Study On The Active Constituents Of Valeriana Officinalis Var

Highly pathogenic avian influenza（HPAI）poses a pressing challenge to humans.Screening anti HPAI compoundscharacterized with high safety and efficacyfromtraditional Chinese medicinesas well asnature products is a highway lead to thedevelopment of novel anti HPAI drugs. In thisstudy, MDCK cellinfected with H5N1virus (A/Tiger/Harbin/01/2002) was selected as screening model, and cytopathiceffect (CPE) method was utilized. The extract of Valeriana officinalis Linn.var.latiofolia miq. dmonstratedremarkable anti HPAI property, so the comprehensiveinvestigations of chemical constituents also anti HPAI activity of this species werecarried out.Various chromatography of silica gel, Sephadex LH20and PHPLC were appliedto separated and purified those65compounds,60of them were successfully identifiedon the basis of physiochemical constants and MS, UV, IR,1D (1H NMR13、C NMR)and2D NMR (1H1H COSY、HSQC、HMBC、NOESY) spectrum. The structures of60compounds were as follows,3monoterpenes: Densispicnins C (9),p menth8ene1,2diol (36) and9hydroxyborneol (45),21sesquiterpenes:Volvalerenal F (1)、Volvalerenal G (2)、Volvalerenic Acid F (3)、Volvalerenal H (4)、Volvalerenic Acid G (5)、Volvalerenal I (6)、Volvalerenal J (7)、Volvalerenal K (8)、Madolin A (12)、Volvalerenl A (14)、Volvalerenl C (30)、Volvalerenl B (42)、MadolinB (46)、Volvalerenal D (50)、Volvalerenic A (54)、Aromadendrane4α,10β diol (31)、15hydroxyspathulenol (32)、1hydroxy1,11,11decahydrocyclopropane azulene10one (33)、4α,10α trimethyl epoxyaromadendrane (49)、Allo romadendrane4α,10β diol (51) and Volvalerenic acid D (52),4steroids: β sitosterol (11),5α stigmanstan3,7dione (17),3β Hydroxy5α,8α epidioxyergosta6,22diene (41)and stigmasterol (55),2triterpenoids: ursolic acid (34) and oleanolic acid (56),6flavonoids:3,5dihydroxy7,4’ dimethoxyflavone (16), kaempferol (39), acacetin (47),1,7dihydroxy3,8dimethoxyxanthone (57), quercetin (58) and quercetin (59),4anthraquinones:1,3dihydroxy9,10anthraquinone (19),1,2dihydroxy9,10anthraquinone (20), citreorosein (37), and emodin (48),5lignans: Neojusticin A (13)、Justicidin A (18)、Pinoresinol (26)、Pinorespiol (40) and8hydroxypinoresinol (43). Compounds19were new compounds. Compounds13,1620,32,36,37,41,45,47,51and57were obtained from the genus Valeriana for the first time. Compounds21,22,29,34,38,39,55,56and5860were isolated from Valeriana officinalis Linn. var.latiofolia miq. for the first time.All those nine novel compounds identified in the study have greatly contributedto the diversity of natural products skeletons of the genus Valeriana and Valerianaofficinalis Linn. var. latiofolia miq. It is interesting that they all classified as terpenes,among them compounds15were bicyclogermacranes,68were aromadendranes andcompound9was iridoids. During the separation process we found silica gelchromatography column eluted with chloroform–methanol solution produceddesirable separation outcome of low polarity compounds like sesquiterpenes.There was a summary of spectrographic characters of bicyclogermacrane andaromadendrane, since both shared a same three membered ring in their molecularskeletons, presenting a pair of characteristic protons (H6, H7). This pair of protonsgenerally shown up at δH00.6and δH0.91.9for aromadendraneand aromadendrane,respectively. Commonly, proton H6presented dd signs and H7dddsigns, however,those signs were often complicated by others. Bicyclogermacrane molecular positionsC1and C5often presented1to2allyl protons signs.In13C NMR spectra, bicyclogermacrane compounds generally showed2or4signs of carbons with double bond, and2signs of protons from cyclopropylmoiety.Both chemical shifts of C1and C10will shift up to6070ppm if an epoxythree membered ring was formed between them. Aromadendrane compounds alsoshowed2signs of protons from cyclopropylmoiety, while rarely presented signs ofcarbons with double bond. If positions C1or C5not substituted, their chemicalshifts were about55and48, respectively.Through CPE activity assay, many compounds displayed anti H5N1activities atvarying levels. Compound X9.4g/mL possessed anti influenza virus potent inMDCK cell infected with H5N1subtype, without obvious side effects on cell viability,metabolism and proliferation. This compound is a promising candidate for thedevelopment of novel anti HPAI drugs. Additionally, compound39also shownsignificant anti H5N1virus property in the tested model at37.5g/mL.