Cymbopogon Citrates Oil Showing Antimicrobial Activity against Microbes of Environmental, Clinical and Food Origin

Mohd. Yaqub Khan*, Poonam Gupta, Vinod Kumar Singh, Sanjay Yadav, Vikas Kumar Verma

Saroj Institute of Technology & Management, Ahimamau P.O. Arjunganj Sultanpur Road, Lucknow-226002,Uttar Pradesh, India

*Corresponding Author E-mail: khanishaan16@yahoo.com

ABSTRACT:

Out of the 1114 strains belonging to 29 genera and 105 species of microbes (molds, yeasts and bacteria) isolated from different sources [clinical cases, environment (water, air, soil, droppings of lizards and birds), food and healthy animals], 38.2% were sensitive to lemongrass oil discs containing 50 µg oil/disc. All molds, yeasts, Lactobacillus acidophilus, Morganella morganii, most of the Bacillus spp. Strains (84.3%), aeromonads (78%), Edwardsiella spp. (73.9%), 53.6% pseudomonads, 53.1% streptococci and 50% of Budvicia aquatica and Leminorella ghirmontii strains were sensitive to lemongrass oil (LGO). On the other hand, all Hafnea alvei, Laclercia adecarboxylata, Xenorhabdus luminescens and majority of Salmonella enterica (98.3%), Citrobacter spp. (93.7%), Providencia spp. and Kluyvera cryocrescens (83.3%), Enterobacter spp. (78.2%), Proteus spp. (78%), Escherichia spp. (77.7%), enterococci (73.7%), Serratia spp. (75%) and Erwinia ananas (75%), Pragia fontium (70.6%), staphylococci (69.8%) and Klebsiella spp. (62.7%) strains were resistant to LGO. MIC of LGO for sensitive strains (tested against discs containing 50 µg LGO) varied from 1 µg to 32 µg /ml while none of the resistant strains had MIC <64 µg LGO/ ml. MIC for yeast strains was the least i.e., 1 µg LGO/ ml. LGO had microbicidal activity on E. coli, S. aureus and Candida albicans. LGO instantly killed C. albicans and E. coli, and S. aureus in 10 min at 1 mg/ ml concentration, indicating of its wide spectrum antimicrobial activity at easily achievable concentrations. Study also indicated that LGO is more effective on enterococci in aerobic instead of microaerophilic growth conditions, it is indicative that in-vivo sensitivity results may differ from in-vitro tests.

KEY WORDS: Lemongrass oil, Antimicrobial activity, Microbes, Microaerophilic growth

INTRODUCTION ^{[1, 2]}

Lemon grass belongs to the section of Andropogan called Cymbopogam of the family Germineae. Due to the production of lemon grass oil as major component, two of the species i.e. Cymbopogan citrates and C. flexuosus are generally called Lemon grass. Medicinal use of lemongrass is known to mankind since antiquity. Its oil has been used to cure various ailments like cough, cold, spitting of blood, rheumatism, lumbago, digestive problems, bladder problems, leprosy, and as mouth wash for the toothache and swollen gums. It is also been claimed to be stimulating, diuretic, anti purgative and sudorrific to reduce fever.

To cure cholera, colic and obstinate vomiting only 3-6 drops of the oil are effective medicine of choice. The oil has been found to posses bactericidal and anti fungal properties, which is comparable to penicillin in its effectiveness. The oil also contains male sex hormone agent. It is also reported to have strong activity against two dermatophytes, namely Trichophyton rubrum and Microsporium gypsum. Similarly pharmacological investigation on the essential oil of C. citratus revealed that it has a depressant effect on the CNS. It has analgesic and antipyretic properties. The extract juice from the lemon grass contains inhibitor of the promotion stage of carcinogenesis induced by cotton oil. It is an oral anti tumor drug for the cancer and in combination with cyclodextrin lengthened the survival time. Gallstone dissolving preparations have been made of oil. The lemon grass contains high percentage of Vitamin C, which is a characteristic of plants used as drug e.g., belladonna and jaborandi. Lemon grass oils show activity towards the phyto pathogenic fungi. A combination of lemon grass oil is given for use on human and domestic animal pathogens. This work was set out in order to investigate the antimicrobial activity of lemongrass extracts against some pathogenic bacteria and fungi and to ascertain the chemical constituents that may be present.

Extraction procedure: ^[3]

Dried plant leaves were extracted by weighing samples of 1 g of finely ground plant material and extracting with 10 mL of acetone hexane, dichloromethane (DCM) or methanol (technical grade- Merck) and boiled water in polyester centrifuge tubes. Tubes were vigorously shaken for 3 to 5 min & shaking machine at high speed. After centrifuging at 3500 rpm for 10 min the supernatant was decanted into pre-weighed, labeled containers. The process was repeated three times to exhaustively extract the plant material and the extracts were combined. The solvent was removed under a stream of air in a fume cupboard at room temperature and the extraction efficiency was quantified by determining the weight of each of the extracts. The antimicrobial activity of the crude extract was screened against four gram-negative bacteria; Neisseria gonorrheae, Salmonella sp., Pseudomonas aeruginosa, Proteus vulgaris and two gram-positive bacteria; Staphylococcus aureus and Streptococcus aerugenosa. (Clinical isolates) obtained from the Public health center laboratories isolates each of gram-negative bacteria Escherichia coli and Salmonella typhi); and grampositive bacteria S. aureus and Streptococcus pneumoniae; and four fungi, Aspergillus niger Aspergillus tamari , Candida albicans and Fusarium oxysporum (standard laboratory isolates), all obtained from the Public health center laboratories.

MATERIALS AND METHODS: ^{[11, 12]}

Determination of Antimicrobial activity of LGO

The antibacterial activity was determined by disk diffusion method and minimum inhibitory concentration (MIC) determination assays methods of National Committee for Clinical Laboratory Standards (NCCLS) and Clinical and Laboratory Standards Institute (CLSI). For disk diffusion test, sterile disks of five mm diameter were soaked in methanolic solution of LGO and dried at room temperature to contain 50µg of the oil. Mueller Hinton agar (MHA; Hi-Media, Mumbai) plates were swabbed with 6-8 hour growth of test bacteria in tryptic soy broth (TSB, Hi-Media) medium or with overnight Sabrauds’ broth (Hi-Media Mumbai) growth of yeast and mold strains, plates were allowed to dry. LGO discs with standard positive control disc (50µg mercuric chloride) and negative control disc (disc soaked in methanol and dried) was placed on the MHA plate. Plates were incubated overnight at 37°C for bacteria and for 48-72 hours at 22°C for yeast/fungi, the inhibition zone around discs was measured in mm.

To determine the effect of growth condition on disc diffusion assay, 8 strains of Enterococcus avium were tested under aerobic and microaerobic growth conditions simultaneously. For microaerophilic condition, plates were incubated in an anaerobic culture jar (Merck, Germany) using gas generating kit, Anaeocult® C (Merck) Cat No. 1.16275.0001. Plates were incubated for 24 h and zone of inhibition was recorded as for the aerobic plates.

Table 1 Antimicrobial effect of lemongrass oil on strains of different genera of microbes ^{[4, 5, 6, 7, 8, 9, 10]}

Microbial strains Tested (Number of species)	Strains Tested	Strains resistant	Strains sensitive	% sensitive strains	% resistant strains
Aspergillus spp.	11	0	11	100.0	0.0
Candida spp.	7	0	7	100.0	0.0
Lactobacillus acidophilus	1	0	1	100.0	0.0
Morganella morganii	3	0	3	100.0	0.0
Penicillium spp.	3	0	3	100.0	0.0
Bacillus spp.	115	18	97	84.3	15.7
Aeromonas spp.	91	20	71	78.0	22.0
Edwardsiella spp.	23	6	17	73.9	26.1
Micrococcus agilis	3	1	2	66.7	33.3
Pseudomonas spp.	28	13	15	53.6	46.4
Streptococcus spp.	32	15	17	53.1	46.9
Budvicia aquatica	8	4	4	50.0	50.0
Leminorella ghirmontii	2	1	1	50.0	50.0
Klebsiella spp.	110	69	41	37.3	62.7
Staphylococcus spp.	43	30	13	30.2	69.8
Pragia fontium	17	12	5	29.4	70.6
Ervinia ananas	12	9	3	25.0	75.0
Escherichia spp.	112	87	25	22.3	77.7
Enterococcus spp.	213	157	56	26.3	73.7
Proteus spp.	41	32	9	22.0	78.0
Enterobacter spp.	55	43	12	21.8	78.2
Kluyvera cryocrescens	6	5	1	16.7	83.3
Providencia spp.	6	5	1	16.7	83.3
Citrobacter spp.	95	89	6	6.3	93.7

For determination of MIC of selected LGO disc sensitive and resistant strains (Table 4) of Klebsiella pneumoniae (CP62, M10, LT 81, LT121), Escherichia coli (E382, C91, P82, P86), Edwardsiella tarda (26P, 1BCY, 56LT1, 59LT3), Bacillus coagulans (CB1, CB6, A12, B17), Staphylococcus aureus (SK10S2, SK5S1, SK6S1, SKE111), Streptococcus mobilis (SV11, SV27NC, SV12, SV36NC), Enterococcus faecalis (SV7, SV20, E31, CV14NC) and Candida albicans (CV1PD, ABY42), agar dilution susceptibility test was performed based on modified method of NCCLS and CLSI. Briefly, LGO dissolved in sterilized dimethyl-sulphoxide (DMSO; 1024 µg /ml) was taken as standard and two fold dilutions were made to achieve 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg /ml concentration of essential oil in molten (at 450C) MHA. Plates were poured and after solidification, the plates were spot inoculated with loopfull (2 μl) of overnight grown bacterial/ yeast cultures. The test was carried out in triplicates and plates were incubated overnight at 37°C for bacteria and 22°C for yeast. After 18 to 24 hours, the MIC was determined.

To determine that LGO is either microbiostatic or microbicidal, LGO dissolved in sterilized dimethyl sulphoxide (DMSO; 100 mg /ml) was mixed with sterilized normal saline solution (NSS) or with brain hear infusion (BHI) medium (Hi-Media) to the final concentration of 1 mg/ ml and 0.01 mg/ ml. In LGO containing BHI medium or NSS, washed (with NSS) cells of overnight grown bacteria (S. aureus SKE111, E. coli 382) and yeast (C. albicans, ABY42) were added at concentration of 42000 colony forming units per ml. Aliquots were drawn at an interval of 1 min for first 10 min and then at an hour interval for 30 h. Aliquots were plated in triplicate for counting the cfu/ ml after serial dilution in NSS. All tests were repeated thrice for conformity.

Table 2 Antimicrobial effect of lemongrass oil on strains of Gram negative bacteria ^{[13, 14, 15, 16, 17, 18]}

Microbial strains Tested (Number of species)	Strains Tested	Strains resistant	Strains sensitive	% sensitive strains	% resistant strains
Aeromonas caviae	12	2	10	83.3	16.7
A. eucranophila	18	10	8	44.	55.
A. hydrophila	18	3	15	83.3	16.7
A. media	9	0	9	100.0	0.0
A. salmonicida ssp. achromogenes	3	2	1	33.3	66.7
A. salmonicida ssp. salmonicida	5	2	3	60.0	40.0
A. salmonicida ssp. smithia	1	0	1	100.0	0.0
A. schubertii	8	0	8	100.0	0.0
A. sobria	3	0	3	100.0	0.0
A. veronii	14	1	13	92.9	7.1
Xenorhabdus luminescens	1	1	0	100.0	0.0
Budvicia aquatica	8	4	4	50.0	50.0
Citrobacter amalonaticus	11	11	0	0.0	100.0
C. diversus	6	6	0	0.0	100.0
C. freundii	78	72	6	7.7	92.3
Edwardsiella hoshiniae	1	1	0	0.0	100.0
Edwardsiella. tarda	22	5	17	77.3	22.7
Enterobacter agglomerans	9	14	9	39.1	60.9
Enterobacter. amnigenus I	9	9	0	0.0	100.0
Enterobacter amnigenus II	3	1	2	66.7	33.3
Enterobacter cancerogenus	1	1	0	0.0	100.0
Enterobacter cloacae	5	5	0	0.0	100.0
Enterobacter gregoviae	11	11	0	0.0	100.0
Enterobacter hormaechei	1	1	0	0.0	100.0
Enterobacter sakazaki	1	1	0	0.0	100.0
Enterobacter spp.	1	0	1	100.0	0.0
Erwinia ananas	12	9	3	25.0	75.0
Escherichia blattae	6	4	2	33.3	66.7
Escherichia coli	96	77	19	19.8	80.2
Escherichia furgusonii	8	4	4	50.0	55.0
Escherichia vulneris	2	2	0	0.0	100.0
Hafnea alvei	4	4	0	0.0	100.0
Klebsiella oxytoca	9	7	2	22.2	77.8
K. pnumoniae ssp. pneumoniae	95	57	38	40.0	60.0
Klebsiella terrigena	6	5	1	16.7	83.3
Kluyvera cryocrescen	6	5	1	16.7	83.3
Leclercia adecarboxylata	1	1	0	0.0	100.0
Leminorella ghirmontii	2	1	1	50.0	55.0
Morganella morganii	3	0	3	100.0	0.0
Proteus mirabilis	12	8	4	33.3	66.7
Proteus myxofaciens	1	0	1	100.0	0.0
Proteus penneri	19	17	2	10.5	89.5

Table 3 Antimicrobial effect of lemongrass oil on strains of Gram positive bacteria and fungi ^{[19- 26]}

Microbial strains Tested (Number of species)	Strains Tested	Strains resistant	Strains sensitive	% Sensitive strains	% Resistant strains
Aspergillus flavus	6	0	6	100.0	0.0
Aspergillus niger	5	0	5	100.0	0.0
Bacillus anthracoides	3	0	3	100.0	0.0
Bacillus badius	7	0	7	100.0	0.0
Bacillus brevis	4	1	3	75.0	25.0
Bacillus circulans	4	0	4	100.0	0.0
Bacillus coaggulans	51	10	41	80.4	19.6
Bacillus laterosporus	1	0	1	100.0	0.0
Bacillus licheniformis	6	6	0	0.0	100.0
Bacillus marcerans	4	0	4	100.0	0.0
Bacillus mycoides	2	0	2	100.0	0.0
Bacillus pentothenticus	16	1	15	93.8	6.3
Bacillus stearothermophilus I	1	0	1	100.0	0.0
Bacillus stearothermophilus II	4	0	4	100.0	0.0
Bacillus subtilis	3	0	3	100.0	0.0
Bacillus spp.	1	0	1	100.0	0.0
Candida albicans	7	0	7	100.0	0.0
Eenterococcus asacchrolyticus	1	0	1	100.0	0.0
Eenterococcus avium	13	6	7	53.8	46.2
Eenterococcus caecorum	32	21	11	34.4	65.6
Eenterococcus casseliflavus	32	26	6	18.8	81.3
Eenterococcus dispar	29	26	3	21.4	78.6
Eenterococcus durans	2	1	1	0.0	100.0
Eenterococcus faecalis	13	8	5	18.8	81.3
Eenterococcus faecium	11	11	0	21.4	78.6
Eenterococcus solitaries	1		1	100.0	0.0
Eenterococcus gallinarum	16	13	3	0.0	100.0
Eenterococcus malodoratus	3	3	0	0.0	100.0
Enterococcus spp.	6	0	6	100.0	0.0

Table 4 Minimum inhibitory concentration of lemongrass oil for different microbes ^{[27- 32]}

Type of strain	Strain number	Results with disc diffusion method	Minimum inhibitory concentration of LGO in µg/ ml
Candida albicans	CV1PD	Sensitive	1
Candida albicans	ABY42	Sensitive	1
Enterococcus faecalis	SV7	Sensitive	16
Enterococcus faecalis	SV20	Sensitive	32
Enterococcus faecalis	E31	Resistant	64
Enterococcus faecalis	CV14NC	Resistant	128
Streptococcus mobilis	SV11	Sensitive	16
Streptococcus mobilis	SV27NC	Sensitive	32
Streptococcus mobilis Streptococcus mobilis	SV15 SV12	Sensitive Resistant	1 64
Streptococcus mobilis	SV36NC	Resistant	64
Staphylococcus aureus	SK10S2	Sensitive	1
Staphylococcus aureus	SK5S1	Sensitive	8
Staphylococcus aureus	SK6S1	Resistant	64
Staphylococcus aureus	SKE111	Resistant	64
Bacillus coagulans	CB1	Sensitive	1
Bacillus coagulans	CB6	Sensitive	4
Bacillus coagulans Bacillus coagulans	A12 B17	Resistant Resistant	64 64
Klebsiella pneumoniae	CP62	Sensitive	16
Klebsiella pneumoniae	M10	Sensitive	32
Klebsiella pneumoniae	LT81	Resistant	64
Klebsiella pneumoniae	LT121	Resistant	124
Edwardsiella tarda	26P	Sensitive	4
Edwardsiella tarda	1BCY	Sensitive	32
Edwardsiella tarda	56LT1	Resistant	128
Edwardsiella tarda	59LT3	Resistant	64
Escherichia coli	E382 (Control)	Sensitive	1
Escherichia coli	C91	Sensitive	8
Escherichia coli	P82	Resistant	128
Escherichia coli	P86	Resistant	128

RESULTS ^[33-35]

Results of antimicrobial activity of LGO using disc diffusion method revealed that 38.2% of 1114 strains of different microbes were sensitive. All molds (Apergillus spp., 11; Penicillium spp., 3), yeasts (Candida albicans, 7), Lactobacillus acidophilus (1) and Morganella morganii (3) strains tested were sensitive to LGO (Table. 1) while for other bacteria results varied with species of the microbes (Table 2, 3). The effect of reduced oxygen and enhanced carbon-di-oxide in incubating chamber was also evident, of the 8 Enterococcus avium strains tested simultaneously under aerobic and microaerobic conditions. Only three stains were resistant under aerobic incubation while six turned resistant under microaerobic incubation. Zone of inhibition also reduced significantly under microaerobic growth conditions.

Among the Gram negative bacteria there was a wide variation in sensitivity of bacterial strains to LGO discs among different genera and different species of a genus (Table 2). Although 78% aeromonads were sensitive to LGO, species wise analysis (Table 2) revealed that all strains of A. media (9), A. schubertii (8), A. sobria (3), A. salmonicida ssp. smithia (1), majority of the strains of A. caviae (10 of 12), A. hydrophila (15 of 18), A. veronii (13 of 14), A. salmonicida ssp. salmonicida (3 of 5) were sensitive to LGO discs. However, majority of the strains of A. salmonicida ssp. achromogenes (2 of 3) and A. eucranophila (10 of 18) were resistant to LGO. Many of the pseudomonads (46.4%) were sensitive but all strains of P. aeruginosa and P. fluorescens were resistant to LGO.

DISCUSSION ^{[36, 37]}

Plant extracts have been used for many thousands of years in food preservation, pharmaceuticals, alternative medicine and natural therapies .It is necessary to investigate those plants scientifically which have been used in traditional medicine to improve the quality of healthcare. Plant extracts are potential sources of novel antimicrobial compounds especially against bacterial pathogens. In vitro studies in this work showed that the plant extracts inhibited bacterial growth but their effectiveness varied with the concentration. The antimicrobial activity of many plant extract has been previously reviewed and classified as strong, medium or weak. In our study, alcohol extract exhibited strong activity against the selected bacterial strains. Several studies have shown that lemon grass had strong and consistent inhibitory effects against various pathogens. Even though earlier studies have reported better antimicrobial activity for lemon grass our study correlates with photochemical components involved in the inhibition of the bacteria. This study indicated that plant extract may possess antibacterial activity and can be exploited as an ideal treatment for future human disease management programs eliminating bacterial spread. Recently, there has been a considerable interest in extracts and essential oils from aromatic plants with antimicrobial activities for controlling pathogens and/or toxin producing microorganisms in foods. Essential oils are natural products extracted from vegetal materials, which because of their antibacterial, antifungal, antioxidant and anti-carcinogenic properties can be used as natural additives in many foods. In general, the levels of medicinal plants and their compounds necessary to inhibit microbial growth are higher in foods than in culture media. This is due to interactions between phenolic compounds and the food matrix and should be considered for commercial applications. The plant extracts and/or essential oil, especially the oil for its citral content, presented positive antibacterial activity for Escherichia coli. Pseudomonas aeruginosa, Streptococcus pneumoniae, S. pyogenes, Neisseria gonorhoeae, Clostridium perfrigens Aeromonas veronii biogroup sober, Enterobacter faecalis, Klebsiella pneumoniae, Salmonella enterica subsp. Enterica sorotipo typhimurium, Serratia marcenscens Proteus mirabilis, Shigella flexneri and Salmonella typhy. Antimicrobial properties of plants are desirable tools in the control of undesirable microorganisms especially in the treatment of infections diseases and in food spoilage. The active components usually interfere with growth and metabolism of microorganisms in a negative manner. Many plants contain non-toxic glycosides that can get hydrolyzed to release phenolics that are toxic to microbial pathogens. Therefore, the compounds detected may be responsible for the antibacterial activity. Similarly various other compound present in several plants showed such antimicrobial activity against disease causing pathogens. In conclusions of this study it is possible to state that the lemon grass bear antimicrobial activity. Comparisons with pertinent data from literature indicate that, according to the methodology adopted in studies on antimicrobial activity, the most diverse results can be obtained. Plant extracts have shown inhibitory effect on the growth of the bacteria studied, although of distinct forms. It is therefore recommended that the nature and the number of the active antibacterial principles involved in each plant extract be studied in detail.

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Received on 30.03.2013 Accepted on 23.04.2013

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