| In order to investigate the possible activity of persimmon tannin(Diospyros kaki L.) against the toxicity of snake venoms and elucidate the structure-activity relationships of persimmon tannin, persimmon tannin was extracted with absolute ethanol from persimmon pulp using Ultrasonic-assisted extraction technique and fractionated by column chromatogram. Three fractions with different degree of polymerization were obtained and their effects on inhibiting enzymes activities from Agkistrodon halys pallas venom protein(Ahp VP),Agkistrodon acutus venom protein(Aa VP) and Naja naja atra venom protein(Naja VP) in vitro were examined. Meanwhile, their inhibition effects on myotoxic activity, hemorrhagic activity, neurotoxicological toxicotyand lethality of Agkistrodon halys Pallas and Agkistrodon acutus venoms and Naja naja atra venom protein(Naja VP) were evaluated in vivo. In addition, a combination of HPLC, MALDI-TOF-MS, Infrared (IR)spectra and circular dichroism(CD) spectrum were used to study the structural feacture of the key fraction accounting for detoxifying effect on snake venoms, and the method for determing the content of persimmon was also included.The results were shown as follows:1. Ultrasonic-Assisted extraction of persmimmon tannin from persimmon pulpThe optimum extraction conditions of persimmon tannin were absolute ethanol containing 1% concentrated HC1 as extraction media with a solid/solvent ratio of 1:8(W/V), extracting at 60℃for 30min and 80w as the extraction power. The yields of total polyphenol and condensed tannin were 24.36 mg/g and 15.23mg/g, separately. The crude extract was purified with macroporous adhesive resin followed by Toyopearl HW-50F column, and three fractions with different degree of polymerization were obtained. 2. Detoxifying effect on snake venoms both in vitro and in vivoDifferent amounts of PT fractions (PT20, PT40 and PT60) were mixed with snake venoms,the enzyme activities of proteolytic enzyme,phospholipase A2, L-amino acid oxidase, acetylcholine esterase and arginine esterase from the supernatants were inhibited significantly, and the inhibiting effect was in a dose-dependent and degree of polymerization-dependent manner.PT 40 showed an improvement in survival of the mice in the test group dose-dependently, and the survival rates of mice caused by Ahp VP、Aa VP and Naja VP that received high dose of PT40 increased significantly(p<0.01), and reached 60%、70% and 40% respectively.PT40 significantly and dose-dependently alleviated the hemorrhagic activity induced by Ahp VP (p<0.01), and the inhibiting effect of PT40 on hemorrhagic spots area were 31.4%、63.3% and 95.6% at the dose of 50μg、100μg、500μg,respectively.Myotoxicity from Naja VP was inhibited statistically and significantly (p<0.01) when the venom mixed with PT40 at ratios of 5:1 and 1:1.The inhibiting effect of PT40 on creatine kinase activities were 4.5%、26.1% and 42% at the dose of 5μg、10μg、50μg, respectively.The histological analysis of muscle suggested that Naja VP cause myonecrosis, characterising by degeneration of muscle cells, but PT40 could relief this damage.Protective effect of PT40 on Naja VP induced neurotoxicity in adult mice was observed. Both open-field and water maze tests suggested that PT40 significantly reversed the behavioral retrogression, such as rearing/learning and cognition, induced by Naja VP.Besides, the cerebellum of venom-treated mice showed translucent vacuoles in the Purkinje cells.The Purkinje cells changes were improved and repaired obviously by PT40 at the dose of 1:3(w/w).3. Charaterization of the structure of PT40The combination of MALDI-TOF/MS analysis and thiolysis HPLC was used to characterize the PT40. A novel terminal unit comprising the flavonol myricetin was identified along with the more common flavan-3-ols catechin and epigallocatechin-3-O-gallate. The extension units were confirmed to be epicatechin, epigallocatechin, (epi)gallocatechin-3-O-gallate, and (epi)catechin-3-O-gallate. PT40 was highly 3-O-galloylated (90%) and it has A-type interflavan linkages in addition to the more common B-type interflavan bonds.The FT-IR spectrum of the carbonyl (C=O) stretching vibration at 1707 cm-1 is due to the carboxylic group; A series of signals noticed in the range of 1000-1650cm-1 and 700-850 cm-1 supported the structure of proanthocyanidin. There is a shoulder signal at 1534 cm-1 and this may be due to the B-ring stretching vibration. Whereas those at 726 and 763 cm-1 were attributed to aromatic ring CH out-of-plane deformation.These absorption bands indicated that the PT40 was of a mixture of prodelphinidin and procyanidin type proanthocyanidins.A strong positive cotton effect in the CD spectrum at 220-240 nm indicated a 4βlinkage of the flavanyl substituent and hence the 4R absolute configuration of PT 404. Determination of condensed tannin content in persimmon tanninIt was found that the content of condensed tannin in persimmon tannin extract was underestimated in several classical methods for determing tannin content, such as BuOH-HCl assay, HCl-Vanillin assay, et al. BuOH-HCl assay for determinating of condensed tannin content in persimmon tannin extracts was optimized. And it was found that the color yield was very low for persimmon tannin even in the optimized condition, and standards used had a great influence on the color yield and determination of the content of persimmon tannin. Some undegaraded fraction was separated from the products of PT40 in BuOH-HCl system, and a combination of thiolysis-HPLC、and Infrared (IR) analyses suggested that there is a-C2H5,at the C-3 ring position. |
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