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Ethnobotanical
Leaflets 14: 248-58, 2010. Phytochemical Screening of Methanolic Extract and
Antibacterial Activity of Active Principles of Hepatoprotective
Herb, Eclipta alba Sunita Dalala*, Sudhir K
Katariab, KV Sastryb and SVS Ranac aDepartment of
Biotechnology, Kurukshetra University, Kurukshetra-136 119, Haryana, India
bDepartment of Zoology(Bioscience), Maharshi Dayanand University,
Rohtak-124 001, Haryana, India cDepartment of Environmental Sciences, *Corresponding author: sunitadalal11@rediffmail.com Issued: March 01, 2010 Abstract Aerial parts of Eclipta alba are used traditionally for the treatment of several diseases of liver, skin and stomach. Methanolic extract and active principle compound of a well known Indian hepatoprotective herb, Eclipta alba was tested for in vitro antimicrobial studies. It was evaluated using zone of inhibition studies and minimum inhibitory concentration. The extract exhibited activity against all six strains studied. Phytochemical screening of the extract revealed the presence of tannins, flavonoids, coumestans, saponins and alkaloids etc. Ethylacetate fraction and further pure isolated wedelolactone showed enhanced antimicrobial activity. Staphylococcus epidermidis, Staphylococcus aureus and Salmonella typhimurium were most susceptible. Shigella flexneri was the most resistant bacterial strain. These results suggest coumestans/wedelolactone as a promising antimicrobial agent. Key words: Eclipta alba, wedelolactone, antimicrobial activity. Introduction Modern
medicine has evolved from folk medicine and traditional system only after
thorough chemical and pharmaceutical screening. The use of synthetic
compounds led to a decline in the use of plants in modern medicine. However,
synthetic medicine can cause side effects and as a result people are more
favorable to use natural compounds obtained from plants. Thus, plants remain
a major source of medicinal compounds. About 20,000 plant species are used
for medicinal purposes (Penos, 1983). Seventy four percent of 119
plant-derived drugs were discovered as a result of chemical studies to
isolate the active substances responsible for their traditional use
(Farnsworth and Soejarto, 1991). So plants, especially the higher
plants contain a variety of substances, which are useful as food additives,
perfumes, and in treatment of various diseases as medicine due to their
versatile therapeutic potential (Mukherjee and Wahile, 2006). The active
secondary metabolites possess various medicinal applications as drugs or as
model compounds for drug synthesis. Phytochemical analysis of plants, used in
folklore has yielded a number of compounds with various pharmacological
activities. In view of the increasing development of resistant
microorganisms, treatment of various diseases caused by microorganisms has
become a major challenge in the human medical field. This may be due on the
one hand, to the synthetic nature of these substances, but also to their
known side effects and in some cases to their unpleasant smell, taste or the
burning sensation felt on the skin. Medicinal plants are important substances
for the study of their traditional uses through the verification of
pharmacological effects and can be natural composite sources that act as new
anti-infectious agents. About 3,000 materials from 2,764 plant species have
been screened for their pharmacological and chemotherapeutic properties (Anon,
1988). Various biological
activities are possessed by E. alba,
such as memory disorders treatment, general tonic,
edema, fevers and rheumatic joint pains treatment, digestion, hepatitis,
enlarged spleen, antioxidant activity and skin disorders (Chopra et al.,
1956; Karnick and Kulkarni, 1990; Karthikumar et al., 2007). Wedelolactone is active principle compound of
this liver disorder treating drug (Wagner et al., 1986). It also
exhibits Trypsin inhibitory effect (Samiulla et al., 2003; Syed et
al., 2003), suppresses LPS-induced caspase-11 expression in cultured
cells by directly inhibiting the IKK complex (Kobori et al., 2004),
treatment of cirrhosis of the liver and infectious hepatitis (Murphy et
al., 1979). The shoot extract of E. alba showed antimicrobial
(Anonymous 1952; Kosuge et al., 1985; Wiart et al., 2004),
antifungal activity (Venkatesan and Ravi, 2004) and weak cytotoxicity against
the M-109 cell lines by alkaloids Verazine (Abdal Kadar et al., 1998),
antiviral activity against Ranikhet disease virus
(Khin et al., 1978), effective against internal and external parasites
(Lans et al., 2001) G.
intestinalis (Sawangjaroen et al.,
2005), antibacterial (Kumar et al., 2007). Since E. alba is a
weed /herb growing in dump, moist puddles distributed in the tropical and
subtropical regions of the world. So besides ethnobotanical evidence, it can
be hypothesized that plants which survive in media rich in microbes most
likely be possessing antimicrobial principles. However, up to date, research has been done to investigate various pharmacological activities and antimicrobial activity of only crude extracts of this traditionally used herb. We report here our findings on antibacterial effects of wedelolactone (Fig. 1), the principle active compound, extracted from E. alba.
Figure 1. Chemical structure of Wedelolactone (R1-OH, R2-CH3). Materials and Methods
Plants of E. alba were collected locally from botanical
garden and surroundings of Qualitative
estimation of primary and secondary plant metabolites All estimations were done following Hunda et al. (1985); Brindha et al. (1981).Different aerial parts of the plant were dried at room temperature, powered, and extracted with methanol (70% v/v) in Soxhlet apparatus for six hours. The extract was filtered and was tested with different alkaloid reagents. Methanol extract The three months old 950 gm lyophilized leaves
were Soxhlet extracted with methanol for 36 h. Ethyl acetate fraction The methanol was removed from
extract and the residues were suspended in water separately and heated on
steam bath below 80 °C for 30 min. After filtration, the aqueous phase was
partitioned with ethyl acetate. The organic phase was dried, filtered and the
solvents were evaporated to yield 6.8 gm light brown powder. Isolation
of Wedelolactone The powder was subjected to fractionation by column chromatography on
silica gel, eluted with the solvent of increased polarity i.e. Non-polar - polar
- highly polar. The coumestans are polar compounds so the solvent combination
found suitable for their elution was Chloroform + Methanol (70 + 30). They
were eluted simultaneously in 37 to 48 fractions. The pooled sample was then
subjected to TLC, the solvent system (Toluene : Acetone : Formic acid :: 11 :
6 : 1 v/v) showed two spots with Rf values 0.39 and 0.28 which
matched with the Rf values of reference wedelolactone and
demethylwedelolactone respectively (Courtesy M/s Natural Remedies, Bangalore,
India). The purified sample of wedelolactone
was put to HPLC for further qualitative analysis using instrument - Thermo Finnigan from Thermo Electron Corp.
Figure 2. The chromatogram of extract showing the peak. Preparation of samples for testing The zone
of inhibition studies were conducted with various extracts diluted with 10%
dimethylsulfoxide (DMSO). Micro organisms Standardized strains
from the American/Microbial type culture collection were used in bioassays. Staphylococcus aureus (ATCC 9144), Salmonella typhimurium (ATCC
23564), Escherichia coli (ATCC 10536), Staphylococcus epidermidis (ATCC 155), Shigella flexneri (ATCC 29508) and Pseudomonas aeruginosa (ATCC 25668) were cultured at 37 °C on nutrient medium in aerobic conditions for
24 h. These bacteria were obtained from the Antimicrobial susceptibility testing MIC of wedelolactone was determined by microdilution technique as described by the National Committee for Clinical Laboratories standards (2000). The MIC was defined as the lowest concentration of the compound to inhibit the growth of microorganism. The bacteria inoculums were prepared in 5 ml nutrient broth and incubated at 37°C. The final inoculums were of approximately 5 x 106 CFU/ml. Controls with 0.5 ml of culture medium with out the samples and other without microorganisms were used in the tests. Tubes were incubated at 37°C for 24 h. The activity was measured as a function of turbidity at 660 nm. Lack of turbidity was further confirmed by pouring suspension aliquot of 0.1 ml into pre-sterilized Petri dishes with nutrient agar medium. The tests were conducted in triplicate. Agar well
diffusion method was carried out by allowing perforation of extract and
wedelolactone dissolved in DMSO at a concentration of 3.5mg/well and 10 mg/ml
respectively. Petriplate containing 30 ml nutrient agar medium were kept for
the solidification before inoculating the microorganism, desired numbers of
holes of uniform diameter of 8mm were made after solidification, using
sterile aluminum borer. 0.2 ml of compound, positive (Gentamycin) and
negative (solvent blank) controls were poured into wells. After incubation
for 24 h at 37 °C
the plates were observed and the compound activity was evaluated by measuring
zone of inhibition (diameter mm). The tests were conducted in triplicate.
Gentamycin (10.0 µg/ml) was used as positive control. The negative control
was 10% DMSO. Results The results of the presence of various primary and secondary metabolites
in methanol extract (Table 1) reported negative for antraquinones throughout
herb. Methanol extract and Ethyl
acetate fraction showed positive signs of antimicrobial activity against all
six strains (Table 2). Wedelolactone exhibited significant antibacterial
activity against the six tested strains (Table 3). S. epidermidis and S
aureus were found to be highly sensitive. The MIC of S. epidermidis, S.
typhimurium, S. aureus, P. aeruginosa, S. flexneri and E. coli was, 15 µg/ml, 25 µg/ml,
20µg/ml, 1250 µg/ml ,1300 µg/ml and 1000 µg/ml respectively. The compound
showed the highest antibacterial activity (ZOI) in S. epidermidis (10.24 mm), followed by S.
typhimurium (9.16 mm), S. aureus(9.14 mm),E.coli (8.60mm) P. aeruginosa (8.00 mm)
and S. flexneri (7.60 mm) as zone of inhibition in
radius. Table 1. Quantitative estimation of the various primary and secondary metabolites.
Table
2. Antibacterial activity
of Methanol extract and Ethyl
acetate fraction against bacterial strains.
Discussion The primary and secondary metabolites were analyzed in methanolic extracts. Anthraquinones were found to be absent in the natural plant. While the alkaloids and reducing sugars were absent in root extract of the natural plant. In an earlier study, the extracts of the leaves of E. alba tested positive for steroid, reducing sugars, alkaloids, phenolics, saponins and tannins, but no anthraquinones and flavonoids were detected (Gopalakrishan and Solomon, 1992). In Gujrat and Table 3. Antibacterial activity of wedelolactone and gentamycin against bacterial strains.
MIC: minimum inhibitory concentration; ZOI: zone of inhibition (Diameter); A: wedelolactone (10.0 mg/ml); B: gentamycin (10.0 µg/ml); Values are mean of three replicates. Karthikumar et al., (2007) reported in vitro antimicrobial activities of ethanolic extract of E. prostrate. It indicated good activity against S aureus 7.2mm (ZOI) and MIC 70µl/ml, and for P. aeruginosa 8.8mm (ZOI) and MIC 65µl/ml, at 50 µg concentration. While the present studies exhibited respective ZOI at 9.14 mm, 8.00mm and MIC 20 µg/ml and 1250 µg/ml respectively. Traditional reports on E. alba indicate that it is one of the herb used for treatment of stomach and digestion disorders, skin diseases and conjunctivitis (Poonam and Singh, 2002).Since S. typhimurium, S.flexneri and S. aureus are pathogens responsible for stomach disorders ,while P.aeruginosa is common in skin flora and S. aureus is responsible for most common bacterial conjunctivitis. The results from the current studies revealed that the wedelolactone could be the main constituent responsible for these treatments as it exhibited good activity against them. Conclusion On the basis of the antibacterial studies of wedelolactone,
it can be suggested that this can be used effectively to treat S.
epidermidis and S. typhimurium
infections. However, the compound must be studied in animal models to
determine the efficacy in vivo against these pathogens and to
elucidate their mechanism of action. In
vivo data may be helpful in determining the real potential usefulness of
this plant for the treatment of infectious diseases. Acknowledgements
The authors wish to thanks Dr. T Velpandian, Associate
Professor Ocular Pharmacology Laboratory at All India Institute of Medical
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