Mode of Antibacterial Activity of Eclalbasaponin Isolated

from 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 of
Eclalbasaponin 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 mass
spectroscopy. The antibacterial activity was evaluated by well diffusion technique, pH
sensitivity, chemotaxis, and crystal violet assays. Eclalbasaponin showed clear zone of
inhibition against both Bacillus subtilis and Pseudomonas aeruginosa and exhibited growth
inhibition at the pH range of 5.59.0. The isolated saponin exhibited its positive
chemoattractant property for both bacterial strains. Results of crystal violet assay and the
presence of UV-sensitive materials in the cell-free supernatant confirmed the cellular
damages caused by the treatment of Eclalbasaponin. The release of intracellular proteins
due to the membrane damage was determined by sodium dodecyl sulfate polyacrylamide gel
electrophoresis. Changes in the cell surface structure and membrane disruption were further
revealed by FTIR and scanning electron microscopy analysis. The present study suggests
that the isolated saponin from E. alba causes the disruption of the bacterial cell membrane
which 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 global
threat and an important concern for the hospitals, long-stay residential centers, and in the
community [1]. Appearance of such problems is due to the extensive use of antibiotics and
selective 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 antibiotics
and lack of new drugs, vaccines, and diagnostic aids made the current antimicrobial agents
inefficient to control various bacterial diseases [2]. The development of new medicines with
different chemical moieties and mode of action may be considered a relevant strategy [3].
Appl Biochem Biotechnol (2013) 171:20032019
DOI 10.1007/s12010-013-0452-3
A. Ray (*)
:
P. Bharali
:
B. K. Konwar
Department of Molecular Biology & Biotechnology, Tezpur University, Tezpur, Assam 784028, India
e-mail: anggana@tezu.ernet.in
Medicinal plants represent as a valuable source for natural products and explored
continuously for therapeutics in the cases of infectious diseases. They can also be a possible
source for new potent antibiotic to which pathogen strains are not resistant [4]. The use of
plant products for pharmaceutical purposes has been gradually increasing in both developing
and developed countries due to growing recognition of natural products and their easily
availability at affordable prices [5]. Thus, the search for new antimicrobial agents from
medicinal plants could be considered as a positive approach, particularly in developing
countries 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. ex
B. D. Jacks. Other sources suggest Eclipta prostate (L.) L. [syn. E. alba (L.) Hassk.]. E. alba
is an annual herbaceous plant commonly known as bhringaraj, belonging to the family
Astereaceae; an erect prostate, leaves are opposite, sessile and lanceolate, much branched,
roughly hairy, rooting at the nodes and commonly found as a common weed throughout
India. Several compounds were isolated and reported such as wedelolactone, dimethyl-
wedelolactone, and stigmasterol [810]. Phyto-active compounds such as saponin are the
important 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]. Because
of its amphiphatic properties, they can decrease both the surface tension and interfacial
tension of the solution system. Saponins are known to interact with cell membranes and are
well-known for their toxic activities against several microorganisms [16]. Due to such
properties, they showed several pharmacological, physiological, and immunological actions
in biological systems. Plant-derived saponins are also known for its use in industrial as well
as for pharmacological formulations [17].
In the last decade, research has been done to investigate the various pharmacological
activities 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 the
mechanisms of underlying antibacterial effects, seven different experiments were performed
to understand the mode of antibacterial action of the isolated saponin from E. alba on
selected 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 shed
dried leaf powder. The mixture was kept under stirring for 12 h at room temperature, filtered
through 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 spraying
was 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 Fourier
transform 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 4,000
and 400 cm
1
on Perkin-Elmer instrument which were calibrated using polystyrene band as a
KBr 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 Novapack
column 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 filtered
under vacuum through a membrane (Millipore, PVDF). The flow was 1.0 ml/min and the
sensitivity was 0.001 AUFS. Mass spectra of the isolated saponin were analyzed by using
mass 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 (MTCC
7815), 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 saponin
isolated from E. alba. The bacterial strains were grown in nutrient broth medium overnight at
37 C. MullerHinton agar medium was prepared and an aliquot of 0.2 ml of each of the fresh
bacterial culture was spread over the MullerHinton agar plate. Wells were made and different
concentrations 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 the
observed 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 bactericidal
concentration (MBC), saponin fraction was diluted 10 times and seeded in a 96-well culture
plate with streptomycin as the positive control and methanol as the negative control. It was
then inoculated with a fresh bacterial culture. Inoculated microplates were incubated at
37 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 the
growth 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 diffusion
technique at different pH values. The pH of the medium was altered by adding 0.1 N HCl or
0.1 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 chemical
gradient motility agar method of Garg and Kanitkar [22] with slight modifications. The test
strains 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 shown
in 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 then
loaded with 200 l of fresh bacterial culture broth. The plates were incubated at 37 C for
24 h. Glucose 1 % (v/v) was used as the positive control and as is known for its excellent
chemo-attractant property.
Crystal Violet Assay
Crystal violet assay is a convenient technique to determine the damages caused by the tested
compound on the cellular membrane. Crystal violet assay for both the Gram-positive and
Gram-negative bacteria after treatment with the saponin of E. alba were carried out using the
protocol as described by Devi et al. [23] with slight modifications. The bacterial cells were
centrifuged at 6,000 rpm for 10 min at 4 C; the pellet was washed twice in PBS and
resuspended in the same buffer. Isolated saponin 1 % (w/v) and streptomycin (1 mg/ml) were
added 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 for
5 min, resuspended in PBS containing 10 g/ml of crystal violet, and incubated at 37 C for
20 min. This was followed by centrifugation at 10,000 rpm for 15 min. The optical density
of cell-free supernatant was measured at 590 nm and the percentage of crystal violet uptake
by the samples was calculated as: optical density (OD) value of the sample/OD value of the
crystal violet solution100.
Loss of Absorbing Material at 260 nm
The release of UV-absorbing material after the treatment with E. alba saponin fraction was
determined 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 pellets
were then washed twice in PBS and treated with saponin. Streptomycin (1 mg/ml) was used
as the positive control and the bacterial cells without any treatment were taken as the
negative control. After 3 h of treatment, the cell suspension was centrifuged at 8,000 rpm
for 10 min and the OD of the supernatant was taken at 260 nm. All measurements were done
in 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. An
aliquot of 1 ml of culture broth was taken in a centrifuge tubes and centrifuged at 8,000 rpm
for 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 were
incubated at 37 C for 2 h. After the treatment, cell suspensions were centrifuged at
10,000 rpm for 10 min. The supernatant was collected, chilled, and 20 % (v/v) trichloroacetic
acid was added drop-by-drop to the supernatant and kept overnight at 4 C to precipitate the
protein in the supernatant. The control samples were prepared in the same way without
treatment. Twenty microliters of the supernatant was taken in centrifuge tubes and mixed
with 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
2006 Appl Biochem Biotechnol (2013) 171:20032019
and kept at 4 C for 1 min and loaded in 12 % SDS-PAGE gel. Staining was done using
Coomasie brilliant blue solution.
Fourier-transformed Infrared Spectroscopy
In the presence of E. alba saponin, structural change in the bacterial cell was confirmed by
FTIR analysis. Isolate saponin from E. alba (1 % w/v) was added in the freshly grown
bacterial culture and kept at 37 C overnight in a shaking condition. After the treatment, cells
were washed with Milli-Q water. The control samples were prepared similarly without the
treatment of plant extract and were subjected to FTIR analysis for having a comparative
analysis 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 min
at 4 C. The bacterial cell pellet were washed two times in 0.1 M Tris-Cl buffer (pH 7.2) and
resuspended in the same buffer. Cells were then treated with 1 % (w/v) isolated saponin
compound for 2 h at 37 C and then washed with PBS (pH 7.2). Both the treated and
untreated cells were fixed in 2 % glutaraldehyde for 2 h at 4 C. The cells were then
subjected to washed with phosphate buffer saline solution and dehydrated for 5 min in each
increasing 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 of
120130 . The scanning electron microscopy (SEM) observations were carried out using a
scanning 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 layer
chromatography (TLC). Among the three separated spots (with R
f
value of 0.08, 0.27, and
0.82), spot with the R
f
value of 0.27 was detected for the presence of saponin after spraying
with anisaldehyde. For the collection of the required fraction in larger quantity, the
methanolic extract was separated in preparative TLC. The fraction (R
f
value 0.27) was then
purified by HPLC. The HPLC chromatogram showed a prominent peak at RT 11.52 min as
shown in Fig. 1. The purified fraction was further characterized by FTIR and mass
spectroscopy. 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 (OCH
3
) asymmetric
stretching. The FTIR spectral data was given in the chart as Table 1 and Fig. 2. The isolated
purified saponin fraction was studied by mass spectroscopy. The compound showing an m/z
value of 620.5 in its negative ion mass spectrum also revealed the molecular formula of
C
32
H
62
O
8
indicating the presence of Eclalbasaponin. The structure of the saponin is derived
from 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 determination
of antibacterial activity using well-diffusion method and the results were shown in Table 2.
Appl Biochem Biotechnol (2013) 171:20032019 2007
Among the tested bacterial strains, distinct inhibition zones were observed in B. subtilis (13
19 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 case
of Gram-positive bacteria B. subtilis, cells were completely killed at a concentration of
93.7 g/ml while in case of Gram-negative bacteria P. aeruginosa, it was 187.5 g/ml of the
saponin fraction. Isolated saponin fraction at a concentration of 187.5 and 375 g/ml arrest
the 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 saponin
compound was also found to be increased. Both Gram-positive and Gram-negative bacteria
showed different behavior with increasing pH. In case of B. subtilis, well loaded with 100 l of
1 % (w/v) saponin showed the highest inhibition zone of 21 mm at pH 6.0; in P. aeruginosa, it
was 14 mm at pH 5.5 as shown in Fig. 5. The well loaded with 100 l (1 mg/ml) of
streptomycin 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 of
Eclalbasaponin
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 OCH
3
stretching
1,097.72 CO stretch
2008 Appl Biochem Biotechnol (2013) 171:20032019
Evaluation of Chemotactic Responses
Bacterial growth was observed towards the well loaded with saponin 1 % (w/v) and glucose
3 % (w/v) showing their positive chemo-attractant property. However, no such growth was
observed 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, the
absorption 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 with
streptomycin, 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 was
measured by UVVis spectrophotometer [24]. The cell suspension supernatant of B. subtilis
after treatment with 1 % (w/v) of Eclalbasaponin shows an OD of 0.292, whereas without
any 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
Appl Biochem Biotechnol (2013) 171:20032019 2009
treatment with the isolated saponin fraction was 0.170 whereas the supernatant without any
treatment 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 released
into the culture supernatant as suggested by disruption of the cellular membrane. The pattern
of protein banding in SDS-PAGE of the bacterial strains were quite different indicating the
differences in the degree of actions and amount of protein released (Fig. 8). In the case of
streptomycin (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 of
the isolated saponin against
bacterial strains (zone of
Inhibition in mm)
The data given here is the mean
of the three independent
experimentsSD
Microorganisms Concentrations (%)
0.5 1 6
Klebsiella pneumonia (ATCC 10031) 10 <10 <10
P. aeruginosa (MTCC 7815) 11 <10 16
E. coli (ATCC 9637) 12 10 <10
B. subtilis (ATCC 11774) 13 14 19
S. aureus (ATCC 11632) 10 13 <10
2010 Appl Biochem Biotechnol (2013) 171:20032019
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 by
changes 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 1,812.76
and 732.82 cm
1
. Such changes in the spectra involved in the alternation in the ester
functional groups present in lipids, fatty acids, proteins, and nucleic acids. These results
are further confirmed by distinct spectral changes in the region between 1,702.84 and
1,380.78 cm
1
. The changes in the frequency at 1,724.05 cm
1
reflect in the alternation in
the ester functional group in the lipids. Changes in the alternation in the polysaccharide units
of the bacterial cell membranes were observed by increase in the frequency between
1,058.73 and 875.52. Further alternation in the phospholipids components of the bacterial
plasma membrane was indicated by a decrease in the frequency at 1,294.0 cm
1
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 change
in the frequency between 1,945.83 and 838.88 cm
1
. Such changes indicate the alternation in
the ester functional group of cellular and macromolecular components which includes lipids,
proteins, fatty acids, nucleic acids, etc. Spectral changes between 1,745.26 and
1,515.28 cm
1
confirmed the alternation in the cellular macromolecules. The major changes
in the ester functional group of lipids were reduced to a peak value of 1,745.26 in
comparison to the control. Other distinct changes in the cellular structure of the bacterial
cells were confirmed by increase in the frequency between 1,049.09 and 823.46 cm
1
Fig. 4 Antibacterial assay of saponin (1 %, w/v) with streptomycin sulfate (1 mg/ml) as positive control
against a B. subtilis and b P. aeruginosa
Appl Biochem Biotechnol (2013) 171:20032019 2011
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 the
alteration in the phospholipids units of bacterial cell membrane.
Study on the Effect of E. alba Isolated Saponin on Bacterial Cell Surface by Scanning
Electron Microscopy
The SEM analysis showed distinct morphological change between saponin-treated and
untreated bacterial cells as shown in Fig. 10ad. Shrinkage of the bacterial cell surface
was observed in the case of treated cells whereas cell surface was normal in the case of
untreated cells. SEM analysis revealed that the bacterial membrane integrity was completely
lost and the membrane disrupted.
Discussion
Antibacterial property of saponin isolated from E. alba and its chemical composition have
been reviewed in various literature [9, 10, 25]. Karthikumar et al. [4] determined the
antibacterial activity of various solvent extracts of E. prostrata leaves and reported the
significant 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
2012 Appl Biochem Biotechnol (2013) 171:20032019
bacteria including both P. aeruginosa and B. subtilis. Similar observation was also reported
by Khanna and Kannnabiran [26] where they conducted the antibacterial activity of saponin
isolated from E. prostate against several bacterial pathogens. There are several reports
available 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 bacteria
has not been studied so far. Therefore, the present study was undertaken to determine the
probable 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 saponin
towards the different bacterial strains such as degradation of saponin by some glucosidase
enzymes produced by Gram-negative bacteria, differences in the cell envelope structure and the
variation in the chemical structure of the saponin. Variation in the saponin structure such as
glycone 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, F
streptomycin (1 mg/ml); c G saponin (1 %, w/v), H P. aeruginosa culture, I streptomycin (1 mg/ml); d J
glucose solution (1 %, w/v), K P. aeruginosa culture, L streptomycin (1 mg/ml)
Appl Biochem Biotechnol (2013) 171:20032019 2013
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. alba methanolic
extract acts as bactericidal and bacteriostatic agent against B. subtilis and P. aeruginosa,
respectively. Such a bacteriostatic agent restricts the bacterial growth by interfering with protein
synthesis, DNA replication, and other cellular metabolism of bacteria. Since complete killing
has 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-positive
bacteria as compared to Gram-negative bacteria used. Our findings were found to be consistent
with the previous reports of Karthikumar et al. [4] and Khanna and Kannabiran [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 saponins
isolated from E. alba. The data given here is the mean of the three independent experimentsSD. b Release of
260 nm absorbing material in the culture supernatant of B. subtilis and P. aeruginosa after treatment with
isolated 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 treatment
with 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, E
supernatant of P. aeruginosa without treatment, F supernatant of P. aeruginosa with treatment (1 %, w/v), G
supernatant of B. subtilis with streptomycin (1 mg/ml)
2014 Appl Biochem Biotechnol (2013) 171:20032019
Their studies also suggests that the sugar moiety (glycone) is not important for the antimicrobial
efficacy while another study conducted by Mandal et al. [30] reported that saponin are
hydrolyzed by the bacterial enzymes to its corresponding aglycone that leads to the decrease
in the antibacterial activity. Saponin may be interacting in a different way with the bacterial cell
walls; however, the specific modes of action are not yet clear [31].
Compounds having chemoattractant property for the pathogen further enhance their
antibacterial activity [22]. Chemotaxis is the phenomenon in which bacteria direct their
movements according to the changes in the chemical composition of their surrounding
environment. The E. alba isolated saponin exhibited an appreciable chemo-attractant prop-
erty for bacteria, which was confirmed further by performing chemical gradient motility agar
assay. To validate the mode of action of saponin on the bacterial strain precisely, further
analysis including crystal violet assay, quantification of UV-absorbing material release,
SDS-PAGE, and FTIR were carried out. The action of saponin on the outer membrane
especially on the permeability was determined by the accumulation of crystal violet. In
normal condition, crystal violet dye penetrates the outer membrane of the bacteria very
leisurely. However, occurrence of damages in the membrane significantly enhances the
uptake 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 the
membrane permeability and enables it to increase the accumulation of dye as compare to the
normal untreated condition. In comparison to the Gram-negative P. aeruginosa, Gram-positive
B. subtilis showed higher accumulation of dye indicating more effectiveness of the saponin
isolated fromE. alba. B. subtlis was the most susceptible bacterium, an observation that may be
due to the presence of single membrane which makes it more accessible to permeation by
saponin component of the E. alba. In contrast, P. aeruginosa showed the least susceptibility to
the 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 after
treatment with E. alba saponin. Bacterial cells without treatment were taken as control
Appl Biochem Biotechnol (2013) 171:20032019 2015
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 does
not alter the membrane permeability rather inhibit the growth of the test bacteria by some other
modes of action such as inhibition of protein synthesis.
The presence of UV-absorbing materials in the saponin-treated bacterial cell suspension
signifies 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 targets
the 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 in
comparison to the P. aeruginosa. Such anomalies are due to the difference in their cell wall
constituents. Formation of such pores is mainly responsible for the loss of viability of the treated
bacterial cells. Several authors such as Miller [32] and Augustin et al. [33] showed that
incubation of digitonine (steroidic saponin with five sugars) with biological membranes caused
their deformation. Such action can cause the rupture of the membrane of the vesicle containing
cholesterol, 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)
2016 Appl Biochem Biotechnol (2013) 171:20032019
The leakage of intracellular bacterial protein into the surrounding media after treatment
with the test compound is another important sign for membrane damage and loss of
membrane integrity [35]. The action of the saponin on the membrane integrity was verified
by analyzing the supernatant of the treated bacterial cultures on SDS-PAGE. The distinct
bands were revealed in both the treated samples. Such banding patterns were not observed
on the control bacterial cells as well as the streptomycin-treated cells. Such observations
clearly established that the saponin isolated from E. alba caused the membrane damage that
further lead to loss of membrane integrity which was responsible for release of intracellular
proteins.
Further confirmation of membrane damage was determined by FTIR spectroscopy. The
technique was used to study spectral changes arising as a result of bacterial injuries [36]. The
IR spectra of the saponin-treated bacterial cells reflects the alternation in biochemical
structures and the cellular constituents, which includes proteins, polysaccharides, fatty acids,
nucleic acids for both the test strains, clearly indicating cellular damages. Majority of the
spectral changes were noted between the frequencies 1,800 and 800 cm
1
. Alternation in
membrane phospholipids was confirmed by the spectral variation between the frequencies
1,720 and 1,250 cm
1
. The alternation in the bacterial cell surface morphology of the treated
cells were studied by scanning electron microscope and morphologically appeared as rough
and shrunken. However, the untreated bacterial cells appeared smooth and normal rod
shaped. Such loss of membrane integrity and damages of cell surface supports the evidence
of 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 be
effective against both Gram-positive and Gram-negative bacteria. The study provides clear
information regarding the mode of antibacterial activity of the Eclalbasaponin against B.
subtilis (ATCC 11774) and P. aeruginosa (MTCC 7815). The isolated saponin was found to
have bactericidal effect at higher concentration. Antibacterial activity was further evident by
scanning electron microscopy images revealing the deformation of the cytoplasmic mem-
brane supporting the action of saponin on the cell membrane. The results were further
confirmed by the enhanced cellular permeability. The work also established the chemo-
attractant property of saponin for both the test strains. Hence, the Eclalbasaponin isolated
from E. alba might be a potential candidate to prove its efficiency as a preventive and
therapeutic drug against both Gram-positive and Gram-negative pathogenic bacteria.
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