Evaluation of Ipomoea reniformis for antimicrobial activity.

 

Ajay Raghuvanshi^1*, D.M. Kar¹, Prabhat Das², Renu Bala².

¹School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan University, Kalinga Nagar, Bhubaneswar, Odisha.

²GRY Institute of Pharmacy, Borawan, Khargone.

 

ABSTRACT:

Scientific validation of much medicinal flora is lacking in India and still many species are used traditionally. From many years Ipomoea reniformis was studied for its traditionally claimed activity and still it is unexplored. The plant can be further explored for the study of traditionally claimed unexplored activities, as well as isolation and identification of active constituents may lead to new findings. In the present study whole plant of I. reniformis was collected, dried and extracted with various solvents according to solubility index, and finally five extracts were studied by well diffusion assay for their antifungal and antimicrobial effects on both gram positive, gram negative bacteria and a fungal species. The study revealed that ethyl acetate extract was having highest activity that is why fractions of the ethyl acetate extract was prepared by various solvents and again the antimicrobial study was done with each fraction. The ethyl acetate extract and fraction exhibited activity against both Gram positive and Gram negative bacteria, while the aqueous extracts elucidated antimicrobial activity against only Staphylococcus aureus. The ethanolic and chloroform extracts showed inhibition of Aspergillus niger. The plant showed antimicrobial activity, evidencing the need to conduct further studies that can identify their active components responsible for the activity.

 

 

INTRODUCTION:

Antibiotics brought about a revolution to control pathogenic diseases and infections. But these synthetic drugs are out of reach to millions of people. Those people who live in remote places depend on traditional healers, whom they know and trust¹. Microbial diseases rank as number one cause for almost half of the deaths in underdeveloped and tropical countries. The frequency of life threatening infections caused by pathogenic microorganisms has increased worldwide and is becoming an important cause of morbidity and mortality in immune compromised patients in developed countries². Secondary metabolites are present in all higher plants. They play an important role in the plants protection against bacteria, virus, fungi and insects³. Microbial infections are considered as the most common causes of food borne diseases worldwide.

 

Examples of food spoiling microorganisms include Pseudomonas aeruginosa, Bacillus subtilis, Lactobacillus sp. Saccharomyces cerevisiae and Aspergillus niger⁴. In fokelore medicine, medicinal plants have been used widely in facilitating antimicrobial activity with high degree of successes. The literature survey revealed that the ethanolic extract of the whole plant, Ipomoea fistulosa exhibited significant activity against a number of Gram positive and Gram negative bacteria except Streptococcus faecalis; the aqueous extract was found to be inactive⁵. The antimicrobial activity of artificially grown sweet potato (Ipomoea batatas) leaves was investigated against both gram positive and gram negative bacteria⁶. Antimicrobial activity of metal complexes prepared from the leaf proteins of Ipomoea carnea was reported⁷. Antimicrobial activity of Ipomoea carnea leaves was reported against both Gram positive and Gram negative bacteria⁸. Considering the plants, as sources for antimicrobial drugs with reference to antibacterial and antifungal agents, a systematic investigation is undertaken to screen the local flora for antimicrobial activity from Ipomoea reniformis plant. In the present study well diffusion method was used to study the antimicrobial activity using the microbial strains (Escherichia coli NCIM 2109; Staphylococcus aureus NCIM 2079, Pseudomonas aeruginosa NCIM 2036; Bacillus subtilis NCIM 2250 and Aspergillus niger NCIM 545).

 

MATERIAL AND METHOD:

Plant material:

The plant material used in this study was whole plant of Ipomoea reniformis, collected from Narmada valley, Maheshwar, Madhya Pradesh, India during Aug 2012 and was authenticated by the Taxonomist Dr. S. K Mahajan, Botany Department, Government P. G. College Khargone M.P.

 

Extraction and Fractionation:

The plant materials were initially rinsed with distilled water and dried on paper towel in laboratory at (37 ± 1°C) for 24 h and milled into coarse powder by a mechanical grinder. The coarse powder was extracted with n-hexane, aqueous, benzene, ethyl acetate, chloroform and ethanol in a soxhlet extractor. The solvent was completely removed by distillation and dried in a vacuum desiccator. The standard extracts obtained were then stored in a refrigerator at 4°C for further use⁹.

 

As followed by the results of antimicrobial activity, dried Ethyl acetate extract was stored in vacuum desiccator and subjected to phytochemical screening ¹⁰.

 

The dried Ethyl acetate extract of plant materials was subjected to column chromatography on silica gel 60, and eluated with solvents according to solubility index. The eluates were collected in grouped based on their thin layer chromatography (TLC) profile to yield 05 fractions, IPR-1 to IPR-5 and the MIC study was carried out. From the result it was clear that maximum activity was shown by ethyl acetate fraction.

 

Phytochemistry:

Little work is done in the field of phytochemical investigation of the plant. Shah et al., have showed the presence of resin, glycosides, reducing sugars and starch while alkaloids are absent. Petroleum ether extract was reported to contain fats and fixed oil while aqueous extract was reported to contain amino acids, tannins (condensed tannins, pseudo tannins), and starch. Chemical investigation of Ipomoea reniformis shows the presence of caffeic, p‐ coumaric, ferulic and sinapic acid esters identified in seeds¹¹.

 

In-vitro antimicrobial activity:

Microbial strains:

All the extracts of Ipomoea reniformis was tested against the following five microbial strains (Escherichia coli NCIM 2109; Staphylococcus aureus NCIM 2079, Pseudomonas aeruginosa NCIM 2036; Bacillus subtilis NCIM 2250 and Aspergillus niger NCIM 545).

 

Well Diffusion Method:

The antibacterial activities of all the extracts (Table 1) and fractions (Table 2 )were determined by well diffusion method.¹² From the obtained results it was clear that the extracts posses good antimicrobial potential.

 

Microbiological media used for bacteria:

For various bacterial strains the media used was Nutrient agar (Hi-media), Composition (G/Litre): Sodium chloride, 5.0; Beef extract 10.0; Peptone 10.0 (pH 7.2).

 

Microbiological media for fungi:

The media used for fungal growth was Potato dextrose agar (Himedia), Composition (G/Litre): Potatoes infusion, 200.0 Dextrose 20.0 (pH 5.2).

 

100μl of each test bacterium was spread with the help of sterile spreader on a sterile Agar plate (Hi Media, Mumbai, India) so as to achieve a confluent growth and was incubated for 24 hours at 37 ± 0.1 °C. Well diffusion method was employed.

 

Concentration of the Extracts/Fractions taken was as:

Test samples of each extracts (200 mg) were dissolved in respective solvents (1 ml). Hi-media antibiotics: Streptomycin (10 microgram), Amphotericin-B (100 units) were used as standard. The count of the bacterial strains and fungal strain was adjusted to yield 1 X 10⁷ to 1 X 10⁸ mL-1 and 1 X 10⁵ to 1 X 10⁶ mL^-1 respectively. The microbes (0.1 ml) were inoculated with a sterile spreader on the surface of solid nutrient agar medium in plates. The agar plates inoculated with test organism were incubated for one hour before placing the extract in the wells on the plates. The bacterial plates were incubated at 37 ± 0.1 °C for 24 hours in a BOD incubator. After incubation all the plates were observed for zones of inhibition and the diameters of these zones were measured in millimeters by vernier calliper. All tests were performed under sterile conditions and in triplicate. Streptomycin (10μg/well) and Amphotericin B (100 unit/well) were used as positive controls.

 

Determination of Minimum Inhibitory Concentration (MIC):

Serial dilutions of concentrations ranging from 0.01 to 200 mg/ml were prepared as follows:

DMSO (2 ml in the first tube and 1 ml in the rest tubes) was filled in tubes. 400 mg of extract was added to the first tube and vortexed to prepare 200 mg/ml test extract solution. 1 ml of this solution was transferred in to other tube containing 1 ml DMSO to prepare the next dilution (100 mg/ml). Similarly, 1 ml of the second dilution was transferred in to the third tubes to prepare the third dilution (50 mg/ml) and the procedure was continued until the last dilution MIC determination was performed using the above serially diluted plant extracts in 96-well micro plates. 25μl of the test extract dilution were transferred from each test tube to wells of 96-well plates. Each well of the plate was loaded with 25 μl of bacterial suspension (adjusted to 0.5 McFarland standards) and 200 μl of broth except wells left for checking sterility. Chloramphenicol was used as a positive control, inoculated wells of antibiotic free broth were used as negative control and un-inoculated wells of antibiotic free broth were used to check sterility. Then the plates were covered with plate sealing tape and incubated at 37°C for 20 hours. Finally, the lowest concentration of the plant extract that showed no visible growth was taken as minimum inhibitory concentration. From the obtained results it was clear that ethyl acetate extract was having least MIC values. (Table. 3). The same procedure was carried out for various fractions obtained from ethyl acetate extract. (Table. 4)

 

Isolation of Compound:

Ethyl acetate fraction was evaluated by thin layer chromatography (TLC). Fraction was eluted with petroleum ether - ethyl acetate (30:70, v/v) and further purified by repeated CC on silica gel using dichloromethane and methanol gradient elution to yield compound (1) (40 mg). Finally, compound (1) was identified by using NMR, IR and Mass spectrometry¹³.

 

Table 1: Antimicrobial activity of Ipomoea reniformis extracts.

Diameter in mm calculated by Vernier Caliper; ‘-’ means no zone of inhibition, NA: Not applicable.

* Readings below 5 mm were not considered.

 

Table 2: Antimicrobial activity of Ipomoea reniformis Fractions.

Diameter in mm calculated by Vernier Caliper; ‘-’ means no zone of inhibition, NA: Not applicable.

* Readings below 5 mm were not considered.

 

MIC Study:

It was clear from the antimicrobial study that ethyl acetate extract has shown most of the activity so it was carried further for fractions. The MIC study of all fractions as shown in table 2 exhibited that ethyl acetate fraction is most potent among all the fractions¹⁴.

 

Data Analysis

Data was analyzed using SPSS software. The zone of inhibition was expressed as mean ± SD. One way ANOVA coupled to Least Significant Difference was employed to compare result between fractions and between bacteria. Result was considered statistically significant at 95% confidence level and P-value < 0.05.

Table: 3. MIC studies of various Extracts of Ipomoea reniformis.

Minimum Inhibitory Concentration (rounded to two decimal place) of I. reniformis solvent Extracts against the tested microbes.

Key: NA = No activity.

 

Table: 4. MIC studies of various fractions of Ipomoea reniformis.

Minimum Inhibitory Concentration (rounded to two decimal place) of I. reniformis solvent fractions against the tested microbes.

Key: NA = No activity.

 

 

IR NMR and Mass study of the compound:

The compound isolated from ethyl acetate fraction was processed for compound isolation and after isolation the Scopoletin NMR study gave following results:

 

 

 

Scopoletin Mass study gave following results:

 

 

 

 

 

 

 

 

Scopoletin IR study gave following results:

Peak cm-1		Functional group
Standard Scopoletin	Isolated compound V
3337.44	3341.44	O-H Alcohol group present
2850.97	2875.05	C-H Group present
1702.9	1703.42	Carbonyl C=O group present
1628.09	1606.75	CH=CH group present
1565.06	1568.83	Benzene ring present
1510.53	1511.16	Benzene ring present
861.46	861.5	Due to distribution of benzene

 

The IR, NMR and mass study showed the presence of alcohol, carbonyl and benzene ring in it. This might be the main cause for the antimicrobial activity of I. Reniformis. Further studies might be carried out to explore the lead molecule responsible for aforesaid activity from this plant.

 

RESULTS AND DISCUSSION:

The present study indicates that the ethyl acetate extract and fractions showed highest antimicrobial activity as compared to the other extracts. Aqueous extract also exhibits indicative activity against S. Aureus. While chloroform and ethanolic extracts also showed antimicrobial activity against the said strains. The study indicates that the plant can be studied for further evaluation of its effectiveness as antimicrobial agent. The IR, NMR and mass study showed the presence of hydrocarbons in it which might be the main cause for its activity against the microbial strains used for the study. Clinical trial needs to be carried out. Further studies might be carried out to explore the lead molecule responsible for aforesaid activity from this plant.

 

CONFLICT OF INTERESTS:

Declared none.

 

ACKNOWLEDGEMENTS:

The authors are grateful to SPS, SOA University, and Bhubaneswar for providing necessary facilities to carry out the research work in the faculty of pharmacy, SOA University.

 

The author is also thankful to NCIM: National Collection of Industrial Microorganisms, National Chemical Laboratory (NCL), Pune 411008 [India] for providing the microbial samples.

 

ABBREVIATIONS:

NCIM: National Collection of Industrial Microorganisms.

NCL: National Chemical Laboratory.

MIC: Minimum inhibitory concentration.

IPR: Ipomoea reniformis

CC: Column Chromatography

NMR: Nuclear Magnetic Resonance

IR: Infra Red

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Received on 22.07.2017         Modified on 17.08.2017

Accepted on 12.09.2017      © RJPT All right reserved