黑水缬草抗老年痴呆活性成分研究
2016-07-09万莹莹王长福王秋红肖阳王知斌匡海学
万莹莹 王长福 王秋红 肖阳 王知斌 匡海学
[摘要]该实验基于前期黑水缬草抗老年痴呆(AD)的作用,对其有效部位进行化学成分研究,采用75%乙醇加热回流的方法对黑水缬草药材进行提取,提取物经萃取和大孔吸附树脂柱色谱分离得到其抗AD的有效部位,采用硅胶、ODS柱色谱以及制备型HPLC等色谱法分离该有效部位并得到9个化合物(1~9),结合质谱及核磁等波谱技术,分别鉴定为6hydroxy7(hydroxymethyl)4methylenehexahydrocyclopenta[c]pyran1(3H)one(1),suspensolide F(2),马钱子苷(3),α莫诺苷(4),β莫诺苷(5),patrinovalerosidate(6),野花椒苷A(7),(-)angelicoidenol2OβDglucopyranoside(8)和citroside A(9),其中化合物6~9为首次从缬草属植物中分离得到,进一步对化合物1~9进行体外抗AD活性研究发现化合物2和6对PC12神经元细胞损伤具有显著的保护作用。
[關键词]黑水缬草;老年痴呆;活性成分;结构鉴定
[Abstract]In this study, the chemical constituentsfrom Valeriana amurensis ADeffective fraction were investigated based on the effect of Valeriana amurensis on Alzheimer′s disease(AD) in previous study Valeriana amurensis was extracted with 75% ethanol and the obtained extract were extracted and subjected to AB8 macroporous resin column to obtain the ADeffective fraction of Valeriana amurensis 9 compounds (19)were isolated with silica gel, ODS column chromatography and preparative HPLC The structures of these compounds were determined as 6hydroxy7(hydroxymethyl)4methylenehexahydrocyclopenta[c]pyran1(3H)one (1), suspensolide F (2), loganin(3), αmorroniside(4), βmorronisid (5), partinovalerosidate (6), zansiumloside A (7), (-)angelicoidenol2OβDglucopyranoside (8), citroside A (9). Compounds 69 were isolated from the valerian genus for the first time and further investigated the antiAD effect of compounds 19 in vitro found that compound 2 and 6 protected the PC12 cells from injury significantly
[Key words]Valeriana amurensis; Alzheimer′s disease; active constituents; structure identification
doi:10.4268/cjcmm20160914
黑水缬草Valeriana amurensis Smir ex Kom为败酱科缬草属植物,在我国东北部以及俄罗斯远东地区分布较广;其根及根状茎入药[1],味辛、甘,性温,具有安神,理气,止痛的功效,传统中医临床用于失眠,癔病,癫痫等神经系统疾病的治疗。前期的研究发现,黑水缬草主要含有挥发油、木脂素、倍半萜及环烯醚萜等类型的成分,并具有镇静安神、抗菌抗病毒以及抗肿瘤等药理活性[25]。而本课题组前期研究首次发现黑水缬草具有抗老年痴呆(AD)的作用,并确定其有效部位为黑水缬草75%乙醇提取物的大孔吸附树脂柱色谱的50%乙醇洗脱组分,该有效部位能够抑制AD小鼠模型神经元损伤[6],本实验在此基础上对其活性成分进行研究,为其药效物质基础的揭示提供了理论依据。
1材料
反相ODS柱色谱(ODSAM) 采用日本YMC公司产品;大孔吸附树脂(AB8)购于南开大学化工厂;制备型色谱柱(HypersilODSII)购买于大连依利特公司。
超导核磁共振光谱仪(Bruker400型 )(TMS为内标);HRESIMS(Xero QTOF)、HPLC(26952996型)及UPLC(ACQUITY型)均为Waters公司;GCMS(7890A5975C型)为Angilent公司。
2方法
21提取与分离将45 kg干燥的黑水缬草药材,以75%乙醇(20∶1)加热回流提取(3 h×3次)得到的提取物(8 21196 g)以水混悬,采用石油醚萃取,将剩余水层萃取物(6 14460 g)经AB8型大孔吸附树脂柱色谱,H2O,50%乙醇和95%乙醇依次洗脱,获得的50%乙醇洗脱组分(1 39190 g)即为黑水缬草抗AD有效部位;取该有效部位(800 g)经二氯甲烷甲醇(15∶1,8∶1,4∶1,2∶1)梯度洗脱,共得7个组分(Fr1~Fr7),Fr2(784 g)经正相硅胶柱色谱二氯甲烷甲醇(40∶1)反复洗脱得到化合物1(54 mg),Fr4(1482 g)经反相ODS柱色谱甲醇水(25∶75)洗脱,并经制备型反相HPLC乙腈水(15%)洗脱分离得到化合物4(23 mg),5(26 mg),8(25 mg),9(35 mg),Fr6(1844 g)经反相ODS柱色谱甲醇水(15∶85)洗脱,并经制备型反相HPLC乙腈水(9:91)洗脱分离得到化合物2(33 mg),3(28 mg),6(41 mg)和7(53 mg)。
22抗AD活性化合物筛选采用MTT法对化合物1~9的抗AD活性进行筛选[7],采用酶标仪检测各浓度化合物(5,12,25 μmol·L-1)对Aβ142所致PC12细胞损伤的保护作用,组细胞活力=A各组细胞/A空白对照组细胞×100%。
3结构鉴定
化合物1无色针状结晶,HRESIMS m/z 199098 9 [M+H]+;1HNMR(CD3OD,400 MHz)δ:429(1H,br s,H3),156(1H,m,H5), 205(1H,dd,J=132, 74 Hz,H5),462(2H,m,H6),231(1H,m,H7),291(1H,t,J=100 Hz,H8),337(1H,m,H9),377(1H,dd,J=48,108 Hz,H10), 370(1H,t,J=81 Hz,H10),496(1H,s,H11), 509(1H,s,H11);13CNMR(CD3OD,100 MHz)δ:1775(C1),737(C3),1443(C4),415(C5),726(C6),522(C7),451(C8),411(C9),621(C10),1139(C11)。将上述数据与文献[8]比较,鉴定化合物1为6hydroxy7(hydroxymethyl)4methylenehexahydrocyclopenta[c]pyran1(3H)one。
化合物2白色无定形粉末,ESIMS m/z 501 [M+Na]+;1HNMR (CD3OD,400 MHz)δ:609(1H,d,J=48 Hz,H1),632(1H,s,H3),298(1H,dd,J=84,167 Hz,H5),194(2H,dd,J=40,78 Hz,H6),431(1H,m,H7),215(1H,ddd,J=48,84,128 Hz,H8),228(1H,m,H9),373(1H,m,H10), 352(1H,m,H10),442 (1H,d,J=126 Hz,H11), 405(1H,d,J=126 Hz,H11),217(2H,m,H13),204(1H,m,H14),091(3H,d,J=68 Hz,H15),091(3H,d,J=68 Hz,H16),424(1H,d,J=80 Hz,Glc H1′),315(1H,m,Glc H2′),330(1H, m,Glc H3′),323(1H,m,Glc H4′),330(1H,m,Glc H5′),362(1H,dd,J=56 Hz,120 Hz,Glc H6′),381(1H,dd,J=20 Hz,120 Hz,GlcH6′);13CNMR(CD3OD,100 MHz)δ:920(C1),1403(C3),1167(C4),331(C5),384(C6),796(C7),841(C8),449(C9),665(C10),699(C11),1733(C12),444(C13),269(C14),228(C15),228(C16),1036(Glc C1′),753(Glc C2′),782(Glc C3′),718(Glc C4′),780(Glc C5′),629(Glc C6′)。將上述数据与文献[9]对照,鉴定化合物2为suspensolide F。
化合物3白色无定形粉末,ESIMS m/z 391 [M+H]+;1HNMR(CD3OD,400 MHz)δ:517(1H,d,J=44 Hz,H1),729(1H,s,H3),301(1H,dd,J=84,160 Hz,H5),213(1H,m,H6), 151(1H,m,H6),311(1H,m,H7),177(2H,m,H8),193(1H,m,H9),086(3H,d,J=68 Hz,H10),359(3H,s,H12),456(1H,d,J=80 Hz,Glc H1′),320~400(4H,m,Glc H4′),376(1H,dd,J=20,120 Hz,Glc H6′), 353(1H,dd,J=56,120 Hz,Glc H6′);13CNMR(CD3OD,100 MHz)δ:978(C1),1522(C3),1141(C4),322(C5),428(C6),751(C7),422(C8),466(C9),154(C10),1696(C11),518(C12),1002(Glc C1′),755(Glc C2′),782(Glc C3′),717(Glc C4′),781(Glc C5′),619(Glc C6′)。将上述数据与文献[10]对照,鉴定化合物3为马钱子苷。
化合物4白色无定形粉末,ESIMS m/z 429 [M+Na]+;1HNMR(CD3OD,400 MHz)δ:579(1H,d,J=93 Hz,H1),748(1H,s,H3),278(1H,dt,J=127 Hz,45 Hz,H5),199(1H,m,H6)、115(1H,m,H6),478(1H,d,J=32 Hz,H7),392(1H,m,H8),173(1H,m,H9),135(3H,d,J=70 Hz,H10),367(3H,s,H12),478(1H,d,J=80 Hz,Glc H1′),320~400(6H,m,Glc H4′);13CNMR(CD3OD,100 MHz)δ:972(C1),1546(C3),1110(C4),321(C5),374(C6),960(C7),742(C8),400(C9),200(C10),1688(C11),520(C12),1002(Glc C1′),751(Glc C2′),785(Glc C3′),717(Glc C4′),781(Glc C5′),629(Glc C6′)。综上,将上述数据与文献[11]对照,鉴定化合物4为α莫诺苷(αmorroniside)。
万莹莹等:黑水缬草抗老年痴呆活性成分研究化合物5白色无定形粉末,ESIMS m/z 429 [M+Na]+;1HNMR(CD3OD,400 MHz)δ:583(1H,d,J=92 Hz,H1),747(1H,s,H3),309(1H,dt,J=128 Hz,46 Hz,H5),186(1H,dt,J=45 Hz,138 Hz,H6), 147(1H,dt,J=37 Hz,134 Hz,H6),520(1H,d,J=32 Hz,H7),451(1H, m,H8),178(1H,m,H9),128(3H,d,J=70 Hz,H10),366(3H,s,H12),478(1H,d,J=80 Hz,Glc H1′),320~400(6H,m,Glc H4′);13CNMR(CD3OD,100 MHz)δ:958(C1),1546(C3),1118(C4),276(C5),347(C6),925(C7),660(C8),406(C9),199(C10),1689(C11),519(C12),1002(Glc C1′),751(Glc C2′),785(Glc C3′),717(Glc C4′),781(Glc C5′),629(Glc C6′)。将上述数据与文献[11]对照,鉴定化合物5为β莫诺苷(βmorroniside)。
化合物6白色无定形粉末,ESIMS m/z 4492 [M+H]+;1HNMR (CD3OD,400MHz)δ:409(1H,dd,J=73,111 Hz,H1),421(1H,dd,J=64,111 Hz,H1),535(1H,br s,H3),517(1H,br s,H3),322(1H,m,H5),223(1H,m,H6),180(1H,m,H6),383(1H,br d,J=45 Hz,H7),238(1H,m,H9),138(3H,s,H10),434(1H,d,J=121 Hz,H11),417(1H,d,J=121 Hz,H11),219(2H,d,J=74 Hz,H13),208(1H,m,H14),097(3H,d,J=66 Hz,H15),097(3H,d,J=66 Hz,H16),431(1H,d,J=81 Hz,Glc H1′),324(1H,m,Glc H2′),337(1H,m,Glc H3′),332(1H,m,Glc H4′),326(1H,m,Glc H5′),371(1H,dd,J=56,120 Hz,Glc H6′),390(1H,dd,J=20 Hz,120 Hz,Glc H6′);13CNMR(CD3OD,100 MHz)δ:642(C1),1154(C3),1472(C4),416(C5),391(C6),813(C7),828(C8),482(C9),232(C10),737(C11),1752(C12),446(C13),269(C14),229(C15),229(C16),1031(Glc C1′),753(Glc C2′),783(Glc C3′),718(Glc C4′),781(Glc C5′),630(Glc C6′)。将上述数据与文献[12]对照,鉴定该化合物6为patrinovalerosidate。
化合物7白色无定形粉末,HRESIMS m/z 463217 2 [M-H]-;1HNMR(CD3OD,400 MHz)δ:385(1H,br d,J=83 Hz,H2),245(1H,m,H3),157(1H,m,H3),157(1H,m,H4),197(1H,m,H5),164(1H,m,H5),197(1H,m,H6),162(1H,m,H6),111(3H,s,H7),128(3H,s,H9),119(3H,s,H10),431(1H,d,J=77 Hz,Glc H1′),313(1H,t,J=84 Hz,Glc H2′),336(1H,m,Glc H3′),326(1H,t,J=93 Hz,Glc H4′),338(1H,m,Glc H5′),397(1H,br d,J=90 Hz,Glc H6′),361(1H,t,J=62,51 Hz,Glc H6′),502(1H,d,J=18 Hz,Api H1″),389(1H,d,J=18 Hz,Api H2″),394(1H,d,J=95 Hz,Api H4″),375(1H,d,J=95 Hz,Api H4″),358(2H,s,Api H5″);13CNMR (CD3OD,100 MHz)δ:737(C1),768(C2),326(C3),354(C4),229(C5),270(C6),250(C7),753(C8),290(C9),294(C10),1019(Glc C1′),751(Glc C2′),783(Glc C3′),720(Glc C4′),771(Glc C5′),688(Glc C6′),1110(Api C1″),781(Api C2″),807(Api C3″),752(Api C4″),659(Api C5″)。將上述数据与文献[13]比较,鉴定化合物7为野花椒苷A。
化合物8白色无定形粉末,HRESIMS m/z 331175 1 [M-H]-;1HNMR(CD3OD,400 MHz)δ:385(1H,br d,J=111 Hz,H2),249(1H,dd,J=89,129 Hz,H3),170(1H,d,J=44 Hz,H3),218(1H,m,H4),404(1H,br d,J=99 Hz,H5),132(1H,br d,J=136 Hz,H6)、102(1H,br d,J=138 Hz,H6),085(3H,s,H7),108(3H,s,H9),092(3H,s,H10),422(1H,d,J=78 Hz,Glc H1′),315(1H,t,J=84 Hz,Glc H2′),333(1H,m,Glc H3′),322(1H,t,J=93 Hz,Glc H4′),328(1H,m,Glc H5′),385(1H,br d,J=111 Hz,Glc H6′),367(1H,dd,J=117 Hz,52 Hz,Glc H6′);13CNMR(CD3OD,100 MHz)δ:512(C1),832(C2),344(C3),538(C4),761(C5),399(C6),215(C7),489(C8),205(C9),135(C10),1031(Glc C1′),753(Glc C2′),784(Glc C3′),719(Glc C4′),780(Glc C5′),630(Glc C6′)。将上述数据与文献[14]比较,鉴定该化合物为(-)angelicoidenol2OβDglucopyranoside。
化合物9白色无定形粉末,HRESIMS m/z 409183 3 [M+Na]+;1HNMR(CD3OD,400 MHz)δ:134 (1H,brd,J=117 Hz,H2),193(1H,br d,J=117 Hz,H2),433(1H,m,H3), 249(1H,dd,J=18 Hz,135 Hz,H4),138(1H,dd,J=18,135 Hz,H4),590(1H,s,H8),220(3H,s,H10),148(3H,s,H11),138(3H,s,H12),116(3H,s,H13),453(1H,d,J=77 Hz,Glc H1′),315(1H,t,J=83 Hz,Glc H2′),335(1H,t,J=86 Hz,Glc H3′),326(1H,t,J=96 Hz,Glc H4′),322(1H,m,Glc H5′),382(1H,dd,J=21,117 Hz,Glc H6′), 363(1H,dd,J=52,117 Hz,Glc H6′);13CNMR(CD3OD,100 MHz)δ:372(C1),500(C2),640(C3),482(C4),789(C5),1193(C6),2131(C7),1015(C8),2008(C9),268(C10),267(C11),302(C12),326(C13),988(Glc C1′),754(Glc C2′),788(Glc C3′),719(Glc C4′),779(Glc C5′),631(Glc C6′)。将上述数据与文献[15]比较,鉴定该化合物为citroside A。
4化合物抗AD活性
体外实验研究表明,5,12,25 μmol·L-1的化合物2和6的细胞活力与模型组比较差异显著(P<005或P<001),因此,化合物2和6对Aβ142所致的PC12神经元细胞损伤具有显著地保护作用,见表1。
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