Studies On The Anti-Alzheimer's Disease Constituents From The Leaves Of Diospyros Kaki Thunb.

Persimmon(Diospyros kaki Thunb.),a member of the family Ebenaceae,is widely distributed in China including Guangxi,Henan,Anhui,Shanxi,Hebei and Zhejiang Provinces,whose leaves(LP)are a traditional plant medicine used for the treatment of ischemic stroke,angina,and internal emorrhage.They are also used as a plant source for manufacturing a variety of functional health food and cosmetics,such as LP tea,to promote maternal health and treat hypertension.In addition,it was recorded in part I of the 2015 edition of the Chinese Pharmacopoeia.Many studies have demonstrated that extracts of LP have beneficial effects involving their antioxidant,neuroprotective,cardio-protective,reducing blood lipid,anti-atherogenic,anti-aging and immune-enhancement activities,as well as their antidiabetic activity.To date,more than 80 compounds have been isolated from LP,including flavonoids,terpenoids,and many other valuable constituents,such as polysaccharides,and polyphenols.We review the advances of research about chemical constituents,pharmacological activities,effective fractions and quality control of LP.Herein,a detailed phytochemical investigation of effective fractions were reported.The dried and milled LP were sequentially extracted with 95%,50%(v/v)EtOH by percolation.The extract solutions were collected and pooled,and concentrated to yield the extracts.The residue was further suspended in H2O and successively extracted with petroleum ether,EtOAc to afford petroleum ether and EtOAc fractions.A part of the EtOAc extract was subjected to macroreticular resin,silica gel,polyamide,Sephadex LH-20,opening ODS column chromatography,and semi-preparative HPLC.46 compounds were isolated from the AcOEt extract,and their structures were elucidated on the basis of spectroscopic data and physico-chemical methods.These compounds included four secoiridoids,eight lignans,nineteen triterpenes,seven flavonoids,and eight-one aromatic alcohols?aldehyde?acids.Nine new compounds were named as persimmonoid A?B(1-2),persimmonin C?D?E?F?G?H?I(13-18;25).The other 37 known compounds were identified as ligustroside(3),oleuropein(4),(+)-medioresinol(5),(+)-syringaresinol(6),(+)-pinoresinol(7),(+)-medioresinol monoglucoside(8),(+)-syringaresinol-?-D-glucoside(9),(+)-pinoresinol-?-D-glucoside(10),(+)-isolariciresinol(11),and(-)-(7'S,8S,8'R)-4,4'-dihydroxy-3,3',5,5'-tetramethoxy-7',9-epoxy lignan-9'-ol-7-one(12),kakisaponin A(19),kakisaponin B(20),2a,3a,19a,23-tetrahydroxyurs-12-en-28-oic acid-O-?-D-glucopyranosyl ester(21),ryobunin C(22),28-O-?-L-rhamnopyranosyl(1?2)-?-D-glucopyranoside tormentic acid ester(23),rotungenic acid 28-O-?-L-rhamnopyranosyl-(1?2)-?-D-glucopyranoside(24),2a,3a,19a,24-tetrahydroxyolea-12-en-28-oic acid-?-D-glucopyranosyl ester(26),2a,3a,19?,24-tetrahydroxyurs-12-en-28-oic acid-O-?-D-glucopyranosyl ester(27),niga-ichigoside F1(28),barbinervic acid(29),19a,24-dihydroxy ursolic acid(30),19a-hydroxyursolic acid(31),quercetin(32),hyperoside(33),kaempferol(34),vitexin(35),astragalin(36),trifolin(37),myricetin(38),tyrosol(39),2-methylbenzoic acid(40),2,4-dihydroxy-6-methylacetophenone(41),3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol(42),vanillic acid(43),protocatechuic acid(44),protocatechualdehyde(45),hydroxytyrosol(46).Among them,twenty-three compounds(3?12,21?24;26?28,39?42,45?46)were obtained from LP for the first time.Taking the content of total flavonoid and the ratio of dry extract yielding as two main reference indexes,orthogonal design was used to determine the optimal alcohol extraction process for persimmon leaves(LP).And we also researched petroleum ether and ethyl acetate extracting conditions of the obtained extractum.In addition,we also investigated the extraction process of LP total triterpene.The optimal process of the alcohol extraction was as follows:ten times amount of 70%alcohol,decocting 2 times,each time 2 hours.The obtained residue was redissolved in water,decolorized and defatted with petroleum ether three times,then extracted three times with ethyl acetate followed by a further evaporation.The ethyl acetate fractions were dried to yield extract(EAPL).The preparation process of LP total flavonoid and total triterpene was reasonable and practical.Asensitive and reliable LC-Q/TOF-MS method was applied to identify the major compounds present in EAPL.32 constituents were 14 flavonoids,16 triterpenoids and 2 others.In addition,HPLC-QQQ-MS/MS method has been developed to allow for the quantification of 7 flavonoids,in EAPL,which revealed that the contents of 7 compositions were significantly different.In order to illustrate the structural features,the contents,the antioxidant and neuroprotective activities of twelve bioactive constituents(4 secoiridoids and 8 lignans),we developed HPLC-QQQ-MS/MS method to simultaneously quantify twelve identified compounds from persimmon leaves(LP)and fruits(FP).As a result,the contents of the seven components of LP and FP were similar,but they were more abundant in the LP.Antioxidant mechanism of this twelve constituents from LP was elucidated by three different methods,including DPPH,ABTS radical cation scavenging activity,and FRAP assay.Neuroprotective evaluation of this eleven isolated compounds used hydrogen peroxide(H2O2)-induced neuronal cell damage in human neuroblastoma SH-SY5Y cells.The results showed that there was an obvious relationship between the structures and their antioxidant activities,and some of these compounds showed statistically significant neuroprotective activities.This study is to examine the effect of EAPL on beta-amyloid(A?25-35)-induced PC 12 cell apoptosis and the protein expression of Bax,Caspase-3 and Bcl-2,exploring the mechanism of the prevention and treatment of Alzheimer disease.The results indicated that EAPL improved the viability of PC 12 cells when treated with A?25-35,and decreased A?25-35 induced the apoptosis in PC 12 cells.EAPL decresed the expression levels of Bax,Caspase-3 in PC 12 cells induced by A?25-35 but increased the expression of Bcl-2 protein.EAPL exhibited significant neuroprotective effects against A?25-35-induced injury of PC 12 cells.The mechanism of the development of AD may be connected with its free radical scavenging activity.In this study,we establish A?1-42 induced rat dementia model to observe the cognitive function improvement of EAPL and discuss the possible mechanism.The results indicated that EAPL has a potent protective effect on AD induced by A?1-42 in rats?The protective effect of EAPL on AD has relationship with its antioxidant and inhibition of JNK/Caspase signaling pathways.