Studies On Chemical Constituents And Biological Activities Of Acorn Kernel And Leaves

Acorn is an edible,bitter,mild,nontoxic fruit of Quercus genus in Fagaceae family.It is widespread in the world and also distributing widely in China.It contains abundant phenolic components and has many pharmacological effects such as antioxidation,antibacterial,hypoglycemia,hypolipidemia and so on.Acorn leaves are the leaves of Quercus(Fagaceae family),which is widely distributed in nature.It has the characteristics of small toxicity,wide range of application,high activity,low price,safe and so on.It is a promising wild resource.Based on the review on the chemical constituents and pharmacological researches of acorn,the chemical constituents were studied and their biological activities were also tested.By means of many chromatographic methods,61 compounds were isolated from acorn and acorn leaves ethanol extract,and their structures were elucidated by spectroscopic and chemical methods as:3β-O-galloyl-oleanolic acid(1),23-acetoxy-3β-O-galloyl-oleanolic acid(2),3β-acetoxy-23-O-galloyl-oleanolic acid(3),3β-O-galloyl-ursolic acid(4),oleanolic acid(5),ursolic acid(6),2α,3β,19α,23-tetrahydroxyolean-12-en-28-oic acid(7),2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid(8),arjunglucoside Ⅰ(9),nigaichigoside F1(10),3β-O-acetyloleanolic acid(11),3β-O-acetylursolic acid(12),3β,23-O-acetylolean-12-en-28-oic acid(13),arjunic acid(14),arjunolic acid(15),arjunglucoside Ⅱ(16),bayogenin(17),3β,23-dihydroxyolean-12-en-28-oic acid(18),3-O-β-D-glucosyl-polygalacic acid(19),rotundic acid(20),methy barbinervate(21),germanicol acetate(22),3-oxo-oleanolic acid(23),lupeol(24),lupinic acid(25),stigmast-4-ene-3,6-dione(26),5α-stigmast-9(11)-en-3β-ol(27),22,23-dihydrospinasterol(28),22,23-dihydrospinasterol-3-O-β-D-glucoside(29),α-spinaterol(30),3-O-β-D-glucopyranosylspinasterol(31),pinoresinol(32),eudesmin(33),gallic acid(34),chlorogenic acid(35),2",3"-diol acetonide-3-O-α-L-rhamnopyranosidekaempferol(36),quercetin-3-O-(2"-O-galloyl)-β-galactopyranoside(37),isoquercitrin(38),quercetin-3-O-α-L-rhamnopyranoside(39),kaempferol-3-O-α-L-rhamnopyranoside(40),myricetin-3-O-α-L-rhamnopyranoside(41),quercetin-3-O-β-D-galactoside(42),kaempferol-3-O-rutinoside(43),quercetin-3-O-rutinoside(44),naringin(45),5-hydroxyl-7,4’-dimethoxy flavone(46),dihydromyricetin(47),4’,5,7-trihydroxyflavanone(48),4’-methoxy-5’,5,7-trihydroxyflavanone(49),catechin(50),myricetin(51),quercetin(52),kaempferol(53),ellagic acid(54),caffeic acid(55),ferulic acid(56),protocatechuic acid(57),3,4-dimethoxy benzoic acid(58),syringic acid(59),3,4,5-trimethoxybenzoic acid(60),gallic acid(61).Among them,compounds 1-3 and 36 were new compounds.1.The antioxidant activity test was carried out on the ethanol extract of acorn and the compounds isolated from it.Acorn ethanol extract have good antioxidant activity,triterpenes with galloyl group showed excellent antioxidant activity.2.In the course of our searching for the compounds with anti-diabetes activity,the inhibiting activities on α-glucosidase and PTP1B of compounds were assayed.Most of the triterpenoids and flavanoids show good inhibition on these two enzymes,summarizing their structure-activity relationships,the triterpenoids with galloyl group show the best inhibition,the flavonoids showed higher inhibitory than flavonoid glycosides.However,it was found that flavonoid glycoside(compound 37),which had galloyl group,could also inhibit a-glucosidase and PTP1B.3.The protective effects of acorn leaves ethanol extracts and their compounds on MIN6 cells against oxidative stress-induced damageand were studied.Compound 37 was found to significantly increase pancreatic beta-cell survival.When the concentration of compound 37 was 100μM,the survival rate of MIN6 cells reached 81.52%,and its EC50 value was 73.09±0.56 μM.The structure-activity relationship showed that compound 37 was a flavonoid compound with galloyl group.4.The anti-proliferation activity of the acorn ethanol extract and their compounds on t-HSC/Cl-6 cells were studied.All the triterpenoids showed stronger anti-proliferation effect on t-HSC/Cl-6 cells than silymarin.In particular,compound 1、2、3、4、5 showed significant anti-proliferation effects on t-HSC/Cl-6 cells,and the IC50 for them were 3.12,9.62,16.13,6.98,11.82μmol/L,respectively.It was found that the triterpenoid derivatives with galloyl group could significant inhibit the proliferation of t-HSC/Cl-6 cells.In the course of studying the chemical constituents of acorn kernel and acorn leaves,the natural products with galloyl group were found and showed good biological activities in many aspects.Therefore,inspired by these researches,we design and synthesize a series of natural galloyl triterpenes.Five kinds of natural triterpenes that in acorn,oleanolic acid,ursolic acid,hederagenin,betulinol,and betulinic acid were selected as substrates for esterification with gallic acid.Their anti-oxidation and anti-fibrosis effects were evaluated.1.Firstly,the hydroxyl groups of gallic acid were protected by acetic anhydride.The intermediate was synthesized by classical esterification reaction with triterpene under DCC/DMAP condition,and the intermediate was directly deacetylation by hydrazine hydrate to obtain the final product.The final product was separated by silica gel column chromatography and semi-preparation HPLC to obtain the target product 1-9.And their structures were elucidated by NMR and HR-ESI-MS as:3β-O-galloyl-20(29)-lupane-28-oic acid(1),3β-hydroxy-28-O-galloyl-20(29)-lupane(2),3β-acetyl-28-O-galloyl-20(29)-lupane(3),3β-hydroxy-23-O-galloyloleanolic acid(4),23-hydroxy-3β-O-galloyl-oleanolic acid(5),3β-acetoxy-23-O-galloyl-oleanolic acid(6),23-acetoxy-3β-O-galloyl-oleanolic acid(7),3β-O-galloyl-oleanolic acid(8),3β-O-galloyl-ursolic acid(9),Among them,compounds 1-5 were new compounds,the structure of 6-9 are consistent with the structure obtained from acorn kernel.2.We tested the antioxidant activity of all the synthesized galloyl triterpenes and substrates.It was found that the antioxidant activity was significantly increased when galloyl group bind to triterpenoids,and the antioxidant activity of some products was even better than Trolox.3.The activation of hepatic stellate cells(HSC)is strictly related to liver fibrosis,hence the acorn extract and all compounds were assessed via their anti-proliferative effects against t-HSC/Cl-6 cells.After the synthesis,the anti-fibrosis activity of the esterification products of triterpenes and GA resulted better,compared to the substrates.We observed that the synthesized products had similar trend between anti-fibrosis and antioxidant activities,which increased after esterification with GA.It was preliminarily assumed that antioxidant activity might be related with anti-fibrosis,and good antioxidant activity might promote anti-fibrosis effects.The synthetic method used to obtaind these galloyl triterpene derivatives is mild and controllable,and can be used to prepare large quantities.Particularly,they as naturally derived may had a better safety profile and are invaluable for further development of antifibrosis drug and antioxidant.