<Insert Table 1: Chemical composition of essential oils extracted by hydrodistillation (HD) and Microwave assisted hydrodistillation (MAHD) from Amomum subulatum seeds>
The compounds were identified as α-pinene, β-pinene, 1, 8-cineol, terpinen-4-ol, and α- terpineol. Results show that there is the dominance of oxygenated compound over the monoterpene hydrocarbons in essential oil extracted by both i.e. MAHD and HD methods. Presence of oxygenated compound in the essential oil is good as those are odoriferous and are mainly responsible for the characteristic aroma of the essential oil[27][28]. Essential oil obtained by MAHD is more concentrated in oxygenated monoterpenes - 1, 8-cineol (89.62), terpene-4-ol (1.42) and α-terpineol (3.10) - as compared to essential oil by HD where the percentage of oxygenated monoterpenes are 88.75 (1, 8-cineol), 1.21 (terpene-4-ol) and 1.50 (α-terpineol). The composition of extracted essential oil by MAHD and HD methods reveals that there is a decrease in the percentage of monoterpene hydrocarbon and increase in oxygenated monoterpene. Increase in yield of the essential oil and greater proportion of oxygenated monoterpenes is possibly due to less thermal and hydrolytic impact as heating is very fast in case of MAHD as compared to HD and lesser use of water in MAHD than in HD[26][16]. Another reason may be as polar compound adsorbs microwave irradiation more radially than others[29][27]. In connection with above theory here we observed that 1, 8-cineol, terpene-4-ol, and α-terpineol are more polar as compared to α and β-pinene.
Optimization of Parameters
3.3.1. Effect of microwave power
In the extraction of Callistemon citrinus skeels by MAHD microwave power is a key factor that influences the efficiency due to the interaction of microwave with the extraction material. Microwave powers of different wattage (160-800 W) level were studied for MAHD extraction of essential oils by keeping time 80 min and temperature 1000c constant. Each experiment was done twice, and the average was taken.
< Insert Figure 2 Influence of microwave power on essential oil yield >
The outcomes of the trials are presented in fig. 2. The result illustrates that there is an increase in the yield of essential oil as we increase the microwave power from 160 to 800 W from 2.1 to 3.3 % respectively. The increase in the yield with the increase in the microwave power is due to the rapid generation of heat in the sample material (mostly polar material in oil) with the absorption of microwave (Thakker, Parikh, & Desai, 2016). We get the highest yield of 3.3 % at the 800 W microwave power.
3.3.2. Effect of extraction time
Fig.3 shows that yield of extracted essential oil at different time interval where the microwave power 800W and the temperature 1000c are kept constant. The rate of extraction at an initial stage of 50 min to 80 min increases rapidly from 2.5 to 3.3 % and then no rises as we increase the time up to 90 min yield is 3.3 %. This shows that more will be extraction time more is extraction yield. As we go from 80 min to 90 there no increase in this means that after some certain period the extraction yield was constant, which is well accordance with the previous studies (M.-T. Golmakani & Rezaei, 2008; Man, Hamzah, Jamaludin, & Abidin, 2012; Ranitha et al., 2014).
< Insert Figure 3 Influence of extraction time on essential oil yield >
3.3.3. Effect of temperature
Fig. 4 shows the yield of extraction at different temperatures having the time and the microwave power was kept constant. The graph shows that the rate of extraction from 700c to 800c there is little increase in yield. As we go beyond 800c the rate of increase in yield is more up to 1000c, as we further increase the temperature there is no further improvement in the yield of extracted oil. Higher extracting temperature is gainful for extraction owed to the improved solubility. The system we used here is closed one and in a closed microwave system the temperature of the water in flask could be increased above the boiling point. The solubility of the essential oil is increased because of increase in temperature and the efficiency of extraction in enhance. higher temperature causes intermolecular interactions and have higher molecular motion. The higher temperature favours opening of the cell matrix and increases the availability of essential oil.
< Insert Figure 4 Influence of temperature on essential oil yield >
Antibacterial Study
In vitro antibacterial study of essential oil extracted by both the methods was done against gram negative and gram-positive bacteria. Microbial growth inhibited by amomum subulatum seed oil extracted by MAHD and HD methods are shown in fig.5.
<Insert Figure 5 Antibacterial activities of a: HD essential oil against E-coli b: HD essential oil against S. aureus c: MAHD essential oil against E-coli and d: MAHD essential oil against S. aureus>
Fig. 6 reveals that oils obtained by both methods have good antibacterial activity against Staphylococcus aureus and Escherichia coli. Zones of inhibition for microwave essential oil and hydrodistilled oil were respectively 1.5 ± 0.2mm and 1.1 ± 0.2mm in the case of Staphylococcus aureus. Similarly, in case of Escherichia coli were 1.2 ± 0.2mm and 1± 0.2mm respectively. In case of both gram-positive and gram-negative bacteria zone of inhibition of MAHD extracted oil was more as compared to that of HD extracted oil. The higher antibacterial activity of MAHD extracted essential oil may be due to the higher amount of oxygenated compounds present[26].
<Insert Figure 6: Zone of inhibition of MAHD and HD extracted essential oil against Escherichia coli and Staphylococcus aureus bacteria>
Antioxidant activity
In this study scavenging activity of both the essential oil was studied against DPPH. DPPH is a stable radical which shows higher absorption at 515nm[30][31].
\begin{equation} DPPH\bullet\ +AH\ \rightarrow DPPH-H+A\bullet\nonumber \\ \end{equation}
Above reaction demonstrate that DPPH radially undergoes reduction by an antioxidant (AH)[30]. DPPH assay is a rapid, trustworthy and reproducible parameters for showing in vitro antioxidant activity of plant extracts because of the ease and convenience of reaction[25][30]. The studied essential oils were able to reduce the stable radical DPPH from deep violet to the light yellow color indicating their antioxidant activity[24][32].