Study of Antibiofilm Activity of Methanolic Extract and Oil of

Cymbopogon citratus and its other Applications

 

Priya Narkar and Prof. Savanta V. Raut

Department of Microbiology, Bhavan’s  College,  Munshi Nagar  Andheri [W], Mumbai-400058

*Corresponding Author E-mail: svrmicro@yahoo.co.in

 

ABSTRACT:

Cymbopogon (lemongrass) is a genus of about 55 species of grasses, of which the type species is Cymbopogon citratus (a natural and soft tea Anxiolytic) native to warm temperate and tropical regions of the Old World and Oceania. It is a tall perennial grass. This study was aimed at evaluating the antimicrobial potential of Cymbopogon citratus and its Essential Oil, in a bid to identify potential natural sources for the synthesis of new drugs to address the growing antimicrobial resistance. Crude Methanolic extract of leaves of Cymbopogon citratus prepared from solvent showed varying levels of bactericidal activity; however both the crude extract and the essential oil of Cymbopogon citratus exhibited most promising activity. MIC was carried out by Microdilution method, MIC of the crude methanolic extract was 1.015mg/ml while of lemongrass essential oil was 0.09 % v/v. Activity guided fractionation was further carried out using various solvents in differing polarity for isolation of active compounds and AST. Ethyl acetate and Acetone fractions showed maximum inhibition than other fractions. Phytochemical analysis showed the presence of the phytocompounds from Ethyl acetate fraction and characterized by U.V Spectrophotometer and LCMS. Anti - biofilm activity was studied, the   activity against Pseudomonas aeruginosa and Streptococcus pyogenes at  concentrations for  MBIC are - Lemongrass Essential oil – 0.01,0.01  % (v/v), Ethyl acetate fraction – 3.9884,0.1992 mg/ml, Acetone fraction- 6.578,0.822 mg/ml. The activity against Pseudomonas aeruginosa and Streptococcus pyogenes at concentrations for MBEC are - Lemongrass Essential oil -2.5, 0.625 % (v/v), Ethyl acetate fraction – 255,255 mg/ml, Acetone fraction- 421,421mg/ml.  Minimum biofilm metabolism inhibition concentration against Pseudomonas aeruginosa and Streptococcus pyogenes found to be were - Lemongrass Essential oil – 0.63, 5% (v/v), Ethyl acetate fraction – 128, 64 mg/ml, Acetone fraction- 6.6, 13.00 mg/ml and Crude extract - 8.2, 4.00 mg/ml respectively. Biofilm Viability Assay was further carried out and Percent reduction for Streptococcus pyogenes was found to be more than Pseudomonas aeruginosa. Anti- biofilm activity was further confirmed by SEM analysis, showing total destruction of the biofilm on treatment. Other applications like antifungal, antidandruff, antiviral and antioxidant activity was further analysed and found to be effective.

 

KEYWORDS: Ficus Cymbopogon citratus, Methanolic extraction, AST, MIC, UV Spectrophotometer, LCMS, Anti- biofilm activity, Minimum biofilm metabolism inhibition, Biofilm viability assay, SEM, antifungal, antidandruff, antiviral and antioxidant activity.

 

 


INTRODUCTION:

Biofilm is the community of micro- organisms living together in amorphous extracellular matrix composed of polysaccharides, extracellular DNA, and proteins. In the nature, we have found that biofilm can develop both on abiotic and biotic surfaces. Because of their complexity, biofilm makes microbial cells inside the matrix confer high level of antibiotic resistance.

 

Biofilm is not only a key factor for survival in diverse environments but also a way of microorganisms to colonize the new sites. The biofilm cells are resistant to all kinds of antimicrobial substances: antibiotics, antiseptics, disinfectants; this type of resistance, consecutive to biofilm formation is, behavioral, and more recently, called tolerance. (Hall-Stoodley L et al, 2004)

 

The problem of antibiotic resistance which has limited the use of cheap and old antibiotic has necessitated the need for continued search for new antimicrobial compounds. Medicinal plant based drugs owe the advantage of being simple, effective and exhibit broad spectrum activity. Therefore, the search for new drug from plant continues to be major source of commercially consumed drugs.

 

Plants produce a large group of bioactive molecules; therefore they have been recognized as a rich source of medicines. In recent year medicinal plants have been checked for their potential against several microbes. In this association, plants continue to be rich supply of curative agents. The significant involvement of plant to the medicine production was promising as large number of phytochemicals and biological studies all over the world.

 Plants are rich in a wide variety of secondary metabolites; such as tannin, terpenoides, alkaloid and flavonoids which has been found in vitro to have an antimicrobial property, generally antimicrobial properties of methanol extracts can be attributed to the presence of secondary metabolites especially flavonoids in first degree, in the second degree terpenes and in the third degree saponins. (Eleyinmi, A.F., 2007)

 

Essential oils are complex mixers comprising many single compounds. Chemically they are derived from terpenes and their oxygenated compounds. Each of these constituents contributes to the beneficial effects. Essential oils have been shown to possess antibacterial, antiviral, insecticidal and antioxidant properties. Some oils have been used in cancer treatment, some other oils have been used in food preservations, aromatherapy and fragrance industries. Essential oils are a rich source of biologically active compounds. There has been an increased interest in looking at antimicrobial properties of extracts from aromatic plants particularly essential oils. Therefore, it is reasonable to expect a variety of plant compounds in these oils with specific as well as general antimicrobial activity and antibiotic potential. Because of the antimicrobial properties showed by essential oils, the aromatherapy has been used for treatment of serious skin diseases, in special, superficial mycoses. (R. Harris, 2002)

 

MATERIALS AND METHODS:

Sample preparation:

The obtained leaves were washed with tap water to remove visible dust and then were dried at R.T for 3 days and then powered and stored under refrigeration at 4şC for further analysis.

 

Preparation of plant extract:

Leaves powder were extracted by Alade and Irobi’s Cold Extraction Method. (Jafari Behboud et al 2012)

 

20 gm powder of leaves was taken in 200 ml Methanol and was kept for 72 hrs at Room Temperature and it was stirred with a glass rod after every 24 hours. After 3 days, the mixture was filtered using the Whatman filter paper no.1.

 

The extract obtained was dried and concentrated in Rotary Vacuum Evaporator. The concentrated extract was stored in the refrigerator (4şC) for further use.

 

Table No.1   Species and sources for standard test cultures

Sr.no.

Test cultures

Source

1

Staphylococcus aureus

MTCC 1144

2

Pseudomonas aeruginosa

MTCC 2488

3

Staphylococcus epidermidis

MTCC 2639

4

Salmonella typhi

MTCC 432

5

Klebsiella pneumonia

MTCC 4032

6

Streptococcus pyogenes

NCIM 2608

7

Candida albicans

MTCC 183

8

Corynebacterium diphtheriae

NCIM 2640

9

Bacillus cereus

NCIM 1272

10

Escherichia coli

MTCC 2641

11

Malassezia furfur

MTCC 1374

12

Asperjillus niger

NCIM 902

 

Determination of Antimicrobial Activity:

In Ethanopharmacology research the antimicrobial susceptibility test (AST) is used to determine the efficacy of potential antimicrobials from biological extracts against a number of different microbial species.

 

The crude extract of the plant along with the essential oil was subjected to antimicrobial assay using: Disc assay method/ Paper disc method and Agar cup method/Agar well diffusion method.

 

Disc assay method/ Paper disc method:

The test employs the use of dried filter paper discs impregnated with known concentration of the extracts against the pathogens seeded on Mueller and Hinton agar media. The zone of inhibition is then measured. Control: A control was set up by dipping the disc in the solvent and checking against same organisms as in the test on Mueller and Hinton agar media. Method- The culture of micro-organisms is plated on the surface of the agar plate allows confluent growth. When a filter paper disc of size 6mm (Whatman Paper 1) is dipped into a solvent extract and placed onto such a surface it allows diffusion of the extract and a concentration gradient is formed around the disc. The extract if possess antibacterial activity will inhibit the growth of micro-organisms around the disc upto a certain concentration.

 

Agar cup method/Agar well diffusion method:

In this technique a known volume of extract is added to the agar well to form continuous concentration gradient. The pattern of growth was altered at a point where the concentration of the extract is inhibitory for the organisms. (Konemann, 1997).

 

Minimum Inhibitory Concentration (MIC):

The MIC was performed using Microtitre plate or broth microdilution technique. It has several advantages over diffusion techniques: increased sensitivity for small quantities of extract which is important if the antimicrobial is scarce as in the case for many natural products; ability to distinguish between bactericidal and bacteriostatic effects and quantitative determination of the MIC. This method can also be used for a wide variety of microorganisms, is inexpensive and produces reproducible results.

 

Determination of the Minimum Inhibitory Concentration:

For determination of Minimum Inhibitory Concentration (MIC), the Microdilution method using 96 well microtitre plates described by the National Committee for Clinical Laboratory Standards (NCCLS) was used. For this microtitre assay, 100 µl of the extract and Cymbopogon citratus essential oil was serially two-fold diluted in sterile Mueller Hinton broth with 0.2 % TTC. Then 50 µl of standardized suspension of the test organism was transferred to each well. The results were observed after 4-5 hrs and after 24 hrs and inhibition of growth was detected visually by colour change to pink when the culture was growing and absence of pink upto concentration that is inhibitory. Medium control, extract control and positive control were also used for elimination of false positive results. The MIC is defined as the lowest concentration of the extract that does not show any growth of the tested microorganism after macroscopic evaluation.

 

Activity guided fractionation:

For successful isolation of the active compounds from the plant, the crude extract was sequentially fractionated with various organic solvents differing in their polarity, from highly polar to non polar using Petroleum ether, Chloroform, Benzene, Ethyl acetate, Acetone, Methanol and Water and each obtained fraction is subjected to bioassay.

 

Antibacterial activity of the fractions:

Antimicrobial activity of each fraction was performed against Escherichia coli and Staphylococcus aureus with solvent controls on St.MHA plates and zone of inhibitions were measured.

 

Preliminary Phytochemical studies:

The fractions that were most effective were subjected to preliminary chemical tests to detect the presence of various phytoconstituents like Tannins, Saponins, Flavonoids, Terpenoids, Alkaloids, Steroid, Coumarins, Phenolic compounds, Anthraquinone, Quinone, Cardiac glycosides using standard methods. (Abegaz B et al,1983)

 

Characterization of the antimicrobial compounds:

Separation and Characterisation of the phytochemical compounds was done using analytical technique like Liquid Chromatography- Mass Spectroscopy at Indian Institute of technology, Powai, Mumbai and UV-Spectrophotometry at Bhavans College, Andheri (West).

 

Application of the fractions obtained from fractionation along with lemongrass oil Antibiofilm activity Biofilm formation:

The biofilm formation of all the test organisms was evaluated as mentioned by Stepanovic et al. (2000) with sligh modification. Cell suspentions (200 µl of suspension containing 1.5 × 108 cfu/ml in 1 % glucose) were seeded into 96- well microplate. After aerobically incubated at 37şC for 48 hrs, the medium was gently removed and the wells were washed three times with potassium phosphate buffer pH 7.5. The biofilm fixation was done by adding 200 µl of methanol and left for 15 min. after the methanol was removed and the microtiter plate was air dried, each well was stained with 200 µl of 1 % crystal violet for 5 min, was washed in tap water and the microtiter plate was completely air dried.

 

Alternative method was also set up where cell suspensions (200 µl of suspension containing 1.5 × 108 cfu/ml in 1 % glucose) were seeded into 5ml of Mueller Hinton broth along with pieces of cover slips to allow growth onto it. After aerobically incubated at 37şC for 48 hrs, the cover slips were gently removed and were washed twice times with potassium phosphate buffer pH 7.5. Gram staining method was further carried out.

 

Minimum Biofilm Inhibitory Concentration (MBIC) [By Microtitre Plate Method]:

Inhibition of Biofilm formation was assessed using a method adapted from Nostro et al. (2007).An aliquot (100µl) from an overnight culture diluted in BHI broth supplemented with 1% (w/v) glucose to 108 cfu /ml was dispensed in each test well of a 96 well plate. In all, 100µl of the test (10- 0.01 % (v/v) for Cymbopogon citratus essential oil, 421- 0.822 mg/ml for Acetone fraction and 510- 0.99 mg/ml for Ethyl acetate fraction) were added into the wells. The negative control was BHI broth only whereas the positive control contained cell cultures alone with no added essential oils. Following24 hr incubation at 37°C, the contents of the well were decanted and each well gently rinsed twice with 300 µl of sterile Phosphate Buffered Saline (PBS) (pH: 7.3 + 0.3).The plates were air dried for 30 mins, stained with 0.1% (w/v) crystal violet for 30 mins at room temperature, washed 3 times with PBS (200µl per well) and dried. The crystal violet was then solubilized using 10% v/v glacial acetic acid and the OD measured at 595nm using a micro plate reader. The MBIC was determined as the essential oil concentration at which the OD < negative control (Pettit, R.K. et al, 2005).Each experiment was performed in duplicates. (Adukwu E.C. et al ,2012) For evaluation of MBIC, analysis was performed at Bhavan’s Research Centre (BRC), Andheri (W). The microtitre plate was analysed by Bio Tek Microtitre Plate Reader, Power wave XS.

 

Minimum Biofilm Eradication Concentration (MBEC) [By Microtitre Plate Method]:

The method used was similar to that described by Kwiecinski et al. (2009). After biofilm formation for 48 hrs, the medium was discarded and the wells gently rinsed twice with PBS. A total of 200 µl of the test (Cymbopogon citratus essential oil, Acetone fraction and Ethyl acetate fraction) was serially diluted and added into the wells ranging from (0.01 - 10% (v/v) for lemongrass essential oil, 0.822-421 mg/ml for acetone fraction and 0.99-510 mg/ml for ethyl acetate fraction ).

 

The plates were then incubated for 24 hrs at 37ş C after which the wells were decanted and washed with 300 µl of Phosphate Buffered Saline (PBS) (pH: 7.3 + 0.3).The plates were air dried for 30 mins, stained with 0.1% (w/v) crystal violet for 30 mins at room temperature, washed 3 times with PBS (200µl per well) and dried. The crystal violet was then solubilized using 10% v/v glacial acetic acid and the O.D measured at 595nm using a micro plate reader. The concentration at which already established biofilms were removed from the bottom of the treated wells was determined as the MBEC. (Adukwu E.C. et al ,2012)

 

For evaluation of MBEC, analysis was performed at Bhavan’s Research Centre (BRC), Andheri (W). The microtitre plate was analysed by Bio Tek Microtitre Plate Reader, Power wave XS.

 

Biofilm metabolism inhibition assay- TTC reduction:

This method is based on reduction of TTC [2, 3, 5- Triphenyltetrazolium chloride] is performed to determine the metabolic activity of the biofilm formed using methods described by Laird et al.(2012). Stock solutions of TTC in Nutrient Broth (0.2 %) were prepared. At the start of the experiment, a fresh solution of TTC was prepared. To determine the Biofilm metabolism assay, the Microdilution method using 96 well microtitre plates described by the National Committee for Clinical Laboratory Standards (NCCLS) was used. For this microtitre assay, 200 µl of the crude extract, Cymbopogon citratus essential oil, Ethyl acetate fraction and Acetone fraction was serially two-fold diluted in sterile Mueller Hinton broth with 0.2 % TTC. Biofilms were formed for 48 hrs in the wells of 96 well plates and then 200µl of the extract and lemongrass essential in TTC solution TTC in Mueller Hinton Broth was added into each test and control well with only TTC in Mueller Hinton, incubated in the dark at 37° C for 24 hrs and the OD measured at 450 nm. (Adukwu E.C. et al ,2012)

The range for Biofilm metabolism inhibition assay were, Cymbopogon citratus Essential oil – 10-0.01 % (v/v), Acetone fraction- 510-0.99 mg/ml, Ethyl acetate fraction – 421- 0.822 mg/ml and Crude Methanolic Extract – 520- 1.0 mg/ml. The results were observed after 4-5 hrs and after 24 hrs and inhibition of growth was detected visually by colour change to pink when the culture was growing and absence of pink upto concentration that is inhibitory. For evaluation of Biofilm metabolism inhibition assay, analysis was performed at Bhavan’s Research Centre (BRC), Andheri (W). The microtitre plate was analysed by Bio Tek Microtitre Plate Reader, Power wave XS.

 

Biofilm viability assay (cfu ml-1):

Biofilm viability was measured using a method adapted from Pettit et al.Following 24 hr exposure of biofilms to the fraction (Crude Methanol Extract, ethyl acetate fraction, acetone fraction and Cymbopogon citratus essential oil), a sterile scraper was used to dislodge each biofilm into the micro-titre wells, and 10 and 100 µl of the well contents removed and spread onto Brain Heart Infusion agar. Plates were incubated for 24 hr at 37°C before enumeration. (Leite JR et al ,1986)

 

 

 

Scanning Electron Microscopy (SEM):

Following preliminary investigations of biofilm formation organisms having higher biofilm OD values compared with the other cultures. 1 cm diameter sterile stainless steel discs (Nisha Engineering Works, Umargaon, India) were immersed in petri plate, containing 5ml of BHI broth supplemented with 1 % (w/v) glucose for 48 hrs. A total of 100 µl of a 108 cfu ml-1 overnight culture was then added and the plates incubated for 48 hrs at 37ş C. After incubation the discs were removed and gently rinsed with sterile PBS to remove loosely attached cells and resuspended in the fraction (Ethyl acetate). After exposure to the fraction the discs were washed 3 times with PBS and fixed with 2.5% (v/v) glutaraldehyde in PBS solution for 2 hrs at 4°C, washed twice with PBS and dehydrated for 10 mins using a graded ethanol series; 30, 50, 70, 90, 100% (v/v).The samples were then dried prior to coating with gold and observed in Scanning Electron Microscope. (Leite JR et al ,1986)

 

For evaluation of Biofilm Eradication, SEM analysis was performed at SAIF, IIT Powai. The steel discs were coated and viewed on a JSM-7600F Scanning Electron Microscope with Resolution1.0nm (15 KV), 1.5nm (1 KV), Accelerating Voltage 0.1 to 30 kv and Magnification 25X to 1,000,000X.

 

Antifungal activity against Aspergillus niger NCIM 902:

The antifungal activity was tested by Disc Diffusion assay against standard fungal culture (Aspergillus niger NCIM 902). Extent of inhibition of fungal growth & diameters of zones of inhibition were noted. Methanol control and standard Flucanazole disc was used to eliminate the possibility of inhibition by methanol solvent.

 

Antidandruff activity against Malassezia furfur MTCC 1374:

The antifungal activity was tested by Disc Diffusion assay against standard fungal culture (Malassezia furfur MTCC 1374). Extent of inhibition of fungal growth & diameters of zones of inhibition were noted. Methanol control and standard Flucanazole disc was used to eliminate the possibility of inhibition by methanol solvent.

 

Antiviral activity, Phage reduction assay:

One loopful of E.coli from overnight culture (108 cfu ml-1) was inoculated into a Nutrient Broth medium (10 ml), mixed and incubated at 37° C for 5 hours. Different concentrations of fraction/oil were prepared .100 µl of extracts + 100 µl of phage in proper dilution (containing 800 PFU) and 500 µl of bacterial suspension were mixed together and then 6 ml of soft agar (top layer) was added and the mixture was poured onto a Nutrient Agar plate and incubated at 25° C for 24 hours. The negative control included all above except the extract solution.

 

Antioxidant Activity:

An aliquot of each sample (0.05 ml) was mixed with 0.5 ml solution of reagent (0.6 H2SO4, 28 mM Sodium Dihydrogen Phosphate, and 4 mM Ammonium molybdate) in 1.5 ml eppendorf tube. The tubes were capped and boiled in a boiling water bath at 95ş C for 90 mins and cooled. The absorbance of each sample was measured at 695 nm against blank in a spectrophotometer. A typical blank contained 0.5 ml of reagent solution and 0.05 ml of buffer and treated in the same manner as test. The antioxidant capacity was expressed as micromoles of Ascorbic acid equivalents of antioxidant capacity.

 

Statistical analysis:

Statistical analysis was conducted using ANOVA. Significance levels was set at p = 005. After assumptions of normality and variances of homogeneity were checked, one way analysis of variance (ANOVA) was performed. Statistical significance was accepted when the probability of the result assuming the null hypothesis (p) is less than 0.05 (level of probability).

 

RESULT AND DISCUSSION:

Antimicrobial aspect: The antimicrobial activity of the extract and its potency was quantitatively assessed by the presence or absence of inhibition zone and zone diameter.  From the extraction method used, it was found , Cymbopogon citratus essential oil showed higher activity  (9 - 59.3 mm) as compared to the Crude Methanolic Extract (8.5 – 14 mm) by disc diffusion method and   (16 – 22 mm) and  (14 – 21 mm) by Agar cup method.

 

By disc diffusion method, in the case of Cymbopogon citratus essential oil Bacillus subtilis showed no zone of inhibition and Corynebacterium diphtheria showed maximum zone of inhibition. The sensitivity of the strains in decreasing order: Corynebacterium diphtheria > Staphylococcus aureus > Staphylococcus epidermidis > Streptococcus pyogenes > Salmonella typhi > Pseudomonas aeruginosa > Candida albicans > Klebsiella pneumonia > Escherichia coli > Bacillus cereus.

 

In the case of crude lemongrass extract: All organisms showed high activity except Escherichia coli and Salmonella typhi which showed a lower activity. The sensitivity of the strains in decreasing order: Corynebacterium diphtheria > Pseudomonas aeruginosa > Staphylococcus aureus> Streptococcus pyogenes> Staphylococcus epidermidis > Candida albicans> Klebsiella pneumonia> Bacillus cereus> Salmonella typhi> Escherichia coli.

 

By agar cup method, in the case of lemongrass essential oil Staphylococcus aureus, Corynebacterium diphtheriae and Bacillus cereus showed maximum zone of inhibition. The sensitivity of the strains in decreasing order: Corynebacterium diphtheria > Staphylococcus aureus > Bacillus cereus> Escherichia coli> Streptococcus pyogenes> Staphylococcus epidermidis > Pseudomonas aeruginosa > Candida albicans> Salmonella typhi> Klebsiella pneumonia.

 

 

In the case of crude Cymbopogon citratus extract, the sensitivity of the strains in decreasing order: Staphylococcus aureus > Corynebacterium diphtheria > Salmonella typhi> Streptococcus pyogenes> Escherichia coli> Staphylococcus epidermidis > Bacillus cereus> Candida albicans> Pseudomonas aeruginosa > Klebsiella pneumonia.

 

Antibacterial activity thus served to be broad spectrum as its activity was independent of the organism being Gram positive or Gram negative.

 

This inhibition effect can be related to its active compounds that include: steroids and terpenoids, alkaloids, citral, geraniol, flavonoids, eugenol, cytronolal, geranyl acetate, beta cariofiln, tannins, phenolic  compounds,   saponin   and  farnsul (C.K. Hindumathy , 2011).

 

According to the considerable antibacterial effect of methanol extract of Cymbopogon citratus grass leaves on pathogenic bacteria especially Gram positive bacteria that are involved in creating variety of nosocomial and malicious infections this extract can be considered as a natural antibacterial herbal product. (S. Isam et al , 2009)

 


 

Fig No.1.Graphical representation of AST of Cymbopogon citratus Essential Oil and Crude Methanolic Extract by Cold Extraction Method


Minimum Inhibitory Concentration (MIC):

The MIC range for crude methanolic extract was 520 - 1.015mg/ml while for Cymbopogon citratus essential oil was 50 - 0.09 % v/v. MIC was carried out by Microdilution method, MIC of the Crude methanolic extract was 1.015mg/ml while of Cymbopogon citratus essential oil was 0.09 % v/v.  The high values of the MIC’s are attributable to the facts that the active components may present in low concentration or there may be some antagonistic components present that serve as growth promoters for the bacteria, thereby necessitating the presence of high amount of the fraction to inhibit the growth.

 

Thus from these findings it can be said that the crude methanolic extract and Cymbopogon citratus essential oil gave the least possible concentration of the extract required to inhibit the organisms.

 

Fractionation:

Since the extract showed promising activity in the bioassay, it was further subjected to Activity - guided fractionation wherein the crude extract was sequentially fractionated with solvents of increasing polarity from Petroleum ether (least polar) to water (most polar), to ensure that a wide polarity range of compounds could be separated and an AST was performed for each fraction Ethyl acetate and Acetone fractions gave the maximum activity i.e. these fractions contain bioactive compounds.

 

UV spectrophotometer:

The absorption spectra in the UV and Visible at the chosen wavelength arise due to certain chromophores present in the analyte molecule that gives rise to distinct parts of the absorption spectra. These specific absorption peaks may relate to known molecular substances that can be identified directly with the liberated data or with a standard compound. By carrying out the absorption of Ethyl Acetate fraction in the UV and Visible wavelength in a UV Spectrophotometer, it was found that the maximum absorption in the UV light was 335 nm while the maximum absorption in the visible range was 445 nm.

 

LCMS analysis:

LCMS was performed which has a broad range, upto 200 m/z. Two peaks were obtained, 1A and 1B. Their retention time along with the peak values was tabulated. Thus the compound of interest may have a molecular weight within the range of 180 – 540. Probable phytochemicals present may be Geranial, Neral, Myrcene, Geraniol, Linalool, Limonene, Citral, Geranyl acetate, Nerol, Citronellol.

 

Table no. 2. LCMS of Ethyl acetate fraction

Sr.no.

Retention time (minutes)

Peak area

(A)

Retention time

(minutes)

Peak area

(B)

1

7.005

202.1

8.562

316.2

2

12.713

186.2

14.408

418.4

3

17.477

284.2

18.353

540.4

4

19.746

524.4

20.522

524.5

5

24.524

420.4

25.780

368.4

 

Minimum Biofilm Inhibition Concentration:

The range for MBIC were, Cymbopogon citratus Essential oil – 10-0.01 % (v/v), ethyl acetate fraction – 421- 0.822 mg/ml, acetone fraction- 510-0.99 mg/ml .The results of  MBIC demonstrate that Cymbopogon citratus Essential oil and extract of Cymbopogon citratus   possesses inhibitory activity  against Pseudomonas aeruginosa and Streptococcus pyogenes at  concentrations for  MBIC - Cymbopogon citratus Essential oil – 0.01,0.01  % (v/v), ethyl acetate fraction– 3.9884, 0.1992 mg/ml, acetone fraction- 6.578,0.822 mg/ml .Significant difference was not observed for MBIC, when treated with Cymbopogon citratus essential oil, Ethyl acetate fraction and Acetone fraction, as determined by the one-way ANOVA for Pseudomonas aeruginosa (p =0.638) and significant for Streptococcus pyogenes (p = 0.0299)

 

 

Minimum Biofilm Eradication Concentration:

The range for MBEC were, Cymbopogon citratus Essential oil -0.01- 10 % (v/v), Ethyl acetate fraction – 0.822- 421 mg/ml, acetone fraction- 0.99 – 510 mg/ml. The results of  MBEC demonstrate that Cymbopogon citratus Essential oil and methanolic extract of Cymbopogon citratus  possesses antibiofilm activity  against Pseudomonas aeruginosa and Streptococcus pyogenes at concentrations for MBEC- Cymbopogon citratus Essential oil- 2.5,0.625  % (v/v), ethyl acetate fraction – 255,255 mg/ml, acetone fraction- 421,421mg/ml. Aiemsaard et al. (2011), previously reported that Cymbopogon citratus Essential Oil possesses anti-biofilm activity , and at low concentrations between 0.06 and 0.125% (v/v)  showed against five strains of Staphylococcus aureus (Adukwu E.C. et al ,2012). However higher concentrations were required for Minimum Biofilm Eradication.  The high values of the MBEC’s are attributable to the facts that the active components are present in low concentration or there are some antagonistic components present that serve as growth promoters for the bacteria, thereby necessitating the presence of high amount of the fraction to inhibit the growth. Thus from these findings it can be said that the extract and Cymbopogon citratus essential oil gave the least possible concentration of the extract required to inhibit the organisms.

 

Significant difference for MBEC was observed, when treated with Cymbopogon citratus essential oil, Ethyl acetate fraction and Acetone fraction, as determined by the one-way ANOVA , for Pseudomonas aeruginosa p =0.0116 and for Streptococcus pyogenes  p = 0.00041.

 

In this study, both MBIC’s and MBEC’s of biofilm were higher than that of the planktonic cell. This indicates that biofilm are more resistant than planktonic cell. Biofilm, not only make the microorganism more resists to antimicrobial agents, there are many advantages of the cells in biofilm have been reported, such as protection from host immune system and increasing of pathogenicity.( Hall-Stoodley L et al, 2004)

 

 


Table No.3. MBIC and MBEC of oil and fractions

Test organism

Cymbopogon citratus

Essential Oil % ( v/v)

Ethyl acetate fraction

(mg/ml)

Acetone  fraction (mg/ml)

MBIC

MBEC

MBIC

MBEC

MBIC

MBEC

Pseudomonas aeruginosa

MTCC 2488

0.01

2.5

4.00

255

6.6

421

Streptococcus pyogenes

NCIM 2608

0.01

0.6

0.2

255

0.8

421

 


Biofilm metabolism inhibition assay:

The range for Biofilm metabolism inhibition assay were, Cymbopogon citratus Essential oil – 10-0.01 % (v/v), Acetone fraction- 510-0.99 mg/ml, Ethyl acetate fraction – 421- 0.822 mg/ml and Crude extract – 520- 1.0 mg/ml.

 

Minimum biofilm metabolism inhibition:

Concentration against Pseudomonas aeruginosa and Streptococcus pyogenes found to be were - Lemongrass Essential oil – 0.63, 5% (v/v), Ethyl acetate fraction – 128, 64 mg/ml, Acetone fraction- 6.6, 13.00 mg/ml and Crude extract - 8.2, 4.00 mg/ml.

 

Inhibition of metabolic activity occurred in the presence of test (Cymbopogon citratus essential oil % (v/v), Acetone fraction (mg/ml), Ethyl acetate fraction (mg/ml) and Crude extract (mg/ml)), after 24 h for Pseudomonas aeruginosa and Streptococcus pyogenes strains no significant difference was observed when treated with Lemongrass essential oil, Acetone fraction, Ethyl acetate fraction and Crude extract, as determined by the one-way ANOVA, for Pseudomonas aeruginosa (p=0.09210) and for Streptococcus pyogenes (p = 0.35538).

 

When the effect of each test was compared for both the strains, there was no statistical significant difference as determined by the one-way ANOVA for Cymbopogon citratus s essential oil (p = 0.5159), Acetone fraction (p = 0.63425), Ethyl acetate fraction (p = 0.9697) and Crude extract (p = 0.21728). 

 

Biofilm Viability Assay:

Biofilm viability was carried out to study the viability of cells after treatment with Crude methanolic extract, Cymbopogon citratus essential oil, Ethyl acetate fraction and Acetone fraction. Ethyl acetate fraction was found to be effective against both Pseudomonas aeruginosa MTCC 2488 and Streptococcus pyogenes NCIM 2608. The Percent reduction of the viable cells was calculated and the percent reduction for Streptococcus pyogenes NCIM 2608 was found to be more than Pseudomonas aeruginosa MTCC 2488.

 


 

Fig. no. 2 Graphical representation of Percent Reduction of Biofilm viability against Ethyl acetate  and Acetone fraction

 

 


SEM:

Ethyl acetate fraction at the MBEC concentration 255 mg/ml (25.5 %) disrupts Pseudomonas aeruginosa biofilm and the total destruction of the biofilm was observed.  In the study carried out by Adukwu E.C. et al. at 0.5% (v/v) Cymbopogon citratus Essential Oil, there was evident damage on the biofilm structure. Kwiecinski et al. (2009) has suggested that TTO treatment of Staphylococcus aureus biofilms causes damage to the extra cellular matrix and damage to the biofilm structure was observed on treatment with 1% (v/v) Cymbopogon citratus Essential Oil suggesting a similar mode of action.


 

B]Test- Treated with Ethyl acetate fraction

 

Fig .No. 2. SEM micrographs of Pseudomonas aeruginosa MTCC 2488

 

 


Antifungal activity:

Cymbopogon citratus essential oil showed activity against Aspergillus niger while no activity was seen for crude methanolic extract of lemongrass, Ethyl acetate fraction and Acetone fraction. The antifungal activity of this oil may be due to the presence of several components known to have biological activities. Studies on the antimicrobial, especially antibacterial and antifungal, activity of lemongrass oil and its components were reported. Their mechanism of action appears to be predominantly on the fungal cell membrane, disrupting its structure causing leakage and cell death; blocking the membrane synthesis; inhibition of the spore germination, fungal proliferation and cellular respiration.

Because of high volatility and lipophilicity of the essential oils, they are readily attached to penetrate into the cell membrane to exert their biological effect . Lemon grass oils show activity towards the phyto pathogenic fungi. (R. Harris, 2002)

 

Antidandruff activity:

Maximum anti dandruff activity was observed against Cymbopogon citratus essential oil. The sensitivity in the decreasing order was: Lemongrass essential oil > Crude methanolic extract > Acetone fraction > Ethyl acetate fraction.

 

Dandruff results from at least three etiologic factors: Malassezia fungi, sebaceous secretions, and individual sensitivity. Malassezia spp are involved in the etiology of pityriasisversicolor, folliculitis, sebborrhoeic dermatitis and dandruff. The genus Malassezia belongs to the basidiomycetous yeasts and is classified in the Malasseziales (Ustilaginomycetes, Basidiomycota). They are normally found in areas rich in sebaceous glands as they are lipid dependent.

A survey of literature reveals that there are many essential oils which possess antifungal activity (Tullio V et al, 2007). With this interest investigated the antidandruff activities of 2 essential oils Eucalyptus globules and Coleus amboinicus  as volatile compounds against Malassezia furfur by exposure to pure essential oils and essential oils diluted with coconut oil by agar well diffusion method. (P. Selvakumar et al, 2012)

 

Antiviral activity:

Antiviral activity of lemongrass was evaluated using plaque reduction assay technique. Lemongrass extract exhibited antiviral activity by reducing the plaque count to 101 time. Since Cymbopogon citratus contains a wide variety of compounds of active polyphenols, including flavonoids these compounds have been identified that exhibited a high level of antiviral activity which have the ability to inhibit the replication cycle of various types of DNA or RNA viruses. Other mechanisms of the most active antiviral compounds from medicinal plants are inhibition of viral adsorption processes to host cell and blocking.

 

Antioxidant activity:

The total antioxidant value of Cymbopogon citratus was evaluated quantitatively by Prieto et al method. The antioxidant value of the Cymbopogon citratus for crude extract utilized for the experiment was 2.24 mg/ml, while for Cymbopogon citratus essential oil, ethyl acetate fraction and acetone fraction was 1.555 mg/ml, 1.400 mg/ml, 1.477 mg/ml respectively. The Antioxidant activity was found to decrease in the following order Crude Methanolic Extract > Cymbopogon citratus essential oil > Acetone Fraction > Ethyl acetate Fraction.

 

The Crude Methanolic Extract of Cymbopogon citratus showed maximum antioxidant activity (in term of Ascorbic acid equivalent).Antioxidant function to reduce oxidative reaction by scavenging and eliminating free radicals. Synthetic antioxidant compounds are commonly processed in foods however, it has been reported to have side effects and carcinogenic. Natural antioxidant from foods have presumed safe and potential in nutritional and therapeutically use.

 

According to the results, it is observed that Cymbopogon citratus essential oil has more activity than Crude Methanolic Extract, Ethyl acetate fraction and Acetone fraction. From the Biofilm viability results it could be it concluded that, Ethyl acetate fraction was found to be effective in reducing the viability of the cells. This is because of the fact that some essential oils contain active components which influence certain metabolic functions of microbial cells. As most components of spice oils belong to the terpenoid family, there has been much speculation on the contribution of the terpene fraction of the oils to their antimicrobial activity. (Conner, D.E. 1993)

 

A study by Jeong and his colleagues conducted on lemon grass plant has concluded that essential oil prepared from Cymbopogon citrates maybe a safe alternative environment inhibition of antimicrobial agents for various uses. It seems that generally antimicrobial properties of methanol extracts can be attributed to the presence of secondary metabolites especially flavonoids in first degree, in the second degree terpenes and in the third degree saponins. (Jeong, M.R. et al, 2009)

 

With the increase in the incidence of resistance to antibiotics alternative natural products of plants could be of interest. Some plants extracts and phytochemicals are known to have antimicrobial properties, which could be importance in therapeutic treatment. In the last few years, various studies have been conducted in different countries demonstrating the efficacy of this type of treatment. Many plants have been evaluated not only for direct antimicrobial activity but also as resistance- modifying agents. ( Matias E. F.F. et al, 2011)

 

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Received on 22.07.2014       Modified on 10.08.2014

Accepted on 25.08.2014      ©A&V Publications All right reserved

Res.  J. Pharmacognosy & Phytochem. 6(4):Oct. - Dec.2014; Page 160-169