Synthesis, Identification Of Aryl Alkyl Glucosides And Assessments Of Flavor-Enhancing And Antifungal Activities

In plant kingdom, many aromatic volatiles present in both free and glycosidically bound form are compartmented with their hydrolytic enzyme such as β-glycosidases. However, volatile aroma components are released, when endogenous enzymes and "aroma precursors" separated within the cellular structure are allowed to encounter each other during during the ageing and stress or act as a response of plant defense. Previously, most researches relative in glycosidic aroma precursors were focused on the isolation and identification of aroma precursors. In this paper, we synthesized and identified aryl alkyl glucosides, and investigated their flavor-enhancing and antifungal activities. The method for the chemical synthesis of glycosides was modified. The purified synthetic glucosides was resolved in water for the cluture of cutting rose flowers and tea shoots for ssessment the possibility of improving fragrance. In addition, the antifungal activities of these glucosides was measured. During aroma-enhancing induce by adding glucosides, the variation of the volatiles was investigated. The differential gene expression of endogenous alcohol acyltransferase during this process was also investigated for discussing the molecular mechanism of ester aroma formation. The major results were listed as follows:1. Improved Helferich method was utilized for synthesizing benzyl alcohol β-D-glucopyranoside (BA-G) and2-phenylethyl β-D-glucopyranoside (2-PE-G). Reaction conditions were optimized by orthogonal analysis based on individual factor experimental design. It turned out that the terminal yeild of two glucosides were optimal when the proportion of alcohol to sugar was1.2:1, catalysis dose was0.6%, reaction time was24h, and reaction temperature was at room temperature. The yield of BA-G and2-PE-G were53.2%and49.8%, respectively.2. Three methods, namely TLC, SGCC and HPLC, were emploied for products purification. The results of HPLC analysis showed that purity of final products of two glucosides was98.2%and98.7%, respectively. The chemical structures of target compounds were identified by LC-MS-MS,’H NMR and13C NMR.3. The synthesized aryl alkyl glucosides were added in water to culture the unscented rose cutting flowers as precursor of aroma. Obvious floral scent could be smelt during the treatment. Solid phase microextraction plus GC-MS techniques was utilized for analyzing volatile compound of the fragrance. It showed that the volatile compounds were mainly the corresponding aryl alcohol and its esters. By dynamical testing of flower blooming, it was found that volatile compounds of alcohol first rised then descended while volatile compound of esters reach its maximum at flower full-open stage. It was also clarified that the regular of emitting volatile compound was independent of light and controlled by its own growth metabolism. During flower blooming, activities of endogenous glucosidase in flowers were increasing firstly and decreasing later, and then reach its maximum at half-open stage. Simultaneously, compared with the control, there was no significant differences on enzymes activities in treated flowers fed with two glucosides respectively.4. Tea seedlings were cultivated in glucoside solution for seven days, and glycosidic aroma precursors were extracted from tea leaves. After further derivatization, the volatile compounds were detected by gas chromatography with electron capture detection detector. Leaves of tea seedling growing in BA-G solution had an increase of total glucoside concentration, and BA-G was much significant. Simultaneously, Benzyl alcohol primeveroside was also formed after cultivation. After cultivated in2-PE-G solution, the concentration of2-PE-G in seedling leaves was increased significantly, and the amount of corresponding diglycoside was also increased at the same time. By HPLC-MS analysis, the diglycoside was identified as the2-phenylethyl vicianoside. It was declared that tea seedling could absorb glucosides effectively from root and transport them to leaves, so as to improve the concentration of aroma precursors of tea leaves.5. Based on qRT-PCR techniques with β-actin gene as the reference, differential gene expression of endogenous alcohol acyltransferase of rose cutting flowers and tea shoots were analyzed before and after glucosides treatment. The results showed that gene expression level of alcohol acyltransferase was correlated to blooming procedure of cutting rose flower, but the level in tea shoots displayed similar trend to the control.6. The antifungal activities of these glucosides and their aglycones towards Colletorichum camelliae Masse were measured. Results showed that the inhibitory rate of benzyl alcohol was84.1%,89.5%,96.4%,100%respectively when the concentration in culture medium was1.7μL/mL,1.8μL/mL,1.9μL/mL,2.0μL/mL accordingly. And the inhibitory rate of BA-G was19.4%,29.0%,41.9%,48.4%respectively. The inhibitory rate of2-phenylethanol was91.4%,95.3%,100%,100% respectively, while the inhibitory rate of2-PE-G glucoside was22.6%,35.8%,46.3%,55.6%respectively. At the same amount-of-substance concentration, the inhibitory rate of aglycones was higher than that of glucosides. The two glucosides showed significant inhibitory activities towards Colletorichum camelliae Masse, and the effect was rising along with the increasing of glucosides contraction in culture medium.