Study On The Therapeutic Effect And Chemical Basis Of Phospholipic Complexes Of Total Flavonoids From Persimmon Leaves For The Treatment Of Atherosclerosis

Persimmon leaves are the fresh or dry leaves of persimmon(Diospyros kaki L.)from the Ebenaceae family.Persimmon leaves were first recorded in Diannan Bencao written by Lan Mao in Ming Dynasty.Studies have shown that the total flavonoids of persimmon leaves(PLF)have the anti-atherosclerosis effect.In this study,the PLF was selected and investigated based on natural product chemistry,pharmaceutics,pharmaceutical analysis,pharmacology and molecular biology to screen its effect on atherosclerosis and its potential mechanism for the treatment of atherosclerosis.To clarify the constituents of the PLF,a reliable and rapid ultra-performance UPLC-Q-TOF/MS method was established.Moreover,phospholipid complexes of the total flavonoids from persimmon leaves(PLF-PC)were prepared to enhance the oral bioavailability of PLF,as well as the effects of PLF and PLF-PC on atherosclerosis in rats at the same dose was compared.Furthermore,a model of oxidized low-density lipoprotein(ox-LDL)-induced injury on human umbilical vein endothelial cells(HUVECs)was established to investigate the anti-atherosclerotic effect of PLF-PC versus PLF.Our results play an important roal in the development of PLF for anti-atherosclerosis.1.Identification of the components in the total flavonoids of persimmon leaves and compounds from plasma after oral administration of the total flavonoids of persimmon leaves.In this part,in order to clarify the constituents of PLF,a reliable and rapid ultra-performance UPLC-Q-TOF/MS method was established.Both positive and negative ion modes were employed to comprehensively analyze the main componds of PLF.Based on their exact masses,fragment information and references,31 compounds were identified from PLF including 20 flavonoids,4 three-stick compounds,6 organic acids and 1 aromatic alcohol compound.These flavonoids were mainly classified into two types of compound:quercetin and its glycosides,kaempferol and its glycosides.The flavonoid glycoside compounds detected in this study often lost m/z 146 and 162 glycosyl radicals,the flavonoid aglycones were mainly lysed by RDA way.Ten compounds were identified from rat plasma after oral administration of PLF including 5 prototypes and 5 metabolites.All the flavonoids were also identified based on their exact masses,fragment information and literatures in the same way.Among them,the major proportion is the combination of sulfuric acid conjugates of quercetin and kaempferol glucuronic acid.Among them only 4 of the prototype flavonoids were detected.The results suggest that the PLF are not easily absorbed into the blood,which will hinder its many pharmacological effects and restrict its application in the field of biomedicine.Therefore,it is of great significance to improve the absorption of PLF.This part provides useful chemical information that could be employed for further study of the pharmacodynamic material basis of PLF anti-atherosclerotic effects.2.Preparation of phospholipid complexes of total flavonoids from persimmon leaves to improve the oral bioavailability of total flavonoids from persimmon leavesAs a traditional Chinese medicine.PLF has been traditionally used in China for treating the coronary heart disease and cerebral arterial sclerosis.However,the oral bioavailabilities of flavonoids are relatively low due to their low liposolubility and poor solubility.Poor bioavailability means the poor therapeutic results.In order to improve bioavailability of PLF.PLF-PC was prepared by solvent-evaporation method.Fourier transform infrared spectroscopy(FT-IR),powder X-ray diffraction(PXRD)and Scanning electron microscopy(SEM)were employed to confirm the formation of PLF-PC.A HPLC-MS method was developed and validated for the determination of quercetin and kaempferol in Sprague-Dawley(SD)rat plasma to investigate the oral bioavailability of PLF-PC versus PLF.The chromatographic separation was achieved on a CAPCELL PAK C18 column(150 mm × 4.6 mm,5 μm).Acetonitrile(containing 0.1%fomic acid,solvent A)-water(containing 0.1%formic acid,solvent B)system was used as the mobile phase using gradient elution.Baicalin was used as the internal standard(IS).There was no significant endogenous peak that could interfere with the analytes and IS.The established HPLC-MS method was validated and applied to study the pharmacokinetics of quercetin and kaempferol after a single oral administration of PLF and PLF-PC using SD rats as the animal model.As a result,the PLF-PC exhibited increased AUC0-36h when compared with the PLF(20.2 ± 2.09 vs.8.33±0.30 mg/L*h for quercetin and 28.10±4.21 vs.8.34±0.32 mg/L*h for kaempferol,showing the improved bioavailability.Compared to the PLF,the relative bioavailability of quercetin and kaempferol in the phospholipid complex group was 242%and 337%,respectively,and both the quercetin and kaempferol exhibited a certain sustained-release characteristics.Our results demonstrated that the PLF-PC could be absorbed more and maintained the effective concentration for a long period of time in blood.3.Effects of phospholipid complexes of total flavonoids from persimmon leaves on experimental atherosclerosis in ratsTo compare the effects of PLF and PLF-PC on atherosclerosis in rats at the same dose.Atherosclerosis(AS)rats was induced by excessive administration of vitamin D3 and cholesterol.After orally administered for 4 weeks,the effect of PLF and PLF-PC on AS was assessed.The levels of LDL-C,TC,HDL-C,TG,ApoB and ApoAl in serum as well as the morphological changes of aorta were evaluated.Moreover,in order to determination of the concentrations of Val,Ile,Lys,Ser,Met and Phe in rat plasma after orally administration of PLF and PLF-PC,we developed and validated a HPLC-MS method in our study.Clarithromycin was used as IS,plasma samples were extracted with methanol(0.01 formic acid).The mobile phase was consisted of methanol and 0.01%formic acid in water.The concentrations of Ser,Lys,Met,Val Ile,and Phe in rat plasma were detected after orally administered PLF and PLF-PC for 4 weeks.The results showed that both PLF and PLF-PC could decrease the serum level of TC,TG,LDL-C,ApoB and increase the level of HDL-C and ApoA1,while the PLF-PC had better therapeutic efficacy in terms of atherosclerosis than PLF at the same dose.Furthermore,both PLF and PLF-PC coulde change the level of Ala,Arg,Lys,Met,Ser,Thr,Tyr and Val in rat plasma in diffident degree.It was observed that the PLF-PC treatment group was more significant compared to PLF treatment group.All of these indicated that PLF-PC exerts better therapeutic potential in the treatment of atherosclerotic disease.4.Cell-Based evaluation the anti-atherosclerotic effect of phospholipid complexes of total flavonoids from persimmon leavesAS is a chronic inflammatory disease of the arterial wall and endothelial cells play a critical role in maintaining cardiovascular homeostasis.In this part,we performed a model of oxidized low-density lipoprotein(ox-LDL)-induced injury on human umbilical vein endothelial cells(HUVECs)to investigate the anti-atherosclerotic effect of PLF-PC versus PLF.We investigated the levels of intracellular adhesion molecule-1(ICAM-1),reactive oxygen species(ROS),superoxide dismutase(SOD),tumor necrosis factor-α(TNF-α),nuclear factor-κB(NF-κB)nitric oxide(NO)and endothelial nitric oxide synthase(eNOS)via assay kits.Our results showed that the cell viability was high in the PLF-PC treatment group than PLF treatment group at the same dose.It was observed that both PLF and PLF-PC may protective role in Ox-LDL-induced endothelial injury by suppressing oxidative stress,while the effect of PLF-PC was more prominent at the same dose.Moreover,both PLF and PLF-PC could increase the levels of SOD,NO and eNOS mean while decrease the levels of TNF-α,NF-κB and ICAM-1,also,the effect of PLF-PC was more remarkable than PLF at the same dose.