Isolation of antimicrobial compound from Sphaeranthus indicus against human pathogens.
Traditionally used medicinal plants were subjected to preliminary antimicrobial activities against several pathogenic and opportunistic microorganisms. It is estimated that there are above 250,000 to 500,000 species of plants on earth. Relatively small percentage i.e. 1 to 10% of plants was used as foods by both humans and other animal species. It is possible that even more plants can be used for medicinal purpose (Schultes, 1978).
The presence of inherent antimicrobial compounds in several higher plants has long been recognized as important factor for disease resistance. Such compounds being degradable and selective in their toxicity are considered as valuable ingredients for controlling plant diseases caused by viruses (Balasaraswathi, 1995), bacteria (Akhtar et al., 1997) and fungi (Bindu et al., 1998).
There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action because there has been an alarming increase in the incidence of new and re-emerging infectious diseases. Another big concern is the development of resistance to the antibiotics in current clinical use (Erturk et al., 2006). In addition, the allopathic medicines are not curing all the human diseases. There are diseases like jaundice, cancer, paralysis, diabetics, skin diseases etc. which are not curable by the allopathic medicines. Hence, the perception of medicare, turned around towards the naturally grown, traditional medicinal plants and also attracted the attention of the Governments in the world. Pharmacological and phytochemical insights into several plants that were similarly used in many countries have led either to the isolation of novel structures for the manufacture of new drugs or to templates that served for the production of synthetically improved therapeutic agent (Geyid et al, 2005). The search for new molecules, nowadays, has taken a slightly different route where the science of ethnobotany and ethnopharmacognosy are being used as guide to lead the chemist towards different sources and classes of compounds (Gurib-Fakim, 2006).
The use of various parts of several plants of medicinal value to cure specific ailments is in vogue from ancient times in our indigenous medicine system. We are fortunate enough to have a rich heritage in the form of information available from tribal areas, gathered by tribal through experience for generations, in the form of folklore medicine. Sometimes, the knowledge on the very rare medicinal plants for controlling some dreaded human diseases and also for other aspects is being kept as a family secret by Siddha, Ayurveda and other medical practitioners. This type of practice vanishes abruptly after certain period of time further giving any benefit to the mankind. Therefore, valuable and in depth knowledge about the medicinal plants in India gathered over a long period of time need to be exploited scientifically to the maximum extent. Medicinal plants are the most exclusive source of life saving drugs for the majority of the world's population. Bioactive compounds currently extracted from plants are used as medicines; many of the plant species that proved medicinal herbs have been scientifically evaluated for their possible medical applications (Patwardhan et al., 2004).
This paper deals with Isolation of effective antimicrobial compound from Sphaeranthus indicus against human pathogens Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, klebsiella sp., Candida albicans and Cryptococcus neoformans.
Materials and Methods
Plant sample and preparation of plant extracts
The plant sample Sphaeranthus indicus was collected from in and around Coimbatore, Tamil Nadu, India. Dried and powdered leaves of S. indicus were extracted by percolation with ethanol (polar solvent), chloroform (medium polar solvent) and petroleum ether (non polar solvent) at the rate of 1:5 at room temperature for overnight (Geyid et al., 2005). The extracts were then filtered with country filter paper and concentrated under vacuum in rotary evaporator to give (as a percentage of powdered plant materials) 6-11% gummy residue. All the extracts were kept in tightly stoppard bottle in a refrigerator until used for the anti-microbial testing.
The pathogens (bacteria and fungi) used in the study was obtained from the PSG hospital. The pathogens used for the study were maintained in respective slants under refrigerated condition (4[degrees]C). The cultures were frequently subcultured in fresh slants and stored for further study.
Agar well diffusion assay
The sterilized Nutrient agar medium for bacteria and PDA medium for fungi was poured into the petri plates and allowed to solidify. Then each Petri plate was divided into four equal quarters using a marker pen. Using a sterile cork borer, wells of 6 mm in diameter were made in each quadrat of the plate containing the media. For each organism, 20[micro]l (2 mg of extract concentration) of the prepared plant sample was loaded in each well using sterilized dropping pipette. Three replicates were maintained for each treatment. For each microorganism, the positive control (Ketoconazole) and the negative control (100% ethanol) (three replications each) were also loaded in separate wells. The plates were incubated for 24 hrs and the observations were recorded. The zone of inhibition (or halo like area) was measured. The Diameter of Inhibition Zone (DIZ) was measured and the mean D1Z was calculated (Iqbal et al., 1998).
Separation of antimicrobial compounds through Primary Thin Layer Chromatography
Different solvent systems (ranging from low polar to high polar) were tested for the effective separation of antimicrobial compounds from chloroform extract of S. indicus. A suitable mobile phase was standardized based on the separation of antimicrobial compounds. The mobile phase was poured in the TLC tank and the spotted TLC plate was kept in this with approximately 0.5 mm immersed in solvent at the bottom. The tank was closed with a glass lid so as to have the chamber completely filled with the solvent vapors. The plates was kept in the TLC tank till the solvent front reached the top of TLC plate. Then, the plate was removed from the tank and kept in open air at room temperature so as to enable the solvent to get evaporated.
The TLC run plates were observed under bright light and the separated spots were marked. The Relative front values were calculated by using the given formula
Rf value = Distance moved by the solute from the origin/ Distance moved by the solvent from the origin
Those bands which are UV visible are observed under UV transilluminator and their Rf Value observed separately.
Agar well diffusion assay for the separated Bands from Primary TLC
Assay was performed similar to the extract, in order to identify which Band have good antimicrobial activity.
Secondary TLC separation and identifying the active secondary Band
The secondary TLC was carried out to partially purify the active band. The Bands which separated in the secondary TLC were subjected to agar well diffusion assay and the secondary active band was identified.
Identification of compounds using GC-MS
GC-MS analysis was carried out by using Perkin Elmer--Clarus 500 GC-MS unit. The analysis was carried out to detect the possible compounds present in the active fractions of secondary band S7 .The column type used was PE-5 (equivalent to DB-5) with a column length of 30m using Carrier gas as Helium .The Flow rate maintained was 1 ml/min with an initial Column temperature of 100[degrees] C and final temperature of 270 [degrees] C. The rate of temperature change in the column was maintained as 5[degrees] C/min. 1 [micro]l volume of sample was taken for injection.
Results and Discussion
The chloroform extract of S .indicus exhibited comparably more activity against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella sp., Candida albicans and Cryptococcus neoformans than the ethanol and petroleum ether extract (Fig 1). The Diameter of Inhibition Zone (DIZ) produced by the chloroform extract was comparable/equal to that of the positive control Chloramphenicol (Bacteria) and Ketaconazole for fungi (Table 1).
EE- Ethanol extract, CE-Chloroform extract, PE-Petroleum ether, negative control-Ethanol, Positive control- Chloramphenicol 10 mg/ml (Bacteria), Ketoconazole 10mg/ ml (Fungi),(-)-No zone of inhibition
Thin Layer Chromatography
In the thin layer chromatographic separation a total of 8 bands were separated using dichloromethane as mobile phase, of which 4 bands were visible and four bands were invisible , which were visualized by placing the plate under UV trans illuminator.(Fig 2).Among the separated bands, Band 7 was active against the pathogens. Band 7 separated into seven secondary bands (5 visible and 2 UV absorbing) using petroleum ether: ethyl acetate (85: 15) as mobile phase which were visualized under UV trans illuminator (Fig 3) . The secondary band S7 showed potent antimicrobial activity when compared to other bands.(Table 2, Fig 4)
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
The secondary Band S7 showed three major peaks in GC-MS analysis (Fig 5).The first peak observed has the retention time of 19.77 min with molecular weight 188. Library match obtained for this compound was isoprenyl-7-(2 methyl-1-properyl)tricyclo heptan. The second peak observed has the retention time of 20.17 min with molecular weight 248.Library match obtained was 7-hydroxyfrullanolide (Fig 6). The thrid peak observed has the retention time of 20.23 min with molecular weight 204.
[FIGURE 5 OMITTED]
[FIGURE 6 OMITTED]
The herb is reported to be useful as a tonic to treat indigestion, asthma, leucoderma and dysentery (Chopra et al. 1956). A novel isoflavone glycoside from leaves (Yadava and Kumar 1999) and a new sesquiterpene glycoside and sphaeranthanolide were isolated from the flowers of S. indicus and it was found to be an immune stimulant (Shekhani et al. 1990). Medicinal information from tribal healers indicated that S. indicus is used to treat skin disease, cough and fever. The bark, ground and mixed with whey, is said to be useful in treating piles.
In the present study the chloroform extract of leaves showed potent activity against the selected human pathogens and the Thin layer chromatographic separation resulted in a active ingredient 7-hydroxyfrullanolide. This compound has been isolated from Sphaeranthus indicus flower part and it is characterized as sesquiterpenes (Rahman et al., 1989).Since this herb has wide range of medicinal value, this compound can be analysed for treatment of other diseases like skin diseases, cancer etc. and a herbal drug can be produced .
In the conclusion , the results from this study reveals that 7-hydroxyfrullanolide extracted from the chloroform extract of the leaves portion of S.indicus exhibit potent antimicrobial activity against the selected pathogens and this compound can be formulated for the treatment of various diseases caused by these selected pathogens.
This research was financially supported by SRM University and M/s BIOFARM , Coimbatore and is gratefully acknowledged.
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Research Scholar, Dept. of Biotechnology, SRM University, Kattankulathur, India
Table 1: Diameter of inhibition zone of sphaeranthus indicus extract pathogens. Mean Diameter of inhibition zone Pathogens EE CE PE Negative Positive control control Escherichia coli -- 2.0 0.5 -- 2.4 Pseudomonas aeuroginosa 1 2.2 1 -- 3 Salmonella typhi -- 2.1 -- -- 3 Klebsiella sp. 1 2.0 1 -- 2.9 Candida albicans 1.2 3.0 2 -- 3 Cryptococcus neoformans 1.8 3.0 -- -- 2.9 Table 2: Diameter Of Inhibition Zone Of Secondary Bands Against Human Pathogens. Mean Diameter of inhibition zone in cm Escherichia Pseudomonas Salmonella coli aeruginosa typhi S1. Bands No. 1 Uv Band S1 - - - 2 Band S2 1 - 1.5 3 Uv Band S3 - - - 4 Uv Band S4 - 2 - 5 Band S5 - - - 6 Band S6 - - - 7 Band S7 2.1 1.5 1.4 8 Negative - - - Control 9 Positive 2.4 3 3 Control Mean Diameter of inhibition zone in cm Klebsiella Candida Cryptococcus sp. albicans neoformans S1. Bands No. 1 Uv Band S1 - - - 2 Band S2 - 1.5 - 3 Uv Band S3 - - - 4 Uv Band S4 3 G G 5 Band S5 - - - 6 Band S6 - - - 7 Band S7 1.4 3.5 3.6 8 Negative - - - Control 9 Positive 2.9 3 2.9 Control Negative control-Ethanol, Positive control- Chloramphenicol 10 mg/ml (Bacteria), Ketoconazole 10mg/ml (Fungi), (-)-No zone of inhibition
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|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Oct 1, 2010|
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