Biomedical Research

DOI: 10.4066/biomedicalresearch.29-17-4016

Abstract

The antimicrobial activity of 100 extracts of twenty plants Namely Symplocos racemosa, Solanum xanthocapum, Phyllanthus urinaria, Blumea lacera, Acorus calamus, Thevetia nerifolia, Bauhinia purpurea, Jatropha gossypifolia, Pithecellobium dulce, Hydrangea integrifolia, Sphaeranthus indicus, Vitex negundo, Zizyphus xylopyrus, Aconitum heterophyllum, Scoparia dulcis, Callicarpa dichtoma, Adhatoda zylamei, Sphaeralcea angustifolia, Swertia chirayita, Aconitum heterophyllum from Uttar Pradesh, India was tested against twelve pathogenic micro-organisms. The 51 extracts of P. urinaria, B. lacera, B. eriantha, T. nerifolia, J. gossypifolia, P. dulce, H. integrifolia, S. indicus, V. negundo, Z. xylopyrus, A. heterophylum, S. dulcis, C. dichtoma, A. zylamei, S. angustifolia, S. chirayita, A. heterophyllum were found to be inhibiting all the bacterial strains tested, in a range of concentrations by microbroth dilution assay and 0.75 to 48.0 mg/disc by disc diffusion assay. The 37 extracts were found to be active against the fungal strains in arrange of 0.75 to 24.0 mg/ml by microbroth dilution assay. Other plant extracts also exhibited variable antimicrobial activity against one or more species of microorganisms. As the extracts of all the plants which were used in this study effectively controlled the growth of many pathogenic bacteria and fungi, they can be used in the treatment of various infectious diseases. Bioactive compounds from these plants can be employed in the formulation of antimicrobial agents for the treatment of various bacterial and fungal infections.

Keywords

Plant extracts, Antifungal, Antimicrobial, Susceptibility testing.

Introduction

Inhabitants in rural areas of Uttar Pradesh (India) depend on medicinal plant preparations for treatment of many diseases, including those of infectious by nature [1-3]. People in this region use these plants as a paste or their extracts to cure themselves of various kinds of diseases [2]. Uttar Pradesh is consisting of porous soil. It gets low rainfall and has variable temperature (2.0-48.0°C), therefore, the region has the characteristic flora of plants [3].

The increasing number of multi-drug resistant microbial strains and the appearance of new strains with reduced susceptibility to antimicrobial raise the spectrum of untreatable infections [4-6]. Therefore, there is need to search new infection-fighting strategies [7,8]. Micro-organisms, animals, and plants are the well-known source of new antimicrobial compounds [9-11]. Systematic screening of the plants which are used to cure various disease conditions in folk fare medicine may result in the discovery of novel effective compounds as shown in Table 1.

Table 1. Selected Indian medicinal used to treat various kinds of human diseases.

Antimicrobials of plant origin have minimum side effects and have an enormous therapeutic potential to heal many infectious diseases [7]. Out of all, 83% drugs in the present market are related to the natural product therefore, natural product is gaining important to treat these microbial infections or to find out new drugs to treat these infections which have the least side effects.

In the present study, various extracts of ten plants from different families were tested against microbial pathogens. The plant species tested were Symplocos racemosa, Solanum xanthocapum, Phyllanthus urinaria, Blumea lacera, Acorus calamus, Thevetia nerifolia, Bauhinia purpurea, Jatropha gossypifolia, Pithecellobium dulce, Hydrangea integrifolia, Sphaeranthus indicus, Vitex negundo, Zizyphus xylopyrus, Aconitum heterophyllum, Scoparia dulcis, Callicarpa dichtoma, Adhatoda zylamei, Sphaeralcea angustifolia, Swertia chirayita, Aconitum heterophyllum. All these plants have their own medicinal importance in the life of rural natives of Uttar Pradesh to treat various kinds of infections.

Materials and Methods

Plant materials

The aerial parts of plants tested were collected from different region of the Uttar Pradesh from the months of October 2012 December 2016. The plant material was dried in shade and used to prepare different extracts.

Preparation of plant extracts

The powdered material of plants was subjected to Soxhlet extraction successively with n-hexane, chloroform, acetone, methanol and water to obtain respective fractions [10]. Solvents were evaporated under reduced pressure using rotavapor and dry fractions were stored at 4°C till use.

Micro-organisms

Clinical isolates of Aspergillus, Candida and bacterial strains namely, A. fumigatus (190/96), A. flavus (223/96), A. niger (56/96), C. albicans (345/96), Salmonella typhi (135/98), Escherichia coli (576/98), Staphylococcus aureus (231/98), Pseudomonas aeruginosa (87/98), Streptomyces pyogene (124/98), Enterobater aerogenes (248/98), Bacillus cereus (272/98) and Klebsiella pneumonae (31/98) from the Microbiology Department, Vallabhbhai Patel Chest Institute, Delhi were used along with the standard strains. Quality control strains of A. fumigatus ITCC 4517, A. flavus ITCC 5192, A. niger ITCC 5405, C. albicans ITCC 4718 obtained from Indian Type Culture Collection, IARI, Delhi. S. typhi MTCCB 733, E. coli MTCCB 82, S. aureus MTCCB 737, P. aeruginosa MTCCB 741, S. pyogenes MTCCB 655, E. aerogenes MTCCB 111, B. cereus MTCCB 1272, K. pneumonae MTCCB 109 were included in each test as recommended by the National Committee for Clinical Laboratories Standards (NCCLS) were purchase from Institute of Microbiology Technology, Chandigarh, India.

Antimicrobial activity

Antimicrobial activity of the various extracts of different plants was determined by the Micro broth dilution Assay as per described in NCCLS document M-27A (NCCLS), 2011 [12]. The fungi and bacterial strains used as inoculum were grown overnight on Sabouraud dextrose agar and nutrient agar respectively at 37°C. Lowest concentration which inhibited any visual growth was considered to be Minimum Inhibitory Concentration (MIC).

Test of antibacterial activity

All the extracts were prepared in a concentration ranging from 0.750 to 48.0 μg/ml. Proper controls were used for each experiment. The bacterial strains used as inoculum were grown at 37°C to get OD 0.6 at 600 nm and used for susceptibility testing. The lowest concentration, which inhibited any visual growth, was considered to be Minimum Inhibitory Concentration (MIC).

Antifungal activity

The susceptibility testing of Aspergillus strains was determined by the microbroth dilution method. Cultures were grown on Sabouraud dextrose agar at 37°C until sporulation occurs, typically 5 d. Stock spores’ suspensions were with yeast nitrogen or broth (pH 7.0) supplemented with 5.0 g glucose per liter and 24% (vol/vol) glycerol and were stored at 4°C until use. The numbers of Colony Forming Units (CFU) per milliliter were determined by plating serial dilutions on Sabouraud dextrose agar plates. For susceptibility tests, the spore suspensions were diluted to achieve 2 × 10⁴ to 2 × 10⁵ CFU/ml in yeast nitrogen broth plus 0.5% glucose (Ph 7.0) and were incubated for 24 h at 35°C to germinate spores. Serial twofold dilution of extracts was made in yeast nitrogen broth plus 0.5% glucose (pH 7.0) in a concentration ranging from 0.75 to 48.0 mg/disc. The cultures were incubated for 48 h at 37°C. MICs were determined at the lowest concentration that inhibited visible fungal growth. Disc diffusion assay was performed as per Indian Pharmacopeia (1996) [13] against pathogenic strains of microorganisms and lowest concentration which forms a zone of inhibition about 6.5 mm (Disc diameter was 6.0 mm) was considered to MIC by disc diffusion assay in this paper [10].

Results and Discussion

The differences among various antimicrobial effects of plant groups are natural depending upon the phytochemical properties of plants and antimicrobial nature of plant metabolites [10]. Uttar Pradesh due to its typical environmental conditions (Temperature to 47ºC and average rainfall) [1,2] and characteristic flora provides rich source of plant materials [11]. The region also has a history of the application of plants in folklore medicines.

100 different extracts of twenty plants used in this study to evaluate their antimicrobial potential against S. typhi, E. coli, S. aureus, P. aeruginosa, S. pyogenes, E. aerogenes, B. cereus, K. pneumonae C. albicans, A. niger, A. fumigatus, and A. flavus using micro-broth dilution as shown in Table 2 as well as disc diffusion assays Table 3.

Table 2. Minimum inhibitory concentration of plant extracts against the microorganisms by micro dilution broth assay.

Table 3. Antimicrobial activity of compounds by disc diffusion method.

In the present study, the concentration of the extract which forms the least zone of inhibition (~6.5 mm: diameter of disc was 6.0 mm) was considered as MIC by disc diffusion assay. The MIC by disc diffusion assay of the bacteria and fungi listed in Table 2. Activity of various extracts was found to be ranged from 0.75 to 24.0 μg/disc against the tested bacterial species by disc diffusion method. Hexane extract of P. urinaria, B. lacerav H. integrifolia P. dulce and chloroform extract of P. urinaria, B. lacera and B. eriantha and water extract of. P. urinaria were not found active against any tested pathogen by DDA and MDA.

The 51 extracts of P. urinaria, B. lacera, B. eriantha, T. nerifolia, J. gossypifolia, P. dulce, H. integrifolia, S. indicus, V. negundo, Z. xylopyrus, A. heterophylum, S. dulcis C. dichtoma, A. zylamei, S. angustifolia, S. chirayita, A. heterophyllum were found to be inhibiting all the bacterial strains tested, in a range of 0.75-24 mg/ml by microbroth dilution assay and 6.24 to 48.0 mg/disc by disc diffusion assay. The 37 extracts were found to be active against the fungal strains in arrange of 1.5 to 24.0 mg/ml by microbroth dilution assay. Other plant extracts also exhibited variable antimicrobial activity against one or more species of microorganisms. T. nerifolia is used with Cannabis sativa to treat urinary infections. B. lacera and B. eriantha are described in Ayurveda as anti-inflammatory, antipyretic and anthelmintic. Roots of these plants are used to cure the mouth disease.

Methanol extract of Blumea lacera, T. nerifolia, J. gossypifolia S. dulcis and C. dichtoma showed activity against all tested pathogen. Water methanol and aacitone extract of B. eriantha active against all tested bacterial stain in the rage of 0.750 mg/ml to 24 mg/ml. Water and chloroform extract of P. urinaria, chloroform extract of B. eriantha, water and hexane extract of P. dulce. Hexane extract of H. integrifolia chloroform extract of heterophylum, Hexane extract of C. dichtoma and extract of S. chirayita does not show any activity.

S. xanthocapum and Bauhinia purpurea showed antifungal activity in water methanol acetone chloroform and hexane extractions. Water, methanol and chloroform extraction of Symplocos racemosa, methanol and acetone extraction of P urinaria. Methanol extraction of B. lacera hexane extraction of B. eriantha. Methanol extraction of T. nerifolia and J. gossypifolia. Water and methanol extraction of H. integrifolia and S. indicus and methanol and chloroform extraction of V. negundo showed antifungal activity in the range of 750 mg/ml to 24 mg/ml.

Conclusion

The available antibiotics become resistant to day by day to the microbial diseases and crucial need to determine new antimicrobial compounds with various chemical structure and novel mechanism of action for new and reemerging infectious disease. In this attempt, folkloric herbal drugs must necessarily to be approved the advantage of modern science and technology to serves further global needs. The drugs derived from herbs have the opportunity of using in medication because of its good antimicrobial activity as well as less toxic effects. An innovative drug should not comprise any toxic side effects, Hence the present study aimed to focus the antimicrobial activity of plants on tested pathogens which showed significant activity.

Acknowledgements

The author gratefully acknowledges Dr. Vinod Kumar Tiwari, Department of Chemistry, Banaras Hindu university, Varanasi India; Prof. Amita Jain, Department of Microbiology King George’s Medical University, Lucknow, India; Dr. Rambir Singh, Institute of Biomedical sciences, Bundelkhand University Jhansi India; Dr. Rajesh Dabur, Department of Biochemistry MDU, Rohtak, Prof. S. L. Kothari and Prof. Ajay Jain, Amity Institute of Biotechnology, Amity University Rajasthan, India and finally with all centers of whole research and technical staff members for their wonderful lab support without any of them this work was not possible for me.

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