| Alpinia zerumbet ’Variegata’ is an excellent aromatic and ornamental plant and its foliage has an intense, unique fragrance. The leaf essential oil of A. zerumbet ’Variegata’ (LEOA) was extracted, and the chemical composition, antioxidant activity, antibacterial activity, anti-quorum sensing activity, isolation and purification of the LEOA were done systematically in this study. The main results were as follows:1. Optimization of LEOA extractionsDifferent extractions were used to extract the LEOA, and the optimal parameters were obtained under the test conditions. The maximum yield of LEOA was 0.216% extracted by hydrodistillation (HD) extraction under the conditions of 1.5 h distillation with the material/liquid ratio of 1:6 and 1.5% NaCl after soaking 30 min. In the organic solvent extraction, the optimal conditions were material/liquid ratio 1 6.5, temperature 50℃ and extracted for 4h by petroleum ether, the yield of LEOA was 0.340% under this condition. The optimal yield of LEOA was 1.40%, and could be obtained at a pressure of 25 MPa, temperature of 30℃, CO2 flow of 15.5 L·h-1 and extracted for 2 h by the supercritical-CO2 extraction (SCE). The results showed that the optimal yield of LEOA in SCE was the highest among the different extractions.2. The chemical composition of LEOAThe gas chromatography-mass spectrometry (GC-MS) was used to identify the compounds of LEOA from different extractions. A total of 60,31,28 and 33 compounds were identified in the HS-SPME (headspace solid-phase microextraction), HD, PEE (petroleum ether extraction) and SCE respectively. Compounds with high content in the HS-SPME included 1,8-cineole (43.50%),p-cymene (11.26%), linalool (5.74%), humulene (5.02%), camphor (4.69%), (E)-methyl cinnamate (3.23%), y-cadinene (3.13%), methyl eugenol (2.76%), humulene oxide II (1.89%) and α-terpineol (1.82%). The major compounds in HD were 1,8-cineole (51.52%), p-cymene (11.26%), camphor (5.74%), D-limonene (3.86%), humulene oxide Ⅱ (3.63%), a-pinene (3.33%), camphene (3.32%) and (E)-methyl cinnamate (2.38%). The principal components of PEE were dihydro-5,6-dehydrokawain (DDK,60.00%), 1,8-cineole (15.50%), camphor (3.11%), p-cymene (2.25%), (E)-methyl cinnamate (2.07%) and caryophyllene oxide (1.07%). The major compounds in SCE included DDK (51.55%),1,8-cineole (8.48%), dodecyl methacrylate (7.45%),1-hexadecene (3.99%), camphor (2.68%), calarene epoxide (2.56%), (E)-methyl cinnamate (2.21%), humulene (1.90%), a-eudesmol(1.62%) and p-cymene (1.09%). The highest content of terpenoid compounds were found in HS-SPME and HD, and the highest amount of aromatic compounds were identified in PEE and SCE.3. The antioxidant acticity of LEOAThe in vitro antioxidant activities of LEOA were determined by different assays in this study. The results showed that the LEOA extracted by SCE and PEE had strong antioxidant activity. It was speculated that the phenolics and terpenoids were the main constituents of antioxidant activity in LEOA. The total phenolic contents of LEOA in HD, PEE and SCE were 2.04,5.72 and 13.91 μg GA/mg respectively. PEE showed the strongest superoxide anion scavenging activity; however, SCE displayed best activities in the DPPH free radical scavenging activity, reducing power and anti-lipid peroxidation.4. The antibacterial acticity of LEOAInhibition zone, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assays were used to evaluate the antibacterial activity of LEOA. The results revealed that LEOA had antibacterial activity against all six tested bacteria, but HD, PEE and SCE showed different antibacterial activities on different bacteria. The strongest inhibition activity of LEOA was evident for Bacillus subtilis, with minimum MIC and MBC. Using the MIC to evaluate the antibacterial activity, the results indicated that the LEOA from PEE had the strongest inhibition activity on B. subtilis, but the antibacterial activities of HD, PEE and SCE on Escherichia coli, Pseudomonas aeruginosa and Serratia marcescens H30 were almost the same. PEE and SCE had better effect on Staphylococcus aureus than HD, while HD showed better effect on S. marcescens MG1.5. The anti-quorum sensing acticity of LEOALEOA showed antibacterial zone and anti-quorum sensing zone on 1Chromobacterium violaceum ATCC31532 and C. violaceum CV026, and could also partly reduced the production of violacein in C. violaceum CV026. The reducing effects on the production of violacein, which were the results of both antibacterial and anti-quorum sensing activities, were better in SCE and PEE than HD. SCE displayed the strongest inhibition activity on swarming motility of the tested bacteria. PEE showed stronger inhibitory activity on swarming motility of S. marcescens H30 and S. marcescens MG1 but weaker effect on P. aeruginosa than HD.6. Isolation, purification and structural identification of the anti-quorum sensing compound from LEOATaken the anti-quorum sensing activity as the indicator, the major active compounds 1 and 2 were isolated from LEOA in SCE and PEE respectively. According to the data of GC-MS and NMR, the two compounds were identified as the same comoound-DDK. |
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