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.