Studies On The Separation, Structure Identification And Bioactivities Of Procyanidins From Sea Buckthorn

Sea buckthom (Hippopha rhamnoides L.) is a conventional herb with a long history of application (more than 1000 years) in Tibetan and Mongolian medicines in the treatment of various diseases. Numerous nutritional and physiological activities have been reported in most part of sea buckthom including the berry, leaves, seed and bark. Many of which have bioactivities such as anticancer, antiradiation, anti-atherosclerotic, enhancing body energy and immunity, anti-inflammation, antiblastic, acesodyne and accelerating regeneration. So far, there has been a great amount of research about the effects of its composition, including vitamine C, carotenoids, flavonoid, fat and polysaccharides etc. Although sea buckthom is a resource of procyanidins, neither the systematic researches of procyanidins structure indentification nor its bioactivities has been reported at home and abroad. Therefore it has very important academic and plactical significance to make comprehensive research on procyanidins of sea buckthorn for its deep exploitation and use.In this study, modem separate and analytical technologe of chromatogram, spectrum and the chemical analysis are used to analysis the component and identify the structure of procyanidins in sea buckthorn bark. While some advanced technologies of modern medical and molecular biological technology were used to study the antioxidation activity in vitro and antigastric activities and its mechanism in vivo. The results are as follows:1. Extraction, purification and determination of content of proanthocyanidinsThe analytical results of proanthocyanidins in different part of sea buckthorn showed that the content of seed and bark is 9.67% and 4.42%, respectively. The extraction rations of proanthocyanidins from sea buckthorn seed was compared using different solvent, temperature, pH value and so on. After investigating the single effect factor and orthorhombic analysis, the optimal parameters of extraction were obtained: 65%ethanol/water (v/v),temperature (21℃),pH(5.1),the ratio of material to extract solvent (1:10, v/v),extract time (90min,×3) .The yield was 5.10%.In order to purify proanthocyanidins from sea buckthom seed by resin, the behavior of static adsorption, dynamic dasoption and desorption of sixteen kinds of resins was compared. D3520 resin is the optimum for proanthocyanidins purification; the optimum elution fraction in the 30% ethanol aqueous get 96.5% proanthocyanidins purity and 30.4% rate of recover.2. Identification and chemical structure characteristics of procyanidins B3 in sea buckthorn barkSBPC, 30% ethanol fraction of sea buckthorn bark procyanidins extract which eluted from D3520 resin, was analyzed by NP-HPLC and RP-HPLC. The results showed that it consisted of monomer, at least three dimmers and trimer of procyanidins. Their molecular weights are 290,578,866 respectively. SBPC was separated into 4 fractions (â… ,â…¡,â…¢andâ…£) with100% methanol eluted from Toyopearl HW-40S gel chromatogram. Fractionâ… was neamed as compoundâ… by the reason that both NP-HPLC and RP-HPLC chromatograms of fractionâ… presented single peak. And the spectrum of compoundâ… provided by DAD totally in character with spectrogram of procyanidins. ESI-MS spectrogram indicated that m/z of pseudomolecular was 577 and the fragment ions were 289 and 425 in negative ion spectum, which accorded with fragment formation of procyanidins B3. When compoundâ… was acid-catalyzed in the presence of excess phloroglucinol, the terminal subunit is catechin via reference standards. The identification of compoundâ… was further confirmed using 1D NMR and 2D NMR technique, and the result indicated that compound was procyanidins B3 composed of two catechin units linked through C4-C8 bonds.3. The antioxidative activity of SBPC and B3 in vitroThis paper investigated the antioxidative activityof SBPC in vitro including the effects of SBPC on hydroxyl radical ('OH), red blood cells (RBC) hemolysis, liver mitochondria swelling and lipid peroxidation in liver homogenate and liver mitochondria. The results showed that SBPC could inhibit .OH and lipid peroxidation. It could protect the biomembrane, reduce RBC hemolysis and lighten the degree of liver mitochondria swelling. So SBPC had very strong antioxidation activities.Effects of SBPC and B3 on scavenging hydroxyl radical (OH) and hydrogen perocide, removing super oxygen-anion free radical and preventing DNA were determined in CuSO₄-Phen-VC-H₂O₂ system, Luminol- H₂O₂ system, Pyrogallol-luminol system and CuSO₄-Phen-VC-H₂O₂-DNA chemiluminescence system respectively. The results showed that SBPC and B3 possessed a good potency to scavenge reactive oxygen (ROS) in different systems. However, the antioxidation activities of B3 were stronger than that of SBPC.4. Prevention effects of SBPC on gastric ulcer in stress ratesThe rat model of acute gastric ulcer was induced by water immersion and restraint stress (WRS). Rats were orally administrated with different dose of SBPC (50,100, 150mg/kgbw) or ranitidine (positive drug, 30mg/kgbw ) before stressed, Gastric lesion index, acidity and pepsin activity of gastric juice, Cortisol, ACTH, epidermal growth factor (EGF), NO in plasma were determined. The expression of epidermal growth factor receptor (EGFR), iNOS and proliferating cell nuclear antigen (PCNA) around ulcer were detected by immunohistochemical method. The apoptotic cells in the gastric mucosa were examined through the methods of terminal deoxynucleatidyl transferase mediated DUTP nick and labeling (TUNEL). The ultrastructural change of parietal cells was observed by. transmission electron microscope. Compared with the control group, the ulcer index, the level of [H⁺] and pepsin activity of gastric juice of SBPC high-dosed group significantly decreased(P〈0.01 ),and the level of Cortisol, ACTH, NO in plasma of SBPC high-dosed group significantly decreased (P〈0.01) , In plasma, the EGF level was dose dependently increased, and inducible Nitric oxide synthase (iNOS) expression in gastric mucosa in high-dose group was significantly decreased (P〈0.01) . The expression of EGFR protein and the PCNA were increased significantly (P〈0.01, P〈0.05, respectively), meanwhile the apoptosis index (AI) was significantly decreased (P〈0.05).The results above Showed that gastric lesion could be depressed in rat when orally administrated with SBPC beforehand. The mechanisms of which are complicated: one of mechanisms is that SBPC could regulate the level of ACTH and Cor which effect the gastric juice secretion. And this assumption could be proved by the observation of the ultrastructural change of parietal cells. Another possible mechanism is SBPC could up-regulated the EGF and EGFR and down regulated NO to promote cell proliferation and inhibit cell apoptosis, which could be accorded with the up-regulator of expression of PCNA simultaneously.5. Effects of SBPC on healing of acetic acid-induced gastric ulcer in ratesChronic gastric ulceration was induced by injecting acetic acid in the subserosa of stomach. Different concertrations of SBPC were orally administrated to gastric ulcers rats. After treatment 7d and 14d, rats were sacrificed respectively. The ulceration was measured by ulcer index (UI). The level of EGF in plasma and PAS position area in gastric mucosal was determined.The expression of EGFR and PCNA around ulcer was detected by immuohistochemical method after treatment 7d. SBPC was found to reduce the size of the ulcers at day 7 and 14 in a dose-dependent manner. Compared with the control, the UI of SBPC group was significantly decreased (P〈0.01) and the level of EGF in the plasma of SBPC group increased significantly (P〈0.01 ), meanwhile the expression of EGFR and PCNA around ulcer in high-dose SBPC stomach were enhanced (P〈0.05). The results implied that SBPC plays an important role in healing of acetic acid-induced gastric lesions possibly by the acceleration of the mucosal repair. Meanwhile SBPC could enhance the defence mechanism by promotion mucous secretion.