Studies On Extraction, Fractionation, And Analysis Of Proanthocyanidins From Red Skin Of Red Locus Seed

The lotus seed (Semen Nelumbinis), mature seeds of herbaceous plant of Nympheaceae Nelumbo Adans, has high natritional value and health care value, and can nourish vigour. But processing lotus seed include drying, grading, shelling, grinding, coring and so on. The powder of red skin,15%of lotus seed, is abandoned because of tasting puckery. It cause a great waste. Consequently, development and use powder of red skin of lotus seed has become an important issue on protection healthy development of lotus seed industry. Powder of red skin of lotus seed is rich in proanthocyanidins. Proanthocyanidins, a great potential on development of natural active substances, have a varirty of biological pharmacological activity and non-toxic. The research used the powder of red skin of lotus seed as experiment material, mainly focused on extraction and fractionation, as well as structure and composition analysis of proanthocyanidins. Results of research can improve the value-added of the process of lotus seed, and promote the development of the lotus seed industry.1. The single experiments and response surface methodology were researched to determin the opitimum extractive conditions of proanthocyanidins from red skin of red locus seed. The single experiments were used to expolre the type of extractant, extractant concentration, different acid, pH, liquid-to-material ratio, extraction temperature, extraction time and extraction times on the extraction of proanthocyanidins.lt resulted three factors (pH, liquid-to-material ratio, and acetone concentration) had more significant effect of the extraction of proanthocyanidins. Response surface analysis of the three factoes get a quadratic polynomial regression model of proanthocyanidins from red skin of locus seed:Y=-54.925+4.52Xi+0.3633X2+1.4117X3-0.015X1X2-0.005X1X3-0.0051X2X3-0.6483X12+0.0005X22-0.0083X32。 Y is yield of proanthocyanidins from red skin of locus seed, Xi is pH, X2is liquid-to-material ratio, X3is acetone concentration. Based on single experiments and response surface analysis, the optimum extractive conditions were ascertained as follows:extraction temperature40℃, extraction time90min, pH2.6, liquid-to-material ratio55mL/g, acetone concentration67%, extraction times one. The yield of proanthocyanidins of this optimized procedure was9.57%.2. Then, based on differences of hydrophilicy and polarity of proanthocyanidins compontnts in the extracts solution, proanthocyanidins from red skin of locus seed were fractionated according to degree of plomerization. The crude proanthocyanidins extract solution was extracted with ethyle acetate. The organic phase contained mainly oligomeric proanthocyanidins, while the aqueous fraction contained the polymeric proanthocyanidins. The chloroform/methanol two-phase was used as a solvent-precipitation method for fractionation of proanthocyanidins water-soluble fraction according to degree of plomerization. Proanthocyanidins water-soluble fraction was dissolved in methanol. Chloroform wasn’t added until30%of chloroform for the total volume. The precipitate was colltected, named proanthocyanidins fraction F4. The filtrate was retained for repeating by adding successively chloroform representing40%and60%of chloroform for the total volume. This resulted in proanthocyanidins fraction F3, F2for the precipitates. Macroporus adsorption resins was used to speration and putification proanthocyanidins ethyl acetate fraction. It resulted the AB-8resin gave the best results.The optimum speration and putification conditions were ascertained as follows:resin volume was5mL, loading flow velocity was1.33mL/min, concentration of proanthocyanidins ethyl acetate fraction was0.99mg/mL. loading volume was20mL, distilled water volume was40mL, the flow rate was2.66mL/min of50%acetone concentration by25mL. The purity of proanthocyanidins ethyl acetate fraction is about91.86%under the opitimum conditions, named proanthocyanidins fraction F1.3. A series of analaysis method such as infrared spectroscopy, reversed phase high performance liquis chromatography(RP-HPLC), reversed phase high performance liquis chromatography coupled with electrospray ionizaton mass spectrometry(RP-HPLC-ESI-MS), and mean degree of polymerization, were used to reveal the composition and chemical structure of four proanthocyanidins fractions(F1, F2, F3, F4) from red skin of lotus seed. Analaysis showed: The infrared spectra of four proanthocyanidins fractions showed the characteristic stretching vibration of proanthocyanidins, and idicated that proanthocyanidins of red skin of lotus seed mainly composed of procyanidins. By RP-HPLC-ESI-MS, three types of monomers, four types of dimers, and two types of tetramer were tentatively identified proanthocyanidins fraction F1. Three types of monomers were catechin, epicatechin, gallocatechin. Four types of dimers and two types of tetramers were both composed of catechin or epicatechin monomers ploymerization. RP-HPLC showed proanthocyanidins fractions F2, F3, F4were indentified including the monomets of catechin and epicatechin. Then,by comparing HPLC chromatogram of F1and F2, F3, F4, proanthocyanidins fractions F2, F3, F4were infered contaning three types of dimers, one of which was more,.as well as an unknown component contains more. The mean degree of polymerization of proanthocyanidins fractions F1, F2, F3, F4were determined, respectively2.12,7.27,7.85,8.16. This showed the ethyl acetate extraction separation and chloroform/methanol two-phase precipitation separation maked a apparent effect of fractionating proanthocyanidins from red skin of locus seed according to degree of plomerization.