COMPARATIVE STUDY OF METABOLITES AND ANTIMICROBIAL ACTIVITIES OF ESSENTIAL OILS EXTRACTED FROM THREE AMOMUM SUBULATUM CULTIVARS

Objective: In Sikkim (India), Seremna is a highly growing cultivar of Amomum subulatum at lower altitudes. Other popular cultivated varieties are Varlangy and Sawney in the same state but at different altitude. In this study, we evaluate the variation in essential oils, metabolites and antimicrobial activities among A. subulatum selected cultivars. Methods: The composition of essential oil of Varlangy, Seremna, and Sawney was analyzed using gas chromatography–mass spectrometry and comparative antimicrobial activity of oils was explored using agar well diffusion and agar dilution method. Results: The Seremna cultivar oil was shown the high content of major constituents (1,8-cineole) in comparison to others. Comparative minimum inhibitory concentrations and minimal bactericidal concentration (MBC) or minimal fungicidal concentration against two Gram-positive (Staphylococcus aureus and Bacillus subtilis), two Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, and two (Candida albicans and Aspergillus niger) fungi were determined. The oil of Seremna showed distinct antibacterial and antifungal activity against all the microorganisms except B. subtilis which showed resistance. Conclusion: The present findings concluded that the high content of the principal compound accelerates the antimicrobial activity of essential oils. The essential oil of Seremna could be a good antimicrobial agent and recommended in the case of infections.


INTRODUCTION
Amomum subulatum (commonly known as large cardamom) is a perennial plant widely cultivated in the sub-Himalayan region of North-Eastern part of India (Sikkim), Northern Uttar Pradesh, Arunachal Pradesh, and Mizoram State belongs to the family Zingiberaceae. The worldwide annual production of large cardamom fruits is ~ 12 × 10 3 mt which is 30% of the total production coming from Sikkim [1]. The capsules/fruits are the main edible source of this plant and worldwide called as Badi elaichi/Kali elaichi (Hindi), Heel kalan (Urdu), Greater/ Nepal cardamom (English), Cardamom (French), Ts"ao-k"ou (China), and Qakilahe kalan (Persian) [2]. The fruits of A. subulatum are mainly used for anti-gonorrhea, kidney stones, respiratory, digestive diseases, and alternative systems of medicine [3]. There are six major cultivars of large cardamom growing in the Sikkim region, i.e., Ramsey, Sawney, Golsey, Varlangy, Bebo, and Seremna [4]. Cultivars such as Seremna, Sawney, and Varlangy are very popular among local growers due to its high productivity and yield [5]. Recent study of bioactive compounds of A. subulatum fruit/seeds has shown the different pharmacological activities such as antioxidants, antiulcer, hypolipidemic agents, antimicrobial activity antidiabetics, and hepatoprotective activity by the different investigators [6][7][8][9]. A broad citation on the antimicrobial properties of essential oils has been carried out by several investigators. Essential obtained from various medicinal plants such as Cinnamomum zeylanicum, Eugenia caryophyllata, Rosmarinus officinalis, and Ocimum kilimandscharicum has been reported to possess significant antimicrobial activities and can serve as a powerful tool to reduce the bacterial resistance [10,11]. Due to lack of technological errors, the essential oil of the plant or bioactive compound of the particular cultivars (Seremna, Sawney, and Varlangy) has not been tested earlier. Several authors considered, the environmental conditions, e.g., type and composition of the soil (edaphic), geographic and genetic variations are the determinant factors influence the secondary metabolites of the essential oils [12][13][14]. Keeping the view in mind for cardamom pharmacological properties, we have studied the Seremna, Sawney, and Varlangy essential oils by gas chromatography-mass spectrometry (GC-MS) and also observed the effect of their essential oils for antifungal and antibacterial activities collected from Sikkim.

Sample collection and extraction of essential oils
The fruits of three different cultivars of A. subulatum such as Sawney, Seremna, and Varlangy were obtained from Spice Board, ICRI (Indian Cardamom Research Institute), Tadong (Sikkim, India), in the month of November 2016. The hydrodistillation method was used for the extraction of essential oil from three cultivars of Sawney, Seremna, and Varlangy [15]. For each cultivar, 50 g fine powdered were taken for extraction of oil from each cultivar separately for the time of 4 h. The extracted samples were dried by mixing the sodium sulfate (anhydrous) and stored at 4°C for further study. Data were recorded and percentage yields of each cultivar oil were calculated as triplicate.

Chemical characterization: Metabolite identifications through GC-MS
For the comparative qualitative and quantitative study, the essential oils of each cultivar were characterized by GC and GC-MS. GC analysis was carried out using H. P-5890 II apparatus, prepared to splitless injector method using the HP-5M column (0.52 μm film thickness and 25 m × 0.32 mm) and FID (flame ionization detector) with 1 ml/min flow rate of carrier gas (N 2 ) and injector temperature (250 and 300°C, respectively) in the form of split ratio 1:30. The column temperature
GC-MS of three cultivars of cardamom, i.e., Sawney, Seremna, and Varlangy was carried out using Hewlett Packard G 1800C Series II GCD, GC-MS system assembled with HP-5MS column (0.25 μm film thickness and 30 m × 0.25 mm) ramping the temperature from 40°C to 260°C was operated between 40 and 450 m/z and mass detector (Ionization energy: 70 eV). The components of essential oil extracted from the Sawney, Seremna, and Varlangy were identified by comparison of their retention indices and mass spectra with standard libraries (16;Wiley6;1989 andNIST02 2008).
The bacteria were cultured at 37ºC on MHA medium and incubated overnight to check its growth in pure culture. Similarly, the fungi were also cultured in SDA medium at 37ºC to further check the fungal hypha growth. To prepare the bacterial and fungal inoculums, the overnight culture of bacteria and fungi was diluted, i.e., 10 8 CFU/ml (0.5 of McFarland) for bacterial and 10 7 CFU/ml for the fungal spores to validate the oil efficiency against these microorganisms. 100 μl inoculum was obtained from the secondary culture of microbes (bacterial and fungal) where spread under sterile condition in laminar hood on MHA and SDA basal media over the entire surface of the Petri plates.

Antimicrobial screening of essential oil
All three essential oils extracted from A. subulatum fruit samples were tested for antimicrobial activity using the well diffusion method [18]. The extracted oil (20 mg/ml) of each cultivar of A. subulatum (Sawney, Seremna, and Varlangy) was prepared using 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich) solution and tested against the bacterium S. aureus and B. subtilis, K. pneumoniae, and E. coli and fungus C. albicans and A. niger strains. The antimicrobial activities of the samples were initially evaluated by modifying agar well diffusion assay. Wells (6 mm) of each plate were filled with 15 μl of samples and incubate the plates for 24 h at 37°C; bacteria, 48 h at 37°C; yeast, and 7 days at 28°C for fungi were observed. Gentamicin (GEN-10 µg/ml) and 5-fluorocytosine (5FC, 10 µg/ml) were used as a positive control; similarly, 10% DMSO was used as a negative control. The experiments were repeated thrice, i.e., triplicate manner. The minimum inhibitory concentration (MIC) and minimum bacteriostatic concentration (MBC) were performed using the agar dilution method [19]. Six different dilutions (20, 10, 5, 2.5. 1.25, and 0.625 mg/ml) were prepared and diluted solution (1 ml) of each cultivar oils was separately mixed to 19 ml of MHA hot solution and poured on the sterile Petri plates. After solidification of MHA media, 1 ml inoculum of each microbe was spread on sterile plates with oil and inoculated at 37°C for further observations in terms of antimicrobial activity. For the MIC, the visible growth was observed after 24 h of incubation and for the MBC, visible growth after 5 days of incubation. Similarly, MIC for fungistatic and MIC fungicide (MICF) was observed after incubation for 3 weeks at room temperature.

RESULTS AND DISCUSSION
Percent oil recovery A. subulatum cultivars Collected cultivars from Sikkim have shown the diverse percentage content of the essential oils Table 1. The results of the present study were shown that Seremna has a higher percentage (2.7 ± 0.10) of essential oil than other cultivars; Varlangy (2.5 ± 0.15) and Sawney (1.7 ± 0.20).
In this study, we have observed that the several similar components reported in the essential oil of Nepal A. subulatum (1, 8-cineole, alpha-pinene, beta-pinene, alpha-terpineol, etc.) where, 1,8-cineole (60.8%) has also been reported as the main components [20]. Joshi et al. studied on the six different cultivars collected from different altitudes of other regions than Sikkim, i.e., Himachal Pradesh, showed that 1,8-cineole (50.55% ± 1.87%-60.46% ± 3.50%) was the main active constituents of A. subulatum essential oils. The percentage of the composition of essential oil is varying due to geographical region and its altitude. In the present study, three cultivars were cultivated in different geographical conditions, Seremna is highly cultivated at low altitude (<900 m), Sawney is cultivated at middle altitude (700-1500 m), while Varlangy is cultivated at high altitude (>1500 m) [21]. In our findings, we have also analyzed that 1,8-cineole (eucalyptol) is main compounds of oil and the percentage varies cultivar to cultivar as well as altitude to altitude. This study is also supported by other groups [22,23]. The concentration and  the percentage of the other components (α-pinene, β-pinene, terpineol, nerolidol, and limonene) were different from the previous reports [24]. This study is the 1 st time reported the chemical composition of Seremna and it was found higher than the other selected cultivars.

Antimicrobial activity
The inhibition zone of different essential oils against selected pathogens is shown in Table 3     The oils of all cultivars were shown a good inhibitory effect against fungal strains which is supported by previously reported studies that the plant parts which have major 1,8-cineole content in the essential oil showed good antibacterial and antifungal activities [25,26]. Oxygenated monoterpene, 1,8-cineole is a non-toxicants be used for the antimicrobial activity [27]. In addition to 1,8-cineole, the other compounds such as α-terpineol, β-pinene, α-pinene, D-nerolidol, and limonene are also contributing in the antimicrobial activity. Seremna essential oil showed the potential bactericidal action against the used bacteria such as K. pneumonia and S. aureus with 5 mg/ml MBC, while, MBC of Sawney oil reached to 10 mg/ml and Varlangy reached to more than 10 mg/ml and these oils were less sensitive against both Gramnegative (K. pneumonia) and Gram-positive strains (S. aureus) strains.
The present study showed that essential oil of A. subulatum was highly active against Gram-positive bacteria and among all selected and among all selected cultivars, Seremna oil was shown comparatively better antibacterial effects than Sawney and Varlangy may be due to the high percentage of the principal compound (1,8-cineole). Seremna oil showed potentially highest fungicide activity against selected fungus (C. albicans and A. niger) with MICF of 2.5 mg/ml and 5 mg/ ml for C. albicans and A. niger, respectively, while MICF of Sawney oil was 5 mg/ml and 10 mg/ml for C. albicans and A. niger, respectively. The essential oil of Varlangy was found to be less sensitive against A. niger and its MICF value reached to 10 mg/ml. In the previous studies, investigators have reported that Gram-positive bacterial are more sensitive to volatile oils when compared to the Gram negative [25,28]. The plants contain 1,8-cineole as a major content showed good antimicrobial activities against gram-positive bacterial and fungal strains [29]. The antibacterial activity of A. subulatum essential oil might be due to the components such as 1,8-cineole, limonene, D-nerolidol, β-pinene, α-pinene, and α-terpineol [30,31].

AUTHORS' CONTRIBUTIONS
Alam A designed the study project, performed experiments, wrote whole manuscript, and involved in throughout manuscript editing and finalization. Majumdar RS guide and approved the designed project helped in throughout project work. Alam P contributed in manuscript editing and finalization according to journal.