Introduction
Amomum subulatum Roxb (Zingiberaceae), commonly known as large cardamom, is a perennial herbaceous plant with subterranean rhizomes which produces several leafy shoots and panicles [1][2]. It contains 1.95 to 3.23% of essential oil having typical characteristic flavor and possesses stimulant, stomachic, alexipharmic and astringent effects[3][4][5].
From ancient time spices are consumed in most of the countries.[5][6] Consumption of spices is based on the common assumption that they are related to taste and aroma[7][6][8]. The major constituent of large cardamom essential oil is 1, 8-cineole (35–80%) while the content of α-pinene is low (traces to five percent)[2][9][10].Beyond their taste and smell, the recent scientific studies have proved that they have biological activities like analgesic, anti-inflammatory, antimicrobial, antimicrobial, antioxidant, antiulcer, cardio-adaptogen, antidiabetic and hypolipidemic activities[5]. Continuous consumption of black cardamom leads to enhancement in fibrinolytic activity, total antioxidant status, and alteration in lipid profile in patients with ischemic heart diseases (IHD)[11][5].
Traditionally essential oil is obtained by homogenization, filtration/ centrifugation, steam-distillation, hydrodistillation, solvent, and Soxhlet extraction methods[12][13][13][14]. These methods have certain disadvantages such as the risk of thermal degradation of thermolabile active compounds as it usually requires long extraction time, also these methods consume more solvents and sometimes have lower efficiency[15][14][16].
Several novel extraction techniques have been introduced and studied in the past years, most of which were better in terms of the period of extraction, efficiency, and usage solvent than the traditional techniques[14]. The new extraction techniques available are microwave assisted hydrodistillation extractions (MAHD), supercritical fluid extraction (SFE), and pressurized solvent extraction (PSE)[13][17].
The advantages in case MAHD are saving of extraction time and solvents, selectivity, lessening environmental influence by emitting less CO2 in the atmosphere and controllable heating process which make MAHD as an important alternative extraction technique particularly in extracting biological materials[18][19][12].
The aims of this study are to investigate the potential of MAHD for the extraction of essential oil from seeds of Amomum subulatum and optimise the factors responsible for a good yield. The optimisation of the operating parameters was done. Besides this, we did a comparison between essential oil obtained by HD and MAHD in terms of the time required for extraction, the composition of extracted oil and the yield. We have also studied the antimicrobial and antioxidant activities of oils extracted by both the methods.
Material and methods
Materials
The large cardamom (Amomum subulatum ) was obtained from retail organic food stores. The standard bacterial strain of Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC 25923) for testing the antimicrobial activity were procured from the National Collection of Industrial Microorganisms (NCIM), Pune, India and were maintained on nutrient agar slants and stored at 4 °C along with regular subculturing every two months. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) for the antioxidant study is procured from Alfa Aesar.
Hydrodistillation
The essential oil was extracted from seeds of cardamom by hydrodistillation. 100g of black cardamom seeds and 200ml of distilled water were taken in a flat bottom flask and submitted to hydrodistillation for 4 h using a Clevenger-type apparatus[1]. The Oil was pale yellow in color. The essential oil was collected, dried over anhydrous sodium sulfate and stored at 40C in the sealed amber vial until further study.
Microwave-assisted extraction
Microwave-assisted extraction was performed using microwave variable in 80W increments fabricated by CATALYST. Schematic of the experimental arrangement is shown in fig. 1. In MAE procedure, 100 g of seeds of black cardamom were inserted into an extraction vessel along with 100 ml of water which is connected to a Clevenger-type apparatus located outside the microwave oven. The essential oil was removed from the aqueous medium after a simple decantation and dried over anhydrous sodium sulfate and stored at 40C in the sealed amber vial until further study.
< Insert Figure 1 Schematic of the fabricated microwave and assembly used in this study >
GC-MS Analysis
A detailed chemical composition analysis of the essential oil ofAmomum subulatum seeds was performed using GC-MS (thermo TRACE 1310 GC coupled with ISQ™ Series Single Quadrupole GC-MS) equipped with DB-5 MS fused silica capillary column (30 m × 0.25 m i.e., film thickness 0.25 µm). Helium was used as carrier gas at a constant flow rate of 1ml/min, splitless injection volume 0.1 ml, and injection temperature was set at 250C, oven temperature progress from 60 to 280C at 20C /min. An electron ionization system with ionization energy of 70 eV was used for GC- MS detection. Identification of essential oil components was based on GC retention time on MS column compared with the mass spectral fragmentation patterns in MS database (National Institute of Standards and Technology).
Optimization of parameters
Essential oil’s quality and quantity generally be influenced by the extraction techniques[20]. The optimization of the experimental conditions is a vital step in set up of ideal operating conditions for MAHD method, as different factors can have impact on the extraction method. In general, microwave power, extraction time, and temperature are considered to be the most important factors [21]. All the selected parameter studied using five levels. Microwave power ranges from 160-800 W (160W difference), time of extraction ranges from 50min to 90min and temperature from 700c to 1100c.
Antibacterial activity
To study antimicrobial effect, we have prepared sterile nutrient broth petri plate. The nutrient broth was prepared by dissolving 13 g of nutrient broth mixed with 28.8g of nutrient agar in 1 L water[22]. The media was autoclaved and allowed to cool to 50 °C. Then, 25 mL of this solution was poured onto the petri plates and allowed to be solidified at room temperature for 30 minutes. On the plates, wells were made into an agar layer with stainless steel bore of approximately 4-5 mm in diameter. Then 25 µL of essential oil sample was loaded into the bore and the plates were kept for 24 h of incubation.
Antioxidant activity ( DPPH radical-scavenging activity)
The scavenging effect on the DPPH radical was determined by using the reported method[23][12]. The different concentrations such as 10, 15, 20 and 25 µl of volatile essential oil in methanol (1 ml) were added to 4 ml of 0.004% methanolic solution of DPPH. The reaction mixture was shaken vigorously and incubated for 30 min in the dark. After 30 min of maturation at 300 c, absorbance was measured at 517 nm by using spectrophotometer against a blank. The Same process was applied to the control (without having any sample) and standards (containing antioxidant ascorbic acid in place of essential oil) for comparison. The reduction in absorption arising out of the presence of sample was measured by comparing with the results of control experiment. All experiments were carried out in triplicates. The annihilation activity of free radicals i.e. % DPPH scavenging effect was calculated using the following equation[24],
% I = {(A0-AT)/ A0} ×100
Where A0 is the absorbance of the control and AT is the absorbance of the test samples.
IC50 values were calculated by plotting the percentage of DPPH radical scavenging capacity against the concentration of essential oils (shown in fig.5). IC50 is the concentration required to inhibit 50 % of DPPH free radicals [25][7].
Results and discussion
Yield and extraction time
The yield of essential oil obtained by MAHD extraction was 3.35% which is much better as compared to the oil extracted by HD method i.e. 3%. The time required to extract the essential oil from Amomum subulatum seeds by HD method (4 h) is more than that of MAHD method (70 min). The lesser extraction time in MAHD is because of the fact that in microwave mass and heat transfer occur in the same direction[26][18]. In case of hydrodistillation, heat is transferred to the surface of the material by conduction, convection or radiation and into the interior of the material by thermal while in microwave extraction heat is manifested through their interaction with the sample[18][26]. This results in rapid heating and hence the time required is less.
Composition of essential oil
The constituents of the volatile oil extracted from Amomum subulatum were analyzed by GC–MS. Table 1 gives the results of GC-MS of extracted oil by MAHD and HD methods. It is seen that there is a total of five components in both the oils.