In vitro Antibacterial Efficacy of Selected Plant Extracts, Streptomycin and their Combination

 

Abhipsa V., Manasa M., Poornima G., Rekha C., Prashith Kekuda T.R.*

Department of Microbiology, SRNMN College of Applied Sciences, NES Campus, Balraj Urs Road, Shivamogga-577201, Karnataka, India

ABSTRACT:

The present study was undertaken to determine antibacterial potential of combination of methanol extract of five plants namely Polyalthia longifolia, Vitex negundo, Tephrosia purpurea, Jasminum arborescens and Abrus pulchellus and an aminoglycoside antibiotic Streptomycin against bacteria. The antibacterial activity of extracts, Streptomycin and their combination was determined against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa by Agar well diffusion method. Extracts, antibiotic and their combination resulted in marked suppression of growth of Gram positive bacteria than Gram negative bacteria. Combining extracts with antibiotic showed higher inhibition of test bacteria when compared to inhibition caused by antibiotic alone. The combined effect resulting from the association of antibiotic with plant extracts against bacteria, as observed in this study, possibly leads to the development of new strategy for the treatment of infectious diseases.

 

 

 

INTRODUCTION:

The substantial increase in antibiotic resistance has become a major and ever increasing problem. Staphylococcus aureus and Pseudomonas aeruginosa have been recognized as most common bacteria which have developed resistance against several antibiotics. P. aeruginosa is a major hospital borne pathogen which is particularly dangerous to patients and population having weak resistance. Nearly 50% of S. aureus strains have developed resistance to antibiotic Methicillin. For most resistant strains, glycopeptide-type drugs (such as Vancomycin) are the only effective agents. However, Vancomycin-resistant S. aureus has been reported. The development of resistance against antibiotics is due to increasing and indiscriminate use of antibiotics ^[1-4].

 

The healing properties of plants are because of the presence of characteristic secondary metabolites namely phenols, flavonoids, alkaloids, etc in them. These metabolites have several bioactivities such as antimicrobial, antioxidant, antihelmintic, anticancer activities etc.

 

The plant derived compounds become the base for the development of drug and may be used for the treatment of diseases. They are safer, have profound therapeutic benefits and have more affordable treatments. There are no or minimum side effects as compared to synthetic drugs and usually have effectiveness beyond the symptomatic treatment of diseases ^[5,6]. The inhibitory efficacy of plant extracts against microorganisms is due to the secondary metabolites present in them. Antimicrobial activities of tannins, flavonoids, saponins, terpenoids, alkaloids, steroids and glycosides have been well documented ^[7-13].

 

 

Bioenhancers are substances which promote or augment the bioactivity or bioavailability or the uptake of drugs in combination therapy. Such bioenhancers have been reported from plants. Studies have revealed that the combination of plant extracts with antibiotics could reduce the concentration of antibiotics required for inhibition of pathogenic microbes ^{[14, 15]}. In this study, we have determined inhibitory efficacy of combination of methanol extracts of five plants namely Polyalthia longifolia, Vitex negundo, Tephrosia purpurea, Jasminum arborescens and Abrus pulchellus and an aminoglycoside antibiotic Streptomycin against bacteria.

 

Table 1: Plant materials used in the study

 

Table 2: Antibacterial activity plant extracts, Streptomycin and their combination

 

MATERIALS AND METHODS:

Collection and identification of plant materials

The plant materials (Table 1) were collected and authenticated by Prof. Rudrappa D, Dept. of Botany, SRNMN College of Applied Sciences, Shivamogga-01, Karnataka. Voucher specimens were deposited in the department herbaria for future reference.

 

Extraction

The collected plant materials were shade dried, powdered mechanically and subjected for extraction in Soxhlet apparatus. A known quantity of powdered material (100gm) was exhaustively extracted with methanol (HiMedia, Mumbai) as solvent. The extract was filtered, concentrated in vacuum under reduced pressure and dried in the desiccators ^[16,17]. The extracts obtained were stored in airtight containers and used for antibacterial assay.

 

Preparation of Extract, Antibiotic and their combination

Extract was prepared in dilute Dimethyl sulfoxide (DMSO) to get a concentration of 50mg/ml of DMSO. Streptomycin (1mg/ml of sterile distilled water) was used as standard antibiotic. Combination of extract and antibiotic was prepared by mixing 1ml of each in a test tube.

 

Antibacterial activity of extract, antibiotic and combination

The inhibitory activity of extracts, antibiotic and their combination was tested against bacteria namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis by Agar well diffusion method ^[18]. Test tubes containing sterile nutrient broth were aseptically inoculated with the pure cultures of test bacteria and incubated at 37^oC for 24 hours. Wells of 6mm were punched in agar inoculated with broth cultures of test bacteria (by swabbing using sterile cotton swabs). The extracts, Control (DMSO), Streptomycin and extract-Streptomycin combination were transferred into the respectively labeled wells. The plates were incubated at 37^oC for 24 hours in upright position and the zone of inhibition was recorded.

 

RESULTS:

The inhibitory effect caused by extracts, antibiotic and their combination as studied by employing agar well diffusion method is shown in Table 2. Results were recorded as presence or absence zones of inhibition around the well. The presence of inhibitory zone around the well was taken as positive for antibacterial activity. The extracts were found to cause inhibition of bacteria tested. Pericarp extract of P. longifolia was shown to inhibit test bacteria to high extent when compared to other extracts. Least inhibitory effect was observed in case of leaf extract of T. purpurea. Among the bacteria tested, Gram positive bacteria have shown more sensitivity to extracts when compared to Gram negative bacteria. E. coli was inhibited to lesser extent by extracts when compared to other bacteria. Inhibition caused by standard antibiotic was higher than that of extracts. The antibiotic caused high inhibition of Gram positive bacteria than Gram negative bacteria. DMSO (control) did not cause inhibition of bacteria. In case of combination of extract and antibiotic, marked inhibition of the bacteria was observed and the inhibition recorded was higher than that of inhibition caused by antibiotic alone in majority of trials. High inhibition of Gram positive bacteria was observed.

 

DISCUSSION:

The use of combination of two or more antibiotics is a standard clinical practice as a result of development of resistance in organisms. Such combinations usually contain antibiotics that have different mode of actions in order to prevent resistance development and to improve therapy. This approach is very useful in expanding the antimicrobial spectrum, to prevent emergence of mutants, minimize toxicity and to obtain synergistic activity ^[6,19]. Even though the combined antibiotic therapy to treat diseases is advantageous, it will not be effective for a long period of time because of possible changes in the susceptibility pattern of bacteria. Therefore, the development of new classes of antimicrobial is of significant importance.

 

Plants produce multidrug resistance inhibitors that enhance the activity of antibiotics. Several studies have revealed the reduction in MIC values of Antibiotics when combined with crude extracts or purified phytochemicals. The sub inhibitory levels of Catha edulis were found to reduce the MIC of tetracycline and penicillin against oral pathogens ^[14]. Polyphenols have been reported to reverse β-lactam resistance in MRSA ^[15]. Phytochemicals have been reported to have resistance modulating abilities on various antibiotics against resistant strains of S. aureus ^[20,21]. Chatterjee et al. ^[3] showed marked in vitro synergisitic effect of doxycycline and ofloxacin in combination with Vangueria spinosa extract against pathogenic bacteria. Olayinka et al. ^[6] observed marked synergistic effect, against wound infection associated bacteria, by combination of extracts of Helichrysum pedunculatum and antibiotics. Ahmed et al. ^[22] showed higher inhibition of S. aureus when tetracycline and penicillin were mixed with extracts of Salvadora persica. The methanol extract of Thespesia populnea flowers showed good synergism with oxytetracyclie. Higher synergism was observed against Shigella ^[23]. Purushotham et al. ^[24] showed higher inhibition of bacteria by combination of extract of Tectona grandis and tetracycline. The results of our study are also consistent with the above studies as the combination of extracts and antibiotic resulted in higher inhibition of test bacteria. Inhibition caused by the combination was higher than inhibition caused by individual treatments.

 

CONCLUSION:

The combined effect resulting from the association of antibiotic with plant extracts against bacteria, as observed in this study, possibly leads to the development of new strategy for the treatment of infectious diseases. Also, the dosage of antibiotic in the combination is lesser than the dosage when used alone. This reduction in the antibiotic dosage could result in lesser side effects and also the cost could be reduced. The combination used in this study could be effective against drug resistant bacteria. Further, toxicity studies and in vivo studies are needed to confirm these findings.

 

ACKNOWLEDGEMENTS:

The authors express thanks to HOD, Dept. of Microbiology, Principal, SRNMN College of Applied Sciences, Shivamogga for providing all the facilities to conduct work. Authors also express thanks to NES, Shivamogga for giving moral support.

 

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Received on 17.05.2012        Modified on 05.06.2012

Accepted on 20.06.2012        © AJRC All right reserved