The plant ( Xylocarpus mekongensis) of the Sundarbans mangrove origin was evaluated for its antibacterial, cytotoxic and antioxidant properties using methanolic and chloroformic leaf, stem and bark extracts, respectively. The methanolic extracts contained higher amount of total phenolics, flavonoids, tannins than the chloroformic extracts and the result was in correlation with their ferric reducing power ability as well. However, the chloroformic bark extract contained more potent DPPH free radical scavenging activity than others. Antibacterial activity of the extracts was determined against both Gram-positive ( Micrococcus and Staphylococcus aureus) and Gram-negative bacteria ( Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhimurium and Salmonella paratyphi) by disc diffusion assay and their zone of inhibitions (ZOI) were measured. Moreover, their minimum inhibitory concentrations (MIC) were determined by tube dilution method. Chloroformic bark and stem extracts showed strong inhibition to growth of P. aeruginosa (ZOI = 19 mm and MIC = 150 μg/ml) and S. aureus (ZOI = 19.5 mm and MIC = 250 μg/ml), respectively. All six extracts were subjected to brine shrimp lethality bioassay for possible measure of cytotoxicity. Concentration dependent increment in percentage mortality of brine Shrimp nauplii produced by the extracts indicated the presence of cytotoxic principles in these extractives. Therefore, Xylocarpus mekongensis showed antioxidant, antibacterial and cytotoxic activities.
The mangroves are a promising source of natural products as several bioactive compounds have been isolated [
Xylocarpus mekongenesis (X. mekongenesis) (Lamk.) L.Mohan (Meliaceae) is commonly known as Passur. It is a glabrous, medium-sized tree that grows generally on the inter-tidal silty but consolidated clay or on the sandy or rocky bay distributed throughout the tropical and subtropical regions of Southeast Asia including the Sundarbans [
The genus Xylocarpus consists of Xylocarpus granatum, X. moluccensis, X. mekongensis, X. rumphii that are ethnomedicinally important mangrove plants. Extracts of leaves, barks, pneumatophore and fruits of these plants have been reported for various ethnomedicinal uses such as fever, malaria, inflammation, dysentery, diarrhoea, cholera, abdominal problems, diabetes, elephantiasis, antimicrobials etc. In recent times, these plants are also reported for their antioxidant, anticancer, antidiabetic, antidyslipidemia, antimicrobials, antidiarrhoeal, antifilarial, antiulcer and cardiotonic properties [
The nature of the bioactivity analysis is permutative due to the uses of various plant parts and employing different solvent systems to extract the bioactive natural products. Therefore there remains the scope to analyze the bioactivity of X. mekongensis. Most of the previous studies on X. mekongensis were carried out with a single solvent system for extraction of plant materials while the present study employed both polar and non-polar solvent systems for sequential extraction of plant materials from three types of plant parts. The objective of this study was to evaluate the pharmacological basis of biological activity on X. mekongensis through antioxidant, antimicrobial and cytotoxic assay of chloroformic and methanolic leaf, stem and bark extracts, respectively.
Folin-Ciocalteu (FC) reagent, aluminium chloride, sodium hydroxide, sodium carbonate, and sodium nitrite were purchased from Merck Specialities Private Limited, Mumbai, India. Ferric Chloride, Ethanol, Methanol, Chloroform were bought from Merck KGaA, Germany. Potassium ferricyanide was purchased from UNI-CHEM chemical reagents, China. Gallic acid, 2, 2-diphenyl-1-picryl hydrazyl (DPPH) and Quercetin were brought from Sigma-Aldrich Chemicals Pvt. Ltd. (Germany). Nutrient agar was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India) and the rest of the chemicals and solvents used were of analytical grade.
The plant sample of X. mekongensis was collected from Dhangmaree, Chadpai range, East zone of the Sundarbans, Khulna, Bangladesh on 16th December, 2011 and collected plant samples were sent to Bangladesh National Herbarium, Dhaka, Bangladesh for taxonomical identification. A voucher specimen (Accession No.: DACB-35372) was also deposited.
The plant materials (bark, stem and leaf) were separated from each other and then cleaned by gentle washing with distilled water followed by air drying for several weeks. The dried material was ground into coarse powder with a motorized plant grinder (capacitor start motor, Wuhu Motor Factory, China). The powder was kept in a dry, cool and dark place in a suitable airtight container until analysis commenced. The dried material was ground into coarse powder with a motorized plant grinder (capacitor start motor, Wuhu Motor Factory, China). The powder was kept in a dry, cool and dark place in a suitable airtight container until analysis commenced. About 120 gm of powdered leaf, 160 gm of powdered stem and bark each was soaked into 440 mL and 500 ml petroleum ether respectively, in three clean, flat-bottomed glass containers for a period of 5 days with occasional stirring and shaking. It was then filtered and after this first filtration, the remaining residues (approx. 115 gm powdered leaf, 157 gm powdered stem and 155 gm bark) were soaked into 400, 470 and 450 ml chloroform respectively, kept for a period of 6 days and then filtered; then final remaining residues (113 gm of powdered leaf, 155 gm of powdered stem and 150 gm of powdered bark) were soaked into 390, 450 and 450 ml methanol respectively, kept for a period of 6 days with occasional stirring and shaking and then filtered. Coarse plant material was separated from the mixture by pouring through a clean cloth filter. These extracts were passed through filter paper, and the filtrates were evaporated, yielding the chloroformic and methanolic extracts, res- pectively.
The free radical scavenging property of extracts were analyzed by 1, 2-diphenyl 1-picryl hydrazyl (DPPH) assay developed by Brand-Williams et al., [
The reducing power of the extract was evaluated according to the method of Oyaizu (1986) [
Total phenolic content of the extracts was determined by using Folin-Ciocalteau assay [
Total flavonoid content of the extracts was determined by using an aluminium chloride colorometric assay [
Total tannins content in plant extract was determined by using Folin-Denis method as described by Polshet- tiwar et al. 2007 [
Seven strains of microorganisms were tested in this study. Two Gram-positive bacteria include Micrococcus spp., S. aereus (ATCC 25923) and five Gram-negative bacteria include E. coli (ATCC 8739), P. aeruginosa (ATCC 27833), S. typhimurum (ATCC 13311), V. cholerae and S. paratyphi. These strains were collected from the Microbiology Laboratory, Khulna University, Bangladesh, as pure cultures were used. The bacterial isolates were cultivated in nutrient broth at 37˚C for 24 hours.
Antibacterial activity of X. mekongensis extracts was tested by disc diffusion method [
The extracts that showed antimicrobial activity in disc diffusion were later tested to determine the MIC value for each bacterial sample by the tube dilution method from [
Brine shrimp lethality bioassay was carried out according to [
The results were expressed as means ± standard deviation (SD). P values < 0.05 were considered as the level of significance. The correlation statistical analysis was performed for observing the correlation among different type of assay data of antioxidant activity. Regression analysis was conducted for analyzing the data obtained from brine shrimp lethality bioassay to study the relationship between different samples and vincristine sulphate. The statistical analysis was carried out using GraphPad Prism Version 6.01 (GraphPad Software, Inc., USA).
The antioxidant capacity expressed as IC50 value, %increase in reducing power, total phenolic content (mgGAE/g), total flavonoid content (mg QE/g), total tannin content (mgGAE/g)of all extracts are shown in
Type of plant extracts | DPPH IC50 (μg/ml) | % Increasing range of reducing power (range) | TPC (mg GAE/g) | TFC (mg QE/g) | TTC (mg GAE/g) |
---|---|---|---|---|---|
CLE | 492.66 | 54.7 - 161.9 | 16.5 ± 0.002 | 42 ± 0.004 | 0.6 ± 0.006 |
CSE | 96.281 | 34.23 - 347.65 | 31 ± 0.003 | 78 ± 0.003 | 9.2 ± 0.011 |
CBE | 25.94 | 92.11 - 444.8 | 27.5 ± 0.006 | 76 ± 0.007 | 40.2 ± 0.010 |
MLE | 113.25 | 13.93 - 89.77 | 26 ± 0.002 | 77 ± 0.004 | 4.6 ± 0.011 |
MSE | 50.71 | 82.72 - 767.79 | 82.5 ± 0.002 | 185 ± 0.007 | 28.6 ± 0.009 |
MBE | 35.842 | 154.36 - 1230.03 | 137.5 ± 0.009 | 200 ± 0.006 | 73 ± 0.009 |
Standard (Quercetin) | 7.65 | 191.69 - 334.55 | X | X | X |
N.B: CLE = Chloroformic leaf extract, MLE = Methanolic leaf extract, CSE = Chloroformic stem extract, MSE = Methanolic stem extract, CBE = Chloroformic bark extract, MBE = Methanolic bark extract, TPC = Total phenolic content, TFC = Total flavonoids content, TTC = Total tannin content, GAE = Gallic acid equivalent, QE = Quercetin equivalent, IC50 = 50% Inhibition concentration.
formic bark and stem extracts of have shown to exhibit better percent (%) increase in reducing power than quercetin. However, initially the percentage of increase in reducing power of all the extracts with minute concentration were relatively lower that the standard trend. Along with the increase of the concentration gradient, methanolic bark extract (MBE) and methanolic leaf extract (MLE) as well as chloroformic bark extract (CBE) supersede and the chloroformic stem extract (CSE) reaches the range of the standard’s trend at their highest concentration. Chloroformic leaf extract (CLE) and MLE remain steady and lower than the standard’s capacity regardless the intensiveness of the concentration.
Phenolic compounds exhibit antioxidant activity by inactivating lipid free radicals or preventing decomposition of hydroperoxides into free radicals. Total phenolic content of the different extracts of X. mekongensis was solvent dependent and expressed as mg GAE/g (
The content of flavonoids was expressed as QE/g of extracts while MBE (200 mg QE/g) contained the highest level of TFC among all extracts, and the order of the flavonoid richness resembles in the order of MBE > MSE > CSE > MLE > CBE > CLE. Total Tannin content of the extracts was shown in
The antibacterial activities of extracts were evaluated by the diameters of the inhibition zone around the disc and MIC; these diameters and MIC values are reported in
CBE and CSE were strongly active against P. aeruginosa (ZOI = 19 mm and MIC = 150 µg/ml), S. aureus (ZOI = 19.5 mm and MIC = 250 µg/ml), respectively with high zone of inhibition and the lowest MIC values. MSE was also strongly active against S. aureus (ZOI = 15.75 mm and MIC = 300 µg/ml) with high zone of inhibition and the lowest MIC value. However, MBE showed no activity against E. coli and S. typhimurum. CLE and MSE also possessed no activity against S. paratyphi and E. coli respectively. Analyzing the results of the antimicrobial activity of X. mekongensis extract, it was found that chloroformic extracts exhibited higher antimicrobial activity compared to methanolic extracts. Some of the phytochemical compounds e.g. glycoside, saponin, tannin, flavonoids, terpenoid, alkaloids, have previously been reported to have antimicrobial activity [
Compared parameters for correlation analysis | Level of Significance at P < 0.05 | R2 |
---|---|---|
Phenol vs. Flavonoid | ** | 0.8998 |
Phenol vs. Tannin | * | 0.7257 |
Flavonoid vs. Tannin | * | 0.5814 |
In the brine shrimp lethality bioassay, the result of cytotoxic potential of extracts in terms of percent (%) mortality of brine shrimps is presented in
Name of the bacteria | Assay Parameters | CLE | MLE | CSE | MSE | CBE | MBE | Ery |
---|---|---|---|---|---|---|---|---|
E. coli | MIC (µg/ml) | 600 | 1000 | 400 | NA | 400 | NA | nd |
ZOI (mm) | 8.25 ± 0.5 | 7.5 ± 0.577 | 10.25 ± 0.5 | - | 10.75 ± 0.5 | - | 15.5 | |
P. aeruginosa | MIC (µg/ml) | 1000 | >1000 | 350 | >1000 | 150 | 250 | nd |
ZOI (mm) | 7.75 ± 0.5 | 7.25 ± 0.5 | 15 ± 0.816 | 6.25 ± 0.5 | 19 ± 0.816 | 14.5 ± 0.577 | 24 | |
V. cholerae | MIC (µg/ml) | 350 | 350 | 350 | 350 | 340 | 320 | nd |
ZOI (mm) | 12.25 ± 0.5 | 12.5 ± 0.577 | 14 ± 0.816 | 11.75 ± 0.5 | 13 ± 0.816 | 13 ± 0.816 | 29 | |
S. typhi | MIC(µg/ml) | 340 | 600 | 350 | 300 | 380 | NA | nd |
ZOI (mm) | 12.5 ± 0.577 | 8.25 ± 0.5 | 14.75 ± 0.957 | 14.5 ± 0.577 | 11 ± 0.816 | - | 25 | |
S. paratyphi | MIC (µg/ml) | NA | 600 | 400 | 350 | 370 | 370 | nd |
ZOI (mm) | - | 8.75 ± 0.5 | 11.5 ± 0.577 | 12.25 ± 0.5 | 12.75 ± 0.957 | 10.25 ± 0.5 | 21 | |
Micrococcus | MIC (µg/ml) | 400 | 470 | 300 | 350 | 430 | 370 | nd |
ZOI (mm) | 10 ± 0.816 | 9.75 ± 0.5 | 9.75 ± 0.5 | 11.25 ± 0.957 | 9.25 ± 0.5 | 10.75 ± 0.5 | 24 | |
S. aureus | MIC(µg/ml) | 400 | >1000 | 250 | 300 | 200 | 450 | nd |
ZOI (mm) | 11 ± 0.816 | 7.5 ± 0.577 | 19.5 ± 0.577 | 15.75 ± 0.957 | 14.75 ± 0.5 | 9.25 ± 0.5 | 23 |
N.B.: MIC = Minimal inhibitory concentration (µg/ml), ZOI = Zone of inhibition (mm) at 500 µg/disc, Ery = Erythromycin, NA = Not ACTIVE, nd = Not determined, (-) = No zone of inhibition.
Types of extracts | LC50 (µg/ml) | Regression equation | R2 |
---|---|---|---|
CBE | 49.29 | y = 47.16x − 29.83 | 0.982 |
MBE | 465.08 | y = 23.13x − 11.70 | 0.975 |
CLE | 117.13 | y = 32.92x − 18.10 | 0.984 |
MLE | 298.79 | y = 26.99x − 16.81 | 0.973 |
CSE | 42.65 | y = 51.31x − 33.63 | 0.99 |
MSE | 549.48 | y = 21.35x − 8.498 | 0.987 |
VS | 26.68 | y = 52.79x − 25.29 | 0.972 |
N.B.: CLE = Chloroformic leaf extract, MLE = Methanolic leaf extract, CSE = Chloroformic stem extract, MSE = Methanolic stem extract CBE = Chlorofornic bark extract, MBE = Methanolic bark extract, VS = Vincristine sulphate.
Percent mortality of CBE and CSE showed comparatively nearer value to the standard, which indicated that these extracts could be potential candidates for determination of antitumor and anticancer properties. The crude extracts resulting in LC50 values less than 250 µg/ml are considered significantly active [
Based on the results of this experimental study, it can be suggested that the extracts of X. mekongensis possess antioxidant, antibacterial, and cytotoxic properties. Further investigation is necessary to fractionate the extract, to identify the bioactive compounds and should be evaluated in vitro and in vivo studies for the biological activity. Such bioactive compounds have the potential to be developed into medicine, nutraceuticals and agrochemicals and last but not least, cosmetics.
The authors would like to acknowledge the financial assistance obtained from British Council, Bangladesh through INSPIRE project for conducting this research.
Mahmuda Akter,Sadia Afrin,Sk. Nazmus Sakib,Rana Biswas,Md. Morsaline Billah,Mohammad Shahedur Rahman,Umme Salma Zohora, (2016) Investigation of Antibacterial, Cytotoxic and Antioxidant Properties of the Mangrove Plant Xylocarpus mekongensis. Advances in Bioscience and Biotechnology,07,205-213. doi: 10.4236/abb.2016.74019