Inhibiting Effectes Of The Pine Pollen On Several Enzymes About Fat Metabolism

Pine pollen (Pinus massoniana) is a kind of natural pollen, which is riched ofnutrition constituent such as proteins, sugers, vitamins, fat, microelements and lifeactive material such as enzyme, flavone, nucleotide and so on. It has abilities ofdiuresis, weight lose, run cardiopulmonary, dispel rheumatism and so on. In this paper,we used pine pollen as the raw materials, the first was to assay the optimal reactionconditions of lipase in the pine pollen; the second was to gain the extractions byisolation and purification the pine pollen with water and ethamol; the third was to testeffects of the different pine pollen components on FAS and CPT-1 activity. Finally, wegeted the conclusion as followed:In order to study the optimal conditions of lipase in pine pollen, we investigatedsome influence factors, including temperature (℃), pH, substrate and metal ions et al.The lipase activity was obtained by assaying the acid value after ended the reactionwith 95% ethanol and the optimal conditions were determined. The result showed thatthe optimum temperature and pH of lipase were 35℃and 7.4 respectively, thesubstrate was glyceryl tributyrate, and Ca2+、Fe3+、Mg2+、Fe2+enhanced the activity oflipase.Using 0.1mg/mL orlistat as a positive control, the effects of pine pollen aqueousextract at concentration 0,10,30,70,150,250 mg / mL on the lipase were tested. Resultsshowed that the extract had little effects on the pancreatic lipase activity comparedwith orlistat except at 70mg/mL had a slight inhibitory effect; 0,1,5,10,20,40 mg / mLof pine pollen ethanol extracts solution dissolved in DMSO showed inhibition ofpancreatic lipase activity compared with 0.1mg/mL of orlistat. According to thesequence of pollen extract solution added the lipase was first combined with inhibitors,thus preventing it combining with substrate. Analyzing with double reciprocalLineweaver-Burk diagram and the characteristics of the Dixon, results showed thepollen ethanol extract on pancreatic lipase inhibition was noncompetitive inhibition. The inhibition constant of pine pollen ethanol extract Ki was 0.02, indicating that theinhibitor with enzyme conjugates was very stable.Pine pollen ethanol extract may get two components (precipitate and supernatant)by centrifugation, the supernatant showed the inhibition of the lipase activity; thensupernatant was added to petroleum ether and divided into two components directly:orange-yellow transparent liquid A1and yellow-brown solid A2. Two substancesboth showed inhibition of pancreatic lipase activity by enzyme activity determination,because A2was darker, affected the titration results, the following A1was isolated andpurified; selection medium-pressure column chromatography as separation method,the mobile phase was ethyl ether and N-hexane. Finally we geted two components: C1and C2. Components C1and C2both showed inhibition of pancreatic lipase activity byenzyme activity determination, but the inhibition of C1was better than C2; resultsshowed that both C1and C2were complex mixture by high performance liquidchromatography (HPLC) analysis.Fatty acid synthase was extracted from rat liver, measured the enzyme activitywhich was 2.1U/mg. Selected the mass concentration of 100μg/mL of pine pollenwater extract and ethanol extract effect on fatty acid synthase, compared with thewater and positive control C75, pine pollen aqueous extract showed inhibition of FAS;while ethanol extract had a significant promotion on FAS activity.We got the serum after centrifugation of the blood from rat heart and measuredCPT-1 enzyme activity which was 72.06IU/mL. Selected the pine pollen waterextraction and ethanol extraction whose mass concentration was 100mg/mLrespectively and acted on CPT-1. We gained that both of them can inhibit CPT-1, withthe comparison of the control and positive control.