Study on the components and biological properties of the essential oil of Amomum aromaticum Roxb. in Ha Giang, Vietnam

The essential oil of Amomum aromaticum Roxb. in Ha Giang was obtained by steam distillation and dried with Na 2 SO 4 . Using the gas chromatography - mass spectrometry (GC - MS) method, 26 components in the essential oil were predicted by comparing their retention times and determining molecular weights. In particular, there were 13 hydrocarbons such as monoterpenes (33.03%), sesquiterpenes (1.08%), and 13 oxygenated components such as aldehydes (52.04%), alcohols (10.42%), ketones (1.98%), and oxide (0.14%). Antimicrobial activity against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa of the essential was identified by the agar diffusion method . The antioxidant activity was determined by using 2,2 diphenyl - 1 - picrylhydrazyl (DPPH) radical percentage inhibition and the observable result was 46.12±0.15%.


Introduction
The Amomum aromaticum Roxb. is planted in mountainous areas of the Xin Man, Hoang Su Phi, Vi Xuyen, Quang Ba, Bac Me districts of the Ha Giang province. Amomum aromaticum Roxb. possesses medicinal properties. It has been used to weld and increases digestion and reduce swelling pain and fever. In particular, in Southeast Asia, Amomum aromaticum Roxb. is used to treat skin diseases, dyspepsia, some digestive tract symptoms, flu, malaria, rheumatoid arthritis and other kinds of infections. Amomum aromaticum Roxb. has also been used to produce medicines to treat fever, malaria, colic and lousy breath (Loi et al., 2004). The components of different varieties of this plant have shown variability (Diao et al., 2014). The features and bioactivities of some Amomum aromaticum Roxb. have been reported (Cui et al., 2017). However, the components and biological properties of Amomum aromaticum Roxb. in Ha Giang-Vietnam have not been evaluated yet.
Therefore, this study is aimed to primarily analyze the components, antioxidant activity and antibacterial activity of the essential oil of Amomum aromaticum Roxb. in Ha Giang-Vietnam, as a basis for applying this essential oil in food processing and preservation.

Materials
The Amomum aromaticum Roxb. was harvested from the Xin Man district of the Ha Giang province in 2020. The essential oil was obtained by steam distillation after drying with Na 2 SO 4 . The sample was stored in the Department of Biotechnology and Food Processing, Hanoi University of Industry, Vietnam.
The tested bacterial strains (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa) were obtained from the School of Biotechnology and Food Technology, Hanoi University of Science and Technology.

Essential oil extraction
The essential oil was extracted by using the rhizome of the plant with water in essential oil distillation equipment Clevenger (Germany) in the ratio of 1:5 (w/v) respectively for 180 mins (Loi et al., 2021).

Gas chromatography-mass spectrometry (GC-MS)
The sample and standards were run parallelly in the GC-MS experiment. Gas chromatography (GC) analysis was performed by using Agilent Technologies HP 6890 Plus Gas chromatograph system equipped with Flame eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER Ionization Detector (FID) and fitted with HP-5MS columns (30 m × 0.25 mm, film thickness 0.25 µm). The temperature was programmed as followed, the column temperature was programmed from 80 to 150°C in 23.5 mins at a rate of 3°C/mins and then from 150 to 220°C in 8.85 mins at a rate of 8°C/mins. The used injector temperature was 230°C. The MS conditions were as follows: ionization voltage was 70 eV, transfer temperature was 250°C, the carrier gas was helium used at a flow rate of 0.5 mL/mins, and the split ratio of the injector was 1:5 (Teresita et al., 2000;Choi and Sawamura 2002;Wang et al., 2014). The MS fragmentation patterns were compared with known patterns of other essential oils and those in the literature by using Wiley (Wiley 9 th Version), NIST 08Libraries (on ChemStation HP). The content of the components is calculated as a percentage of the chromatographic peak area.

Determination of antibacterial activity using agar diffusion method
Antibacterial activity was roughly determined by the agar diffusion method. A volume of 50 µL of the essential oil was put into wells on the plates containing tested bacterial strains. The activity was roughly estimated by the diameter of the antibacterial round (mm), which was calculated by the formula D-d(mm), wherein D was the diameter of the antibacterial round (mm) and d was the hole diameter (mm) (Cui et al., 2017).

Determination of antioxidant activity using free radical scavenging activity
The free radical scavenging activity of the essential oil was measured by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Teresita et al., 2000;Molyneux et al., 2004;Satyal et al., 2012). A 0.5 mM solution of DPPH in methanol and 0.005 M acetate buffer (pH 5.5) were prepared. An aliquot of 0.1 mL of the essential oil solution was added to the tube containing 2 mL of acetate buffer, 1.9 mL of methanol and 1 mL of DPPH solution. In the blank tube, DPPH was removed, in the control tube, 1 mL of DPPH was added to the tube containing 2 mL acetate buffer and 2 mL methanol. The mixture was shaken immediately after adding DPPH and allowed to stand at room temperature in the dark. The decrease in absorbance at 517 nm was measured after 32 mins until the reaction reached a plateau. Vitamin E with the concentration of 0.5 mM was used as a positive control and its free radical scavenging activity was performed in parallel in the same experiment. These experiments were run in duplicate.
The inhibitory percentage of DPPH was calculated as follows: Wherein A o is the value of absorbance of the control at the wavelength of 517 nm; A is the value of absorbance of the sample at the wavelength of 517 nm, and A b is the value of absorbance of the blank at the wavelength of 517 nm.

Components of the essential oil of Amomum aromaticum Roxb. in Ha Giang-Vietnam
GC-MS of the sample was performed to determine the components of the essential oil. Based on the retention times and molecular weights of the sample and the standards (the GC profile was not shown here), 26 components and their percentages in the essential oil were evaluated and shown in Table 1. Table 1 showed that twenty-six components were predicted in the essential oil of Amomum aromaticum Roxb. in Ha Giang -Vietnam. Thirteen out of twenty-six were hydrocarbons (such as monoterpenes: 33.03% and sesquiterpenes: 1.08%) and the rest were oxygenated hydrocarbons (aldehydes: 52.04%, alcohols: 10.42%, ketones: 1.98% and oxide: 0.14%). The results provided additional evidence to show varied percentages of the components of the essential oils of Amomum aromaticum Roxb. in Ha Giang-Vietnam. Notably, the amounts of aldehydes and alcohols in the essential oil were higher than those of the oil in Nepal (Satyalet al., 2012). The differences were probably due to geographical conditions such as the soil factors, weather, climate, growing conditions and harvesting time (Diao et al., 2014).

The antibacterial activity of the essential oil of Amomum aromaticum Roxb.in Ha Giang-Vietnam
The agar diffusion method was used to estimate the antibacterial potentials of the essential oil of Amomum aromaticum Roxb. in Ha Giang-Vietnam. The tested microorganisms used in this experiment were S. aureus, E. coli, E. faecalis and P. aeruginosa. The diameters of antibacterial activity rounds of the essential oil against these bacteria were shown in Table 2. The results showed that the essential oil of Amomum aromaticum Roxb. in Ha Giang-Vietnam possessed antibacterial activity against all of the four microorganisms tested. Among them, the antibacterial activity against Staphylococcus aureus was the highest. The activity of the essential oil of Amomum aromaticum Roxb. in this research is similar to that of the essential oils of the leaves of Liquidambar formosana Hance in Bac Giang as these essential oils were found to possess antibacterial activities against all of the four tested microorganisms eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER (Loi et al., 2015). This result is also consistent with the research result by Diao et al. (2014).

The antioxidant activity of the essential oil of Amomum aromaticum Roxb. in Ha Giang-Vietnam
The results of the study are shown in Table 3. The DPPH free radical scavenging activity of the essential oil of Amomum aromaticum Roxb. was 46.12±0.15% and this value was slightly higher than that of 0.5 mM vitamin E (41.18±0.26%). These activities of the essential oils of the leaves of Liquidambar formosana Hance in Bac Giang and Citrus sinensis peel were found to be 41.13±0.22% and 45.32±0.18%, respectively (Matook et al., 2006;Loi et al., 2015). Therefore, the DPPH free radical scavenging activity of the essential oil of Amomum aromaticum Roxb. is higher than the leaves of Liquidambar formosana Hance in Bac Giang and Citrus sinensis peel. This research result was also consistent with the research result of Teresita et al. (2000).