An investigation was carried out to study the antibacterial activity of Sphaeranthus indicus from leaf, stem and root extracts by the sequential cold maceration method against selected laboratory bacterial pathogens such as Bacillus subtilis , Staphylococcus aureus , Klebsiella pneumoniae , Escherichia coli and Pseudomonas putida by the agar well diffusion method. Zone of inhibition measured (mm) was compared with standard antibiotics such as Tetracycline, Erythromycin and Ampicillin. The organic solvents such as ethanol, methanol, petroleum ether, chloroform as well as distilled water extracts were employed. Among all the extracts, tested ethanolic leaf extracts have showed more antibacterial activity against Klebsiella pneumoniae . Phytochemical screening methods were also done to identify the major secondary metabolites in the present species such as alkaloids, flavonoids, phenols, steroids and tannins. This study concluded that Sphaeranthus indicus had the sufficient antibacterial activity due to the presence of various secondary metabolites.
Sphaeranthus indicus (Linn.) is one of the important herbaceous medicinal plants belonging to the family Asteraceae. It is commonly known as “Boddasoram” in Telugu and “East Indian globe thistle” in English [
Phytochemical investigation of plants is an interesting area of research, leading to the isolation of several novel compounds. The therapeutic value and phar- macological action of a drug was due to the presence of certain chemical constituents such as various forms of glycosides, tannins, phenolic compounds, lipids, fixed oils and volatile oils, resins, various kinds of alkaloids gums, mucilages, pectin etc. These phytochemicals are of immense importance to mankind [
The plant material of S. indicus (Linn.) for this experiment was collected from the Herbal garden of Dravidian University, Kuppam, Andhra Pradesh, India, and also from the fields in the surroundings of the Dravidian University campus.
The plants were collected from the field and initially rinsed with distilled water to free from soil particles and dried on paper towel under shade for one week and stored in airtight containers at room temperature. The dried leaves were coarsely powdered in a blender before subjecting for extraction.
The plant extracts were prepared by sequential cold maceration method using ethanol, methanol, petroleum ether, chloroform and distilled water as a solvent
[
Phytochemical analysis of all the evaporated solvent extracts was conducted following the procedure of Indian pharmacopoeia [
200 mg of plant material was dissolved in 10 ml of methanol and filtered. For 2 ml filtrate and 1% HCl + steam, 1 ml filtrate + 6 drops Mayer’s reagent/Wagner’s reagent/Dragendorff’s reagent was added. Creamish precipitate/brownish red precipitate/orange precipitate indicated the presence of respective alkaloids.
200 mg of plant material was dissolved in 10 ml of distilled water and filtered. For 2 ml filtrate + 2 ml FeCl3 was added. Blue/black precipitate indicated the presence of tannins.
200 mg of plant material was dissolved in 10 ml of ethanol and filtered. For 2 ml filtrate + conc. HCl + magnesium was added. Ribbon pink/tomato red color indicated the presence of flavonoids.
(Liebermann-Burchard reaction)
200 mg of plant material was dissolved 10 ml of chloroform and filtered. For 2 ml of filtrate + 2 ml of acetic anhydride + conc. H2SO4 was added. Blue/green ring indicated the presence of steroids.
1 ml of each solvent extracts dissolved in alcohol/water was separately treated with 1 ml of neutral FeCl3. The change in colour indicated the presence of phenols.
The test bacterial strains used for the study were B. subtilis, S. aereus, P. putida, E. coli and K. pnemoniae. Specific growth conditions were maintained. The bacteria used for the study were obtained from Department of Microbiology, Sri Venkaterwara University, Tirupati. All the cultures were maintained at 4˚C in nutrient agar slants.
A loop full of the test culture was taken from respective strains of agar slants and sub cultured in to fresh tubes containing nutrient broth and incubated for overnight at 37˚C. The obtained cultures was centrifuged at 5000 rpm for 15 min. Bacterial suspension was added to fresh media which gives final concentration of 107 cfu /ml [
The antibacterial activity was tested by agar well diffusion method [
The minimum inhibitory concentration (MIC) was determined by macro broth dilution method [
Preliminary investigation of phytochemical analysis of selected crude extracts revealed the presence of various compounds such as alkaloids, flavonoids, phenols, tannins and steroids. Alkaloids, flavonoids detected in the extracts are compounds that have been documented to possess a variety of medicinal properties and health promoting effects. Phenolics are the largest group of phytochemicals and have been said to account for most of the antioxidant activity of plant extract. These classes (such as alkaloids, phenols, tannins, steroids and flavonoids) of compounds are known to have curative activity against several pathogens and therefore could suggest the use traditionally for the treatment of various illnesses [
The most of the phytochemicals classified as secondary metabolites are produce mainly by the shoot part of the plant, often their function in the plant is unknown, but certain phytochemiclas have structural, functional and general defence against plant pathogens so the preliminary phytochemical studies received pronounced importance, because the crude drugs posses varied composi-
Type of extract | Alkaloids | Flavonoids | Phenols | Steroids | Tannins |
---|---|---|---|---|---|
LEAF | |||||
Ethanol | ++++ | ++ | +++ | ++ | +++ |
Methanol | +++ | ++ | ++ | + | ++ |
Chloroform | + | + | ++ | - | ++ |
Petroleum ether | +++ | - | +++ | + | +++ |
Aqueous | ++ | - | ++ | - | - |
STEM | |||||
Ethanol | +++ | + | ++ | - | +++ |
Methanol | ++++ | ++ | + | ++ | ++ |
Chloroform | ++ | - | ++ | + | + |
Petroleum ether | +++ | + | ++ | ++ | - |
Aqueous | + | - | - | - | - |
ROOT | |||||
Ethanol | +++ | ++ | ++ | ++ | ++ |
Methanol | ++ | ++ | + | ++ | ++ |
Chloroform | - | + | - | + | - |
Petroleum ether | + | - | + | + | ++ |
Aqueous | - | - | + | - | - |
tion of secondary metabolites [
In India, 70% of its population resides in villages. In spite of the accessibility to western medicine, people in these villages still continue to depend on herbal remedies, for treatment of their health problems. Plant species have long been the principal ingredients of traditional medicine and their use dates back to the beginning of human civilization. As compared to synthetic antimicrobial agents, plant based antimicrobials are cost effective, affordable and exhibit lesser side effects. As microbes are rapidly evolving their defence mechanism, so does the resistance develops against many of the antibiotics which were once effective. The search for new antimicrobial compounds has always been a need.
Zone of inhibition was measured (mm) compared with standard antibiotics such as ampicillin, erythromycin and tetracycline. The organic solvents such as ethanol, methanol, petroleum ether, chloroform and aqueous extracts were employed. Among the solvent extracts used ethanol leaf extract shows very high activity (3.0 - 11.0 mm one of inhibition) against all the tested organisms. All the stem extracts (ethanol, methanol and petroleum ether) were also shows high activity (3.5 - 9.0 mm of inhibition) when compared to root extracts. Aqueous extracts which showed less activity or else poor activity. Ethanol root extracts shows a moderate to less activity (2.0 - 7.0 mm of zone of inhibition) against all the tested organisms. Aqueous leaf extract showed antimicrobial activity (4.5 mm of zone of inhibition) against K. pneumoniae and it does not have any effect on other organisms tested. Methanol extract showed (9.0 mm of zone of inhibition) against K. pneumoniae. The obtained results of the crude extracts were compared with the standard antibiotics such as Tetracycline, Erythromycin and Ampicillin.
All the tested organisms are highly sensitive to the ethanol leaf extract (6.5 - 11.0 mm) than the standard antibiotics such as tetracycycline, erythromycin and ampicillin, which showed more or less activity (5.0 - 12.5 mm) on the tested organisms compared with ethanol leaf extract. In present investigation, the overall leaf extracts show high inhibitory activity against all the tested bacterial strains followed by stem and root extracts. Medicinal plants are one of the most important sources of drugs. The use of different parts of several medicinal plants to cure specific human ailments has been in vogue from ancient times. Natural products drug discovery will be more holistic, personalized and involve wise use of ancient and modern therapeutic skills in a complementary manner so that maximum benefits can be occurred to the patients and community [
The present study reveals that the active principles present in the aerial parts of plant are very active against all the tested bacterial strains compare to roots. Based on earlier reports, among the great variety of secondary compounds found in plants, phenolics and terpenoids represent the main antimicrobial agents. Plants based antimicrobial drugs has enormous therapeutic potential as they can serve the purpose with lesser side effects.
MIC was determined by macro broth dilution method [
Type of extract/ antibiotic used | Zone of inhibition (mm) | ||||
---|---|---|---|---|---|
Bacillus subtilis | Staphylococcus aureus | Escherichia coli | Pseudomonas putida | Klebsiella pneumoniae | |
LEAF | |||||
Ethanol | 7.5 | 8.0 | 6.5 | 7.0 | 11.0 |
Methanol | 6.0 | 9.0 | 8.0 | 5.5 | 9.0 |
P. ether | 4.0 | 6.0 | 3.0 | 4.0 | 7.5 |
Chloroform | 5.0 | 4.5 | 4.0 | 3.0 | 7.0 |
Aqueous | - | - | - | - | 4.5 |
STEM | |||||
Ethanol | 3.5 | 5.0 | 5.0 | 7.0 | 7.5 |
Methanol | 5.0 | 6.5 | 4.0 | 6.0 | 9.0 |
P. ether | 4.0 | - | 3.5 | - | 5.0 |
Chloroform | - | 3.5 | 2.5 | 1.5 | 4.0 |
Aqueous | - | - | - | - | - |
ROOT | |||||
Ethanol | 7.0 | 3.5 | 3.0 | 5.5 | 5.0 |
Methanol | 2.5 | 4.0 | 4.5 | 1.5 | 3.0 |
P. ether | - | 4.0 | - | 2.0 | 3.5 |
Chloroform | 4.5 | 2.5 | 3.0 | 3.5 | - |
Aqueous | - | - | - | - | - |
STANDARD ANTIBIOTICS | |||||
Tetracycline | 12.5 | 9.0 | 9.5 | 10.5 | 9.5 |
Erythromycin | 9.0 | 8.5 | 8.0 | 7.5 | 5.0 |
Ampicillin | 11.0 | 10.5 | 8.5 | 5.0 | 8.0 |
Type of extract | MIC (μg/ml) | ||||
---|---|---|---|---|---|
Staphylococcus aureus | Bacillus subtilis | Escherichia coli | Pseudomonas putida | Klebsiella pneumoniae | |
LEAF | |||||
Ethanol | 3.50 | 6.00 | 7.50 | 3.50 | 9.50 |
Methanol | 17.50 | 13.50 | 9.00 | 12.50 | 11.00 |
Chloroform | - | 5.50 | 7.00 | 6.50 | 7.00 |
Petroleum ether | 7.00 | 12.50 | 6.50 | 4.00 | 5.50 |
Aqueous | 2.00 | 3.50 | 7.50 | 6.50 | 3.00 |
STEM | |||||
Ethanol | 4.00 | 13.50 | 12.50 | 7.00 | 2.50 |
Methanol | 8.00 | 11.00 | 9.50 | 25.50 | 11.50 |
Chloroform | - | 10.00 | 8.50 | 12.00 | 7.00 |
Petroleum ether | 3.50 | - | 2.0 | 5.50 | 4.00 |
Aqueous | 3.50 | 3.00 | - | 2.00 | 3.00 |
ROOT | |||||
Ethanol | 8.50 | 17.50 | 8.50 | 6.00 | 7.50 |
Methanol | 14.00 | 15.00 | 20.00 | - | 11.50 |
Chloroform | 9.50 | - | 9.50 | 25.00 | - |
Petroleum ether | - | 10.50 | - | - | 8.50 |
Aqueous | - | - | - | - | - |
extracts followed by stem and root extracts.
In conclusion, the ethanolic extracts of S. indicus showed the abroad spectrum of activity against all the tested organisms which were responsible for some common bacterial infections. However, the results were encouraging enough to practice fractionation of this extracts and to find out the functional properties of phytochemical compounds in order to ascertain useful potential antimicrobial compounds.
Harathi, K., Giribabu, D. and Naidu, C.V. (2017) Phytochemical Evaluation and in Vitro Antibacterial Activity of Sphaeranthus indicus (L.)―An Important Antijaundice Medicinal Plant. American Journal of Plant Sci- ences, 8, 1011-1021. https://doi.org/10.4236/ajps.2017.85067