白花敗醬草化學(xué)成分研究

白花敗醬草化學(xué)成分研究

白色斑點(diǎn)是白色斑點(diǎn)科植物的白醬汁。全草廣泛分布于我國南方省份，藥物來源豐富。敗醬始載于《神農(nóng)本草經(jīng)》,具有清熱解毒,利濕排膿,活血化瘀,鎮(zhèn)心安神的功效。常用于治療闌尾炎,肺膿癰,結(jié)核瘰疬,癰腫瘡毒,腸炎,痢疾,肝炎,扁桃體炎,眼結(jié)膜炎,產(chǎn)后瘀血腹痛,咯血,衄血,神經(jīng)衰弱,心臟神經(jīng)官能癥等。彭金詠等對(duì)白花敗醬草脂溶性部位化學(xué)成分進(jìn)行了研究。為了充分利用和開發(fā)白花敗醬草的豐富資源、闡明其藥效物質(zhì)基礎(chǔ)尤其是抗腫瘤活性成分,筆者對(duì)其成分進(jìn)行了研究,從其95%乙醇提取物的石油醚和乙酸乙酯萃取物中分離得到了五個(gè)化合物,分別鑒定為:熊果酸(Ⅰ)、β-谷甾醇(Ⅱ)、山柰酚-3-O-β-吡喃半乳糖苷(Ⅲ)、山柰酚-3-O-β-D-吡喃半乳糖苷(6-1)-α-L-鼠李糖苷(Ⅳ)和Patrinalloside(Ⅴ)。除化合物Ⅱ外,其它化合物均為從該植物中首次分到。1儀器和檢測(cè)方法WATERS高效液相(WATERS型RPC18柱(5.0mm×150mm);DAD紫外檢測(cè)器;日本島津LIBRORAEC-200型熔點(diǎn)測(cè)定儀;Perkin-Elmer577型IR光譜儀;Shell/JB2300型細(xì)胞培養(yǎng)箱;DG-3022A型酶聯(lián)免疫檢測(cè)儀;柱層析硅膠(200-300目)和GF254為青島海洋化工廠產(chǎn)品;其余所用試劑均為分析純。白花敗醬購自華東醫(yī)藥有限公司,經(jīng)浙江中醫(yī)藥大學(xué)張如松研究員鑒定為PatriniavillosaJuss.。2熊羧酸的分離純化取干燥的白花敗醬全草7.0kg,加10倍量的95%乙醇加熱回流提取3次,每次2h,過濾,減壓回收乙醇,依次用石油醚、乙酸乙酯、正丁醇萃取,萃取液減壓濃縮,得石油醚、乙酸乙酯和正丁醇三個(gè)萃取部位。其中石油醚部位用硅膠柱分離,以石油醚-乙酸乙酯反復(fù)梯度洗脫,根據(jù)TLC檢測(cè)流份,分離得到了熊果酸(Ⅰ)40mg。乙酸乙酯部位用氯仿-甲醇系統(tǒng)梯度洗脫,得到β-谷甾醇(Ⅱ)55mg、山柰酚-3-O-β-D-吡喃葡萄糖苷(Ⅲ)26mg、山柰酚-3-O-β-D-吡喃葡萄糖苷-(6-1)-α-L-鼠李糖苷(Ⅳ)27mg和patrinalloside(Ⅴ)15mg。3半干合成酶系統(tǒng)成像法檢測(cè)半干合酶系統(tǒng)中dmso-3.將生長(zhǎng)旺盛的乳腺癌細(xì)胞用胰蛋白酶消化后,調(diào)整細(xì)胞濃度為2×105個(gè)/ml;置于37℃、5%CO2、相對(duì)濕度100%培養(yǎng)箱中培養(yǎng)24h后,加入測(cè)試藥液,繼續(xù)培養(yǎng)3d后,每孔加5mg/mlMTT溶液(用生理鹽水配制),在培養(yǎng)箱中溫育4h,每孔加入200μlDMSO,于微量振蕩器振蕩以使甲臜完全溶解,最后用酶標(biāo)儀在570nm波長(zhǎng)處測(cè)吸光度A值,計(jì)算其半數(shù)抑制濃度IC50。4結(jié)果4.1對(duì)照品檢測(cè)rf值化合物Ⅰ:白色結(jié)晶性粉末,mp234～236℃,Liebermann-Burchard反應(yīng)呈紫紅色。MSm/z:456(M+),438(M-H2O),423(M-H2O-CH3),410,300,249,248,(100);233,219,207,203(248-COOH),189,133,119,95,81,55,43。1H-NMR(CDCl3)δ:5.29(1H,br,H-12),3.25(1H,m,H-3),1.28(6H,s,2×CH3),1.11(3H,s,CH3),1.01(6H,s,2×CH3),0.96(3H,s,CH3),0.82(6H,d,J=6.5Hz,2×CH3)。13C-NMR(CDCl3)δ:41.9(C-1),27.9(C-2),78.0(C-3),30.5(C-4),55.3(C-5),18.2(C-6),33.0(C-7),41.9(C-8),48.1(C-9),39.0(C-10),23.1(C-11),125.5(C-12),138.2(C-13),47.5(C-14),27.6(C-15),24.0(C-16),48.1(C-17),52.9(C-18),39.4(C-19),39.4(C-20),29.4(C-21),36.7(C-22),23.0(C-23),15.2(C-24),16.5(C-25),18.2(C-26),23.0(C-27),180.4(C-28),16.5(C-29),20.5(C-30)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道相符,因此鑒定化合物Ⅰ為熊果酸?；衔铫?白色片狀結(jié)晶,mp137～138℃。EI-MSm/z:414[M,100]+,396,330,329,273,255。經(jīng)與對(duì)照品對(duì)比較,混合熔點(diǎn)不下降TLC檢測(cè)Rf值一致。1H-NMR(400MHz,CDCl3)δ:0.67,0.81,0.82,0.85,0.94,1.01(各3H,s,6×Me),3.52(1H,m,3-H),5.35(1H,brs,6-H)。13C-NMR(400MHz,CDCl3)δ:11.9(C-18),12.0(C-29),18.8(C-21),19.1(C-27),19.4(C-19),19.8(C-20),21.1(C-11),23.1(C-28),24.3(C-15),26.1(C-23),28.3(C-16),29.2(C-25),31.7(C-2),31.9(C-7,C-8),36.2(C-22),36.5(C-10),37.3(C-1,C-26),39.8(C-12),42.3(C-13),42.3(C-4),45.9(C-24),50.2(C-9),56.1(C-17),56.8(C-14),71.8(C-3),121.7(C-6),140.8(C-5)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故化合物確定為β-谷甾醇(β-sitosterol)?；衔铫?黃色針狀結(jié)晶(甲醇),mp194.0～195.5℃(dec),三氯化鐵-鐵氰化鉀反應(yīng)陽性,示有酚羥基存在;鹽酸鎂粉反應(yīng)陽性,推測(cè)為黃酮類化合物;Molish反應(yīng)陽性。FAB-MSm/z:447.0([M-H]+,409,307,153(100),64,32。1H-NMR(400MHz,CDCl3):B環(huán)質(zhì)子:δ8.10(2H,d,J=8.0Hz,2′,6′-H)與δ6.90(2H,d,J=9Hz,3′,5′-H):A環(huán)質(zhì)子:δ6.42(1H,d,J=2.0Hz,8-H)與6.18(1H,d,J=2.0Hz,6-H);糖質(zhì)子:δ5.16(1H,d,J=8.0Hz,C-1″-H),δ4.00～3.32(6H,C2-C6-H)。13C-NMR(400MHz,CDCl3)δ:157.1(C-2),134.2(C-3),178.2(C-4),160.2(C-5),98.5(C-6),164.1(C-7),93.4(C-8),157.7(C-9),103.6(C-10),161.7(C-4′),131.0(C-2′,6′),114.7(C-3′,5′),101.7(C-1″),71.6(C-2″),73.6(C-3″),68.6(C-4″),75.7(C-5″),60.6(C-6″)。以上數(shù)據(jù)與文獻(xiàn)中化合物山柰酚-3-O-β-D-吡喃半乳糖苷吻合,故確定其為山柰酚-3-O-β-D-吡喃半乳糖苷(kaempferol-3-O-β-D-galactopyranoside)?；衔铫?黃色針晶(甲醇),mp246.0～247.0℃,三氯化鐵-鐵氰化鉀反應(yīng)陽性,示有酚羥基存在;鹽酸鎂粉反應(yīng)陽性,推測(cè)為黃酮類化合物;Molish反應(yīng)陽性,說明為苷類化合物。HRFAB-MS:分子式為C27H30O15,m/z:593([M-H]+,447([M-146]+,153(100),64?；衔铫襞cⅢ相比,質(zhì)譜中具有447([M-146]+,氫譜及碳譜中也均多出一個(gè)甲基五碳糖,同時(shí)與化合物Ⅲ中的半乳糖碳譜數(shù)據(jù)相比C-6向低場(chǎng)位移至66.0,C-5略微向高場(chǎng)位移至74.0,說明甲基五碳糖連接于半乳糖的C-6位,甲基五碳糖碳譜數(shù)據(jù)與文獻(xiàn)中鼠李糖一致,氫譜中甲基五碳糖端基氫化學(xué)位移為4.54(1H,s),端基碳構(gòu)型為α型,通過HMBC證明半乳糖連于山柰酚的C-3位,故而化合物Ⅳ為山柰酚-3-O-β-D-吡喃半乳糖(6-1)-α-L-鼠李糖苷,通過1H-1HCOSY、HSQC及HMBC對(duì)化合物Ⅳ的氫譜和碳譜數(shù)據(jù)進(jìn)行了歸屬。13C-NMR(400MHz,CD3OD)δ:158.0(C-2),134.3(C-3),178.1(C-4),161.6(C-5),98.6(C-6),164.7(C-7),93.5(C-8),157.1(C-9),104.1(C-10),121.3(C-1′),131.1(C-2′,6′),114.7(C-3′,5′),160.3(C-4′);半乳糖:104.1(C-1″),71.6(C-2″),73.7(C-3″),68.8(C-4″),74.0(C-5″),66.0(C-6″);鼠李糖:100.5(C-1ue087),70.7(C-2ue087),70.9(C-3ue087),72.5(C-4ue087),68.3(C-5ue087),16.6(C-6ue087)。1H-NMR(400MHz,CD3OD)δ:6.23(1H,d,J=2.0Hz,H-6),6.42(1H,d,J=2.0Hz,H-8),8.11(2H,d,J=8.0Hz,H-2′,6′),6.91(2H,d,J=8.0Hz,H-3′,5′),5.06(1H,d,J=8.0Hz,gal-H-1″),4.54(1H,s,rha-H-1ue087),4.00～3.32(10H,gal-H-2″-H-6″andrha-H-2″-H-5″),1.20(3H,d,J=6.5Hz,rha-C5ue087-CH3)。化合物Ⅴ:白色結(jié)晶(氯仿-甲醇),mp63℃,與氨基酸共同加熱形成藍(lán)紫色沉淀;與Shear試劑反應(yīng),產(chǎn)生紫色然后變成棕色。FAB-MSC21H34O11m/z:462(M+),377,298.1,299.1。1H-NMR(400MHz,CDCl3)δ:6.35(s,H-3),6.19(d,J=4Hz,H-1),4.58(d,J=8Hz,H-1′),4.33(m,H-7),4.12(J=12Hz,2H-11),3.94(t,J=3Hz,H-3′),3.85(dd,J=1.7,11.0Hz,H-6′),3.80(dd,J=7.4,11.0Hz,1H-10),3.71(dd,ABX,J=5.6,10.9Hz,H-6′),3.47(dd,J=3.0,9.4Hz,H-4′),3.22(m,H-5),2.23(-CH2-),2.22(dt,J=5.3,8.6Hz,H-9),2.03-2.09(m,2H,H-6,-CH<),2.02(m,H-8),2.00(m,H-6),0.96(d,J=6.6Hz,XH,2X)。13C-NMR(400MHz,CDCl3)δ171.1(C=O),139.9(C-3),115.6(C-4),102.2(C-1′),91.9(C-1),74