Material Basic Of Caesalpinia Sappan L. On The Inhibiting Activity Of Protein Non- Enzymatic Glycosylation

Diabetes Mellitus?DM?,characterized by high blood glucose,is an endocrine metabolic disease that seriously endangers human health.Studies have shown that dicarbonyl compounds were significantly higher in the blood of diabetics.Dicarbonyl compounds are highly reactive toxic compounds,can accelerate protein non-enzymatic glycosylation reaction in vivo.Advanced glycation endproducts?AGEs?and its receptor RAGE play an important role in diabetics,especially in the pathogenesis of diabetes vascular disease.Therefore,it is an important way to control the development of diabetic complications by effectively inhibiting the generation of AGEs of protein non-enzymatic glycosylation,and finding the highly efficient and low-toxic AGEs generation inhibitors.To explore the relationship between homoisoflavonoids and protein non-enzymatic glycosylation,Caesalpinia sappan L.,the family of Leguminous is rich in homoisoflavonoids compounds was choosen for further research.In this paper,the effective constituents of the AGEs inhibitors were studied by using the model of the protein non-enzymatic glycosylation reaction system in vitro.The activity results show that the wood 70%ethanol extract and the parts of macroporous adsorption resin separation has obvious inhibitory effect on protein non-enzymatic glycosylation,especially the 30%ethanol fraction.The compounds were isolated and purified from 30%ethanol fraction by chromatographic column on ODS and preparative HPLC.Their structures were elucidated on the basis of chemical and spectroscopic methods,including MS,1D and 2D NMR(¹H-NMR,¹³C-NMR,HSQC,HMBC,¹H-¹H COSY and NOESY)spectral techniques.Six homoisoflavonoids were isolated and identified as the brazilian?1?,protosappanin B?2?,protosappanin A?3?,sappanone B?4?,protosappanin E-1?5?,protosappanin E-2?6?.In this acticle,we found that brazilian and protosappanin B have significant capture effect on dicarbonyl compounds.With the increase of drug concentration,the capture rate increased significantly.Depending on the result,we established a rapid and effient method for seperation of those two active compounds by using the macroporous adsorption resin column chromatography combined with semi-preparative high performance liquid chromatography.Based on the purity and recovery of the brazilian and protosappanin B,the separation and preparation conditions of 10%effective separation site were optimized.The optimal condition:column,Megres C₁₈?250 mm×20 mm,i.d.,10?m?;mobile phase:CH₃OH?H₂O=25?75?v?v?;feed volume 300?L;flow rate,6mL/min;detection wavelength,254 nm and 280 nm;feed concentration,100 mg/mL.Under this condition,brazilian with purity of 96.7%and the recovery of 95.1%could be obtained,the purity and recovery of protosappanin B were 99.9%and 83.6%.In this paper,the influence of different chromatographic column,different ratio and different pH of mobile phase to the peak were discussed.The best high performance liquid phase analysis condition was found:column,Megres C₁₈?250 mm x 4.6 mm,i.d.,5?m?;mobile phase,CH₃OH?H₂O=50?50?v?v?;flow rate,1 mL/min;The detection wavelength is 254 nm.According to the best analysis conditions,the preparation of semi-preparative high performance liquid phase separation was conducted,and a compound was isolated from 95%separation,and it was identified as brazilein?7?.This topic systematically studied the chemical composition of Caesalpinia sappam L.,for the first time found its extract has the effect of inhibiting AGEs formation and capturing dicarbonyl compounds and based on the reaction model in vitro,and reveal its material base.For the developed with independent intellectual property rights of the high efficiency and low toxicity AGEs inhibiters lay the basis for drug,which has important academic value and application prospect.