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Membrane-Active Antibacterial Compounds in Methanolic Extracts of Jatropha curcas and their Mode of Action against Staphylococcus aureus S1434 and Escherichia coli E216.

Byline: Namuli Aidah Norhani Abdullah Ehsan Oskoueian Chin Chin Sieo and Wan Zuhainis Saad

Abstract

This research presents the antibacterial potential and mode of action of related active compounds of kernel meal leaves stem bark root bark and root wood extracts of Jatropha curcas Linn. plant on Staphylococcus aureus S1434 and Escherichia coli E216. At double MIC (minimum inhibitory concentration) value cell viability of S. aureus S1434 was inhibited by all extracts but only kernel meal and root wood extracts inhibited E. coli E216. At half MIC the 24 (decrease in cell viability after 24h) for S. aureus S1434 was 69 and 66% while that of E. coli E216 were 44 and 42% in the presence of kernel meal and leaves extract respectively. However at double MIC less than 5% of viable cells of S. aureus S1434 were detected in the leaves and root bark extracts after 5h. Conversely less than 5% of the viable cells of E. coli E216 were detected in the presence of kernel meal and root wood extract after 7.5h. Loss of 260nm absorbing compounds and proteins from bacterial cells was directly proportional to the time of exposure of cells to the extracts. All extracts caused bacterial cells to lose their ability to tolerate salt (NaCl) at double MIC value. The loss of 260nm absorbing compounds proteins and the loss of tolerance to NaCl suggest that leaves root bark and kernel meal damaged the bacterial cell membrane. The analysis of bioactive compounds by GC-MS confirmed the presence of acetic acid hexadecanoic acid citric acid 9-octadecenoic acid as the major membrane-active antibacterial compounds.Copyright 2014 Friends Science Publishers.

Keywords: Antibacterial mechanism; Minimum inhibitory concentration; Membrane damage; XTT assay.

Introduction

Jatropha curcas Linn. (J. curcas) belongs to the family of Euphorbiaceae and it is a drought resistant shrub which is widely grown in Central and South America South-east Asia India and Africa. It has gained importance in Malaysia as a source of seed oil for biofuel production. In many African and Asian countries J. curcas plant has been initially considered a traditional herb to cure various ailments ranging from simple fevers to infectious diseases including sexually transmitted diseases (Pandey et al.2012).The leaves have been used against cough and as an antiseptic after birth (Debnath and Bisen 2008). The strong antimicrobial activities of the branches render it suitable as a chewing stick in Nigeria (Kayode and Omotoyinbo 2009). The kernel and the oil are used as purgative and to treat syphilis (Thomas et al. 2008). The ethnomedical practice in West African showed the application of leaves in differentforms to cure various ailments like fever mouth infections jaundice guinea worm sores and joint rheumatism (Thomas et al. 2008; Aiyelaagbe et al. 2011). The roots of J. curcas have been used after decoction as a mouthwash for bleeding gums toothache eczema ringworm and scabies and to cure dysentery (Carels 2009; Aiyelaagbe et al. 2011).Recently Viswanathan et al. (2012) reported the stigmasterol AY-amyrin friedelin and R (+) 4-hydroxy-2- pyrrolidinone as antimicrobial compounds present in the methanolic extract obtained from the leaf of Jatropha tanjorensis. Similarly another study counducted by Oskoueian et al. (2011) reported the phenolics flavonoidstogether with saponins and phorbol esters as antibacterial compounds detected in the methanolic extract of Jatropha curcas kernel. These information and evidences support the fact that this plant possesses antibacterial activity.Despite of the reports indicating the antibacterial potential of Jatropha curcas plant information on the mechanism of antibacterial action of extract and related bioactive compounds are still lacking. Hence this study was conducted to determine how methanolic extracts of different parts of J. curcas Linn. plant affect pathogenic bacterial species like Staphylococcus aureus S1434 and Escherichia coli E216 and what are the active antibacterial compounds present in the extracts.

Materials and Methods

Collection of Plant Materials

Ripe Jatropha curcas Linn. seeds were obtained from the Malaysian Agricultural Research and Development Institute (MARDI) whereas the whole plant was freshly collected from Universiti Putra Malaysia farm (GPS location of30'26.91"N latitude and 10142'13.24"E longitude). Avoucher specimen (SK1764/2010) was deposited in the Phytomedicinal Herbarium Institute of Bioscience Universiti Putra Malaysia Serdang Selangor Malaysia.

Preparation of Extracts

The leaves stem bark root bark root wood and kernel seeds were separated manually. The materials were cleaned with sterile distilled water air dried and finely ground using a grinder mill. Ground kernels were defatted in a Soxhlet apparatus using petroleum ether (boiling point of 40-60C) for 16 h at 40C (AOAC 1990). The kernel meal residue was dried in the oven at 50C to remove the solvent. Thirty two grams of each sample were placed in 800 mL of methanol and refluxed at 50C for 60 min (Chen et al.2007). The extracts were filtered through Whatman filter paper No. 1 and were then evaporated to dryness using a rotary evaporator (Buchi) at 40C. The residues obtained were dissolved in 1% dimethyl sulfoxide (DMSO). Different concentrations (half one and double MIC) ofmethanolic extracts of various plant parts used were derivedfrom minimum inhibition concentration (MIC) values previously determined by broth microdilution (Namuli et al. 2011). The MIC values for the leaves stem bark root bark root wood and kernel against S. aureus were 3.130.39 0.78 1.37 and 1.56 mg/mL respectively. The MIC values for the leaves stem bark root bark root wood and kernel against E. coli E216 were 0.78 3.13 1.95 14.06 and12.50 mg/mL respectively.

Bacterial Species

The clinical isolates S. aureus S1434 and E. coli E216 were obtained from the Institute for Medical Research Malaysia.

Preparation of Inocula

A 24 h bacterial culture was centrifuged at 12000A-g for10min at 4C and the pellet harvested was washed twice using phosphate buffer solution (PBS) at pH 7.0. After washing the pellet was dissolved in PBS and the optical density (OD) of the suspension was adjusted to 0.8 at 600nm using a spectrophotometer (Ultrospec(R) 2100 pro).

Cell Viability

Cell viability was assessed using calorimetric method based on 23-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H- tetrazolium-5-carboxanilide (XTT) reduction assay (Moriarty et al. 2005). The experiment was performed in sterile McCartney bottles containing 2 mL inoculum and extract (at half one and double MIC values) and were filled with broth to a final volume of 20mL. Bacterial growth without plant extract and blank without inoculum were used as controls. The bottles were incubated in a water bath shaker at 37C and 120 rpm. One millilitre of each sample was removed at 0 2.5 5 7.5 and 24 h and processed for the XTT reduction assay.A solution of 0.5 mg/mL XTT was prepared using PBS. The solution was filter sterilized using 0.2 m filter and stored at -70C. Prior to each assay an aliquot of XTT was thawed and menadione (10 mM prepared in acetone) was added to a final concentration of 50 M. Each 200 L sample in a vial was centrifuged at 8000xg for 5 min and thesupernatant was removed. Two hundred micro-litre of XTTwas added to the pellets. The samples were vortexed and poured into a 96 microtitre plate which was then incubated in the dark for 2h at 37C to allow XTT reaction with the cells. The OD resulting from the change in XTT colour (yellow to orange) was measured at 492nm using a microtiter plate reader (Mindray). The final OD was obtained by subtracting the OD values of the blank. The experiment was carried out in triplicates and the decrease in cell viability after 24 h was calculated as follows: equation

Salt Tolerance

Preliminary tests were carried out to determine the salt tolerance of S. aureus S1434 and E. coli E216 (Sampathkumar et al. 2003) because different bacterial species tolerate different salt concentrations some are halo- tolerant while others are not. The concentrations of NaCl at75 and 20 g L-1 were then selected for S. aureus S1434 and E. coli E216 respectively. The bacterial cells were treated with methanolic extracts and were then tested for theirability to tolerate salt. Suspensions of bacteria were treatedwith different plant extracts in broth at half one and double MIC values for 1 h (Carson et al. 2002). The cells from the broth were collected by centrifugation (12000A-g) and washed once with PBS. The cells were re-suspended in 1.0 mL of PBS and 200 L of this suspension was added to (i)2.0 mL of PBS and (ii) 2.0mL of PBS containing NaCl at the concentrations of 75 or 20 g L-1. After mixing the OD680 was measured using a spectrophotometer (Ultrospec(R) 2100 pro). The experiment was carried out in triplicates. The tolerance to NaCl was indicated by the reduction in OD680 which was calculated by subtracting the mean value of three measurements in PBS from the mean value of three measurements in PBS containing 75 or 20 g L-1 of NaCl for the respective test organisms. In each case the change in OD obtained was expressed as a percentage of the mean value obtained with PBS alone (Sampathkumar et al.2003).

Cellular Leakage

Leakage of cytoplasmic contents was determined by usingUltrospec(R) 2100 pro spectrophotometer (Carson et al.2002; Sampathkumar et al. 2003). The OD of the cell filtrates after exposure of the bacterial cells to the crude extracts was measured at 260nm (for nucleic acids and aromatic amino acids) and 595nm (for proteins). A portion of the extract was added to fresh sterile water in a 20 mL bottle in an amount which would achieve concentration of half one and double MIC values after addition of 2 mL of the inoculum. Bacterial growth without plant extract and blank without inoculum were used as controls. Then 4 mL of each test sample were removed immediately after addition of inoculum at 30 and 60 min of incubation. The samples were filtered through a 0.2 m membrane filter into a sterile test tube. The absorbance at 260nm (OD260) was measured for nucleic acids placed in quartz cuvettes. The mean OD260 was expressed as proportion of the initial OD260. The presence of DNA in the cell free filtrates obtained after60min was assessed by running 1.0 mL aliquots of the phenol-chloroform-isoamyl alcohol (25:24:1)(v/v) concentrated supernatants on a 0.8% agarose gel. The remaining filterate was used to quantify the amount of proteins by Bradford assay (Bradford 1976). A hundred micro-litres of each filterate and 5 mL of Bradford reagent was added to a sterile test tube. The mixture was vortexed and incubated for 5 min at room temperature and absorbance measured at 595nm. The standard curve of bovine serum albumin was used to as a reference. The experiment was carried out in triplicates.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

The method of Hossain and Rahman (2011) was used to characterize the chemical composition of extracts using a GC-MS. In this study Schimadzu QP2010PLUS system was used. Six micro-liter of each extract were injected and analysed on a BPX-5 SGE ultra-low-bleed 5% phenyl polydimethylsiloxane capillary column (30m A- 0.25mm i.d.A- 0.25m film thickness). The purge time of 1.0min withsplitless injection. The helium gas was used as carrier at a flow rate of 1mL min-1. The temperature of the column wasadjusted at 50C for 3min followed by increasing 5C min-1 upto to 80C and then at 10C min-1 upto 340C. The temperature of inlet and detector was 250C and 340Crespectively and the solvent delay was 4 min. The peakswere identified based on computer matching of the massspectra with the National Institute of Standards and Technology (NIST 08 and NIST 08s) library and according to the published data.

Gas Chromatography (GC) Analysis

The concentration of acetic acid present in the extracts of different parts was determined by Gas Chromatography (Shimadzu GC-14A). The 20 mM 4methyl-n-valeric acid was used as an internal standard. A volume of 0.5mL(each extract) was added to 0.5 mL of the internal standard. The column used was packed with 10% (w/v) PEG 600 on Shimalate TPA 60/80. The temperature of oven wasmaintained at 160C the FID at 230C and the injector port at 230C. The carrier gas was nitrogen (20 mL min-1). One microlitre of sample was injected into the column. The experiment was carried out in triplicates.

Statistical Analysis

General linear models (GLM) procedure of SAS in a completely randomized design (CRD) was used to analyse the data and the means were compared with Duncan's Multiple Range test. The differences were considered significant when the p value was less than 0.05.

Results

Cell Viability

Fig. 1 shows the effect of different J. curcas plant part extracts at half MIC on the cell viability of S. aureus 1434 and E. coli E216. In both species the 24 was significantly (Pless than 0.05) reduced when compared to the control. However more reduction in 24 was observed with S. aureus 1434 compared to that of E. coli E216. The 24 for S. aureus S1434 was 69 and 66% in the presence of kernel meal and leaves extract respectively whereas 24 for E. coli E216 was 44 and 42% in the presence of kernel meal and leaves extract respectively.As observed in Fig. 2 different J. curcas plant part extracts at MIC value showed varying effects on the cell viability of S. aureus 1434 and E. coli E216. With S. aureus S1434 each extract except the root wood showed a decrease in the number of viable cells between 0 and 5h. The viable cells of E. coli E216 significantly decreased (Pless than 0.05) between 0 and 7.5h in the presence of root wood and kernel meal extracts. However no decrease in cell viability was observed in the presence of leaves stem bark and root bark extracts.The effect of different J. curcas plant part extracts at double MIC on the cell viability of S. aureus 1434 and E. coli E216 is shown in Fig. 3. With S. aureus S1434 less than 5% of viable cells were detected in the leaves and root bark extracts after 5h but after 24 h in the presence of stembark and kernel meal extract. Conversely less than 5% of the viable cells of E. coli E216 were detected in the presence of kernel meal and root wood extract after 7.5 h whereas increase in cell viability was observed even after 24 h in the presence of stem bark root bark and leaves extract.

Salt Tolerance

Fig. 4 shows the salt tolerance of S. aureus S1434 and E. coli E216 cells in PBS containing 75 or 20g/L NaCl

Table 1: Absorbance at 260nm of cell-free filtrates of S. aureus S1434 and E. coli E216 cells exposed to different

concentrations of various plant part extracts

Concentration Bacterial species###Proportion of initial OD260 (after 30min)###Proportion of initial OD260 (after 60min)

(mg/mL)###L###SB###RB###RW###KM###C###L###SB###RB###RW###KM###C

Half MIC###S. aureus S1434###1.05a###1.05a###1.11a###1.03a###1.10a###1.03a###1.16ab###1.09b###1.13b###1.12b###1.28a 1.06b

###a###a###a###a###a###a###bc###c###b###c###a

###E. coli E216###1.06###1.04###1.09###1.02###1.09###1.01###1.11###1.04###1.24###1.03###1.40###1.01c

###b###b###a###b###a###b###ab###b###a###b###a

MIC###S. aureus S1434###1.07###1.06###1.15###1.04###1.17###1.03###1.18###1.09###1.33###1.16###1.32###1.06b

###E. coli E216###1.09ba###1.04b###1.11ba###1.06b###1.18a###1.01c###1.13c###1.04cd###1.22b###1.10c###1.44a 1.01c

Double MIC S. aureus S1434###1.08b###1.09b###1.22a###1.10b###1.23a###1.03b###1.19b###1.14bc###1.42b###1.17bc###1.41a 1.06c

###b###bc###b###bc###a###c###a###c###a###b###a

###E. coli E216###1.10###1.04###1.11###1.06###1.21###1.01###1.50###1.13###1.50###1.25###1.53###1.01c

respectively following 1h of treatment with different extracts at half MIC value. Both species showed significant differences (Pless than 0.05) in the percent OD except the root wood extract (S. aureus S1434) and stem bark (E. coli E216) which showed no significant difference (Pless than 0.05) compared to the control. The bacterial cell membrane of S. aureus S1434 and E. coli E216 cells were more damaged by root bark and root wood extracts respectively.The salt tolerance of S. aureus S1434 and E. coliE216 cells in PBS containing 75 or 20 g L-1 NaClrespectively following 1h of treatment with different extracts at MIC value is shown in Fig. 5. Both species showed significant differences (Pless than 0.05) in the percent OD. The bacterial cell membrane of S. aureus S1434 was more damaged by kernel meal extract and least affected by the root wood extract whereas E. coli E216 cells were more damaged by kernel meal and root wood extracts.Fig. 6 shows the salt tolerance of S. aureus S1434 andE. coli E216 cells in PBS containing 75 or 20 g L-1 NaCl

Table 2: Proteins concentrations in cell-free filtrates of S. aureus S1434 cells exposed to different concentrations of

different plant part extracts

Concentration Bacterial species###Protein concentration (gmL-1) (after 30min)###Protein concentration (gml-1) (after 60min)

(mg/mL)###L###SB###RB###RW###KM###C###L###SB###RB###RW###KM###C

Half MIC###S. aureus S1434###148.15ab 148.15ab 174.07ab 131.48b 216.67a 0 338.89a 246.30b 329.63a 318.52a 337.04a 0

###E. coli E216###150.00bc 120.37c 157.41b 114.82c 168.52a 0 227.78b 192.59c 331.48a 155.56d 340.74a 0

MIC###S. aureus S1434###174.08b 161.11b 325.93b 148.15b 335.19a 0 342.59b 318.52b 485.18a 331.48b 488.89a 0

###E. coli E216###175.93b 124.07b 162.96b###142.59 194.44 0 340.74a 198.15b 361.11a 270.37ab 355.56a 0

Double MIC S. aureus S1434###172.22c 181.48c 298.15b 244.45bc 457.41a 0 514.82b 477.78b 657.41a 496.29b 694.45a 0

###E. coli E216###335.19ab 162.96c 290.74b 166.67c 372.22a 0 437.04a 429.63a 433.33a 412.96a 440.74a 0

respectively following 1 h of treatment with different extracts at double MIC value. The leaves stem bark and root bark extracts showed no significant difference (Pless than 0.05) against S. aureus S1434 whereas stem bark and root bark extracts showed a significant difference (Pless than 0.05) against E. coli E216. Cell membranes of both S. aureus S1434 and E. coli E216 cells were most affected by the kernel meal extract.

Cellular Leakage

Table 1 shows the OD260 after 30 and 60min of treatmentof S. aureus S1434 and E. coli E216 cells with different concentrations of various plant part extracts expressed as a proportion of the initial OD260. After 60 min the OD260 of filtrates of cells exposed to double MIC value (of different plant part extracts) was significantly (Pless than 0.05) higher than that for control values. Generally for both species after 30 and 60 min all extracts (at half MIC value) except kernel meal showed slight or no significant (Pless than 0.05) difference when compared to the control. Table 2 shows the amount of proteins present in the cell free filtrates after 30 and 60min of treatment of S. aureus S1434 and E. coli E216 cells with different concentrations of different plant part extracts. For both species no protein was detected in cell filtrates of thecontrol. Generally the results showed that increasing the time of exposure and the concentrations of plant part extracts had significant (Pless than 0.05) effect on cell leakage. However more proteins were released from S. aureus S1434 cells compared to E. coli E216 cells by all extracts at various times except the leaves extract (after 30 min).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

The main organic compounds detected by GC-MS in the methanolic extracts are shown in Table 3. Oxalic and acetic acids were found to be the major compounds detected in the leaves stem bark root bark and root wood extracts. In addition the stem bark also contained citric acid. The fatty acids which were identified in all the extracts except root bark were hexadecanoic acid (palmitic acid) and 9- octadecenoic acid (oleic acid) in the kernel meal.

GC Analysis

The concentration of acetic acid in the different part of the plant was analysed using GC and leaves stem bark root bark root wood and kernel meal showed the values of 7.356.11 5.22 12.91 2.74 mg acetic acid g-1 dry weight respectively (Table 4).

Table 3: The GC-MS analysis of methanolic extract of various parts of J. curcas plant

Plant part###Main compoundsa###Area %###

Leaves###Oxalic acid###43.2

###Acetic acid###31.5

###Hexadecanoic acid###25.3

Stem bark###Oxalic acid###28.2

###Acetic acid###25.6

###Citric acid###20.12

###Hexadecanoic acid###18.7

Root bark###Oxalic acid###79.3

###Acetic acid###26.4

Root wood###Acetic acid###55.3

###Oxalic acid###19.8

###Hexadecanoic acid###17.4

Kernel meal###9-octadecenoic acid###37.8

###Hexadecanoic acid###21.2

###Oxalic acid###18.9

###Acetic acid###15.7

Table 4: The acetic acid content of different plant parts analysed by GC

###Leaves###Stem Root###Root###Kernel

###bark bark###wood###meal

Concentration (mg g-1 DW) 7.35###6.11 5.22###12.91###2.74

Discussion

As regards cell viability the E.coli cells were less affected compared to those of S. aureus S1434 because E. coli species possess effective permeability barriers comprising of the outer membrane which restrict penetration of antimicrobial compounds and also possess efflux systems that extrude the antibacterial agents out of the cell (Tenover2006). Both bacterial species showed bacteriostatic activity when extracts were used at half or MIC and showed bactericidal activity when double MIC value was used.The results of the salt tolerance test demonstrated that S. aureus species could better tolerate higher NaCl concentration (75 g L-1) than that of E. coli (20 g L-1). In contrast the halophilic bacteria such as S. aureus can withstand hyper osmotic conditions by increasing the solutes inside their cells like glycine betaine ectoine proline glutamate and trehalose (Ikeuchi et al. 2003). Infact the outer membrane determines the bacteria's abilityto tolerate any particular change in a set of ionicconditions (Brown and Turner 1963) thus loss of tolerance to salts or other potentially toxic compounds may reveal membrane damage or weakening (Iandolo and Ordal 1966). In the present study it is proposed that the extracts caused membrane damage during the one hour of incubation by increasing the membrane permeability and this caused the decrease in absorbance since plasmolysis did not occur when the cells were placed in PBS containing NaCl. It has been reported that in a plasmolysed cell protoplast the scattering of light increases and this is what eventually results in increase in OD (Korber et al. 1996). In the current study the absorbance (OD680) decreased (interpreted as loss in tolerance to salt) with increase in the concentration of different plant part extracts.A marked leakage of cellular material indicates irreversible damage to the cytoplasmic membrane (Hugo and Longworth 1964). Antibacterial agents such as polymyxins which is an antibiotic (Tenover 2006) and Psidium guajava plant extract (Henie et al. 2009) have been reported to disrupt the membrane by increasing bacterial membrane permeability and causing leakage of bacterial contents. The data obtained in the present study suggested that nucleic acids and amino acids leaked out of the bacterial cells. However the agarose gel electrophoresis experiments did not show any DNA being present in the cell free filtrates. This could be attributed to the fact that the amount of DNA that leaked out of the cell was too little to be detected by agarose gel electrophoresis as it relies on visual detection and the compounds detected at 260nm were not DNA but RNA and other aromatic amino acids that absorblight at 260nm (Cleaves and Miller 1998). The suggestions were confirmed when the compounds that absorb at 260nmwhich had leaked out of the S. aureus MF 31 cell afterheating were RNA (Iandolo and Ordal 1966).The bactericidal or bacteriostatic effect observed with different concentrations of various extracts against the tested microorganisms could be attributed to the presence of organic and fatty acids detected in the extracts as shown in Table 3 and 4. The interaction of these hydrocarbons with the hydrophobic structures of bacteria has been reported to result in antimicrobial activity (Sikkema et al. 1995; Cowan 1999; Vaquero et al. 2007). The antibacterial activity of 9-octadecanoic acid and hexadecanoic acid against S. aureus and E. coli has been reported by Pu et al. (2010). Similarly acetic and hexadecanoic acids as the main antibacterial compounds have been reported in the aqueous extract of pine needles (Feng et al. 2010). The result of Koga et al. (1996) indicated that organic acids have a higher bactericidal effect than fatty acids. According to Ryssel et al. (2009) acetic acid possesses excellent bactericidal effects toward Proteus vulgaris Pseudomonas aeruginosa and Acinetobacter baumannii.Consequently this study revealed that different J. curcas plant extracts especially the root bark leaves andkernel meal methanolic extracts induce antibacterial actionthrough cell membrane damage and oxalic acid acetic acid hexadecanoic acid citric acid and 9-octadecenoic acid are found to be the active-membrane antibacterial compounds.

Acknowledgments

The grant provided by the Ministry of Higher Education of Malaysia under the Fundamental Research Grant Scheme (Project No. 01-11-08-660FR) is acknowledged.

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