeter, severally. The lowest suppression zone of the 80 % methanol infusions of the foliage against Staph. aureus in this survey conform with the reported opposition of this bacterium toward the methanolic infusion of Ludwigia adscendens studied by Ahmed et Al. ( 2005 ) .
Furthermore, Kl. pneumoniae was susceptible to merely the 80 % methanol infusion of the root with a diameter suppression zone of 8.8 ± 0.5 millimeter. Ps. aeruginosa, Staph. Aureus, and Sh. boydii were besides more sensitive to the 80 % methanol infusion of the root in comparing to other infusions. DMSO did non demo an repressive consequence on any of the bacterium tested. The consequences besides showed that the suppression zones of the 80 % methanol infusion of the foliage against Staph. epidermidis and Ps. stutzeri were significantly higher ( p & A ; lt ; 0.05 ) than that of the positive control, Vibramycin. The diameter suppression zone ( 15.70 ± 1.05 millimeter ) of Ps. stutzeri inhibited by the same infusion was besides larger than that of another positive control, Chloromycetin ( 12.9 ± 1.5 millimeter ) .
Different correlativities between TPC and diameter suppression zones of 11 bacteriums used in this survey were observed ( with R values runing from -0.134-0.848 ; Table 2 ) . The weak correlativity might be associated with the absence of an repressive consequence of the ethyl acetate infusion of the root with higher TPC than its 80 % methanol infusion. This determination suggests the minor part of TPC to the antibacterial activity of the 80 % methanol infusion of the root. The highest correlativity ( r = 0.848 ) was observed between TPC and the diameter suppression zone of B. licheniformis. The interactive consequence of phenolic compounds in the 80 % methanol infusions of the foliage and root can be considered a chief subscriber to this antibacterial activity. Research conducted on assorted stray phenolic compounds from works infusions has proved effectual bactericidal activities at minimum repressive concentrations. Plant phenoplasts were found to exercise their bactericidal activities by interrupting the membrane fluidness, which consequences in the outflow of K + ions, a major cytoplasmatic action of turning bacterial cells, involved in several cardinal maps of bacterial cells. Potassium escape is an early index of membrane harm ( Ultee et al. , 1999 ) . By contrast, the lowest and reverse correlativities ( R = -0.134 ) were determined between the TPC and the diameter suppression zone of Kl. pneumoniae. The non-engagement of phenolic compounds in suppressing this bacterium is suggested. Therefore, the consequences revealed that the antibacterial activity of phenolic compounds may be influenced by their sums and types, every bit good as the species and strains of tried bacteriums.
As shown in Table 3, the lowest MIC and MBC of the infusions were 62.50 µg/mL and 125.00 µg/mL, severally, exhibited by the 80 % methanol infusion of the foliage against B. spizizenii and E. coli, and the 80 % methanol infusion of the root against Ps. aeruginosa. The consistent MIC of the three 80 % methyl alcohol infusions used in this survey against Strep. mutans ( 125.00 µg/mL ) was much lower than that of the aqueous infusion of the full works tested by Chen et Al. ( 1989 ) . This determination indicates the higher efficiency of the 80 % methyl alcohol in pull outing anti-Streptococcus mutans compounds from different parts of L. octovalvis than H2O. However, none of the infusions demonstrated a comparable MIC and MBC values with the positive controls. Comparing the diameter of suppression zones and MIC and MBC values obtained from the highest antibacterial infusion in this survey, L. octavalvis studied by Aliyu et Al. ( 2008 ) exhibited higher activity against Staph. aureus, E. coli and Kl. pneumoniae than its closely related species, L. suffruticosa. The consequences obtained in this survey indicate that fluctuations in the antioxidant and antibacterial activities of the infusions are dependent upon both the parts of L. octovalvis and the type of pull outing dissolvers. These consequences agree with the suggestion of Oloke and Kolawole ( 1998 ) that bioactive constituents of different species and parts of workss have different solubility degrees in different pull outing dissolvers.
The consequences reveal that the infusions from L. octovalvis can be potentially used as antioxidant and antibacterial agents. The pull outing dissolvers and works parts affect the TPC, antioxidant, and antibacterial activities of the infusions. Despite a strong correlativity between TPC and antioxidant activities, moderate to weak correlativities between TPC and antibacterial activities were observed. Our findings besides verified the greater medicative value of the polar infusion of the foliage compared with that of the root and root. The active constituents of this tried works against Gram-negative bacteriums are presently unknown and necessitate farther purification and elucidation of its construction.
This survey was supported by an inducement grant from the Universiti Sains Malaysia ( Grant figure: 1001/PBiologi/822151 ) .