GC-MS Analysis of Commercially Available Allium sativum and Trigonella foenum-graecum Essential Oils and their Antimicrobial Activities

Food preservation and safety is drawing more attention globally due to the increasing prevalence of food-borne diseases. The natural methods of food preservation are considered safer compared to methods using synthetic preservatives. The essential oils with natural preservative properties could be useful for food safety and preservation. The objective of this study was to analyze the chemical composition of commercially available Allium sativum and Trigonella foenum-graecum essential oils by gas chromatography-mass spectroscopy (GC-MS). The antimicrobial activities of Allium sativum and Trigonella foenum-graecum essential oils were determined by agar well diffusion technique. The GC-MS analysis of Garlic essential oil (GEO) revealed that, Allyl methyl trisulfide (13.10%), Di-allyl sulfide (9.47%) and Di-allyl tetrasulfide (4.38%) were the major components, while methanolic extract of Fenugreek essential oil (FEO) showed limonene (12.92%), Silane trimethylphenyl (10.71%), carvone (4.57%) and Trigolline (0.38%) as major components. The results of our study showed a significant antimicrobial activity of GEO and FEO against the tested microbial strains, which indicates the presence of broadspectrum antimicrobial constituents in GEO and FEO. However, further studies are needed for individual bioactive components and safety aspects for their application in food preservation.


INTRODUCTION
The role of essential oils (EOs) in food preservation has been extensively studied due to the undesirable effects of synthetic preservatives used in food industries 1 . Furthermore, rise in foodborne diseases worldwide put people at the risk of health hazards, calling for more effective, safe and natural source of food preservation. To improve the food safety, one of the options in modern time is to study and investigate the antimicrobial activities of the bioactive compounds and use them in food industry. Antimicrobial properties of naturally occurring bioactive components restrict the use of chemical antimicrobial agents, which may possess a potential human health hazard 2 . The main bioactive components of EOs are mono and sesquiterpene, which are believed to be responsible for their biological activity; therefore, the identification of these bioactive components from various plant sources has become meaningful task. Gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) is used exclusively for the qualitative analysis of the volatiles compounds 3 . Additionally, the antimicrobial properties of volatile components are known for decades and because of their potential microbial growth inhibition activity, these volatile components are being investigated as a substitute to synthetic chemical preservatives in food industries 4 . Since ancient times, herbs and spices are added in food and food products, not only as seasoning agent but also as a method of preservation 5 . Plant materials such as Garlic essential oil (GEO) and Fenugreek essential oil (FEO) with antibacterial properties could have a possible application in food preservation, for example, several studies have proved that garlic (Allium sativum) possesses a significant antimicrobial activity. Antimicrobial activity of garlic extracts has been reported against bacteria and fungi 6 . GEO have been reported to contain antibiotic, immunomodulatory, antioxidant, anti-inflammatory, cardiovascular-protecting and hypoglycemic effects 7,8 . Garlic can be used as an effective source for food preservation and also as a natural herbal antibiotic 9 . Moreover, fenugreek seed (Trigonella foenum) oil has been known for their antimicrobial properties against food borne pathogens, thus it could be potentially useful in increasing shelf life of food products. FEO has been reported to have antimicrobial, anti-diabetic, anticancer, anti-fertility, and anti-parasitic activity 10 .
Thus the growing demand of natural ingredients like essential oils in food preservation appears as a viable and healthy alternative to synthetic preservatives. Based upon our literature survey, we did not find any reports on GC-MS analysis as well as antimicrobial activity of commercially available essential oils. Therefore, this work was designed to evaluate the chemical composition and antimicrobial activity of commercial sample (GEO and FEO) against various microbial pathogens.

MATERIALS AND METHODS Sample Collection
Garlic essential oil (GEO) and fenugreek essential oil (FEO) were procured from the local market of Ha'il, Kingdom of Saudi Arabia in December 2018. Selected samples were chosen based upon literature survey and their possible application in food industry. Quality of the oils was ascertained to be more than 98% pure.

Gas chromatography mass spectrometry (GC-MS)
GC-MS (Thermo Scientific, Triple quadropole MS, TSQ 8000) analysis were performed for the GEO and FEO using two fused silica capillary column TG-5MS, (30 m x 0.25 mm x 0.25µm). Moreover, temperature for detector and injector were fixed at 250°C as well as 220°C and helium (1 mL/min) as a carrier gas was employed for this study. Samples (1µL) dissolved in methanol were introduced into column which was initially fixed at 50°C for 1 min and concurrently raised to 280°C by slowly raising temperature of 5°C/ min. Both the samples were run for 30 minutes and analysis of obtained chromatograms was performed. Identification and characterization of various compounds were made by comparing relative retention time (RT) and mass spectra of samples with reference standards by using National Institute of Standards and Technology (NIST) library database 5 .

Test Organisms
Micro-organism used in this study was procured from ATCC (American Type Culture Collection from LGC Promochem, Banglore INDIA as well as MTCC strains were procured from

Culture medium and inoculum preparation
Pure test organisms of all the selected micro-organism (Bacteria and fungus) were subcultured onto fresh plates of Mueller-Hinton agar (Hi Media laboratories) for 24 h and Saboraud dextrose agar (Hi Media laboratories) for 5-7 days at 37°C for bacteria and fungi, respectively. All the test organisms were incubated as specified for each organism for a period of 18-24 h 11 .

Agar well diffusion assay
Antimicrobial activity of GEO and FEO were analyzed by using agar well diffusion assay techniques 5 . Muller-Hinton Agar and Sabouraud Dextrose Agar plates were used for antibacterial and antifungal activity, respectively. 100µL standard bacterial and fungal inoculums were spread over the sterile plates and subsequently 8mm diameter wells were borer over the respective agar plates. Afterwards, 100µL GEO and FEO were filled into the Muller-Hinton agar and Sabouraud Dextrose agar plate wells and kept at room temperature for 1 hour for proper diffusion and incubated for 37C for 24 hours and 30 C for 3-5 days respectively 12 . All the samples were prepared in triplicates, essential oils having antimicrobial activity inhibited the microbial growth and the clear zones were formed. The zones of inhibition were measured in millimeters 13 .

Statistical analysis
All the experimental results were carried out in triplicates and expressed as mean ± SEM (Standard Error of Means) of three independent experiments (n = 3).

RESULTS AND DISCUSSION Characterization of chemical constituents using Gas Chromatography-Mass Spectrometry
Identification of various chemical components present in GEO and FEO were determined by GC-MS. The full scan GC-MS chromatograms are presented in Fig. 1A and 1B. Based upon the GC-MS chromatogram analysis, major components identified are presented in table 1. The identification of bioactive components are determined by comparing the chromatogram peak obtained in our samples with reference peaks as mentioned in National Institute of Standards and Technology (NIST) Library 5 . Furthermore, reference peaks were directly compared with the retention time and mass spectral data obtained in GEO and FEO sample chromatogram. According to GEO chromatogram ( figure 1A), nineteen distinctive peaks were analyzed by GC-MS. Moreover, the major bioactive components analyzed by GC-MS were found to be Allyl methyl trisulfide (13.10%), Diallyl sulfide (9.47%), c-Sitosterol (6.15%), Diallyl tetrasulfide (4.38%) and Allyl methyl disulfide (3.40).. The results of the current study were comparable to previous studies [14][15][16] . Previously, allicin degradation has been proved responsible for the presence of sulphide compounds in oils. Moreover, formation of allicin in garlic occurs due to release of allinase enzyme after crushing garlic bulb. Subsequently, allicin is converted into alliin and because allicin is very unstable compound, it suddenly undergo reactions to form sulphur derivative components 17 . The GC-MS analysis of Chinese commercial GEO sample, which reports diallyl disulphide (45.1-63.2%) as highest components compared to other diallyl sulfide (4.5-11.4%), and diallyl tetrasulfide (6.3-10.5%) 18 . However, our results showed that GEO sample had highest amount of Allyl methyl trisulphide followed by diallyl sulphide and diallyl tetra sulphide. The differences obtained in sulphide content could be due the geographical as well as the process of distillation to obtain the essential oils.

Antimicrobial activity of essential oils
Both Garlic and Fenugreek essentials oils were investigated for in-vitro antimicrobial activity and both GEO and FEO showed positive antibacterial activity against Gram-negative, Gram-positive bacteria and few fungal strains tested in this study 21 . Antimicrobial activities of tested microorganisms are presented in Table 2 &  Table 3. GEO showed antimicrobial activity against all the tested strains. Among the fungal strains, Aspergillus flavus showed the maximum inhibition zone (18.3 ± 0.29) followed by Candida albicans (18.1 ± 0.41). Saccharomyces cerevisiae showed least zone of inhibition (12.5 ± 0.29). Clotrimazole (50µg/ml) were tested against the stated fungal strains and their inhibition zone found to be in the range of 26.4 ± 0.42 -35.2 ± 0.47. Micrococcus luteus showed the highest sensitivity (20.8 ± 0.28) followed by Escherichia coli, Bacillus cereus, Enteococcus hirae and Listeria monocytogenes were the least sensitive (5.2 ± 0.21) to GEO. Tetracycline (50µg/ml) antibacterial activity varied from (9.6 ± 0.48 -31.1 ± 0.38). Clotrimazole and tetracycline antimicrobial standards were compared with GEO, which showed t GEO had a broad antimicrobial potential. The antibacterial activity of GEO has been attributed to the presence of allicin 21 . Additionally, allicin contains thiosulfnate group (-S(O)-S-group) found in the GEO extract is proved to possess antimicrobial properties 22 . One of the previous studies have revealed that, SH group of cellular proteins react with -S(O)-S-group, for the production of mixed disulfides 23 . Furthermore, the antimicrobial activity of GEO has been reported mainly because of the presence of organosulfur compounds such as allicin, ajoene and diallyl sulfides 24 . In addition, Mousumi & Prabir also reported that, garlic   varying from (1.5 ± 0.06 -6.8 ± 0.26). The antimicrobial activity of GEO and FEO could be due to the collective effect of tannins, phenolic compounds, flavonoids, alkaloids, and terpenoids present in oil 29 .

CONCLUSIONS
The antimicrobial activity against the broad range of tested microorganism indicates the presence of wide spectrum of antimicrobial compounds in Garlic and Fenugreek essential oils. Additionally, antimicrobial components showed by GC-MS/MS analysis could be a good source of food preservation rendering the growth of microbes. In conclusion, it is suggested that these plant-derived products could be valuable to find out natural bioactive compounds. More importantly, these can be incorporated in the list of food preservation system due to their antimicrobial activity and lesser side effects. Hence, essential oils and their chemical components can be recommended for food preservation.