Int J Biol Med Res.

2010; 1(4): 341-346

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Volume 3, Issue 4, Sep 2010 ISSN: 0976:6685

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Original Article

Antioxidant, cytotoxic and antimicrobial properties of Eclipta alba ethanol extract.

a b c d e
Nazim Uddin , Atiar Rahman *, Nazim Uddin Ahmed , Sohel Rana , Rasheda Akter ,
f
A M Masudul Azad Chowdhury
a, *b, &D
Department of biochemistry and molecular biology, University of chittagong, chittagong-4331, Bangladesh
c&e
Bangladesh council for scientific and industrial research (BCSIR), chittagong, Bangladesh
f
Department of genetic engineering and biotechnology, University of chittagong, chittagong-4331, Bangladesh

ARTICLE INFO ABSTRACT

Keywords: This study was subjected to investigate antioxidant, cytotoxic and antimicrobial (antibacterial
Kesohraj and antifungal) properties of Eclipta alba ethanol extract. The antioxidant and cytotoxic
Oxidative Stress properties of the extract were assessed by DPPH free radical scavenging method and brine
Antifungal shrimp lethality bioassay, respectively. Disc diffusion technique and food poisoning technique
Fluconazole
were used to determine the antibacterial and antifungal activity of the extract, respectively.
Ethnobotanical.
DPPH free radical scavenging effect of extract was compared with standard antioxidant
ascorbic acid. IC50 value was found 1.34ìg/ml for extract and 1.03ìg/ml for ascorbic acid. LC50
value of the extract in brine shrimp lethality bioassay was found 94.3ìg/ml. Large zone of
inhibition were observed in disc diffusion antibacterial screening against gram negative
Salmonella typhi, Shigella sonnei, Escherichia coli, Salmonella paratyphi, Pseudomonas sp (1) &
Pseudomonas sp (II), and gram positive Bacillus subtilis, Bacillus cereus, Bacillus megaterim &
Staphylococcus aureus at the concentration of 1mg/disc. The extract concentration 250ìg/disc
showed no zone of inhibition to any bacterial strain but 500ìg/disc showed a moderate zone of
inhibition (8mm) against Salmonella typhi. In antifungal assay, the maximum 51.52% of anti-
mycotic activity was observed against Aspergillus ochraceus. Fluconazole was used as standard
antifungal agent. Extract was found nontoxic in acute toxicity test in mice.
c Copyright 2010 BioMedSciDirect Publications IJBMR -ISSN: 0976:6685. All rights reserved.

1. Introduction
Eclipta alba (synonym is E. prostata), locally known as Kesohraj
belonging to the family of Asteraceae, is an evergreen herb of
native Asia [1] albeit of its availability in tropical and subtropical
regions of the world in rainy season [2]. E. alba is a common plant
abundantly grows throughout Bangladesh. It grows well in moist
places especially in paddy fields both as an erect or prostrate
annual herb. The leaves of E. alba are opposite, sessile, lanceolate
and densely arranged on both sides of the stem and rooting at the
nodes and the flower-heads are white [3]. E. alba has great
traditional importance of being used as a medicinal resources in
Bangladesh. Various parts of the plant are used by the rural people
for several human illnesses.
This herb shows seasonal and varietal variation in the quantity
of secondary metabolites. It is traditionally used as promoter of
* Corresponding Author : Dr. Md. Atiar Rahman,
Department of Biochemistry and Molecular Biology,
University of Chittagong,
Chittagong-4331,
Bangladessh, Tel: +88-01711709084, Fax: +88-031-726310, Ex-4334.
E-mail: atiarh@yahoo.com,
c
Copyright 2010 BioMedSciDirect Publications. All rights reserved.
hair growth, blackener of hair and hepatoprotective [4, 2]. Its
aerial parts are administered in jaundice [5-8]. The herb is also
used in gastritis and respiratory disorders like cough and asthma.
It is also antihypertensive and effective in conditions of anemia [9].
All the parts of E. alba and its chemical constituents have been used
in different therapeutic cases. It contains mainly coumestans i.e.
wedelolactone (I) and demethylwedelolactone (II), polypeptides,
polyacetylenes, thiophene-derivatives, steroids, triterpenes and
flavonoids. Coumestans are known to possess estrogenic activity
[10]. Wedelolactone possesses a wide range of biological activities
and is used for the treatment of hepatitis and cirrhosis [2],
bacterial infection and hemorrhagic condition [11]. It also exhibits
trypsin inhibitory effect [12], suppresses LPS-induced caspase-11
expression in cultured cells by directly inhibiting the IKK complex
[13]. It is occasionally used in other infectious diseases of liver
[14].
Diuretic, hypotensive and hypocholesterolemic effect of E. alba
in mild hypertensive subject were reported by Rangineni V. et al
and some other pharmacological activities reported
neuropharmacological profile of E.alba (Linn.) Hassk [15].
Antihyperglycemic activity of E. alba leaf on alloxan-induced
diabetic rats [16] and analgesic studies on total alkaloids and
alcohol extracts of E. alba [17] have already been documented.
 MD. Nazim Uddin et al. / Int J Biol Med Res. 2010; 1(4): 341-346
Eclalbatin, a triterpene saponin from E. alba, DNA-damaging
steroidal alkaloids from E.alba of suriname rainforest [18],
Chemical constituents of E. alba (L.) Hassk [19], Phenolics and
other constituents from E. alba [20], Ecliptal, a new terthienyl
from E. alba [21] have been isolated.
Although the antibacterial and antioxidant properties of
individual part, especially leaves, as well as the ethnobotanical
profile of E. alba and its related species have been documented
[22], antioxidant and antibacterial properties of whole E. alba
extract are still unexplored. Screening of antifungal properties has
also been studied to some fungal strains like Candida tropicalis,
Rhodotorula glutinis and Candida albicans [23] whereas other
strains of fungi have been incorporated in our research to evaluate
the antifungal activity of the same extract with different
concentration. This research attempts to make an extensive study
on antioxidant (free radical scavenging), antimicrobial
(antibacterial and antifungal) and cytotoxic effect of the whole
plant extract. The study also initiates to determine the minimum
inhibitory concentration and acute toxicity test of the extract
which are the first report on the whole plant extract of E. alba .
2. Materials and Methods
2.1. Plant materials
The plant was collected from Chittagong, Bangladesh in the
month of May-June'2009. The plant was taxonomically identified
by Dr. Shaikh Boktear Uddin (Associate Professor, Department of
Botany, University of Chittagong, Bangladesh).
2.2. Preparation of plant extract
The fresh E. alba were washed with distilled water, minced
into small pieces, air dried at room temperature for about 10 days,
ground into powder (750g) and extracted with ethanol, being
stirred and macerated at room temperature (23±50C) for 15 days .
The ethanol was evaporated under reduced pressure below 500C
through rotatory vaccum evaporator (RE200 Sterling, UK). The
concentrated extract (48g blackish-green) was stored at 40C until
use.
2.3. In vitro assay for antioxidant activity
The antioxidant activity of E. alba extract was assessed in
comparison to standard antioxidant ascorbic acid (Sigma,
Germany) on the basis of scavenging effect of the stable 2,2-
diphenyl-1-picrylhydrazyl (DPPH) free radical according to
established procedure [24]. Standard ascorbic acid solution (1ml)
and different concentrations (10, 50,100,200,400,600 and 800
µg/ml in methanol) of 1ml of E. alba solution were mixed with 3
ml of 0.002% DPPH solution. The mixtures were kept in dark for
30 minutes to measure the absorbance at 517 nm using UV-
Visible Spectrophotometer (UV 3600, Shimadzu Corporation,
Japan). Lower absorbance of the reaction mixture indicated
higher free radical-scavenging activity.
2.4. Assay for cytotoxicity
Cytotoxic activity of plant extract was determined by Brine-
Shrimp lethality bioassay as described [25]. Shrimp eggs were
added to the artificial “sea water” (23g salt per liter water) in the
larger compartment of an unequally divided tank which was
darkened by covering it with Aluminum foil. The chamber was
kept under illumination using a table lamp for 48h for the eggs to
hatch into shrimp larvae. The illuminated compartment attracts
shrimp larvae (nauplii) through perforations in the dam. 20
shrimp larvae were added to 5 ml of sea water in 5 test tubes and
500, 250, 125, 62.5 31.25 and 25 ìg/ml solution of extracts were
added to these test tubes. This procedure repeated three times. A
342
control containing 5 ml of dimethyl sulfoxide (DMSO) solvent was
used for each solvent. The test tubes were maintained under
illumination. After 24 hours, survivors were counted with the aid
of a 3X magnifying glass. The LC50 values were calculated from
Probit Chart using computer software “BioStat-2007”.
2.5. Assay for antibacterial activity
The antibacterial activity of E. alba crude extracts performed
by disc diffusion method. In this method sterilized filter paper
discs were used. Dried and sterilized paper discs were treated
separately with desired concentration of previously prepared
ethanol solution of the crude extract using a micropipette dried in
air under aseptic condition and was placed at equidistance in a
circle on the seeded plate. The concentration of 250 ìg/disc, 500
ìg/disc and 1mg/disc of crude extract were used. These plates
were kept for 4-6 hours at low temperature and the test materials
diffused from disc to the surrounding medium by this time. The
plates were then incubated at 370C for 24 hours.
2.6. Determination of MIC
Minimum inhibitory concentrations (MIC) of crude extracts
of the E. alba were determined by macrodilution method
introduced by Shafiqur Rahman [26]. The crude extract was
dissolved in 30% DMSO to obtain 10% (w/v) solution. For MIC test
of the selected bacteria, the extract was first diluted in sterilized
Mueller-Hington broth to the highest concentration of
m 10,000m g/ml and then dilution were performed at concentration
of 5000 m g, 4000 mg/ml, 3500m g/ml, 2500 /ml, 2000 gm /ml, 1500
gm /ml, 1000 gm /ml, 750 gm /ml, 500 gm /ml and 250 gm /ml in
screw caped tube containing broth medium. Bacterial
suspensions of the test organism were prepared in sterilized
Mueller-Hington broth. Then 1 ml of the dilution was added to
each sterilized screw capped tube containing 1 ml of compound
suitably diluted in the sterilized broth medium to give final volume
of 2 ml. Culture medium free of samples and microorganisms were
used as control in the tests. Tubes were incubated at 350C for 20-
24 hours and growth was indicated by turbidity.
2.7. Assay for antifungal activity
2.7.1. Preparation of fungal inoculum
For fungal inoculums, Potato dextrose agar (PDA) pour plates
were prepared. At the center of these plates 5 days old test fungi
were transferred and incubated at (25±2)0C. After 5 days of
incubation they were ready to use.
2.7.2. Procedure for anti-fungal activity
The poisoned food technique [27] was used to assay anti-
fungal activity. Plant extract was dissolved in ethanol to obtain
definite concentration. From this, required concentration of
extract was taken by sterilized pipette in a sterilized petriplate and
then 15 ml medium was poured into the petriplate and mixed well
and allowed to solidify. Inoculation was done at the center of each
plate with 5 mm mycelium block for each fungus. The mycelium
block was prepared with the help of cork borer from the growing
area of a 5 days old culture of the test fungi on PDA. The blocks
were placed at the center of each petriplate in an inverted position
to get greater contact of the mycelium with the culture medium.
The inoculated plates were incubated at(25+_ 2) 0 C.
The experiment was repeated for three times. Proper control (PDA
without extract) was also maintained. After 5 days of incubation (3
days for Macrophomina phaseolina) the diameter of fungal
colonies were measured. The average of three measurements was
taken as colony diameter of the fungus in mm. The percentage
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343

inhibition of mycelial growth of the test fungus was calculated by Table 1. DPPH free radical scavenging effect of E. alba extract and
the following formula: I= (C-T)/C X 100, Where, I=Percentage of Ascorbic acid (Vit C)
Inhibition, C=Diameter of the fungal colony in control, T=Diameter
Concentration Log conn Absorbance % Scavenging IC50 g/ml
of the fungal colony in treatment (ìg/ml) activity
2.8.Acute toxicity test E.alba Vit C E.alba Vit C E.alba Vit C

Ethanol crude extract of E.alba was injected intraperitoneally Control - 0.6972 0.6972 - -
in mice at various dose levels namely 500 mg/kg, 1g/kg, 2g/kg, 10 1 0.2364 0.2315 66.093 66.796
3gm/kg and 4gm/kg of body weight. Five mice in each dose group
15 1.176 0.2259 0.1557 67.599 77.668
were closely observed for 24 hours for any mortality and next ten
days for any delayed toxic effect. 20 1.3 0.219 0.1184 68.589 83.018
25 1.39 0.1923 0.0683 72.418 90.204 1.34 1.03
3. Results
30 1.48 0.1492 0.0558 78.600 91.997
DPPH free radical scavenging activity of the E. alba extract and
40 1.6 0.0835 0.0191 88.024 97.260
ascorbic acid is shown in Table 1. Both ascorbic acid and extract
50 1.78 0.0782 0.012 88.784 98.279
showed a dose dependent activity. However, extract showed very
strong DPPH free radical scavenging effect in comparison to
Table 2. Brine shrimp lethality of E. alba ethanol extract.
ascorbic acid. Among seven different concentrations used in the
Dose Log Total Alive Death % Lethality Actual % Probit
study (10 to 50ìg/ml) 50ìg/ml showed the highest scavenging ( ìg/ml) Dose
activity 88.784% (Table 1). Percent (%) scavenging activity was
plotted against log concentration and from the graph IC50 25 1.398 20 20 0 0.0 0.01 2.840

(Inhibition concentration 50) value was calculated by linear 31.25 1.495 20 18 2 10.0 0.10 3.729
regression analysis(Table3). IC50 value of ascorbic acid and extract 62.5 1.796 20 12 8 41.7 0.40 4.763
was found 1.03ìg/ml and 1.34ìg/ml respectively (Table 1). 125 2.097 20 12 12 61.7 0.60 5.252

In Brine shrimp lethality bioassay, six different 250 2.398 20 3 17 83.3 0.85 6.011
concentrations (25, 31.25, 62.5, 125, 250 and 500µg/ml) of E. alba 500 2.699 20 0 20 100.0 0.99 7.519
extract were used to determine its cytotoxicity (Table 2). The
extract showed lethality in a dose dependent manner. More Actual percent and probit were calculated using statistical
exclusively, 0%, 10.00%, 41.70%, 61.70%, 83.30% and 100% of software “Biostat 2007
mortality were observed at the extract concentration of 25, 31.25,
Table 3. Calculation of LC50 value, regression equation and
62.5, 125,250 and 500µg/ml, respectively. “BioStat-2007”
confidence limit by probit analysis.
computer software was used to calculate the probits for each
concentration. Log10 LC50 95% confidence Regression Chi square
LC50 (ìg/ml) limit (ìg/ml) equation
Calculated tabulated
1.97 94.3 74.2-121.3 Y = -1.2857 + 3.1834 * X 1.32
Table 4: In vitro antibacterial activity of E. alba extract.

Bacterial type Test organism Diameter of zone of inhibition (mm)

E alba extract
Tetracycline
250 (ìg/disc) 500 ìg/disc) 1mg/disc (30 ìg/disc)

Bacillus subtilis - - 18 30

Staphylococcus aureus - - 16 35
Gram (+)ve Bacillus cereus - - 17 30

Bacillus megaterium - - 16 20
Salmonella typhi - 8 14 28
Salmonella paratyphi - - 18 27
Pseudomonas sp.(1) - - 15 21
Gram (-)ve Pseudomonas sp.(II) - - 15 27
Shigella sonnei - - 17 31
Escherichia coli - - 10 26
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344

Antibacterial activity of E. alba extract was evaluated on four [31, 32]. The quantitative determination of ascorbic acid explored
Gram positive and six Gram-negative bacteria by disc diffusion that high quantity of ascorbic acid was found to be 9.83mg/100g
method using tetracycline (30 ìg/disc) as standard antibiotic disc. [34] in E. alba which plays the key role in showing free radical
Plant extract at the concentration of 250ìg/disc showed no zone scavenging activity of this plant. Moreover, as plant phenolics
of inhibition but at the concentration of 500 ìg/disc showed small constitute one of the major groups of compounds acting as
zone of inhibition against Salmonella typhi. Crude extract at the primary antioxidants or free radical terminators. E.alba extract
concentration of 1mg/disk showed 14, 17, 10, 18, 15, & 15mm contains 30.4mg/1gm [33] of total phenols. The phenols contain
diameter zone of inhibition against Salmonella typhi, Shigella hydroxyls that are responsible for the radical scavenging effect
sonnei, Escherichia coli, Salmonella paratyphi, pseudomonas sp.(I) mainly due to redox properties [34]. The high quantity of ascorbic
& pseudomonas sp.(II), respectively and 18, 17, 16 & 16 mm against acid and phenolic content in E. alba can explain its stronger free
Bacillus subtilis, Bacillus cereus, Bacillus megaterium & radical scavenging activity. This study suggests that the selected
Staphylococcus aureus (Table 4). On the other hand, standard plant extract can be used as a source of antioxidants for
antibiotic tetracycline (30ìg/disc) showed significant pharmacological preparations which is very well evidenced by the
antibacterial activity against all of the tested gram (+)ve and gram present work.
(-)ve bacteria (Table 4).
Brine shrimp lethality is a general bioassay which is indicative
Table 5. MIC of E. alba extract against different bacterial strains of cytotoxicity, antibacterial activities, pesticidal effects and
various pharmacologic actions [35]. Probits are calculated to
Bacterial type Test bacterial stains Source MIC of E. alba delineate the trends of mortality. Actually, the notion of probit was
extract (µg/ml) introduced by Chester Ittner [36] to transfigure the data, such as
Gram (+)ve Bacillus subtilis BTCC 2500 the percentage of a pest killed by a pesticide, into a 'probability
Staphylococcus aureus BTCC 3500
unit' (or probit) through plotting the killed percentages against
the logarithm of the doses to obtain more or less a straight line.
Bacillus cereus BTCC 3500 However, probits in these experiments were plotted against
Bacillus polymyxa BTCC 2500 corresponding log concentration of extract and from the plot LC50
(log concentration 50) value was calculated by regression analysis
Bacillus megaterium BTCC 3500
(Table 3). LC50 value of E. alba ethanol extract was found 94.3
Gram (+)ve Klebsiella sp. ICDDR'B 3500
µg/ml with 95% confidence limit where the lower and upper
Salmonella typhi ICDDR'B 4000 limits were 74.2 and 121.3 µg/ml (Table 3). The LC50 value found in
Shigella flexneri ICDDR'B 4000 this study to be significant (94.3ìg/ml) in suggesting the ethanol
Shigella sonnei ICDDR'B 4000 extract of E. alba has high potentiality to kill cancer cells as well as
pests [35].
Proteus sp. ICDDR'B 5000

Results of antibacterial assay (Table 4) implied that the gram-

The MIC values of the crude extract obtained from E. alba are
summarized in Table 5. The range of MIC values of ethanol extracts
against bacteria was 3000µg to 5000µg. The lowest MIC (2500µg)
was recorded against Bacillus subtilis and the highest MIC (5000
µg) recorded against Proteus sp. E.alba showed a significant
degree of anti-fungal activity described in Table 6. The maximum
anti-mycotic activity 51.52% was shown against Aspergillus
ochraceus.
4.Discussion
Free radicals from oxidative stress are involved in many
disorders like atherosclerosis, angina pectoris,
neurodegenerative diseases and cancer. Antioxidants due to their
scavenging activity are useful for the management of those
diseases. DPPH stable free radical method is a sensitive way to
determine the antioxidant activity of plant extracts [28-29]. The
odd electron in the DPPH free radical gives a strong absorption
maximum at 517 nm and is purple in color. The color turns from
purple to yellow when the odd electron of DPPH radical becomes
paired with hydrogen from a free radical scavenging antioxidant to
form the reduced DPPH-H. The resulting decolorization is
stoichiometric with respect to number of electrons captured. The
therapeutic potential of natural medicinal plants as an antioxidant
in reducing such free radical induced tissue injury, suggests that
many plants have antioxidant activities that can be therapeutically
useful [30].
Ascorbic acid, the standard antioxidant in this study, acts as a
chain breaking scavenging agent that impairs the formation of free
radicals in the process of intracellular substances formation
throughout the body, including collagen, bone matrix and tooth
positive bacteria were more susceptible to the extract than the
gram negative bacteria. This is possibly because of the presence of
their outer membrane that serves as an effective barrier in gram-
negative species [37, 38]. Almost equal diameter of zone of
inhibition both for extract and standard signifies that the test
organisms are sensitive to the plant extract. Moreover,
Staphylococcus aureus and Bacillus subtilis were the most
susceptible gram (+)ve bacteria, an observation that may be
attributed to the presence of a single membrane of the organism
which makes it more accessible to permeation by active principles
of E. alba extract. In contrast, Salmonella typhi and Escherichia coli
showed the least susceptibility to the extract. This may be due to
the fact that these organisms have intrinsic resistance from a
restrictive outer membrane barrier and transenvelope multidrug
resistance pumps (MDRs) [39]. The results of present research
highlight the fact that the organic solvent extracts exhibited
greater antimicrobial activity because the antimicrobial
principles were either polar or non-polar and they were extracted
only through the organic solvent medium [40, 41]. The present
observation suggests that the organic solvent extraction was
suitable to verify the antimicrobial properties of medicinal plants
and they are supported by many investigators [42-44]. The
present study justifies the claimed uses of E. alba in the traditional
system of medicine to treat various infectious diseases caused by
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345
the microbes. The obtained results provide a support to use of the
studied plant in traditional medicine. Based on this, further
chemical and pharmacological investigations to isolate and
identify minor chemical constituents in E. alba and to screen other
potential bioactivities are recommended.
Plant natural compounds are important source of fungi toxic
compounds and they may provide a renewable source of useful
fungicides that can be utilized in anti-mycotics drugs against
infection of Aspergillus ochraceus. The effect of extract against
Aspergillus niger was also higher implying that this plant can be
utilized as anti-mycotics drugs against infection of Aspergillus
niger in patients with pulmonary tuberculosis [45]. No anti-
mycotic effect was found against Aspergillus ustus at the
concentration of 2mg/ml. Fluconazole was used as an standard
antifungal agent to compare the potentials of extract (Table 6).
There are, however, alarming reports of opportunistic fungal
infections [46] which describe that the resistance of the organisms
increased due to indiscriminate use of commercial anti-microbial
drugs commonly used for the treatment of infectious disease. This
situation forced the researchers to search for new anti-microbial
substance from various sources including medicinal plant [46].
Our research findings revealed that medicinal plant E. alba can
play a vital role in combating fungal resistance. In the acute
toxicity assay of ethanol extract of E. alba, no death of mice was
observed at higher (4 g/kg) dose level. The doses did not show any
abnormalities.
Table 6: Antifungal activity of E. alba extract.
Name of Organism % of inhibition
Ethanol extract Fluconazole
2mg 3mg 4mg 100µgm
Aspergillus niger 36.84 38.94 47.36 64.21
Aspergillus ustus - 20 28 40
Aspergillus ochraceus 41.37 44.82 51.52 37.93
5. Conclusion
The results of the study demonstrate that the ethanol extract
of E. alba exhibits very potential antioxidant and cytotoxic effect in
experimental models. The extract also showed moderate
antibacterial and antifungal activity against specific strains of
respective organisms. Other studies of our laboratory pointed that
various secondary metabolites like alkaloids, flavonoids, tannins,
saponins, which are present in this plant, can contribute to these
biological activities of E. alba extract. These can be strong
scientific evidence to use this plant as a useful source of both
biological and pharmacological references. However, further
studies are still necessary to elucidate a mechanistic way how the
plant contributes in these properties. Phytochemical investigation
is also proposed to isolate the active fraction and eventually the
pure compound from this plant.
Acknowledgement
The authors were thankful to Mr. Seikh Aftab Uddin, Assistant
Professor, Institute of Marine Science and Fisheries, University of
Chittagong, Bangladesh, to provide all supports in progress of the
cytotoxic works.
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