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Studies On Extraction, Purification, Ingredient And Functionality Of Procyanidins From Pine Needles

Posted on:2014-08-16Degree:MasterType:Thesis
Country:ChinaCandidate:R MaFull Text:PDF
GTID:2250330425951890Subject:Botany
Abstract/Summary:PDF Full Text Request
Proanthocyanidins (PC), which are consist of monomer flavan-3-ol and flavan-3-ol polymer, belongs to polyphenols. As a kind of bioflavonoid in the plant, PC has abundant natural sources. PC is the most effective natural antioxidants on scavenging free radicals and can effectively prevent the occurrence of various diseases. High level of PC has been found in pine needles, which are widely distributed in many provinces of China. Therefore, it’s of great significance to exploit new by-products with high value and make full use of pine needles. Using slash pine needles as materials, the present study aimed to establish an efficient procyanidins extracting method and analyze its main bioactivities. The main results were summarized as following:The optimum PC extracting conditions determined by response surface methodology (RSM) were as follows:ethanol concentration35%, extraction temperature85℃, extracting time2h. Under this condition, the average PC content of three extracting experiments was3.41%±0.0015, which is close to3.52%, the theoretical value calculated through the regression equation. And the purity of crude extract was12.1%±0.0021.Crude PC extracted from pine needles was further purified by macroporous resin, eleven different resins were screened for a high adsorption rate of PC. The highest adsorption rates were sequenced in HPD700, followed by HPD400, NKA-9, HPD100A, HPD600, D101, XDA-1, HPD100, DA-201-C, AB-8, HPD300. Three resins, HPD700, HPD400, NKA-9, which has high adsorption rate were chosen for further screening. Among the3resins, through a comprehensive evaluation based on the adsorption equilibrium time, desorption rate and desorption equilibrium time, HPD700was chosen as the optimal resin for PC purification, And ethanol at35%concentration was screened as the optimum PC desorption solvent. The purity of the refined PC was95.21%±0.0068, or7.8folds higher in purity as compared with the crude extraction.Refined PC were then analyzed by TLC experiments. After the optimization, the expanded matter and proportion were set as:toluene (3/7), acetone (3/7), acetic acid (1/7). With the upper mixed solution as expanded matter, the Rf value of monomer proanthocyanidins on GF254silica gel was0.6. By comparing with the Rf of grape seed proanthocyanidins standard and epicatechin standard, the components of refined PC were similar to the ones in grape seed proanthocyanidins and the monomer proanthocyanidin in PC extracted from pine needles was deemed to be epicatechin.Refined PC had strong effects on DPPH and O2-radicals scavenging capacity. The IC50values obtained from DPPH radical scavenging experiments for refined PC, BHA and Vc were10.88μ.g/mL,5.04μ.g/mL and4.43μg/mL, respectively. Correspondingly, in O2-radical scavenging experiments, the IC50values for refined PC and Vc were43.62μg/mL and30.67μg/mL, respectively. The refined PC also exhibited strong inhibitory ffects on tyrosinase, with an inhibitory rate as66.07%. Comparatively, the inhibitory rate caused by arbutin was85.00%.The crude extracting of pine needle soluble in ethyl acetate were isolated and purified by column filled with silica gel and sephadex LH-20. Two components (A and B) were isolated through silica gel column chromatographic. Based on the spectral data of1H-NMR and13C-NMR, the substance A was identified as Di(2-ethylhexyl)phthalate. And component B were further purified by sephadex LH-20column chromatographic, which was identified as (-)-epicatechin based on NMR spectrum. The content of (-)-epicatechin was100.8mg/100g.Epicatechin isolated from pine needles had strong effects on DPPH and O2-radicals scavenging capacity. The IC50values obtained from DPPH radical scavenging experiments for refined epicatechin, vitamin C and BHA were5.55μg/mL,5.04 μg/mL and4.43μg/mL, respectively. Correspondingly, in O2-radical scavenging experiments, the IC50values for epicatechin isolated from pine needles and Vc were54.30μg/mL and30.67μg/mL. It may be deduced that epicatechin played an important role in DPPH·scavenging capacity in PC extracted from pine needles.
Keywords/Search Tags:pine needles PC, separation, purification, component analysis, antioxidant
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