Studies On Isolation, Identification And Antioxidant Activity Of Oligomeric Proanthocyanidins From Lotus Seed Peel

Lotus seeds are the seeds of plants in the genus Nelumbo. Xiangtan is the main area to plant Lotus seeds in China. In recent years, with the developing of planting scale, the yield of lotus seeds is increasing rapidly and annual yield can arrived at one hundred thousand tons. Due to the lotus seeds are normally sold de-peeled in the supermarket, the lotus seed peel depeeled with the machine become the by-product in the process of lotus seeds and they are either thrown or used for feeding livestock. In fact, the recent research showed the lotus seed peel contained high concentration proanthocyanidins. Hence, recovering proanthocyanidins from the lotus seed peel and applying in humans’ daily life will be a meaningful and effective way to make wealth from waste. The main research results were shown as follows:1Vanillin assay was carried out in HC1medium to determine the content of proanthocyanidins in the lotus seed peel.Several parameters affecting the precision and accuracy of vanillin assay including HC1concentration, vanillin concentration, reaction time, reaction temperature, sunlight were studied. The results showed that the proper reaction conditions were as the following:the medium comprised of0.5ml of sample,3ml of4%vanillin solution in methanol, and1.5ml of concentrated HC1in methanol, and the reaction was carried out at30℃for20min (measured at500nm). Assessment of the method by statistics proved its high stability, reproduction quality, and recovery.2Based on the uniform design and support vector regression, UD-SVR, a novel experimental design and analysis approach was applied. It was used to optimize the extraction process including five independent variables for the proanthocyanidins from the lotus seed peel. The optimization results by UD-SVR showed the proanthocyanidins yield increased from2.9%in the initial scheme and3.98%in the optimal scheme of single factor to5.87%after two rounds of uniform design and testing of36schemes. The optimal scheme was solid:liquid of1:57, acetone concentration of67%, pH value of2.7, temperature of37℃and extration time of90mins. 3In order to obtain the high purity proanthocyanidins, ethyl acetate solvent extraction combining with macroporous resin AB-8was used to purify the extraction firstly. Then polyamide chromatograph was optimized to further purify. The results showed that the suitable velocity of loading, the concentration and capacity of sample loading and the velocity of flow are16BV/h,0.48mg/ml,8BV and24BV/h, respectively. Four different acetone concentration10%,30%,50%and70%were used to elute the polyamide. The four fractions M1、M2、M3、M4were obtained, and the rocovery rate were12.32%、19.84%、54.41%、2.30%, respectively. The purity were78.47%、84.61%、96.26%、34.15%, respectively。4After eluting with10%,30%and50%acetone solvent, M1, M2, M3were obtained and further analyzed by infrared spectru, RP-HPLC and RP-HPLC-MS/MS. In conclusion, infrared spectrum confirmed that the procyanidins, consisted of catechin units, was the mainly construction in the extraction of proanthocyanidins from the lotus seed peel. Compounds were further-confirmed by RP-HPLC and RP-HPLC-MS/MS. Through the above mentioned methods, the basic components of proanthocyanidins had been concertained including the monomer flavanoid ([M-H]" m/z289) and ([M-H]" m/z305), four kinds of isomeric compounds of procyanidin dimers ([M-H]-m/z577) and two kinds of tetramers ([M-H]-m/z1153). Moreover, the MS/MS spectrum showed fragmentation of dimers all include m/z of407and289, one of tetramers include m/z of1001,865,695,575and287, and the other include865,739,575,423and287.5Compounds F2, F4and F6were further identified by NMR:compound F4was (+)-catechin, compound F2was epicatechin-(4β→8)-catechin (procyanidin B1) and compound F6was epicatechin-(4β→8)-epicatechin (procyanidin B2). In addition, compared with the standard sample, other compounds were comfirmed including F1of (+)-gallocatechin, F3of catechin-(4α→8)-epicatechin (procyanidin B4) and F7of (-)-epicatechin.6The research on the antioxidate capacity of PMR, catechin and VC showed: Firstly, total antioxidate capacity, the antioxidative capacity on linolic acid and scavenging capacity on DPPH, O2-and OH-radicals of proanthocyanidins PMR were all higher than VC, but lower than catechin. Secondly, all the antioxidants have higher scavenging capacity on DPPH·system than OH-and O2-. In addition, PMR was found to active Nrf2-ARE signal pathway and result in activation of Nrf2through its dissociation from Keapl. The expression of cytoprotective target genes like antioxidant proteins HO-1is then transactivated in response to the stress.