Antibiotic/Antibacterial activity of Vitex negundo, Duranta repens ...

British Biotechnology Journal 6(1): 16-22, 2015, Article no.BBJ.2015.023 ISSN: 2231–2927

SCIENCEDOMAIN international www.sciencedomain.org

Antibiotic/Antibacterial activity of Vitex negundo, Duranta repens, Acorus calamus and Piper nigrum Burhan M. Padder1*, Shagufta Yasmeen2 and Mudasar Ganaie1 1

Department of Botany, University of Kashmir Hazratbal Srinagar 190006, India. Department of Biotechnology and Microbiology, Karnatak University Dharwad. 580003, India.

2

Authors’ contributions This work was carried out in collaboration between all authors. Author BMP managed the literature searches, designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Author SY performed the practical work and along with author MG managed the analyses of the study. All authors read and approved the final manuscript. Article Information DOI: 10.9734/BBJ/2015/14534 Editor(s): (1) P. Mary Anupama, Department of Chemical Engineering and Biotechnology, Anil Neerukonda Institute of Technology and Sciences, India. Reviewers: (1) Pralhad S Patki, Medical Services and Clinical Trials, R&D Center, The Himalaya Drug Company, Bangalore 562 123, India. (2) Anonymous, India. (3) Anonymous, India. (4) Anonymous, Portugal. (5) Anonymous, Brazil. Complete Peer review History: http://www.sciencedomain.org/review-history.php?iid=805&id=11&aid=7780

Original Research Article

Received 4th October 2014 nd Accepted 22 December 2014 Published 14th January 2015

ABSTRACT Plants produce a diverse nature of secondary metabolites having different biological activity. Some of these chemicals exhibit antimicrobial activity. In the present study the plant extracts of Vitex negundo, Duranta repens, Acorus calamus and Piper nigrum were tested individually as well as synergistically for antibiotic/ antibacterial activities. It was found that the plant extract of V. negundo was most effective against both S. mutans MTCC 890(MIC value 0.37) and P. aeruginosa MTCC 0741 (MIC value 0.75) than other three extracts of D. repens, P. nigrum, A. calamus whereas the plant extract of P. nigrum was least effective against both S. mutans MTCC 890(MIC value 3.12) and P. aeruginosa MTCC 0741(MIC value 6.25).

Keywords: Antimicrobials; plant extracts; secondary metabolites; individual; synergistic activity. _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected];

Padder et al.; BBJ, 6(1): 16-22, 2015; Article no.BBJ.2015.023

included: leaves of Duranta repens and Vitex negundo, seeds of Piper nigrum, rhizome of Acorus calamus. Standard drugs used to compare with plants extracts were:

1. INTRODUCTION Antibiotics are clearly one of the greatest triumphs of medical science, but they rely upon a limited range of targets. Microbes are now developing resistance to these modes of action. Unfortunately, the problem of antibiotic resistance has now reached a crisis point. Dealing with this crisis requires both intensified educational campaigns to promote wise use of existing antibiotics and redoubled effort towards the design of new ones. The increasing prevalence of multidrug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the spectra of untreatable bacterial infections and adds urgency to the search for new infection fighting strategies. Plants are rich in a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids and flavonoids which have been found in vitro to have antimicrobial properties [1,2]. Despite the abundant literature on the antimicrobial properties of plant extracts, none of the plant derived chemicals have successfully been exploited for clinical use as antibiotics [3]. Although all parts of Vitex negundo are used as medicine in the indigenous system of medicine, however leaves are the most potent for medicinal use [4,5]. It is used for treatment of eye-disease, toothache, inflammation, leucoderma, enlargement of the spleen, skin-ulcers, in catarrhal fever, rheumatoid arthritis, gonorrhea, and bronchitis. They are also used as tonics, vermifuge, lactagogue, emmenagogue, antibacterial, antipyretic and antihistaminic agents. Its extract has also shown anticancer activity against Ehrlich ascites tumour cells. The leaves of V. negundo are reported to possess pesticidal, antifungal and antibacterial properties [6,7].

a) Ampicillin (25 mcg/disc) on Pseudomonas aeruginosa b) Erythromycin (15 mcg/disc) on Streptococcus mutans c) Ciprofloxacin (10 mcg/disc) on Pseudomonas aeruginosa The chemicals used for the experiments were of AR standards which include: Petroleum ether, DMF-- Dimethyl Formamide used as a solvent to dissolve plant extract and tested on microorganisms at various concentrations. Antibiotic zone scale was used for measuring zone of inhibition. Solvents used were Hexane, Benzene, Methanol and water in the ratio of 2:6.5:1:0.5. Analytical plate silica gel was used as adsorbent. The pure cultures of the strains were maintained at low temperature in the refrigerator. Subculturing of the pure culture was made by different streaking methods like quadrant streak, Zigzag streaks etc. and incubated at 37°C for 24 hours. After incubation the plates of pure cultures were stored in refrigerator. Nutrient agar medium was used for Pseudomonas aeruginosa and Brain Heart Infusion medium was used for Streptococcus mutans. The extraction of the components of the plants was carried out in following steps: The plant materials i.e. leaf, seeds and rhizome of the respective plants were collected in an appropriate quantity (50gram per sample).The leaves of the Duranta and Vitex were shade dried for about 8-10 days. The temperature at drying place was nearly 22-25°C. At the same time the dried pepper seeds and rhizome of Acorus calamus were powdered. The dried leaves were pulverized and passed through 60– 80 mesh size sieves. These powdered materials of the plants were further subjected to Soxhlet extraction.

It is in this context that the present study has been carried out to evaluate the individual as well as the synergistic antimicrobial properties of Vitex negundo, Duranta repens, Acorus calamus and Piper nigrum. The rationale behind this study is based on the fact that antimicrobials of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious diseases.

After 36 hours of Soxhlet extraction the solvent was collected and subjected to evaporation to get pure extract. The quantity of plant extract obtained was weighed. The plant extracts were stored in sealed bottles and kept in refrigerator until used for further analysis. The known quantity of plant extract was dissolved in known volume of DMF(100 mg per ml) and different concentrations of extract were made as follows:

2. MATERIALS AND METHODS The pure cultures of Pseudomonas aeruginosa (MTCC 0741) and Streptococcus mutans (MTCC 890) microbial strains were procured from MTCC, Chandigarh, India. Plant parts selected 17


100 µg, 200 μg, 300 µg, 400 μg etc. Along with the individual antimicrobial activity the synergistic activity of the extracts of plants was also tested. Comparison between the individual and synergistic action of the plant extracts was also worked out. The combinations were made in the ratio of 1:1. a) b) c) d)

The antimicrobial potential of all the 4 plant extracts was evaluated for the test organisms Streptococcus mutans (MTCC-890), Pseudomonas aeruginosa (MTCC 0741) as given in Table 1.

3.1 Effect of Vitex negundo aeruginosa and S. mutans

Vitex negundo with Piper nigrum Vitex negundo with Acorus Calamus Duranta repens with Piper nigrum Duranta repens with Acorus calamus

2.1 Preparation of Culture Spread Plate Technique

Plates

on

P.

P. aeruginosa was sensitive at the concentration of 300 µg and 400 μg with inhibition zones measuring 8 mm and 10 mm whereas S. mutans was sensitive at concentration of 300 µg and 400 μg with inhibition zones measuring 7 mm and 9 mm.

by

3.2 Effect of Duranta repens aeruginosa and S. mutans

Nutrient agar and brain heart infusion media was prepared and sterilized in Autoclave at 15lbs for 20mins and cooled. The sterile liquid media was poured in the sterilized, labelled petri dishes and allowed to solidify. A suspension 0.1ml of each S. mutans and P. aeruginosa was spread on the brain heart infusion agar and nutrient agar media respectively. Each of the petri dishes was labeled in such a way that they are divided as four parts for four different concentrations of plant extracts i.e., 100 µg, 200 μg, 300 µg and 400 μg.

on

P.

S. mutans was sensitive at concentrations of 200 µg, 300 μg and 400 µg with inhibition zones measuring 7 mm, 9 mm, and 10 mm whereas P. aeruginosa showed sensitivity at concentrations of 300 μg and 400 µg with inhibition zone measuring 9 mm and 11 mm.

3.3 Effect of Piper nigrum aeruginosa and S. mutans

Disc diffusion method was carried out to test inhibition effect of extract. The sterile discs of 6mm diameter were placed on each part of the plates. The discs were loaded with appropriate volume of plant extract to get the concentration of 100 µg, 200 μg, 300 µg, and 400 μg. On the other hand the respective volume of DMF was loaded on the discs placed on the agar plates containing test organisms, which served as control. The plates were incubated at 37°C for 24 hours and the results taken by measuring inhibition zone by zone measuring scale. Synergistic action was also tested on each of organisms by making known concentration of combined extracts of plant in the ratio 1: 1.

on

P.

S. mutans showed sensitivity at concentration of 300μg and 400µg with inhibition zones measuring 7 mm and 10 mm respectively while as P. aeruginosa showed resistance at all the concentration.

3.4 Effect of Acorus calamus on P. aeruginosa and S. mutans S. mutans showed sensitivity at concentration of 200 μg, 300 µg and 400 μg with inhibition zones measuring 7 mm, 8 mm and 10 mm whereas P. aeruginosa showed sensitivity at concentration of 200 µg, 300 μg and 400 µg with inhibition zones measuring 7 mm, 8 mm, 10 mm and 11 mm.

3. RESULTS

4. EFFECT OF SYNERGISTIC ACTIVITY OF PLANT EXTRACTS ON P. aeruginosa And S. mutans

The leaves of Vitex negundo and Duranta repens after drying have shown in general 70% reduction in their weight. The total time taken for the extraction was 26 to 32 hours. For every 50gm of the dried plant material taken the yield obtained after Soxhlet extraction is as follows:

To study the synergistic activity, following combinations of plant extracts were made: 1. 2. 3. 4.

Vitex negundo------------3.50gm Duranta repens-----------1.50gm Piper nigrum--------------1.79gm Acorus calamus-----------3.06gm 18

Vitex negundo and Acorus calamus Vitex negundo and Piper nigrum Duranta repens and Acorus calamus Duranta repens and Piper nigrum


Table 1. Individual activity of plant extracts on S. mutans and P. aeruginosa S. No

Plant extract

1

V. negundo

Concentration of stock solution (mg/ml) 20

2

D. repens

20

3

P. nigrum

20

4

A. calamus

20

5

DMF

20

Total volume loaded on disc(µ1) 5 1 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20

The antimicrobial effects of above mentioned combination of plant extracts in 1:0.5 ratios did not show any inhibitory effect on the test organisms at all concentrations (100 μg, 300 µg and 400 μg). Whereas the antibacterial effect of combined plant extracts in 1:1 ratio have shown considerable inhibitory effect on the test organisms at all concentrations (200 µg, 300 μg, 400 µg and 500 μg). The results obtained are summarized in Table 2.

Respective amount of extract present (μg) 100 200 300 400 100 200 300 400 100 200 300 400 100 200 300 400 -

Diameter of inhibition zone (mm) S. mutans P. aeruginosa 7 9 7 9 10 7 10 7 8 10 -

8 10 9 11 8 10 11 -

zones measuring 8 mm, 10 mm, 12 mm, 11 mm and 11 mm whereas P. aeruginosa was sensitive at concentrations of 200 µg, 300 μg, 400 µg and 500 μg with inhibition zones measuring 8mm, 10 mm, 11 mm and 12 mm.

4.3 Effect of Duranta repens and Piper nigrum on P. aeruginosa and S. mutans S. mutans was sensitive at concentrations of 200 µg, 300 μg, 400 µg and 500 μg with inhibition zones measuring 10mm, 11mm, 12mm and 13mm and P. aeruginosa was sensitive at concentrations of 200 µg, 300 μg, 400 µg and 500 μg with inhibition zones measuring 10 mm, 11 mm, 14 mm and 15 mm.

4.1 Effect of Vitex negundo and Acorus calamus on P. aeruginosa and S. mutans S. mutans was sensitive at concentrations of 200µg, 300μg, 400µg and 500μg with inhibition zones measuring 10mm, 11mm and 12mm. For P. aeruginosa was sensitive at concentrations of 200µg, 300μg, 400µg and 500μg with inhibition zones measuring 10mm, 12mm, 13mm and 16mm.

4.4 Effect of Duranta repens and Acorus calamus on P. aeruginosa and S. mutans S. mutans was sensitive at concentrations of 200 µg, 300 μg, 400 µg and 500 μg with inhibition zones measuring 8 mm, 9 mm, 10 mm and 12 mm and P. aeruginosa was sensitive at concentrations of 200 µg, 300 μ, 400 µg and 500 μg with inhibition zones measuring 11 mm, 12 mm, 12 mm and 13 mm.

4.2 Effect of Vitex negundo and Piper nigrum on P. aeruginosa and S. mutans S. mutans was sensitive at concentrations of 200 µ, 300 μg, 400 µg and 500 μg with inhibition 19


oil of V. negundo was found to be active against P. aeruginosa. The leaf extracts of V. negundo showed antimicrobial activity against P. aeruginosa. Aqueous, chloroform and methanol extracts of V. negundo were tested against P. aeruginosa NCIM 2036, of these only aqueous extract failed to show inhibitory effect. The petroleum ether extract of leaves of V. negundo showed inhibitory activity against P. aeruginosa and S. mutans at a concentration of 300 µg. The inhibitory effect was found to be more on P. aeruginosa than S. mutans at this concentration. The petroleum ether extract of D. repens exhibited considerable inhibitory activity against S. mutans at a concentration of 200 μg and this P. aeruginosa at 300 µg. This shows that S. mutans is more susceptible than P. aeruginosa. Similar results have also been reported by [8,9,10,11].

4.5 Effect of standard Antibiotics on P. aeruginosa and S. mutans Ampicillin, Ciprofloxacin and Erythromycin were used as standard drugs to compare the effects of plants extracts. Ampicillin showed least inhibitory effect against P. aeruginosa. Ciprofloxacin was effective against P. aeruginosa and shown inhibition zone of 15 mm. Erythromycin was effective against S. mutans with an inhibition zone measuring 27 mm, the results are best summarized in the following table.

5. DISCUSSION From the present studies, it was summarized that the petroleum ether extracts of the selected plants showed considerable antimicrobial activity against P. aeruginosa and S. mutans. The flower

Table 2. Synergistic activity of plant extracts on S. mutans and P. aeruginosa S. No

Plant extract combination

1.

V. negundo +P. nigrum

2.

V. negundo

Conc. of each extract (mg/ml) 40

Total volume loaded on disc (µl) 10 15 20 25

40

10 15 20 25

3.

D. repens + P. nigrum

40

10 15 20 25

4.

D. repens +A. calamus

40

10 15 20 25

Respective amount of each plant extract in the disc (µg) V. negundo P. nigrum 200 200 300 300 400 400 500 500 V. negundo A. calamus 200 200 300 300 400 400 500 500 D. repens P. nigrum 200 200 300 300 400 400 500 500 D. repens A. calamus 200 200 300 300 400 400 500 500

Diameter of inhibition zone (mm) S. mutans 8 10 11 11

P. aeruginosa 8 10 11 12

10 11 11 12

10 12 13 16

10 11 12 13

10 11 14 15

8 9 10 12

11 12 12 13

Table 3. MIC values recorded for S. mutans and P. aeruginosa S. no. 1 2 3 4

Plant extract S. mutans 0.37

V. negundo (P. ether) D. repens (P. ether) P. nigrum (P. ether) A. calamus (P. ether)

20

Strain (mg/ml) P. aeruginosa 0.75

0.75

0.65

3.12

6.25

1.5

6.25


Table 4. Antibacterial activity of standard antibiotics on S. mutans and P. aeruginosa S.no 1 2 3

Name of the standard antibiotic Ampicillin Ciprofloxacin Erythromycin

Concentration (mcg / disc) 25 10 15

Organism tested P. aeruginosa P. aeruginosa S. mutans

Diameter of inhibition zone (mm) 32 15 27

Control: DMF served as control and was inactive against both test organisms

The Ethyl Acetate extract of P. nigrum was tested against Gram +ve and Gram –ve bacteria. It was observed that Gram –ve bacteria were not inhibited by Ethyl Acetate extract of P. nigrum at concentrations of ≤1000 μg/ml. P. aeruginosa was the most susceptible bacterial strain compared to P. nigrum tested with MIC of 5 µg/ml when extracted through Ethanol; these results are in accordance with those reported by [11] in Piper regnelli.

aeruginosa with an inhibition zone of 15 mm which is similar to the inhibitory effect shown by combined plant extracts of D. repens + P. nigrum and V. negundo + A. calamus with an inhibition zone of 15 mm and 16 mm respectively. Erythromycin was found to be effective against S. mutans than compared to any of the plant extracts showing an inhibition zone of 27 mm. Also the MIC values for S. mutans and P. aeruginosa were carried out and are recorded in Table 3.

The petroleum ether extract of P. nigrum did not show inhibitory effect of P. aeruginosa, but it showed inhibitory effect on S. mutans at concentration of 300 μg and 400 µg. This shows that S. mutans is more susceptible than P. aeruginosa. Findings of our study are similar to those reported by by [12]. A weak antimicrobial activity was found against some Pseudomonas sps. [13]; P. aeruginosa was found to be very much susceptible to A. calamus extract at concentrations of 200 μg, 300 µ and 400 μg and same results were observed in case of S. mutans.

6. CONCLUSION From the plant extracts that were investigated V. negundo was most effective against both S. mutans (MIC value 0.37) and P. aeruginosa (MIC value 0.75) when compared to other three extracts of D. repens, P. nigrum, A. calamus. The plant extract of P. nigrum was observed to be effective against both S. mutans (MIC value 3.12) and P. aeruginosa (MIC value 6.25) was most susceptible. DMF served as control and was inactive against both test organisms. A variation in the degree of antibacterial activities of the extracts on the tested organism which is presumed to be due to different active compounds present in these plants. Also chemical constituents of these extracts may have a casual role in in vivo prevention of disease caused by bacteria, fungi and yeast also beingresponsible in the multi-chemical defense against microbial attack.

Apart from the individual antimicrobial studies of plant extracts, combined extracts from the selected plants were screened for synergistic activity. The combined activity of A. calamus and P. nigrum with Vitex negundo and D. repens in 1:0.5 ratios did not show any inhibitory effect; whereas antimicrobial effect was observed in 1:1 ratio with the combined extracts. The extract of P. nigrum did not show any inhibitory effect on tested organisms but the combination of P. nigrum with V. negundo and D. repens showed considerable inhibitory effect. The results are also supported by [14] who reported the presence of phytochemicals in Vitex negundo (L.) hydroalcoholic extract of antimicrobial activity.

COMPETING INTERESTS Authors have interests exist.

declared

that

no

competing

REFERENCES 1.

From the Table 4 above, the findings reveal that the standard antibiotic Ampicillin showed least inhibitory effect on P. aeruginosa, whereas the plant extracts except P. nigrum have shown considerable inhibitory effect on P. aeruginosa. Ciprofloxacin has shown inhibitory effect on P.

2.

21

Cowan MM. Plant products as antimicrobial agents. Clin. Microbiol Rev.1999;12(4):564-582. Lewis K and Ausubel FM. Prospects for plant-derived antibacterials. Nat. Biotechnol. 2006;24(12):1504-1507.


3.

Gibbons S. Anti-staphylococcal plant Linn. Extracts Indian J. Pharm. Sci. natural products. Nat. Prod. Rep., 2008;70(4):522-526. 2004;21:263-277. 10. Loganathan V, Sreekanth N, Senthil 4. Raghavendra H, Lakshmanashetty, Kumar B, Ubaidulla U, Sivaprasada Reddy Vijayananda B, Nagaraj, Madhumathi G, MV, Rajasekran A, Manimaran S. Hiremath, Vadiapudi Kumar. Invitro Preliminary phytochemical and antioxidant activity of Vitex negundo L. leaf antibacterial screening of Vitex negundo extracts. Chiang Mai J. Sci. Linn. Phytomedica. 2004;5:69-72. 2010;37(3):489-497. 11. Perumal Samy, Ignacimuthu, Sen 5. Singh Pradeep, Mishra Garima, Jha KK, entomology research institute, Chennai. Kumar Vipin, Khosa RL. Chemical 1997;(10):117-119. composition and antimicrobial activity of 12. Pessini GL, Dias Filho BP, Nakamura CV, essential oil of leaves of Vitex negundo L., Cortez DA. Antibacterial Activity of extracts International Journal of Chemical and neolignans from Piper regnelli (Miq.) Technology Research. 2010;2(3):1688C. D. C.Yunck var. pallescens E pub. 1690. 2004;98(8):1115-1120. 6. Rasaga CY, Morales E, Rideout JA 13. Vasinauskiene M, Radusiene J, Zitikaite T, Antimicrobial compounds from Vitex Survillience E. Antibacterial activities of negundo, Philipp J. Sci. 1999;128:21-9. essential oils from aromatic and medicinal 7. Merlin RC, Catherine L. Preliminary plants against growth of phytopathogenic phytochemical screening and antibacterial bacteria Agronomy Research. activity on Vitex negundo. International 2006;(4):437-440. Journal of Current Pharmaceutical 14. Amirtharaj R Vijy, Mohammed Reyaz A, Research. 2011;3(2):99-101. Kumar J Arun, Kaarthikeyan M, 8. Chandramu Rao, Manohar, David GL Saivishwathdindu Kumar, Senthil N. Krupadanam, Reddy Danshavantha. Preliminary phytochemical studies and In Isolation, Characterization and biological vitro cytotoxic activities on Vitex negundo activity of betulinic acid and ursolic acid (L.) International Journal of Research in from Vitex negundo, Phytotherapy Pharmaceutical and Biomedical Sciences. Research. 2004;17(2):129-134. 2011;2(4):1800-1804. 9. Khokra SL, Prakash Jain, Aneja, Yogita Dhingra. Essential oil composition and Antibacterial Studies of Vitex negundo _________________________________________________________________________________ © 2015 Padder et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history.php?iid=805&id=11&aid=7780

22