Quantitative Analysis Of Batatasins And Their Derivatives From Chinese Yam (Dioscorea Opposita Thunb.)

Batatasins, as one of the most important factors in the dormancy of yams bulbils, were firstly found in yams by Hashimoto in 1972. Further investigation demonstrated that they also exist in the other plant species, such as Dioscorea cayenesis Lam, D. sansibarensis Pax, and D. composite Hemsl. Previous pharmacological studies suggested that Batatasins possess a variety of beneficial health effects, such as anti-inflammatory, antifungal, radical scavenging and immune system modulator etc.To date, few analytical methods have been reported to determine the Batatasins in Chinese yam, which were mainly ascribed to the shortage of Batatasins standards. In order to provide further insights into the Batatasins and their biological activitives, a reliable and global strategy, which could quantitative determination of the Batatasins effectively, is in great demand. In this thesis, the approach of high performance liquid chromatography (HPLC) and quantitative 1H NMR (q-NMR) were used for the simultaneous determination of Batatasins and/or their derivatives. The main contents are as follows:1. Synthesis and Characterization of Batatasins standardsBatatasin Ⅰ, Ⅲ, Ⅳ, Ⅴ were synthesized via Wittig reaction and stilbene reduction procedure as key steps. Briefly, functionally-substituted benzyl alcohols, which were prepared according to the methylation, benzylation and ester reduction procedures, were reacted with phosphorus tribromide to obtain Br-substituted intermediates respectively, which were submitted to the Wittig olefination reaction and catalytic hydrogenolysis to get the Batatasin Ⅲ, Ⅳ, and Ⅴ. Batatasin I with a phenanthrene skeleton was prepared by the following strategy, where the intermediate 4-benzyloxy-3,3’,5-trimethoxystibene was irradiated with UV light in the presence of catalytic iodine and then adopted to the reduction procedure under a hydrogen atmosphere. All compounds were characterized by 1H NMR,13C NMR and FT-IR spectrum.2. HPLC method for the simultaneous determination of BatatasinsHigh performance liquid chromatography (HPLC) was applied for the simultaneous determination of Batatasins and their derivatives. The chromatographic separation was performed on a Cosmosil 5C18-PAQ column (20× 250 mm, Nacalai Tesque) with methanol and water elution (52:48, v/v) at a flow rate of 1.0 mL min-1. The column temperature was maintained at 35℃ and the UV detection was performed at 210 nm. The detection limits and quantification limits ranged within 0.217-1.56 μg/mL and 0.651-4.70 μg/mL, respectively. The validation of the proposed approach was acceptable, with 85.5-104.7%accuracy in recovery test. Finally, the proposed method was applied to practical samples from Chinese yam and yielded highly reproducible data in the repetition tests.3. q-NMR method for the simultaneous determination of BatatasinsAn analytical method using quantitative nuclear magnetic resonance (q-NMR) spectroscopy was applied for the quantification of Batatasin I and its derivative (5,7-dimethoxyphenanthrene-2,3-diol) using 1,3,5-Trioxane as internal standard. q-HNMR was carried out with the following parameters:irradiation frequency,400 MHz; number of scans,4; acquisition time,4 s; relaxation delay,60 s; pulse width, 12.4 μs; spinning off. The results indicated that the content of Batatasin I and 5,7-dimethoxyphenanthrene-2,3-diol in the peel of yam were 563.1 μg/g and 287.1 μg/g respectively. Validation of the method was performed in terms of specificity, precision, accuracy and stability. Quantitative results were compared to those obtained with the HPLC method and were found to be in reasonable agreement. NMR analysis did not rely on the use of reference compounds and enables significantly faster analysis compared to HPLC determination. Thus, NMR represented a feasible alternative to HPLC-based methods and could be a useful and practical tool for determining the level of Batatasins and its derivatives.