Antimicrobial screening of ethnobotanically important stem bark of medicinal plants

Abstract

Background:

The stem barks are the rich sources of tannins and other phenolic compounds. Tannins inhibited the growth of various fungi, yeast, bacteria and virus. Hence, ten stem barks of ethnomedicinally important plants were screened for antibacterial and antifungal activities against human pathogenic strains.

Methods:

Air-dried and powdered stem bark of each plant was extracted with 50% aqueous ethanol, lyophilized and the dried crude extracts were used for the screening against 11 bacteria and 8 fungi. Antibacterial and antifungal activities were performed according to microdilution methods by NCCLS.

Results:

The plants Prosopis chilensis, Pithecellobium dulce, Mangifera indica showed significant antibacterial and antifungal activities against Streptococcus pneumonia, Enterobacter aerogenes, Klebsiella pneumonia and Candida albicans with MIC of 0.08mg/ml. Pithecellobium dulce bark also showed significant antibacterial activity against Bacillus cereus.

Conclusion:

The bark of Pithecellobium dulce has more or less similar activity against the known antibiotic and may be considered as potent antimicrobial agent for various infectious diseases.

INTRODUCTION

Infectious diseases are the world's leading cause of premature deaths, killing almost 50,000 people every day; especially the burns remain a large public health issue and approximately 50-75% of hospital deaths are reported to be due to secondary infections.[1] Another important factor is that drug resistance to human pathogenical bacteria has been increasing not only in the developing countries but throughout the world due to indiscriminate use of antibiotics. The drug resistance bacterial and fungal pathogens have further complicated the treatment of infectious diseases in immunocompromised AIDS and cancer patients. In the present scenario due to emergence of multiple drug resistance to human pathogenic bacteria and fungi, especially the antibiotic penicillins, cephalosporins and chloromphenical types involve the enzymic inactivation of the antibiotic by hydrolysis or by the formation of an active derivative. This has opened a new vista for the search of new antimicrobial substance.

Search for newer drugs from plant has been increasing day by day due to the emergence of new diseases and alarming side-effects of synthetic drugs. Several plant species are being used by ethnic groups for the treatment of various diseases like dysentery, skin diseases, asthma, malaria, and a horde of other indications.[2–4] Natural products of higher plants may give a new source of antimicrobial agents. Tannins are water-soluble polyphenols and their presence in plants mostly located in dead or dying cells. They exert an inhibitory effect on many enzymes due to protein precipitation and hence they may contribute a protective function in barks and heartwood.[5] The antimicrobial activities of tannins are well documented. The growth of many fungi, yeast, bacteria, and viruses are inhibited by tannins.[6] Generally barks are considered a rich source of tannins. This paper present the antimicrobial screening of stem barks of ten medicinal plants. The selection of the plants for evaluation was based on traditional uses.

MATERIALS AND METHODS

The stem bark of medicinally important plants viz. P. chilensis (Molina) Stuntz, Mangifera indica L., Ceiba pentandra (L.) Gaertn, Senna siamea, Tectona grandis, Semecarpus anacardium, Holoptelea integrifolia, P. dulce, Cedrella toona, and Strychnos nux-vomica were collected from the field. The herbarium were prepared and deposited in the pharmacognosy and ethnopharmacology division herbarium wide voucher specimen numbers as shown in Table 1.

Table 1.

Details of the stem bark material procured along with their medicinal properties

Air-dried and powdered stem bark of each plant was percolated with 50% aqueous ethanol (v/v) at 25°C±2°C for 24 h (consecutively three times). The extracts were decanted, filtered with Whatman No. 1 filter paper, and concentrated under reduced pressure and low temperature (below 45°C). The concentrated extracts were lyophilized and the dried crude extracts were used for the screening. The test microorganisms used for the antimicrobial screening were 11 bacteria and 8 fungi and procured from Institute of Microbial Technology (IMTECH) Chandigarh, India. Micrococcus luteus MTCC (106), Bacillus subtilis MTCC (121), Bacillus cereus MTCC (430), Enterobacter aerogenes MTCC (111), Escherichia coli MTCC (443), Klebsiella pneumonia MTCC (109), Proteus mirabilis MTCC (1429), Pseudomonas aeruginosa MTCC (424), Staphylococcus aureus MTCC (96), Salmonella typhimurium MTCC (98), Str. pneumonia MTCC (2672), Candida albicans MTCC (183) and Cryptococcus albidus MTCC (2661), Trichophyton rubrum MTCC (296), Aspergillus niger MTCC (16404), A. flavus MTCC (1973), A. spinulose MTCC (16919), A. terreus MTCC (1782), and A. nidulans MTCC (11267).

Stock bacterial inocula suspensions were obtained from 6-12 h culture on Mueller Hinton Broth (MHB, Oxoid) at 37°C. These final suspensions served for the inocula preparation. The cell density of each suspension was determined by NCCLS[7] using a counting chamber and then adjusted to 0.5 McFarland turbidity at the concentration of 10⁵-10⁶ CFU/ml by dilution with MHB. The fungi were grown on Sabouraud Dextrose Agar (SDA, Oxoid) at 28-30°C for 3±7 days, to induce conidia formation. Then, the culture was washed with 2 ml of peptone water (HiMedia Lab. Pvt. Ltd. India) to prepare a suspension. This suspension was transferred to a sterile tube and kept for 5 min. The upper homogenous suspensions were transferred in a new sterile tube, and added appropriate quantity of Mycological peptone (MP. HiMedia Lab. Pvt. Ltd. India). This suspension was adjusted microscopically about 10⁴ CFU/ml.

Antibacterial activity was performed according to microdilution method by NCCLS[8] and Zgoda and Porter[9] with slight modifications. Briefly, extract was dissolved 2.5% dimethyl sulphoxide (DMSO, Sigma) and filtered through 0.2 micron nonpyrogenic filter and serially diluted (twofold) with 2.5% DMSO to give a range 0.08 – 50 mg/ml. Tests were performed in sterile U bottom 96 - well by dispensing in to each well 95 μl of MH broth and 5 μl of inoculums (0.5 McFarland turbidity). Here 100 μl of test extract was finally added to each appropriate well. The final volume in each well was 200 μl. Standard antibiotic - gentamicin (Sigma) was used as positive control. The plates were covered with sterile sealer and incubated at 37°C for 18-24 h. To indicate bacterial growth, 40 μl of 0.2 mg/ml p-iodonitrotetrazolium violet (INT, Sigma) solution was added to each well and incubated for further 30 min. Inhibition of bacterial growth was visible as a clear well and the presence of growth detected by the presence of pink red color.

The method used for antifungal activity was M27-T described by the NCCLS[10] with some modifications. The culture medium was mycological peptone. Ketoconzole (Sigma) was used as standard. Concentration range was 0.08-50 mg/ml. After inoculation, the plates were incubated at 28-30°C for 24-96 h. The minimum inhibitory concentration (MIC) values of extract/compound of antifungal agent were estimated by lack of visual turbidity (matching the negative growth control).

RESULTS AND DISCUSSION

The ethnobotanical data of these barks and their distributions have been compiled in Table 1. The inhibitory concentration of aqueous ethanolic (50%) extracts of all the bark has been established.

The results of the present work indicate that bark selected have significant antibacterial activity as compared to antifungal. Very weak activity was observed against C. albidus and A. spinulose in comparison to known antibiotics [Table 2]. Some of the extracts like P. chilensis and P. dulce gave very low MIC values and inhibited the growth of S. pneumonia, K. pneumonia, and E. aerogenes with a concentration of 0.08 mg/ml. The extract of P. dulce bark show promising activity against B. cereus, E. aerogenes, K. pneumonia, Str. pneumonia, and C. albicans [Table 2]. It is interesting to note that this bark has significant inhibition against K. pneumonia and E. aerogenes as compared to known antibiotic gentamicin, which is considered more potent antibiotic against gram negative bacteria. The extracts of H. integrifolia and S. nux-vomica showed better antibacterial activity against B. cereus and the extracts of P. chilensis and S. anacardium possesses similar inhibition against B. subtilis as compare to the positive control.

Table 2.

MIC values of aqueous ethanolic extracts (50%) of ten stem bark against microorganisms by microdilution method (conc. mg/ml)

The stronger and broader spectrum of antifungal activity was also observed in the extract of M. indica and P. dulce against C. albicans with MIC values of 0.08 mg/ml [Table 2]. The ongoing results indicated that the plant species assayed possess more antibacterial property and explain the use of these plants in folk medicine for the treatment of various diseases, which may be caused by bacterial infection and also suggest the importance of the ethnobotanical claims for the development of bioactive compounds.

From the present screening, it has been concluded that the bark of P. dulce is more potent antimicrobial agent and has more or less similar activity against the known antibiotic. Further, the detailed phytochemical research is required to identify the active principal responsible for aforesaid activities.