eclalbasaponin_ 4 qtr

Mode of Antibacterial Activity of Eclalbasaponin Isolatedfrom Eclipta alba

A. Ray & P. Bharali & B. K. Konwar

Received: 17 September 2012 /Accepted: 20 August 2013 /Published online: 8 September 2013# Springer Science+Business Media New York 2013

Abstract The present study was undertaken to evaluate the mode of antibacterial activity ofEclalbasaponin isolated from Eclipta alba, against selected Gram-positive and Gram-negative bacteria. The probable chemical structure was determined by using various spec-troscopic techniques such as Fourier transform infrared spectroscopy (FTIR) and massspectroscopy. The antibacterial activity was evaluated by well diffusion technique, pHsensitivity, chemotaxis, and crystal violet assays. Eclalbasaponin showed clear zone ofinhibition against both Bacillus subtilis and Pseudomonas aeruginosa and exhibited growthinhibition at the pH range of 5.59.0. The isolated saponin exhibited its positivechemoattractant property for both bacterial strains. Results of crystal violet assay and thepresence of UV-sensitive materials in the cell-free supernatant confirmed the cellulardamages caused by the treatment of Eclalbasaponin. The release of intracellular proteinsdue to the membrane damage was determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis. Changes in the cell surface structure and membrane disruption were furtherrevealed by FTIR and scanning electron microscopy analysis. The present study suggeststhat the isolated saponin from E. alba causes the disruption of the bacterial cell membranewhich leads to the loss of bacterial cell viability.

Keywords Eclipta alba . Eclalbasaponin . Bacillus subtilis . Pseudomonas aeruginosa

Introduction

Currently, the emergence of antibiotic and multidrug-resistant bacteria becomes a serious globalthreat and an important concern for the hospitals, long-stay residential centers, and in thecommunity [1]. Appearance of such problems is due to the extensive use of antibiotics andselective pressure on the bacterial strains. They not only acquire resistance through mutation,but also through plasmid spreading via different strains. The increased use of various antibioticsand lack of new drugs, vaccines, and diagnostic aids made the current antimicrobial agentsinefficient to control various bacterial diseases [2]. The development of new medicines withdifferent chemical moieties and mode of action may be considered a relevant strategy [3].

Appl Biochem Biotechnol (2013) 171:20032019DOI 10.1007/s12010-013-0452-3

A. Ray (*) : P. Bharali : B. K. KonwarDepartment of Molecular Biology & Biotechnology, Tezpur University, Tezpur, Assam 784028, Indiae-mail: [email protected]

Medicinal plants represent as a valuable source for natural products and exploredcontinuously for therapeutics in the cases of infectious diseases. They can also be a possiblesource for new potent antibiotic to which pathogen strains are not resistant [4]. The use ofplant products for pharmaceutical purposes has been gradually increasing in both developingand developed countries due to growing recognition of natural products and their easilyavailability at affordable prices [5]. Thus, the search for new antimicrobial agents frommedicinal plants could be considered as a positive approach, particularly in developingcountries where the frequency of infectious diseases is increasing in a fast ratio [6].

Eclipta alba (L.) Hassk. [7] is traditionally known for its various medicinal properties.According to the International Plant Names Index, the plant has been named as E. alba L. exB. D. Jacks. Other sources suggest Eclipta prostate (L.) L. [syn. E. alba (L.) Hassk.]. E. albais an annual herbaceous plant commonly known as bhringaraj, belonging to the familyAstereaceae; an erect prostate, leaves are opposite, sessile and lanceolate, much branched,roughly hairy, rooting at the nodes and commonly found as a common weed throughoutIndia. Several compounds were isolated and reported such as wedelolactone, dimethyl-wedelolactone, and stigmasterol [810]. Phyto-active compounds such as saponin are theimportant and principle constituent of E. alba and has been reported by several authors [11,12]. Reports have shown that saponin possess significant antibiotic, antifungal, antiviral,hepatoprotective, anti-inflammatory, and antiulcer activities [13]. These compounds gener-ally occur as glycosides of steroids or triterpenic aglycon and a sugar chain [1416]. Becauseof its amphiphatic properties, they can decrease both the surface tension and interfacialtension of the solution system. Saponins are known to interact with cell membranes and arewell-known for their toxic activities against several microorganisms [16]. Due to suchproperties, they showed several pharmacological, physiological, and immunological actionsin biological systems. Plant-derived saponins are also known for its use in industrial as wellas for pharmacological formulations [17].

In the last decade, research has been done to investigate the various pharmacologicalactivities and antimicrobial activity of the crude extracts of the traditionally known herb E.alba [1820]. In depth, studies with purified compounds are very scanty. To study themechanisms of underlying antibacterial effects, seven different experiments were performedto understand the mode of antibacterial action of the isolated saponin from E. alba onselected Gram-positive and Gram-negative bacterial strains.

Materials and Methods

Extraction and Isolation of Saponin from E. alba

The methanol extract of E. alba was prepared by adding 200 ml of methanol to 20 g of sheddried leaf powder. The mixture was kept under stirring for 12 h at room temperature, filteredthrough Whatman no. 1 filter paper, and the solvent was evaporated using rotary evaporator.The dried extract was dissolved in methanol and applied on thin layer chromatogram(2020 cm) using solvent system chloroform/toluene (7:3, v/v). Anisaldehyde sprayingwas done in order to identify the saponin compound.

Characterization of the Isolated Saponin Fraction

The structure of the isolated saponin fraction from E. alba was characterized by Fouriertransform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC),

2004 Appl Biochem Biotechnol (2013) 171:20032019

and mass spectroscopy. The FTIR spectra of the compound were recorded between 4,000and 400 cm1 on Perkin-Elmer instrument which were calibrated using polystyrene band as aKBr pellet. The HPLC analysis of the compound was performed using a liquid chromato-graph (Waters, model 600E) with a 486 UV variable wavelength detector and Novapackcolumn C-18 (5 m, 1503.9 mm). The mobile phase consisted of a gradient mixture,methanol/water (70:30, v/v). The solution was degassed in an ultrasound bath and filteredunder vacuum through a membrane (Millipore, PVDF). The flow was 1.0 ml/min and thesensitivity was 0.001 AUFS. Mass spectra of the isolated saponin were analyzed by usingmass Lynx 4.1 SCN 714 in SAIF, Central Drug Research Institute, Lucknow, India.

Collection of Test Organisms

The bacterial strains including Bacillus subtilis (ATCC 11774), Staphylococcus aureus(ATCC 11632), Klebsilla pneumoniae (ATCC 10031), Pseudomonas aeruginosa (MTCC7815), and Escherichia coli (ATCC 9637) were used for the experiment.

Determination of Antibacterial Activity

The well diffusion technique was used for the determination of antibacterial activity of saponinisolated from E. alba. The bacterial strains were grown in nutrient broth medium overnight at37 C. MullerHinton agar medium was prepared and an aliquot of 0.2 ml of each of the freshbacterial culture was spread over the MullerHinton agar plate. Wells were made and differentconcentrations of saponin were separately introduced into the marked wells. Streptomycin(1 mg/ml) was kept as the positive control. The plates were incubated at 37 C for 24 h and theobserved zones of inhibition were measured using transparent metric ruler.

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

To determine the minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC), saponin fraction was diluted 10 times and seeded in a 96-well cultureplate with streptomycin as the positive control and methanol as the negative control. It wasthen inoculated with a fresh bacterial culture. Inoculated microplates were incubated at37 C for 24 h. Viability of the treated cells was determined by MTT [3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium] assay [21]. The experiments were performed in triplicates.The MIC was determined as the lowest concentration of saponin required to inhibit thegrowth of each organism. The concentration at which no growth was observed was deter-mined as MBC.

pH Sensitivity Assay

The effect of pH on the antibacterial activity of the saponin was determined by well diffusiontechnique at different pH values. The pH of the medium was altered by adding 0.1 N HCl or0.1 N NaOH. The bacterial strains were tested under pH range of pH 5.59.0.

Chemotaxis Assay

The chemotactic activity of the isolated saponin was evaluated by using the chemicalgradient motility agar method of Garg and Kanitkar [22] with slight modifications. The teststrains were grown in the nutrient broth of the organisms overnight at 37 C. After the

Appl Biochem Biotechnol (2013) 171:20032019 2005

solidification of the media, three long rectangular wells (5 cm8 mm) were made as shownin Fig. 6. Both the wells on either side were loaded with saponin (1 % w/v) and streptomycin(1 mg/ml), and plates were then left undisturbed for 60 min. The middle well was thenloaded with 200 l of fresh bacterial culture broth. The plates were incubated at 37 C for24 h. Glucose 1 % (v/v) was used as the positive control and as is known for its excellentchemo-attractant property.

Crystal Violet Assay

Crystal violet assay is a convenient technique to determine the damages caused by the testedcompound on the cellular membrane. Crystal violet assay for both the Gram-positive andGram-negative bacteria after treatment with the saponin of E. alba were carried out using theprotocol as described by Devi et al. [23] with slight modifications. The bacterial cells werecentrifuged at 6,000 rpm for 10 min at 4 C; the pellet was washed twice in PBS andresuspended in the same buffer. Isolated saponin 1 % (w/v) and streptomycin (1 mg/ml) wereadded to the cell suspension supernatant and incubated for 2 h at 37 C for the treatment,respectively. After the treatment, cells were harvested by centrifuging at 10,000 rpm for5 min, resuspended in PBS containing 10 g/ml of crystal violet, and incubated at 37 C for20 min. This was followed by centrifugation at 10,000 rpm for 15 min. The optical densityof cell-free supernatant was measured at 590 nm and the percentage of crystal violet uptakeby the samples was calculated as: optical density (OD) value of the sample/OD value of thecrystal violet solution100.

Loss of Absorbing Material at 260 nm

The release of UV-absorbing material after the treatment with E. alba saponin fraction wasdetermined by the optical density (OD) at 260 nm as per the protocol of Zhou et al. [24].Both the bacterial strains were cultured in nutrient broth for an overnight period at 37 C.The fully grown cultures were centrifuged at 8,000 rpm for 10 min. The bacterial cell pelletswere then washed twice in PBS and treated with saponin. Streptomycin (1 mg/ml) was usedas the positive control and the bacterial cells without any treatment were taken as thenegative control. After 3 h of treatment, the cell suspension was centrifuged at 8,000 rpmfor 10 min and the OD of the supernatant was taken at 260 nm. All measurements were donein triplicates.

Detection of Membrane Disruption by SDS-PAGE

To confirm the membrane damage in the bacterial cell due to the treatment with saponin,sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Analiquot of 1 ml of culture broth was taken in a centrifuge tubes and centrifuged at 8,000 rpmfor 5 min at room temperature, washed twice, and resuspended in TrisHCl (pH 7.2).Saponin (1 % w/v) and streptomycin (1 mg/ml) was added to the cell suspension and wereincubated at 37 C for 2 h. After the treatment, cell suspensions were centrifuged at10,000 rpm for 10 min. The supernatant was collected, chilled, and 20 % (v/v) trichloroaceticacid was added drop-by-drop to the supernatant and kept overnight at 4 C to precipitate theprotein in the supernatant. The control samples were prepared in the same way withouttreatment. Twenty microliters of the supernatant was taken in centrifuge tubes and mixedwith 5 l sample buffer (1 M TrisHCl pH 6.8, 50 % glycerol, 10 % SDS, 10 % -mercaptoethanol, 1 % bromophenol blue). Further, samples were heated at 100 C for 3 min


and kept at 4 C for 1 min and loaded in 12 % SDS-PAGE gel. Staining was done usingCoomasie brilliant blue solution.

Fourier-transformed Infrared Spectroscopy

In the presence of E. alba saponin, structural change in the bacterial cell was confirmed byFTIR analysis. Isolate saponin from E. alba (1 % w/v) was added in the freshly grownbacterial culture and kept at 37 C overnight in a shaking condition. After the treatment, cellswere washed with Milli-Q water. The control samples were prepared similarly without thetreatment of plant extract and were subjected to FTIR analysis for having a comparativeanalysis with that of the plant extract treated cells.

Scanning Electron Microscopy

Cultured bacterial strains at the exponential phase were centrifuged at 5,000 rpm for 10 minat 4 C. The bacterial cell pellet were washed two times in 0.1 M Tris-Cl buffer (pH 7.2) andresuspended in the same buffer. Cells were then treated with 1 % (w/v) isolated saponincompound for 2 h at 37 C and then washed with PBS (pH 7.2). Both the treated anduntreated cells were fixed in 2 % glutaraldehyde for 2 h at 4 C. The cells were thensubjected to washed with phosphate buffer saline solution and dehydrated for 5 min in eachincreasing concentrations of acetone (30, 50, 70, 90 % (v/v)) and 1 min in 100 % (v/v)acetone. All samples were air-dried and coated with platinum to an approximate thickness of120130 . The scanning electron microscopy (SEM) observations were carried out using ascanning electron microscope JEOL JSM Model 6390 LV with a probe diameter of 5060 .

Results

Identification and Characterization of the Isolated Saponin from E. alba

The saponin fraction present in the methanolic extract of E. alba was separated on thin layerchromatography (TLC). Among the three separated spots (with Rf value of 0.08, 0.27, and0.82), spot with the Rf value of 0.27 was detected for the presence of saponin after sprayingwith anisaldehyde. For the collection of the required fraction in larger quantity, themethanolic extract was separated in preparative TLC. The fraction (Rf value 0.27) was thenpurified by HPLC. The HPLC chromatogram showed a prominent peak at RT 11.52 min asshown in Fig. 1. The purified fraction was further characterized by FTIR and massspectroscopy. The IR spectra exhibited absorption at 3,410.97 (OH), 2,925.352,857.04(aliphatic CH stretching), 1,414.63 (CH) bending, and 1,166.26 (OCH3) asymmetricstretching. The FTIR spectral data was given in the chart as Table 1 and Fig. 2. The isolatedpurified saponin fraction was studied by mass spectroscopy. The compound showing an m/zvalue of 620.5 in its negative ion mass spectrum also revealed the molecular formula ofC32H62O8 indicating the presence of Eclalbasaponin. The structure of the saponin is derivedfrom mass spectra of the compound as shown in Fig. 3.

Determination of Antibacterial Activity of Saponin Isolated from E. alba

Both Gram-positive and Gram-negative strains were treated with saponin for the determinationof antibacterial activity using well-diffusion method and the results were shown in Table 2.


Among the tested bacterial strains, distinct inhibition zones were observed in B. subtilis (1319 mm) and P. aeruginosa (1116 mm) against 0.5, 1, and 6 % (w/v) of saponin compound,respectively, as shown in Fig. 4. Streptomycin (1 mg/ml) was taken as the positive control.

Determination of MIC and MBC

The potency of isolated saponin was quantified by determining MIC and MBC. In the caseof Gram-positive bacteria B. subtilis, cells were completely killed at a concentration of93.7 g/ml while in case of Gram-negative bacteria P. aeruginosa, it was 187.5 g/ml of thesaponin fraction. Isolated saponin fraction at a concentration of 187.5 and 375 g/ml arrestthe growth of B. subtilis and P. aeruginosa, respectively, which determine their MBC.

pH Sensitivity Assay

As the pH of the medium gradually increases, the antibacterial activity of the isolated saponincompound was also found to be increased. Both Gram-positive and Gram-negative bacteriashowed different behavior with increasing pH. In case of B. subtilis, well loaded with 100 l of1 % (w/v) saponin showed the highest inhibition zone of 21 mm at pH 6.0; in P. aeruginosa, itwas 14 mm at pH 5.5 as shown in Fig. 5. The well loaded with 100 l (1 mg/ml) ofstreptomycin showed the highest inhibition zone of 32 mm in all the tested pH values.

Fig. 1 High-performance liquid chromatography spectra of the Eclalbasaponin

Table 1 FTIR spectral data ofEclalbasaponin Wave number (cm

1) Type of bond

3,410.97 OH stretch

2,925.352,857.04 Aliphatic CH stretch

1,720.33 C=O stretching (for ester)

1,414.63 CH bending

1,166.26 OCH3 stretching

1,097.72 CO stretch


Evaluation of Chemotactic Responses

Bacterial growth was observed towards the well loaded with saponin 1 % (w/v) and glucose3 % (w/v) showing their positive chemo-attractant property. However, no such growth wasobserved in the case of streptomycin as shown in Fig. 6.

Effect of E. alba Isolated Saponin on Alternation of Membrane Permeability

The uptake of crystal violet by B. subtilis and P. aeruginosa were 44.7 and 47.02 %,respectively, without treatment. After 1-h treatment with 1 % (w/v) saponin fraction, theabsorption capacity increases up to 97.67 and 62.8 %, respectively, as shown in Fig. 7a.Different behavioral response was also observed in the case of bacterial cells treated withstreptomycin, showed highest absorption of crystal violet by B. subtilis, followed by P.aeruginosa which indicates alteration in the cell membrane permeability.

Effect of Saponin on Leakage of 260 nm Absorbing Material from Test Bacteria

The release of UV-absorbing materials is an evidence of cell lysis and its concentration wasmeasured by UVVis spectrophotometer [24]. The cell suspension supernatant of B. subtilisafter treatment with 1 % (w/v) of Eclalbasaponin shows an OD of 0.292, whereas withoutany treatment the OD was 0.099. In case of P. aeruginosa, the OD of the supernatant after

Fig. 2 FTIR spectra of the Eclalbasaponin isolated from E. alba


treatment with the isolated saponin fraction was 0.170 whereas the supernatant without anytreatment was 0.074 and are shown in Fig. 7b.

Saponin-induced Membrane Disruption and Release of Intracellular Proteins

On treatment with 1 % (w/v) of isolated saponin, cellular proteins were found to be releasedinto the culture supernatant as suggested by disruption of the cellular membrane. The patternof protein banding in SDS-PAGE of the bacterial strains were quite different indicating thedifferences in the degree of actions and amount of protein released (Fig. 8). In the case ofstreptomycin (1 mg/ml), the effect was found to be less as compared to the saponin.

Fig. 3 Mass spectra of the Eclalbasaponin isolated from E. alba

Table 2 Antibacterial efficacy ofthe isolated saponin againstbacterial strains (zone ofInhibition in mm)

The data given here is the meanof the three independentexperimentsSD

Microorganisms Concentrations (%)

0.5 1 6

Klebsiella pneumonia (ATCC 10031) 10

FTIR Analysis of Molecular Alternations Induced by Saponin on Test Strains

The changes in the molecular level of the bacterial strains after treatment were analyzed bychanges in the IR spectra as shown in Fig. 9a,b. In case of P.aeruginosa, as shown in Fig. 9a,the treated cells with 1 % (w/v) of saponin cause the changes in frequency between 1,812.76and 732.82 cm1. Such changes in the spectra involved in the alternation in the esterfunctional groups present in lipids, fatty acids, proteins, and nucleic acids. These resultsare further confirmed by distinct spectral changes in the region between 1,702.84 and1,380.78 cm1. The changes in the frequency at 1,724.05 cm1 reflect in the alternation inthe ester functional group in the lipids. Changes in the alternation in the polysaccharide unitsof the bacterial cell membranes were observed by increase in the frequency between1,058.73 and 875.52. Further alternation in the phospholipids components of the bacterialplasma membrane was indicated by a decrease in the frequency at 1,294.0 cm1 in compar-ison to the control untreated cells.

In case of B. subtilis, as shown in Fig. 9b, the treated cells were showing a distinct changein the frequency between 1,945.83 and 838.88 cm1. Such changes indicate the alternation inthe ester functional group of cellular and macromolecular components which includes lipids,proteins, fatty acids, nucleic acids, etc. Spectral changes between 1,745.26 and1,515.28 cm1 confirmed the alternation in the cellular macromolecules. The major changesin the ester functional group of lipids were reduced to a peak value of 1,745.26 incomparison to the control. Other distinct changes in the cellular structure of the bacterialcells were confirmed by increase in the frequency between 1,049.09 and 823.46 cm1

Fig. 4 Antibacterial assay of saponin (1 %, w/v) with streptomycin sulfate (1 mg/ml) as positive controlagainst a B. subtilis and b P. aeruginosa


indicating the alternation in the polysaccharide components of the bacterial cells membrane.Similarly, decrease in the frequency at 1,257.36 as compared to the control referred to thealteration in the phospholipids units of bacterial cell membrane.

Study on the Effect of E. alba Isolated Saponin on Bacterial Cell Surface by ScanningElectron Microscopy

The SEM analysis showed distinct morphological change between saponin-treated anduntreated bacterial cells as shown in Fig. 10ad. Shrinkage of the bacterial cell surfacewas observed in the case of treated cells whereas cell surface was normal in the case ofuntreated cells. SEM analysis revealed that the bacterial membrane integrity was completelylost and the membrane disrupted.

Discussion

Antibacterial property of saponin isolated from E. alba and its chemical composition havebeen reviewed in various literature [9, 10, 25]. Karthikumar et al. [4] determined theantibacterial activity of various solvent extracts of E. prostrata leaves and reported thesignificant antimicrobial activity against a variety of Gram-positive and Gram-negative

Fig. 5 The effect of pH on the antibacterial activity of saponin (1 %, w/v) against a B. subtilis and b P.aeruginosa. Streptomycin sulfate (1 mg/ml) was used as a control


bacteria including both P. aeruginosa and B. subtilis. Similar observation was also reportedby Khanna and Kannnabiran [26] where they conducted the antibacterial activity of saponinisolated from E. prostate against several bacterial pathogens. There are several reportsavailable in support of antimicrobial activity of plant extracts due to the presence of saponin[27]. However, its mechanism of action on both Gram-positive and Gram-negative bacteriahas not been studied so far. Therefore, the present study was undertaken to determine theprobable mode of saponin action isolated from E. alba against the test pathogens.

There may be several reasons for the differences in the antibacterial activity of saponintowards the different bacterial strains such as degradation of saponin by some glucosidaseenzymes produced by Gram-negative bacteria, differences in the cell envelope structure and thevariation in the chemical structure of the saponin. Variation in the saponin structure such asglycone side chains in terms of number, chemical composition specific point of attachment to

Fig. 6 Petri plates containing chemical gradient motility agar exhibiting chemotaxis phenomenon of B.subtilis (a and b) and P. aeruginosa (c and d) toward saponin (1 %, w/v) isolated from E. alba and glucose(1 %, w/v). Streptomycin sulfate (1 mg/ml) was used as standard chemo repellant. a A Saponin (1 %, w/v), B B.subtilis culture, C streptomycin (1 mg/ml); b D glucose solution (1 %, w/v), E B. subtilis cell culture, Fstreptomycin (1 mg/ml); c G saponin (1 %, w/v), H P. aeruginosa culture, I streptomycin (1 mg/ml); d Jglucose solution (1 %, w/v), K P. aeruginosa culture, L streptomycin (1 mg/ml)


the steroid or triterpenoid nucleus is critical to the saponin biological effects [28]. In this study,MIC and MBC experiments revealed the minimum concentration at which E. albamethanolicextract acts as bactericidal and bacteriostatic agent against B. subtilis and P. aeruginosa,respectively. Such a bacteriostatic agent restricts the bacterial growth by interfering with proteinsynthesis, DNA replication, and other cellular metabolism of bacteria. Since complete killinghas occurred at higher concentration, it is marked as bactericidal in nature at higher concentra-tion. The degree of growth inhibition by the saponin was much higher towards Gram-positivebacteria as compared to Gram-negative bacteria used. Our findings were found to be consistentwith the previous reports of Karthikumar et al. [4] and Khanna andKannabiran [26]. Avato et al.[29] reported that the aglycone part of the saponin is responsible for their antibacterial activity.

Fig. 7 a Uptake of crystal violet dye by B. subtilis and P. aeruginosa cells after treatment with saponinsisolated from E. alba. The data given here is the mean of the three independent experimentsSD. b Release of260 nm absorbing material in the culture supernatant of B. subtilis and P. aeruginosa after treatment withisolated saponins from E. alba. The data given here is the mean of the three independent experimentsSD

Fig. 8 SDS-PAGE sowing the released bacterial cell proteins of B. subtilis and P. aeruginosa after treatmentwith saponin. A Supernatant of B. subtilis without treatment, B supernatant with B. subtilis with treatment(1 %, w/v), C supernatant of B. subtilis with streptomycin (1 mg/ml), D protein molecular weight marker, Esupernatant of P. aeruginosa without treatment, F supernatant of P. aeruginosa with treatment (1 %, w/v), Gsupernatant of B. subtilis with streptomycin (1 mg/ml)


Their studies also suggests that the sugar moiety (glycone) is not important for the antimicrobialefficacy while another study conducted by Mandal et al. [30] reported that saponin arehydrolyzed by the bacterial enzymes to its corresponding aglycone that leads to the decreasein the antibacterial activity. Saponin may be interacting in a different way with the bacterial cellwalls; however, the specific modes of action are not yet clear [31].

Compounds having chemoattractant property for the pathogen further enhance theirantibacterial activity [22]. Chemotaxis is the phenomenon in which bacteria direct theirmovements according to the changes in the chemical composition of their surroundingenvironment. The E. alba isolated saponin exhibited an appreciable chemo-attractant prop-erty for bacteria, which was confirmed further by performing chemical gradient motility agarassay. To validate the mode of action of saponin on the bacterial strain precisely, furtheranalysis including crystal violet assay, quantification of UV-absorbing material release,SDS-PAGE, and FTIR were carried out. The action of saponin on the outer membraneespecially on the permeability was determined by the accumulation of crystal violet. Innormal condition, crystal violet dye penetrates the outer membrane of the bacteria veryleisurely. However, occurrence of damages in the membrane significantly enhances theuptake of the dye. After the treatment with saponin against both bacteria, significant enhance-ment in the uptake of the dye was observed. The result suggested that the saponin alters themembrane permeability and enables it to increase the accumulation of dye as compare to thenormal untreated condition. In comparison to the Gram-negative P. aeruginosa, Gram-positiveB. subtilis showed higher accumulation of dye indicating more effectiveness of the saponinisolated from E. alba. B. subtliswas the most susceptible bacterium, an observation that may bedue to the presence of single membrane which makes it more accessible to permeation bysaponin component of the E. alba. In contrast, P. aeruginosa showed the least susceptibility tothe saponin compound. This may be due to the fact that P. aeruginosa has intrinsic resistance

Fig. 9 IR spectra represent the alternation in the molecular level of a B. subtilis and b P. aeruginosa cells aftertreatment with E. alba saponin. Bacterial cells without treatment were taken as control


from a restrictive outer membrane barrier. However, against the positive control streptomycin(1 mg/ml), the accumulation of the dye was much lower which indicated that the antibiotic doesnot alter the membrane permeability rather inhibit the growth of the test bacteria by some othermodes of action such as inhibition of protein synthesis.

The presence of UV-absorbing materials in the saponin-treated bacterial cell suspensionsignifies the lysis of cells and formation of nonselective pores on the cell membrane [24].Releasing of bacterial intracellular components in the medium suggests that the saponin targetsthe cell membrane and causes damages by forming pores. In the case of Gram-positive bacteria,B. subtilis shows higher UV absorbance indicating that the compound is more effective incomparison to the P. aeruginosa. Such anomalies are due to the difference in their cell wallconstituents. Formation of such pores is mainly responsible for the loss of viability of the treatedbacterial cells. Several authors such as Miller [32] and Augustin et al. [33] showed thatincubation of digitonine (steroidic saponin with five sugars) with biological membranes causedtheir deformation. Such action can cause the rupture of the membrane of the vesicle containingcholesterol, whereas the membranes free from cholesterol remained undamaged [34].

Fig. 10 Scanning electron microscopic images of saponin treated and untreated cells of B. subtilis (a and b)and P. aeruginosa (c and d)


The leakage of intracellular bacterial protein into the surrounding media after treatmentwith the test compound is another important sign for membrane damage and loss ofmembrane integrity [35]. The action of the saponin on the membrane integrity was verifiedby analyzing the supernatant of the treated bacterial cultures on SDS-PAGE. The distinctbands were revealed in both the treated samples. Such banding patterns were not observedon the control bacterial cells as well as the streptomycin-treated cells. Such observationsclearly established that the saponin isolated from E. alba caused the membrane damage thatfurther lead to loss of membrane integrity which was responsible for release of intracellularproteins.

Further confirmation of membrane damage was determined by FTIR spectroscopy. Thetechnique was used to study spectral changes arising as a result of bacterial injuries [36]. TheIR spectra of the saponin-treated bacterial cells reflects the alternation in biochemicalstructures and the cellular constituents, which includes proteins, polysaccharides, fatty acids,nucleic acids for both the test strains, clearly indicating cellular damages. Majority of thespectral changes were noted between the frequencies 1,800 and 800 cm1. Alternation inmembrane phospholipids was confirmed by the spectral variation between the frequencies1,720 and 1,250 cm1. The alternation in the bacterial cell surface morphology of the treatedcells were studied by scanning electron microscope and morphologically appeared as roughand shrunken. However, the untreated bacterial cells appeared smooth and normal rodshaped. Such loss of membrane integrity and damages of cell surface supports the evidenceof the antibacterial action of the saponin isolated from E. alba.

Conclusion

Our findings have shown that the Eclalbasaponin isolated from E. alba are found to beeffective against both Gram-positive and Gram-negative bacteria. The study provides clearinformation regarding the mode of antibacterial activity of the Eclalbasaponin against B.subtilis (ATCC 11774) and P. aeruginosa (MTCC 7815). The isolated saponin was found tohave bactericidal effect at higher concentration. Antibacterial activity was further evident byscanning electron microscopy images revealing the deformation of the cytoplasmic mem-brane supporting the action of saponin on the cell membrane. The results were furtherconfirmed by the enhanced cellular permeability. The work also established the chemo-attractant property of saponin for both the test strains. Hence, the Eclalbasaponin isolatedfrom E. alba might be a potential candidate to prove its efficiency as a preventive andtherapeutic drug against both Gram-positive and Gram-negative pathogenic bacteria.

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Mode of Antibacterial Activity of Eclalbasaponin Isolated from Eclipta albaAbstractIntroductionMaterials and MethodsExtraction and Isolation of Saponin from E. albaCharacterization of the Isolated Saponin FractionCollection of Test OrganismsDetermination of Antibacterial ActivityMinimum Inhibitory Concentration and Minimum Bactericidal ConcentrationpH Sensitivity AssayChemotaxis AssayCrystal Violet AssayLoss of Absorbing Material at 260&newnbsp;nmDetection of Membrane Disruption by SDS-PAGEFourier-transformed Infrared SpectroscopyScanning Electron Microscopy

ResultsIdentification and Characterization of the Isolated Saponin from E. albaDetermination of Antibacterial Activity of Saponin Isolated from E. albaDetermination of MIC and MBCpH Sensitivity AssayEvaluation of Chemotactic ResponsesEffect of E. alba Isolated Saponin on Alternation of Membrane PermeabilityEffect of Saponin on Leakage of 260&newnbsp;nm Absorbing Material from Test BacteriaSaponin-induced Membrane Disruption and Release of Intracellular ProteinsFTIR Analysis of Molecular Alternations Induced by Saponin on Test StrainsStudy on the Effect of E. alba Isolated Saponin on Bacterial Cell Surface by Scanning Electron Microscopy

DiscussionConclusionReferences

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