(Cymbopogon flexuosus Steud) Wats Essential Oils in ...

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Antibacterial Properties of Lemongrass (Cymbopogon flexuosus Steud) Wats Essential Oils in Single form and Combination of Honey Against Drug Resistant Pathogenic Bacteria a

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Deepak Ganjewala , Ruchika Mittal , Ashish Kumar Gupta , Martha Premlatha & Reetika Dawar

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Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125, Noida-201303, UP, India b

Amity Institute of Virology and Immunology, Amity University Uttar Pradesh, Sector 125, Noida-201303, UP, India c

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Microbiology Department, Indraprastha Apollo Hospitals, New Delhi, India Published online: 04 Dec 2014.

To cite this article: Deepak Ganjewala, Ruchika Mittal, Ashish Kumar Gupta, Martha Premlatha & Reetika Dawar (2014) Antibacterial Properties of Lemongrass (Cymbopogon flexuosus Steud) Wats Essential Oils in Single form and Combination of Honey Against Drug Resistant Pathogenic Bacteria, Journal of Biologically Active Products from Nature, 4:4, 278-285, DOI: 10.1080/22311866.2014.933083 To link to this article: http://dx.doi.org/10.1080/22311866.2014.933083

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TBAP 4 (4) 2014 pp 278 - 285

278 ISSN Print: 2231-1866 ISSN Online: 2231-1874

Antibacterial Properties of Lemongrass (Cymbopogon flexuosus Steud) Wats Essential Oils in Single form and Combination of Honey Against Drug Resistant Pathogenic Bacteria Deepak Ganjewala 1*, Ruchika Mittal 1, Ashish Kumar Gupta 1, Martha Premlatha 2 and Reetika Dawar 3 Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125, Noida-201303, UP, India 2 Amity Institute of Virology and Immunology, Amity University Uttar Pradesh, Sector 125, Noida-201303, UP, India 3 Microbiology Department, Indraprastha Apollo Hospitals, New Delhi, India

Downloaded by [14.141.144.5] at 04:11 19 January 2015

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Received 02 December 2013; accepted in revised form 21 March 2014

Abstract: Antibacterial potential of four lemongrass essential oils (EOs) and their major oil constituent, citral in single form and in combination of honey were evaluated against multi drug resistant (MDR) bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus. The antibacterial potentials determined using agar well diffusion method has revealed that Krishna EOs exhibited most potent bactericidal effects than other Eos against all MDR bacteria used. The diameter of zone of inhibition of Krishna EOs ranged from 44-50 mm/50 μL of EOs. However, EOs of Pragati and Suvarna were selectively effective against S. aureus while that of Nima against A. baumannii and P. aeruginosa. Citral expressed better antibacterial potential against all the MDR bacteria with zone of inhibition 38-45 mm/50 μl. The combination of all four different EOs with honey showed pronounced antibacterial activity exclusively against S. aureus. The combination of Suvarna EOs and honey showed 22 % increase in antibacterial activity against A. baumannii than the EOs alone. The combination of citral and honey showed significantly lower antibacterial activity as compared to citral alone against all MDR bacteria used. The minimum inhibitory concentrations of EOs determined were 4.5 μg/ml. Thus, the study revealed enhanced antibacterial potential of lemongrass EO when they are used with honey against drug resistant S. aureus. Key words: Antibacterial, citral, essential oil, honey, lemongrass, multi drug resistant, zone of inhibition. Introduction Treatment of infectious diseases caused due to microbes has become the most challenging problem in the modern era. It is reported that infectious diseases causes approximately one half of all deaths in tropical countries 1,2. The literature surveyed indicated that pathogenic microbes have evolved resistance mechanism against most of the available antimicrobial drugs and among MDR *Corresponding author (Deepak Ganjewala) E-mail: < [email protected] >

bacterial pathogens, became a major public health concern in the 21st century emergence and spread of Acinetobacter species 5, Pseudomonas aeruginosa 6-9 and methicillin resistant Staphylococcus aureus (MRSA) 2 are recognized as the most difficult healthcare associated infections to control and treat. Staphylococcus aureus (MSRA) has developed resistance mechanisms against multi drugs including β-lactam antibiotics, amino© 2014, Har Krishan Bhalla & Sons


Deepak Ganjewala et al., / TBAP 4 (4) 2014 278 - 285 glycosides, lincosamides, tetracyclines, and also quinolones and rifampin. The increasing resistance of bacteria to best antibiotics and antivirals drugs available has necessitated new search for powerful alternative drugs from plants. Plants produce plethora of compounds, such as terpenoids or isoprenoids, alkaloids, flavonoids and phenolics which possess many useful medicinal and pharmaceutical properties. Given their medicinal values plants and plant products are used for the treatment of many infectious diseases and may be of great use in primary healthcare as therapeutic remedies 11-13. Essential oils (EOs) are the most important class of terpenoid compounds consisted of complex mixtures of cyclic and acyclic monoterpenes which own awesome biological activities. They are reported to have therapeutic properties, such as chemoprevention, cancer suppression, antidiabetic activity, lowering serum cholesterol and triglycerides and antimicrobial activities against bacteria, virus, fungi and yeast 14. A number of EOs with potent antibacterial properties has been reported to be used in food to control natural spoilage processes and prevent the growth of microorganisms. They are also used as additives to drugs and cosmetics 15. Essential oils are quite safe and effective due to their non toxic nature and minimal or no side effects therefore their applications may be promoted for prevention and treatment of human systemic diseases, including infectious diseases 16. Cymbopogon flexuosus commonly known as East Indian lemongrass is an elite member of the genus Cymbopogon that yield an essential oil (EOs) upon hydro-distillation of its leaves. Lemongrass EOs is mainly characterized by the presence of very high amount of citral in the EOs. Citral is an acyclic monoterpene aldehyde which imparts lemon like aroma to the lemongrass EOs. Lemongrass EOs due to their unique lemon like aroma are widely used in favours, fragrance, cosmetics, deodorants and food 17. Lemongrass EOs and citral possesses many useful bioactivities of medicinal and pharmaceutical importance, thus used extensively for the treatment of various disorders 17,18. Cymbopogon EOs are reported to possess antimicrobial, antifungal, antiyeast,

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insecticidal, antiparasitic, antiviral and antiprotonzoan, anticancer/cytotoxic activities 1925 . Most of these activities of EOs is mainly attribute to major ingredient, citral 24,26. Previously published review articles have described the chemical composition and bioactivities of the EOs of Cymbopogon spp. and its major monoterpene aldehyde constituent citral 26,27 . Despite a number of reports published on chemical composition and bioactivities of lemongrass EOs and constituents, only little work has been carried out on possible combination of lemongrass EOs with antibiotics or other products such as, honey against multi drug-resistant bacteria. For a very long time honey is well known for its beneficial medicinal properties such as, antimicrobial and wound healing for very long time 28. Previous reports on potent bioactivity of honey against antibiotic-resistant bacteria have motivated us to undertake this study to evaluate antibacterial potential of lemongrass EOs in combination of honey against MDR bacteria. Previously, researchers have reported the synergetic effects of honey on EOs and many other plants extracts suggesting their prospects as an alternative potent antimicrobial therapy 29-34. The present study was undertaken to determine the antibacterial activities of the EOs of four lemongrass varieties, Krishna, Neema, Pragati and Suvarna, their major oil constituent, citral in the single form and in combination of honey against clinical pathogens A. baumannii, P. aeruginosa, and S. aureus. The study showed that combination of each of lemongrass EOs with honey was found significantly more effective than Eos alone against S. aureus. Material and methods Chemicals Commercial honey was purchased from Patanjali Center, Haridwar, authentic citral from Sigma-Aldrich, Germany and nutrient agar media (NAM) from Fisher Scientific, India. Plant Material Lemongrass (Cymbopogon flexuosus Steud) Wats varieties Krishna, Neema, Pragati and Suvarna tillers grown in the Organic farm house


Deepak Ganjewala et al., / TBAP 4 (4) 2014 278 - 285

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of Amity University Campus, Noida, India were harvested at the age of one month. Tillers were brought to laboratory washed with plenty of water and cut into small pieces and their EOs oil were extracted by steam distillation in a mini Clevenger apparatus 35. The yield of EOs oil were 0.5-1 % (v/w) of the fresh weight. The essential oils were collected in small vials and stored in a refrigerator at 4°C.

20 μl/ml DMSO. The bacterial suspension culture (0.5 McFarland standards) was seeded on NAM plates and four wells were made in the plates with the help of cork borer (6mm). Fifty microliter of diluted samples were added into four separate wells in the plate. The plates were incubated at 37°C for 24 h. The diameter of zone of inhibition was measured and recorded. Experiment was performed in triplicates.

Microorganisms and their preservation Three MDR bacteria A. baumannii, P. aeruginosa, and S. aureus were obtained from Clinical Microbiology Laboratory, Apollo Indraprashtha Hospitals, New Delhi, India. The bacterial culture was inoculated in nutrient agar media (NAM) plates. The pure colonies were sub-cultured and preserved in agar slants at 4°C and 50 % glycerol stock at -20°C for further use.

Results and discussion In the present study we have evaluated antibacterial activity of lemongrass EOs and its major constituent, citral against three multi-drug resistant bacteria A. baumannii , P. aeruginosa and S. aureus in terms of diameter of zone of inhibition (mm) using well diffusion method. Also, evaluated the effects of honey on antibacterial properties of lemongrass EOs and citral against MDR bacteria. Antibiotic sensitivity test performed using standard antibiotics has revealed that S. aureus was resistant against methiciliin and ampicillin, P. aeruginosa to ampicillin, vancomycin and gentamicin, while the A. baumannii only against ampicillin. Essential oils obtained from four lemongrass varieties viz., Krishna, Nima, Pragati, and Suvarna were found to be highly effective against the MDR bacteria used (Table 1). However, Krishna EOs among others had strongest antibacterial activity against all the tested MDR bacteria with measured values of diameter of zone of inhibition ranged from 4450 mm/50 μL of EOs. Pragati and Suvarna EOs possessed strong antibacterial activity against only S. aureus, but not to A. baumannii and P. aeuruginosa. Essential oils of Krishna and Neema showed strong antibacterial activity against P. aeuruginosa and A. baumannii with zone of inhibition (45-50 mm/50 μL of EOs), while less effective against S. aureus. Neema EOs, however, had least antibacterial effects against S. aureus with measured value of zone of inhibition 30 mm/50 μL of EOs. The combination of EOs and honey displayed similar antibacterial activity of those EOs alone against A. baumannii and P. aeruginosa. The antibacterial potential of all the EOs against S. aureus significantly increased in presence of

Determination of antibacterial activity Antibacterial activity of EOs, citral and honey separately and in combination of honey were determined by agar well diffusion method. The culture plates were prepared by pouring 20 ml sterilized NAM into pre-sterilized Petri dishes. One milliliter of inoculums suspension (1.0 x 105 cfu/ml) was spread uniformly over the agar medium using sterile glass rod. Wells in the agar plates were made by using sterile cork borer (6 mm). Fifty microliter of each EOs samples, citral and honey were separately added to wells in the plates. In other set of experiment, 50 μl of each EOs and citral mixed with 50 μl honey were added separately to the wells in the agar plates. The plates were incubated at 37°C for 24 h. The mean diameters of zone of inhibition (mm) was measured and recorded. Standard antibiotics, gentamicin and amplicillin (10 μg/ml) were used as positive control and dimethyl sulphoxide (DMSO), as a control. Determination of minimum inhibitory concentration (MIC) The MIC of the lemongrass EOs against the test organisms was determined using the agar well diffusion method. Lemongrass EOs were diluted with DMSO in order to prepare dilution from 5-

35 35 35 35 45 45 45 45 38 64 44 72 64 44 30 50 50 20 20 20 20 38 38 38 38 ND 48 50 42 42 48 48 40 40 25 25 25 25 40 40 40 40 ND 48 42 40 44

EOs: Essential oil; H: honey; ND: not detected

EOs

50 45 44 36 Krishna Nima Pragati Suvarna Honey

S. aureus EOs+ H Citral Citral +H Zone of inhibition (mm) P. aeruginosa EOs EOs +H Citral Citral +H EOs A. baumannii EOs +H Citral Citral +H Essential oils

Table 1. Antibacterial activities of lemongrass essential oils, major constituent citral in alone and combination of honey against the multi drug resistant bacteria



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honey but remained unchanged against A. baumannii and P. aeruginosa (Table 1). However, Suvarna EOs mixed wit honey showed 22 % increased activity against A. baumannii (Table 1). The diameter of zone of inhibition of EOs of Krishna, Nima and Pragati in combination of honey recorded against S. aureus was significantly greater (44-72 mm) than those of EOs alone (30-50 mm) indicating a significant 44-46 % increase in the antibacterial activities of EOs. However, combination of Suvarna EOs and honey showed only 28 % (in terms of zone of inhibition) increase in the antibacterial potential against S. aureus. Citral exhibited very similar trends of antibacterial activities to those EOs in single form against MDR bacteria used. The measured values of diameter of zone of inhibition of citral ranged from 38-45 mm/50 μL of citral (Table 2). However, when citral was mixed with honey, its antibacterial potential noticeable declined against all the three MDR bacteria. The diameter of zone of inhibition of mixture of citral and honey reduced reduced to 20-35 compared to an average of 38-45 mm of citral alone against all the three bacteria used. The antibacterial potential of mixture of cirtal and honey dropped significantly by 38 and 47 % against A. baumannii and P. aeuriginosa, respecti-vely as compared to the citral alone. Susceptibility test with standard antibiotic, ampicillin and gentamicin showed zone of inhibition which ranged from 0.0 to 10 mm. From these results, lemongrass EOs, honey and their synergy is comparable to the standard antibiotics. The minimum inhibitory concentrations (MICs) of the EOs against the test microorganisms were also determined by the agar plate methods. The MIC values varied between 4.5 and 18.0 μg/ml of EOs against the MDR bacteria used in the study. As per the MIC data, A. baumannii was highly sensitive to Krishna and Suvarna EOs, P. aeruginosa to Nima and Pragati EOs while the S. aureus to the EOs of Pragti (Table 2). These results are in accordance with many previously published reports on EOs of aromatic plants having antimicrobial activity against bacteria and fungi. These reports have revealed that the antimicrobial activity of EOs can be


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Table 2. Minimum inhibitory concentration (MIC) of lemongrass essential oils against the multi drug resistant bacteria

A. baumannii

μg/ml*) MIC (μ P. aeruginosa

S. aureus

4.5 18 18 4.5

9 4.5 4.5 9

9 9 4.5 18

Essential oils Krishna Nima Pragati Suvarna


* Volume of the essential oils was multiplied by their specific density 0.9 to get the g/ml values.

attributed to their major constituents attributed to their manor constituents mainly monoterpenoids 36. Our previous study also revealed the similar antibacterial activities of the EOs from four varieties of lemongrass viz., Krishna, Cauveri, Nima and Cheerharit and major EOs constituents such as, citral, geraniol and geranyl acetate against Escherichia coli, Salmonella typhi, Bacillus aureus and S. aureus 24. Among all the tested EOs, Krishna EO showed strongest antibacterial activity against S. aureus with diameter of zone of inhibition 48 mm/10 μl EOs 24. A study on anti-bacterial activity screening of the 91 Eos including the lemongrass EOs carried out by Chao et al 37 has also revealed the strong antibacterial potential of lemongrass EOs against S. aureus with diameter of zone of inhibition ~ 60 mm/30 μl EOs. Dahiya and Purkayastha 2 also reported the considerable antibacterial activity of methanol and ethanol extracts of lemongrass against S. aureus (MRSA), however with lower zone of inhibition 11-13 mm values than reported here. Thus, the antibacterial activity profiles of the lemongrass EOs and citral studied here are in full agreement with the outcomes of the previously published reports 2,24,37. The antibacterial potential of lemongrass EOs and mixtures of EOs and honey might be attributed to the major EOs constituent citral and medicinal properties of the honey. The results of antibacterial activity of the lemongrass EOs alone and in combination of honey are strongly supported by many previously published research papers on antimicrobial properties of honey against various micro-

organisms 38-40 In the present study, lemongrass EOs has shown significantly higher antibacterial activity against S. aureus when applied with honey. However, this combination of EOs with honey did not show any increase in the antibacterial activity against A. baumannii and P. aeruginosa. Here, we could not study the mechanism of antibacterial action of the lemongrass EOs, but results of the present study together with previously published reports indicated that citral is most likely be responsible for the antibacterial action of the lemongrass EOs.but results of the present study together with previously published reports indicated that citral is most likely be responsible for the antibacterial action of the lemongrass EOs. Essential oil constituents mainly monoterpenoids display antibacterial properties through their effects on permeability of the bacterial cell membrane that results in the leakage of the vital intracellular components of the bacteria outside the membrane. The loss of intracellular components cause imbalance in the equilibrium of inorganic ions and possible impairment of bacterial enzyme system and cell respiration 41. The hydrophobic nature of the EOs is also reported highly detrimental to the lipids of bacterial cell wall rendering them more permeable 42 . The characteristic feature of the bacteria such as, the cell wall structure which plays major role in the resistance of bacteria against antibiotics, or any other compounds might be responsible for their markedly varied responses to a particular drug/ antibiotic. Perhaps this could be a reason for the differences in response of MDR bacteria to the


Deepak Ganjewala et al., / TBAP 4 (4) 2014 278 - 285 lemongrass EOs and citral alone, and in combination of honey. In the present study, S. aureus was found most susceptible to all the EOs, citral and mixture of EOs and honey. The outer lipopolysaccaride wall of Gram-negative bacteria acts to prevent the entrance of toxic agents. Other factors like functional groups, configuration and chemical structure play a role in the activity of the constituents comprising EOs against bacteria 41 . Several properties of honey like high sugar concentration, low pH, hydrogen peroxides, presence of antibacterial substance (inhibine), proteineceous compounds, or other unidentified compounds makes it a strong antimicrobial agent 43 . Honey causes shrinkage and disruption of the bacteria 28. Thus, positive effects of honey seen on antibacterial activity of lemongrass Eos against methicilin resistant, S. aureus were expected. The effectiveness of mixture of lemongrass EOs and honey in descending order was S. aureus > P. aeruginosa > A. bumanniii. The study could not explain why the mixture of lemongrass EOs and honey resulted in a

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significant increase in antibacterial potential only against S. aureus and not the other bacteria used. Most likely it could be synergy between lemongrass EOs and honey which enhanced antibacterial potential against S . aureus. At present, antibacterial screening of lemongrass EOs against other MDR bacterial strains is in progress with emphasis on elucidation of their mechanism of action. The study explored possibility of applications of lemongrass EOs in combination of honey as an alternative drugs for the treatment of human diseases, particularly infectious diseases caused by multidrug resistant bacteria. Acknowledgements Corresponding author of this article is grateful to Dr. Ashok Kumar Chauhan, Founder President and Atul Chauhan, Chancellor, Amity University, Uttar Pradesh, Noida, India for providing necessary facilities and support. Also, research grant from Council of Scientific and Industrial Research (CSIR), New Delhi is duly acknowledged.

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