Extraction And Antibacterial Activity Of K. Galanga And C. Zedoaria Essential Oil

Both K.galanga and C.zedoaria are Zingiberaceae plants.Their rhizome of plants is used to be medicine.They are perennial aromatic plants as edible food and traditional medicine.They are not only spices and flavors,but also used for the treatment of a variety of diseases.One of the main components of K.galanga and C.zedoaria is essential oil,including terpenoids,aromatic compounds,aliphatic compounds and so on.They have pharmacological activities such as anti-oxidation,anti-tumor and anti-angiogenic properties.Essential oil is an important substances in traditional Chinese medicine（TCM）,so the extraction and isolation technology,composition structure identification,biological activity screening of essential oils are very significant in the field of drug development and the TCM modernization.The methods include steam distillation,mechanical pressing,organic solvent extraction and supercritical CO₂ extraction to extract essential oil.In this thesis,response surface method（RSM）optimized the condition of steam distillation to extract essential oil for high productivity.Subsequently,the chemical compositions of essential oils extracted from K.galanga and C.zedoaria,which were extracted by steam distillation and supercritical CO₂extraction methods,was detected by gas chromatography-mass spectrometry combined technology（GC-MS）to compare.At the same time,we also compared the different chemical composition of essential oils to extract by steam distillation between K.galanga and C.zedoaria from different areas.Then,essential oils of K.galanga and C.zedoaria were separated and purified to obtain main composition by column chromatography.Antibacterial activity and mechanism of K.galanga and C.zedoaria essential oil on Escherichia coli,Salmonella typhimurium and Staphylococcus aureus were discussed in the thesis.In summary,this thesis showed the conclusion.1.The optimal conditions for K.galanga to extract essential oil by BBD design and model verification were the liquid to material ratio was 9.67,soaking time was 17.97 h and extraction time was 10 h,respectively.The total extraction rate was 2.446%through optimal condition.Analysis of variance（ANOVA）test was 99.92%confidence,and identified independent variables and their interaction polynomial regression equation were significant（p<0.0001）.Similarly,the optimum conditions of essential oil from C.zedoaria were as follows:liquid to material ratio,ultrasonic time,and extraction time were 10.22,16.51 min and 12 h,respectively.The total extraction rate was 0.601%.ANOVA test model was 99.23%confidence.2.In this study,we detected the chemical constituents of essential oils from K.galanga and C.zedoaria by GC-MS,and compared the chemical constituents and contents of essential oil extracted by different extraction methods from different batches.The results showed that the main chemical components of K.galanga essential oil in the same area did not have significantly changed,while the chemical composition of K.galanga essential oil from different areas had differences.So it told us that the content of essential oil had a certain correlation with the region.The extraction method also effected the yield of essential oil.The first batch of Guangxi K.galanga was extracted by supercritical CO₂ method to get yield of essential oil which was more than 5%,while the yield was 1.23%by steam distillation.It can be seen that supercritical CO₂ extraction method is an ideal method to improve the yield of essential oil.The essential oil composition of C.zedoaria was complex.Although the essential oil of C.zedoaria from different origins had same compounds,the content was significant difference.Steam distillation and supercritical CO₂ method were used to extract the essential oil of C.zedoaria.There were 10 common compounds in the detection by the two methods.It is noteworthy that chemical compositon was extracted by different methods to have significant difference and unique compounds.Because two kinds of Sichuan C.zedoaria came from different region and time,there was different chemical composition of essential oils.3.Two compounds were isolated from the essential oil of K.galanga and C.zedoaria by silica gel column chromatography,gel column chromatography and recrystallization.The two compounds were identified as ethyl p-methoxycinnamate and germacrone by physicochemical property and spectral analysis.4.The MICs results showed that K.galanga essential oil inhibited E.coli,S.typhimurium,and S.aureus and MICs values respectively were 2 mg/m L,1 mg/m L and 2.5mg/m L.Similarly,three kinds of bacteria were inhibited by ethyl methoxycinnamate and the MIC values respectively were 4 mg/m L,2.5 mg/m L and 5 mg/m L.C.zedoaria essential oil and gemmazone had poor inhibition against three bacteria to compare K.galanga essential oil,and the MIC values both were more than 5 mg/m L.The results of growth curve showed that the antibacterial effect of K.galanga essential oil was dose-dependent,and the strong resistance to E.coli and S.typhimurium.5.K.galanga essential oil inhibited bacterial biofilm formation.The results showed that K.galanga essential oil inhibited the biofilm formation of the three bacteria and MB_IC50（the lowest concentration inhibiting 50%biofilm formation）values respectively were 2.5 mg/m L,1 mg/m L and 5 mg/m L against E.coli,S.typhimurium and S.aureus.6.The results showed that three kinds of bacteria were treated with K.galanga essential oil to get ultrastructural of bacteria by SEM,the three kinds of bacteria appeared morphological change,and did not maintain the regular and complete morphology,the cell membrane and cell wall were destroyed.7.K⁺and Mg²⁺are the most important ions in maintain normal activity of bacterial cells and also important salts of osmotic pressure balance.A large number of K⁺and Mg²⁺leaked out of the bacterial cells indicated that the cell membranes have been damaged.The results showed that K.galanga essential oil increase the cell permeability of E.coli,S.typhimurium and S.aureus to result the leakage of small molecule substances.The results of bacterial proteins and nucleic acids leakage test showed that K.galanga essential oil damaged cell membrane of the three kinds of bacteria to result the leakage of macromolecular substances DNA,RNA and proteins.8.The experiments of the effects of K.galanga essential oil on bacterial cell proteins showed that the protein bands of the three strains became weaken or even disappeared when bacteria was treated with essential oil.It was shown that K.galanga essential oil apparently destroyed cell membrane to result the loss of some intracellular proteins and reduction of the content of intracellular soluble proteins.