陶瓷葉化學(xué)成分的研究

陶瓷葉化學(xué)成分的研究

山茶是科植物的山茶葉。主產(chǎn)于我國(guó)江蘇、浙江、云南、四川等地區(qū)。民間用于治療吐血、衄血、腸風(fēng)下血、赤痢、痔瘡出血、婦人血崩及乳糜血尿等疾病。國(guó)外報(bào)道山茶葉中的鞣質(zhì)成分對(duì)動(dòng)物的皮膚癌和腸癌有明顯的抑制作用。為了開(kāi)發(fā)利用山茶植物資源,尋找其活性成分,本實(shí)驗(yàn)對(duì)購(gòu)自云南的山茶葉的化學(xué)成分進(jìn)行了系統(tǒng)研究,從中得到11個(gè)化合物,分別鑒定為槲皮素(quercetin,1)、(-)表兒茶素[(-)epicatechin,2]、大花新哨吶草素(特利馬素Ⅱ,tellimagrandinⅡ,3)、小木麻黃素(strictinin,4)、1,2,3,4,6-五-沒(méi)食子?；?β-D-葡萄糖(1,2,3,4,6-penta-O-galloyl-β-D-glucose,5)、英國(guó)櫟精(pedunculagin,6)、榛子素A(heterophylliinA,7)、木麻黃素(casuariin,8)、山茶鞣質(zhì)A(camelliatnninA,9)、山茶鞣質(zhì)B(camelliatanninB,10)、山茶鞣質(zhì)C(camelliatanninC,11)。以上化合物均為首次從山茶葉中分離得到。1afq旋轉(zhuǎn)薄膜蒸發(fā)儀BrukerAM600型NMR測(cè)試儀;ShimadzuUV180型紫外測(cè)定儀;JEOLGEXHX100MS儀;HL—2自動(dòng)流份收集儀恒流泵,產(chǎn)于上海市滬西機(jī)器廠(chǎng);AFQ旋轉(zhuǎn)薄膜蒸發(fā)儀,產(chǎn)于天津玻璃儀器廠(chǎng)。DiaionHP-20大孔吸附樹(shù)脂日本三菱公司;MCI-gelCHP-HW-40,日本TOSOH公司;SephadexLH-20凝膠,AmershamPharmaciaBiotech公司。山茶葉于2005年12月購(gòu)于云南省昆明市中藥材市場(chǎng),由哈爾濱商業(yè)大學(xué)藥學(xué)院中藥鑒定教研室張德連教授鑒定,該藥材為山茶科植物山茶CamelliajaponicaL.干燥葉,樣品現(xiàn)存于哈爾濱商業(yè)大學(xué)中藥標(biāo)本室。2化合物結(jié)構(gòu)鑒定山茶葉2kg,用70%丙酮破碎提取,濾過(guò),提取液于40℃真空濃縮后,用乙醚、醋酸乙酯萃取。得到乙醚提取物13g,醋酸乙酯提取物45g。醋酸乙酯提取物用DiaionHP-20柱色譜分離,以乙醇-水(20%→40%→60%→80%)梯度洗脫,得到20%乙醇提取物23.6g。取乙醇提取物20g,用ToyopearlHW-40柱色譜分離,以甲醇-水(3∶7→4∶6→6∶4→7∶3)梯度洗脫,得到4個(gè)組分(Fr.Ⅰ.1866mg;Fr.Ⅱ4218mg;Fr.Ⅲ1700mg;Fr.Ⅳ1300mg)。Fr.Ⅰ經(jīng)MCI-gelCHP-20P柱色譜反復(fù)分離得到化合物1(134mg)、2(130mg)、4(89mg);Fr.Ⅱ經(jīng)ToyopearlHW-40柱色譜反復(fù)分離得到化合物7(69mg),8(123mg)、10(110mg)、11(105mg);Fr.Ⅲ經(jīng)SephadexLH-20柱色譜反復(fù)分離得到化合物9(100mg)、3(56mg)、5(47mg);Fr.Ⅳ經(jīng)ToyopearlHW-40、SephadexLH-20反復(fù)柱色譜分離,得到化合物6(66mg)。正相HPLC采用WatersM4液相色譜儀,柱ZorbaxSIL(150mm×4.6mm,5μm),檢測(cè)波長(zhǎng)280nm,室溫,體積流量2.5mL/min,流動(dòng)相:環(huán)己烷-甲醇-四氫呋喃-甲酸(55∶33∶11∶1)-草酸450mg/L。反相HPLC采用ShimadzuLC-6A液相色譜儀,柱LichirospherRp-18(250mm×4mm,5μm),檢測(cè)波長(zhǎng)280nm,溫度40℃,流動(dòng)相:磷酸緩沖液-乙醇-醋酸乙酯(85∶10∶5),體積流量1.1mL/min。3fab-msm:表兒茶素,c-3-t,d5,3,5,9,10.化合物1:淡黃色無(wú)定形粉末(丙酮),其分子式為C15H10O7,FAB-MSm/z:325[M+Na]+;1H-NMR(Me2CO-d6+D2O,300MHz)δ:6.23(1H,d,J=2.1Hz,H-6),6.49(1H,d,J=2.1Hz,H-8),6.95(1H,d,J=8.7Hz,H-5′),7.63(1H,dd,J=2.1,8.7Hz,H-6′),7.76(1H,d,J=2.1Hz,H-2′),13.02(1H,s,5-OH),10.12(1H,brs,7-OH),9.90(3H,brs,3,3′,4′-OH);13C-NMR(Me2CO-d6+D2O,126MHz)δ:147.1(C-2),135.9(C-3),176.9(C-4),156.4(C-5),99.7(C-6),165.1(C-7),95.3(C-8),162.4(C-9),105.2(C-10),122.5(C-1′),114.6(C-2′),146.2(C-3′),148.2(C-4′),112.4(C-5′),119.3(C-6′)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為槲皮素。化合物2:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,其分子式為C15H14O6,FAB-MSm/z:313[M+Na]+;1H-NMR(Me2CO-d6+D2O,300MHz)δ:4.85(1H,s,br,H-2),4.18(1H,m,H-3),2.83(1H,dd,J=4.5,16.5Hz,H-4),2.71(1H,s,dd,J=3.0,16.5Hz,H-4),6.00(1H,s,d,J=2.5Hz,H-6),5.90(1H,s,d,J=2.5Hz,H-8),7.03(1H,s,d,J=2Hz,H-2′),6.77(1H,s,d,J=8.0Hz,H-5′),6.81(1H,dd,J=2.0,8.0Hz,H-6′)。13C-NMR(Me2CO-d6+D2O,126MHz)δ:79.2(C-2),66.7(C-3),28.7(C-4),99.5(C-4a),157.4(C-5),95.9(C-6),157.3(C-7),95.3(C-8),156.8(C-8a),131.6(C-1′),115.0(C-2′),145.2(C-3′),145.1(C-4′),115.3(C-5′),119.0(C-6′)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為(-)表兒茶素。化合物3:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性,其分子式為C41H30O26,FAB-MSm/z:961[M+Na]+;1H-NMR(Me2CO-d6+D2O,600MHz)δ:7.09,6.99,6.96(2H,s,galloyl),6.20(1H,d,J=8.0Hz,Glu-1),5.58(1H,dd,J=8.0,9.5Hz,Glu-2),5.83(1H,t,J=9.5Hz,Glu-3),5.20(1H,t,J=9.5Hz,Glu-4),4.54(1H,dd,J=6.0,9.5Hz,Glu-5),5.36(1H,dd,J=6.0,13.0Hz,Glu-6),3.87(1H,d,J=13.0Hz,Glu-6)。13C-NMR(Me2CO-d6+D2O,126MHz)δ:93.8(Glu-1),71.8(Glu-2),73.3(Glu-3),70.8(Glu-4),73.1(Glu-5),63.0(Glu-6),166.8,168.2,166.7,166.2,166.0(estercarbonyl)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為特利馬素Ⅱ。化合物4:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性,其分子式為C27H22O18,FAB-MSm/z:657[M+Na]+;1H-NMR(Me2CO-d6+D2O,600MHz)δ:7.15(2H,s,galloyl),6.70,6.55(1H,s,HHDP),5.71(1H,d,J=8.0Hz,Glu-1),3.67(1H,dd,J=8.0,9.0Hz,Glu-2),3.80(1H,dd,J=9.0,10.0Hz,Glu-3),4.88(1H,t,J=10.0Hz,Glu-4),4.08(1H,dd,J=6.5,10.0Hz,Glu-5),5.30(1H,dd,J=6.5,13.5Hz,Glu-6),3.75(1H,d,J=13.5Hz,Glu-6)。13C-NMR(Me2CO-d6+D2O,126MHz)δ:92.0(Glu-1),75.8(Glu-2),77.2(Glu-3),69.0(Glu-4),73.1(Glu-5),63.0(Glu-6),166.8,166.4,166.1(estercarbonyl)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為小木麻黃素。化合物5:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性,其分子式為C41H32O26,FAB-MSm/z:963[M+Na]+;1H-NMR(Me2CO-d6+D2O,600MHz)δ:7.15,7.10,7.05,6.99,6.96(2H,s,galloyl×5),6.29(1H,d,J=8.5Hz,Glu-1),5.67(1H,dd,J=8.5,10.0Hz,Glu-2),6.00(1H,t,J=10.0Hz,Glu-3),5.56(1H,t,J=10.0Hz,Glu-4),4.54(1H,ddd,J=2.0,5.0,10.0Hz,Glu-5),4.58(1H,dd,J=2.0,12.0Hz,Glu-6),4.31(1H,dd,J=5.6,10.0Hz,Glu-6),13C-NMR(Me2CO-d6+D2O,126MHz)δ:93.4(Glu-1),74.9(Glu-2),74.4(Glu-3),69.5(Glu-4),72.1(Glu-5),62.9(Glu-6),166.9,166.5,166.1,165.8,165.4(estercarbonyl)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為1,2,3,4,6-五-沒(méi)食子?；?β-D-葡萄糖。化合物6:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性,其分子式為C34H24O22,FAB-MSm/z:807[M+Na]+;1H-NMR(Me2CO-d6+D2O,600MHz)δ:6.63,6.62(1Hintotal),6.58,6.58(1Hintotal),6.53,6.49(1Hintotal),6.31,6.30(1Hintotal),5.40(1H,d,J=3.9Hz,Glc-1α),5.01(1H,d,J=8.1Hz,Glu-1β),5.02(1H,dd,J=3.9,10.2Hz,Glu-2α),4.81(1H,dd,J=8.1,9.0Hz,Glu-2β),5.41(1H,d,J=10.2Hz,Glu-3α),5.18(1H,dd,J=9.0,10.2Hz,Glu-3β),5.04(1H,t,J=10.2Hz,Glu-4α),5.03(1H,t,J=10.2Hz,Glu-4β),4.55(1H,m,Glu-5α),4.16(1H,m,Glu-5β),5.20(1H,dd,J=6.9,13.2Hz,Glu-6α),5.24(1H,dd,J=6.9,13.2Hz,Glu-6β),3.74(1H,d,J=13.2Hz,Glu-6α),3.81(1H,d,J=13.2Hz,Glu-6β)。13C-NMR(Me2CO-d6+D2O,126MHz)δ:91.4(Glu-1α),95.1(Glu-1β),75.4(Glu-2α),78.1(Glu-2β),75.6(Glu-3α),77.4(Glu-3β),69.6(Glu-4α),69.5(Glu-4β),67.1(Glu-5α),72.6(Glu-5β),63.4(Glu-6α),63.5(Glu-6β)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為英國(guó)櫟精?；衔?:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性。FAB-MSm/z:809[M+Na]+;C34H26O26,1H-NMR(Me2CO-d6+D2O,600MHz)δ:7.24,7.03(2H,s,galloyl×2),6.62,6.48(1H,s,HHDP),6.43(1H,d,J=4.0Hz,Glu-1),4.22(1H,dd,J=4.0,10.0Hz,Glu-2),5.56(1H,t,J=10.0Hz,Glu-3),5.06(1H,t,J=10.0Hz,Glu-4),4.57(1H,dd,J=6.5,10.0Hz,Glu-5),5.26(1H,dd,J=6.5,13.5Hz,Glu-6),3.75(1H,d,J=13.5Hz,Glu-6),13C-NMR(Me2CO-d6+D2O,126MHz)δ:92.9(Glu-1),70.3(Glu-2),74.3(Glu-3),70.6(Glu-4),70.5(Glu-5),63.3(Glu-6),165.4,167.2,167.7,168.3(estrcarbonyl)。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為榛子素A?；衔?:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性,其分子式為C34H24O22,FAB-MSm/z:807[M+Na]+;1H-NMR(Me2CO-d6+D2O,600MHz)δ:6.73,6.50,6.36(1H,s,HHDP),5.54(1H,d,J=4.80Hz,Glu-1),4.64(1H,dd,J=2.44.8Hz,Glu-2),5.46(1H,d,J=2.4Hz,Glu-3),5.07(1H,dd,J=2.4,8.1Hz,Glu-4),4.08(1H,dd,J=3.0,8.1Hz,Glu-5),4.68(1H,dd,J=3.0,12.3Hz,Glu-6),3.81(1H,d,J=12.3Hz,Glu-6),13C-NMR(Me2CO-d6+D2O,126MHz)數(shù)據(jù)見(jiàn)表1。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為木麻黃素?；衔?:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性,其分子式為C49H36O27,FAB-MSm/z:1057[M+H]+;1H-NMR(Me2CO-d6+D2O,600MHz)δ:6.26,6.47,6.65(1H,s,HHDP×2),5.03(1H,br,s,Ec-2),4.24(1H,br,s,Ec-3),2.85(1H,br,dd,J=3.5,16Hz,Ec-4),2.73(1H,br,d,J=16Hz,Ec-4),5.91(1H,s,Ec-6),7.18(1H,br,s,Ec-2′),6.93(1H,br,d,J=8Hz,Ec-5′),6.83(1H,br,d,J=8Hz,Ec-6′),4.71(1H,br,s,Glu-1),4.93(1H,br,s,Glu-2),4.99(1H,br,d,J=3Hz,Glu-3),5.07(1H,br,dd,J=3.0,7.0Hz,Glu-4),3.81(1H,d,J=7.0Hz,Glu-5),4.48(1H,br,d,J=12.0Hz,Glu-6),3.38(1H,br,d,J=12Hz,Glu-6)。13C-NMR(Me2CO-d6+D2O,126MHz)數(shù)據(jù)見(jiàn)表1。以上數(shù)據(jù)與文獻(xiàn)報(bào)道一致,故確定其為山茶鞣質(zhì)A?；衔?0:淺棕色無(wú)定形粉末(丙酮),與三氯化鐵試劑反應(yīng)顯藍(lán)色,明膠試劑反應(yīng)陽(yáng)性,其分子式為C49H36O27,FAB-MSm/z:1057[M+H]+;1H-NMR(Me2CO-d6+D2O,600MHz)δ:6.47,6.53,6.80(1H,s,HHDP×2),4.78(1H,br,s,Ec-2),4.15(1H,m,Ec-3),2.82(1H,br,dd,J=3.5,16.0Hz,Ec-4),2.67(1H,br,d,J=16.0Hz,Ec-4),5.98(1H,s,Ec-8),7.01(1H,d,J=1.5Hz,Ec-2′),6.74(1H,d,J=8.5Hz,Ec-5′),6.79(1H,dd,J=1.5,8.5Hz,Ec-6′),4.66(1H,d,J=1.0Hz,Glu-1),4.82(1H,dd,J=1.0,2.5Hz,Glu-2),5.21(1H,t,J=2.5Hz,Glu-3),5.27(1H,dd,J=2.5,8.0Hz,Glu-