Abstract: The resistance of different bacteria to the current antibacterial agents, toxicity of the antibacterial agents and the cost of the treatment has led to the development of new active molecules against the bacteria. Since ancient times medicinal plants have been used for the treatment of bacterial infections. The roots of the plant Inula racemosa has been used as folk medicine in East Asia and Europe. However, no systematic data is available on the antibacterial activity profile of the different constituents of Inula racemosa. In the present studies, attempt has been made for isolation of root constituents of Inula racemosa (Compositae) and evaluation of its antibacterial activity. The constituents were isolated and purified by column chromatography. The structure of the isolated constituents were confirmed by spectral analysis and were used for the determination of the antibacterial activity of Inula racemosa against various microorganisms. The constituent alantolactone showed maximum antibacterial activity as compared to other constituents and ethyl acetate extract of the roots.
INTRODUCTION
Bacterial infections have posed a great challenge in front of us. The emergence of bacterial resistance to the currently available antibacterial agents necessitates the further research in the discovery of new safe and effective antibacterial agents (Davis, 1994). The medicinal plants have shown a promising alternative for the treatment of infectious diseases. In the antibacterial research, the vast majority, 78%, of the new chemical entities are natural or natural product-derived molecules (Newman et al., 2003). Inula racemosa (Compositae) has been used as traditional medicine in East Asia and Europe (Okuda, 1986). In China, Inula racemosa has been prescribed for abdominal pain, acute enteritis and bacillary dysentery (Tsewang, 1994). The roots are widely used as indigenous medicine, as an expectorant and in veterinary medicine as a tonic (Chopra et al., 1956). Native Americans used this plant for treatment of tuberculosis (Moerman, 1986). Inula racemosa blocks the adrenaline induced hyperglycemia, medicinal uses of plant are well documented in the literature (Tripathi and Chaturvedi, 1995). Inula racemosa can be prescribed in combination with guggul (Commiphora Mukul) for curing myocardial ischemia (Singh et al., 1993). The onset of ischemia was significantly delayed when the rabbits were pretreated with Inula racemosa and severity was less pronounced as compared to controls (Dwivedi and Somani, 1988).
Phytochemical investigation of the plant showed the presence of alantolactone, isoalantolactone, dihydroalantolactone, dihydroisoalantolactone, sitisterol, daucosterol, inunolide, aplotaxene, phenylacetonitrile and isoinunal (Wang et al., 2000).
Some of the sesquiterpene lactones of Inula racemosa has the pharmacological activities (Tripathi et al., 1988). Alantolactone and isoalantolactone are the major constituent of Inula racemosa, they possess anti-fungal and antihelmintic activities (Satyawati et al., 1987). The plant thus has a better pharmacological activity profile, therefore systematic investigation of the antibacterial activity of the plant may lead to the development of novel antibacterial agents. Use of currently available antibacterial agents is limited for many reasons such as poor solubility, low potency, emergence of resistant strains and toxicity. Available literature indicates that the constituents of Inula racemosa and the plant extract have not been studied systematically for their antibacterial activities.
Hence, the aim of the present study was to isolate and study the antibacterial activity of the different constituents and ethyl acetate extract of the plant Inula racemosa.
MATERIALS AND METHODS
Plant Material
The dried roots of the Inula racemosa were obtained from local market
of Pune, India. The plant material was authenticated from Botanical Survey of
India, Pune, India.
Chemicals
n-Hexane, ethyl acetate, methanol, chloroform, benzene, toluene and dimethyl
formamide were purchased from Merck India Ltd., Mumbai, India. Nutrient agar
was purchased from Hi-media, Mumbai, India. Streptomycin (Nicholas, India) was
purchased from the local market of Pune, India. Silica gel chromatographic grade
for the separation was obtained from Rankem, India. All other chemicals used
were of high purity.
Bacterial Strains
Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus,
Klebsiella pneumoniae, Salmonella typhi, Serratia marcescens,
Shigella dysenteriae and Staphylococcus aureus were obtained from
the Microbiology Department, University of Pune, Pune, India. All the strains
were maintained on nutrient agar medium.
General Experimental Procedure
Melting points were determined in a open capillary (Gallenhamp melting point
apparatus) and were uncorrected. IR spectra was recorded on Shimadzu 8400 FT
infrared spectrometer. 1H NMR was recorded on Varian-Mercury (300
MHZ) FT NMR spectrophotometer with chemical shift data reported in ppm. Mass
spectra were recorded on GC-MS Shimadzu (QP 5050) Mass spectrophotometer.
Extraction and Isolation
The dried and hard roots were first chopped into small pieces and crushed in
mortar pestle. This crushed material was further grinded in blender to make
a fine powder. The root powder was soaked in different solvents, like n-hexane,
ethyl acetate, methanol, chloroform, benzene, toluene, etc. and subjected to
Thin Layer Chromatographic (TLC) analysis in various solvents The TLC plates
were observed under UV as well as in iodine chamber. Ethyl acetate extract has
shown the presence of maximum spots on TLC plate and hence has been selected
for the extraction of the root constituents of the plant.
The dried roots (200 g) of Inula racemosa were extracted with ethyl acetate at room temperature for 48 h. This extract was then filtered and evaporated under reduced pressure to obtain a viscous mass (6 g). Some quantity of ethyl acetate extract was preserved for antibacterial activity and remaining extract was chromatographed over silica gel using hexane as eluent (Tan et al., 1998). A total of twenty fractions were (50 mL each) collected. Major constituents were identified by using spectral techniques like IR, NMR and GC-MS spectroscopy. The extraction of the plant resulted in the isolation of three known constituents. The isolated constituents were then subjected to the evaluation of their antibacterial activity.
Antibacterial Activity
Antibacterial activity of the isolated constituents and the crude extract was
determined by the well diffusion method (Rios et al., 1988; Mosquera
et al., 2004). The test constituents and the ethyl acetate extract were
dissolved in dimethyl formamide (DMF). The microbial cultures were grown at
37°C for 24 h and then approximately diluted by 0.9% w/v sterile saline
solution to obtain a cell suspension of 105 cfu mL-1.
Diluted inoculum (0.2 mL-1, 105 cfu mL-1) of
test microorganisms were spread on nutrient agar plates. Wells of 6 mm diameter
were punched into the agar medium and filled with 20 μL each of three isolated
constituents, ethyl acetate extract at a concentration of 10 mg mL-1.
The plates were incubated for 18-24 h at 37°C. The antibacterial activity
was evaluated by measuring the zone of inhibition. The antibiotic streptomycin
at 100 μg mL-1 was used in the test system as positive control.
The measured Zone of Inhibition (ZOI) was noted. The average of the zone of
inhibition was obtained from three replicates of the isolated constituents as
well as ethyl acetate extract.
Minimum Inhibitory Concentration Assay (MIC)
The Minimum Inhibitory Concentration (MIC) values were determined only with
those bacteria that showed inhibitory zones greater than 20 mm (Demo et al.,
2005). B. cereus and P. aeruginosa have shown the maximum (>20
mm) zones hence have been selected for MIC assay. The dilutions 5, 3, 2, 1 and
0.1 mg mL-1 were prepared in DMF. The assay was carried out and zone
of inhibition was measured. The MIC values were determined as the lowest concentration
of the constituent which completely inhibited the growth (Mazzanti et al.,
2000).
RESULTS AND DISCUSSION
Spectral data showed that the major constituents structurally resembled to
that of alantolactone, isoalantolactone, and dihydroalantolactone. The plant
Inula racemosa represent a broad spectrum of activity. All the isolated
constituents and ethyl acetate extract inhibited the growth of bacteria at effective
concentration of 200 μg well-1 (10 mg mL-1). Ethyl
acetate extract was found to show inhibition against all the tested microorganisms
except Serratia marcescens and Shigella dysenteriae. The isolated
constituents of the plant showed moderate to high activity as antibacterial
agent with two Gram positive and six Gram negative bacteria. The results of
the antibacterial activity are reported in Table 1.
| Table 1: | Antibacterial activity of the isolated constituents from the root of Inula racemosa |
| Maximum inhibition >20 mm, Moderate inhibition 16-20 mm, Poor inhibition 10-15 mm | |
The data analysis in Table 1 indicates that the tested constituents, ethyl acetate extract showed the significant results when compared with the standard streptomycin while alantolactone shown maximum inhibition of Pseudomonas aeruginosa and Bacillus cereus and moderate inhibition against the remaining tested bacteria. Of all the constituents of Inula racemosa the alantolactone had shown moderate inhibition of Shigella dysenteriae. In the earlier reports Inula racemosa has been used in China against bacillary dysenteriae (Tsewang, 1994). As our results suggest that the ethyl acetate extract of the plant has poor activity than the purified alantolactone constituents, which indicates the effectiveness of alantolactone in bacillary dysenteriae.
Isoalantolactone shows moderate inhibition against two bacteria Pseudomonas aeruginosa and Bacillus cereus and was found to show poor inhibition with the remaining bacteria. Dihydroalantolactone has shown poor inhibition against all the tested bacteria. The MIC values of alantolactone for Bacillus cereus and Pseudomonas aeruginosa was found to be 100 μg mL-1. These values shows the potential of the isolated constituents in low concentration which is comparable with the standard antibiotics. The data indicates that among the isolated constituents, alantolactone shows better activity profile as compare to the isoalantolactone, dihydroalantolactone and ethyl acetate extract.
The antibacterial activity profile of the isolated constituents when compared with the ethyl acetate extract shows that the activity depends on the pure form of the constituents. The results indicate that the degree of antibacterial activity is based on the varying chemical nature of the isolated constituents. The structural specificity and stereochemical aspects of the constituents might be playing a vital role in the better activity profile. The resistance of the bacteria to the current antibiotics necessitate the further studies on the isolated constituents to find out their safety and efficacy profile.
CONCLUSION
The present systematic investigation of the antibacterial activity of the constituents of Inula Racemosa have lead to the identification of more active antibacterial agents. The isolated sesquiterpene lactones represents a broad spectrum of activity against Gram positive and Gram negative bacteria. In the present study, the antibacterial activity profile of alantolactone against Shigella dysenteriae indicates its use in bacillary dysenteriae. The detail investigation of the alantolactone for the exact mode of action will be a major step in developing a novel therapeutic molecule for the antibacterial discovery.