| Obesity has become one of the greatest threats to human health in this century, and had been recognized as the largest global chronic disease by WHO. At least 3400,000 adults died each year due to overweight or obese. In the face of this grim situation, substantial researches for treating obesity had been made by researchers in the past decades. Results showed that suppression of pancreatic lipase activity by drugs has become an effective method for the treatment of obesity. Orlistat is a weight-loss drug belonging to PL inhibitor. However it still has some side effects. Thus, to develop the safe, long-term use’s, no obvious side effects’drugs of weight loss has become the hot research area. Screening of PL inhibitors from resource-rich natural plants is one of the most effective strategies, and researchers screen the compounds which can interact with PL by immobilized enzyme. This method reduces the blindness of the separation and activity screening of natural products, save time and greatly improve the hit rate of screening.Former studies found that extracts of Forsythia suspensa leaves can inhibit PL activity, in vivo experiments proved that it can reduce weight of obese rats, decrease total lipid and triglyceride content in serum. However, the active compounds are uncertain. In this paper, immobilized PL was firstly synthesized. Second, PL inhibitors of F. suspensa leaves were systematic screened by the immobilized enzyme. Third, inhibitive abilities of other several natural plants on PL were preliminarily studied. The main research contents and results were listed as follows:1. With the carboxyl magnetic beads as carrier, the immobilized enzyme was synthesized according to the formation of an amide bond between the carboxyl group of the magnetic beads and primary amino groups of PL. and was characterized by IR. Then, immobilized enzyme screening model was verified by hesperidins of positive inhibitor. The results showed that hesperidins could be specially bundled with immobilized enzyme, the immobilized pancreatic enzyme screening model could be used for screening the compounds which could specially interact with PL in natural products.2. F. suspensa leaves were firstly extracted with 95% ethanol, and chlorophyll was removed by petroleum ether, then the fraction was systematically extracted to obtain subfractions, and their inhibitive abilities on PL were detected. Results showed that the remaining precipitate had the best inhibitive ability on PL in all subfractions, the inhibition rate of 1.5mg/mL was 81.48%. So this subfraction was selected as screening object of immobilized enzyme. In addition, a new method for the extraction of chlorophyll — cloud point extraction coupled with ultrasonication method was found.3. The remaining precipitate fraction of F. suspensa was systematically screened by immobilized enzyme screening model, the screened-out compounds were identified and enzyme inhibition activity was detected. Eventually a total of 9 compounds which could interact with PL were screened out, and they included 3 flavonoids (rutin, hesperidin and kaempferol-3-O-rutinoside),2 polyphenols (chlorogenic acid and coffee acid), 2 lignans (forsythin and arctigenin) and 2 phenethyl alcohol (forsythiaside A and its isomer). In these 8 compounds which were identified, forsythin had the promoting ability on PL, the remaining 7 compounds possessed the ability of inhibiting PL, of which, kaempferol-3-O-rutinoside was the best effective inhibitor, the IC50 was 2.9±0.5μmol/L. The mechanisms included competitive inhibition, competitive promotion, noncompetitive inhibition and uncompetitive inhibition.4. Other 11 natural plants were tested about their inhibitive abilities on PL, the chloroform and n-butanol fractions of Cassia tora seed were preliminarily screened by immobilized enzyme screening model. Eventually a total of 11 compounds which could interact with PL were screened out in chloroform fraction of C. tora seed. |
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