| Cinnamomum camphora(L.) Presl(Lauraceae), commonly known as one of Lauraceae species with the greatest economic value, widely distributes in the south of the Yangtze River. Camphor seed contains 40% of oil(CSO) in which the content of medium chain fatty acids, C10 and C12, is up to 90%, with the prevention and treatment of arterial thrombosis, blood fat and antibacterial effects. By the enzymatic method, CSO was used to obtain medium chain fatty acid glycerol esters(MCFA-GEs), which have effects of lowering postprandial lipid, losing weight and bacterial inhibition. Camphor tree seeds could be obtained easily, but now they were discarded generally as waste materials, resulting in a huge waste of resources. So the systematic research of CSO MCFA-GEs is particularly important.Camphor seeds were treated using physical squeezing method, deodorization and decoloring to obtain refined CSO. After being catalyzed by the immobilized lipase Lipozyme RM IM, CS-GEs were obtained and then separated by the column chromatography to get the camphor tree seed oil triglycerides(CS-TAG), the camphor tree seed oil diacylglycerol(CS-DAG) and the camphor tree seed oil monoglycerides(CS-MG). In order to further analyze functional activities of CS-TAG, CS-DAG and CS-MG, the filter method, the minimum bacteriostasis concentration and the bacteriostatic activity assays were carried out for their antibacterial active to E.coli, S.typhimurium, L.monocytogenes and S.aureus. The electric conductivity, the determination of the content of soluble protein and nucleic acid of culture media were analyzed for the antibacterial mechanism of CS-GEs. We also studied the chemical composition and the antibacterial activity for the camphor leaf essential oils from 5 different camphor chemotypes, including camphor-type, linalool-type, borneol-type, camphor-type and cineole-type.1. After enzyme catalyzation, CSO MCFA-GEs use TLC analysis, components to a migration value CS-TAG(0.94), CS-FFA(0.83), CS-1,3-DAG(0.53), CS-1,2-DAG(0.50), CS-MG(0.07). Further separating the components by column chromatography, TLC analysis was performed to obtain the component purities 92.7%, 98.2% and 97.8% of CS-TAG, CS-DAG, CS-MG. Furthermore, RP-HPLC-ELSD was carried to verified the components purities of 95.1%, 92.4% and 94.6%.2. For CS-TAG, CS-DAG and CS-MG, the antimicrobial susceptibility was obtained using the filter paper method, the determination of minimum inhibitory concentration(MIC) was through 96-well plate method, and the antibacterial vitality experiment was used to determine the antibacterial aging. The results showed that CS-TAG, CS-DAG and CS-MG exhibit antibacterial effects to four kinds of bacteria. For CS-TAG and CS-DAG, the bacteria inhibition zone diameters ranged from 15.03 mm to 17.76 mm, with high sensitiveness. The bacterial inhibition zone diameter for CS-MG is between 22.21 mm to 25.37 mm, with extreme sensitiveness. According to results of the 96-well plate method, the MIC of CS-TAG were 4.0 mg/m Lto G+ bacteria, as well as 6.0 mg/m L to G- ones. The MIC of CS-DAG was 2.0 mg/m L to G+ bateria, and 4.0 mg/m L G- ones. The MIC of CS-MG was 1.0 mg/m L to G+ bateria, and 2.0 mg/m L to G- ones. For CS-TAG, CS-DAG and CS-MG, the bacteriostatic activity to G+ is stronger than to G-, and the bacteriostatic activity to 4 kinds of bacteria were above 0.60 in 3~48 h. For the same bacteria, the antibacterial abilities ordered as CS-TAG< CS-DAG < CS-MG.3. For the antibacterial assays, the leakage in the bacterial cells was determined by the electric conductivity and the amount of proteins, nucleic acids, and then confirmed by scanning electron microscopy for the bacterial surface morphology change. The results showed that CS-TAG, CS-DAG, CS-MG acts on 4 kinds of bacteria tested was found when 3~15 h fast bacilli conductivity increased, rise in the 18~24 h broth conductivity rises slowed, until 48 h balance. Soluble proteins, bacteria nucleic acid content determination results show that CS-TAG, CS-DAG and CS-MG of microbial protein, nucleic acid content increased, CS- MG increased most, least CS-TAG. Scanning electron microscopy observation of bacteria, CS-TAG, CS-DAG and CS-MG bacteria morphology change is by bacteria deformation or lysis. Therefore, CS-TAG, CS-DAG and CS-MG may through the change of the bacterial cell membrane permeability, lead to a bacteria were the leakage of intracellular proteins, nucleic acids, reducing the bacteriostatic action.4. Camphor tree leaf essential oil obtained by simultaneous distillation extraction method, and then by GC-MS analysis of 5 chemotypes camphor oil composition. From different camphor of camphora-type, linalool-type, cineole-type, isoborneol-type and borneol-type leaf oil were identified 71 kinds of known compounds. The most abundant compositions in the five essential oils were bicyclo [2.2.1] heptan-2-one,1,7,7-trimethyl-,(1R)(41.02%) in camphora-type essential oil, Nerolidol(22.52%) in isoborneol-type, eucalyptol(41.95%) in cineole-type, 1,6-Octadien-3-ol,3,7-dimethyl-(51.23%) in linalool-type and borneol(40.67%) in borneol-type. 71 known compounds were identified from the leave essential oil of 5 chemotypes camphor, including 5 kinds of compounds(alcohols, ketones, ethers, alkenes, oxide and derivatives of these 4 substances). The isoborneol-type essential contained 34.93% of 5 kinds of compounds, and the percentages were larger than 81% for those of other chemotypes, followed by borneol> cineole> camphora> linalool>> isoborneol. For these essential oils, their antibacterial effects increased with their concentrations in the culture medium. The essential oils of linalool-type, camphora-type, cineole-type, and isoborneol-type showed higher inhibitory effect on gram-positive bacteria than on gram-negative bacteria.The above research shows that highly pure CS-TAG, CS-DAG and CS-MG could be obtained by the column chromatography from the enzymatic hydrolysate. These three components show antibacterial activities, with the inhibition effect of G+ stronger than that G-. For the same bacteria, the antibacterial abilities ordered as CS-TAG< CS-DAG < CS-MG. The antibacterial effect of these CS-GEs could be the result of the change of membrane permeability, as indicated by the increase of the electric conducity, as well as the contents of proteins and nuclei acids in culture media. Furthermore, the morphological observation also verified the hypothesis. We also observed the differences of chemical compositions and antimicrobial activities of leaf essential oils of five chemo-types camphor, suggests that camphor oil bacteriostatic activity of leaves may have certain correlation with its chemical composition. |
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