普洱茶普洱茶百科普洱茶工艺

普洱茶化学成分分析与研究论文

摘要:普洱茶是以云南特有的大叶茶Camellia sinensis (Linn)var. assamica (Masters)Kitamura的晒青毛茶。普洱茶分为2种:一种是鲜茶叶加工后没有经过发酵,即晒青毛茶简单压制成饼的生茶;另一种是将生茶储存10年以上,经过自然发酵或者经人工渥堆发酵而成的

普洱茶是以云南特有的大叶茶Camellia sinensis (Linn)var. assamica (Masters)Kitamura的晒青毛茶。普洱茶分为2种:一种是鲜茶叶加工后没有经过发酵,即晒青毛茶简单压制成饼的生茶;另一种是将生茶储存10年以上,经过自然发酵或者经人工渥堆发酵而成的熟茶。不同的发酵工艺产生了风味不同的普洱熟茶产品。普洱茶自然油润、条索肥壮、陈香显著,除作为饮品外,还具有多种的保健作用,由于这些独特品质,使得历经千年的普洱茶愈发充满活力[1]。利用现代研究方法对普洱茶进行了较系统的研究,发现其提取物具有降血脂[2]、抗氧化[35]、抗癌[6]、保肝[7]、抗糖尿病[89]等作用。为了进一步研究普洱茶的化学成分及活性作用,本文对普洱茶水提物的乙酸乙酯部分进行了分离与结构鉴定,从中分离鉴定得到13个化合物,6个首次从该植物中分离得到,1个为新的天然产物。

1 材料

Boetius显微熔点测定仪(温度未校正); Jasco P2000型旋光仪;Mercury400型和INOVA500型核磁共振仪(美国Varian公司,TMS为内标);Agilent 1100 LC/MSD Trap SL型液相色谱,质谱联用仪(Agilent公司);德国诺尔制备型高效液相色谱仪,YMC PackODS型制备柱(4.6 mm×250 mm,5 μm);创新通恒中压液相色谱仪,Sephadex LH20(Pharmacia公司);RPC18 (40~60 μm,Merck公司);柱色谱和薄层色谱硅胶均为青岛海洋化工厂生产;溶剂均为分析纯,提取用水为去离子水,制备色谱用水为市售娃哈哈纯净水。药材为云南西双版纳产台地普洱生茶,由本所马林副研究员鉴定为大叶茶C. sinensis var.assamica,标本保存于本所标本室,编号IDS2441。

2 提取分离

普洱生茶10 kg,分别用20,20,10倍开水,依次浸泡20,20,10 min,减压浓缩后冻干得3 360 g,浸膏用20 L水悬浮,等量乙酸乙酯萃取3次,回收乙酸乙酯得浸膏1 450 g。乙酸乙酯浸膏经等量硅胶(160~200目)拌样,通过柱色谱,以氯仿甲醇(1∶0~0∶1)梯度快速洗脱,分成6个部分(A1~A6)。A1反复重结晶得化合物1 (57 g),A2(127 g)经硅胶(160~200目)柱色谱,以氯仿甲醇(9∶1~0∶1)梯度洗脱分成4个亚流分(B1~B4),其中B1部分结晶析出化合物2 (890 mg),B2(27 g)经中压制备色谱(RPC18柱),甲醇水(5∶95~1∶0)梯度洗脱再分成7个亚流分(C1~C7),C1重结晶析出化合物3(2.5 g),C3经制备色谱反复纯化得化合物4 (85 mg),5(27 mg),6(496 mg);C4经NKA树脂,Sephadex LH20,半制备液相等色谱方法分离纯化,得化合物7 (47 mg);C5经Sephadex LH20和反复半制备液相等色谱方法分离纯化,得化合物8 (14 mg),9 (46 mg),10 (211 mg);C6部分经过Sephadex LH20和半制备液相等色谱方法分离纯化,得化合物11 (21 mg),12(13 mg),13 (23 mg)。

3 结构鉴定

化合物1 白色针状结晶(氯仿),mp 235~236 ℃。ESIMS m/z 195[M+H]+;1HNMR(CDCl3,300 MHz)δ:3.98(3H,s,1CH3 ),3.39(3H,s,3CH3),3.57(3H,s,7CH3),7.50(1H,s,8NH)。以上光谱数据与文献[10]报道的咖啡因基本一致,经与咖啡因对照品共薄层,Rf 相同,且混合熔点不下降,故鉴定该化合物为咖啡因。

化合物2 白色粉末,mp 287~289 ℃。ESIMS m/z 181[M+H]+;1HNMR(D2O,400 MHz)δ:5.07(1H,s,1NH),3.48(3H,s,3CH3),3.95(3H,s,7CH3),7.92(1H,s,8NH)。以上光谱数据与咖啡因的氢谱比较缺一个甲基信号,多1个活波氢信号,与文献[10]报道的可可碱比较,鉴定该化合物为可可碱。

化合物3 白色针状结晶(甲醇),mp 235~237 ℃。ESIMS m/z 171[M+H]+;1HNMR(DMSOd6,400 MHz)δ:6.90(2H,s,H2,6);以上光谱数据与文献[11]报道的没食子酸基本一致,与没食子酸对照品共薄层,Rf 一致,且混合熔点不下降,故鉴定该化合物为没食子酸。

化合物4 白色粉末,mp 153~155 ℃,[α]20D +4.0(c 0.252,CH3OH)。ESIMS m/z 289[M-H]-; 1HNMR(DMSOd6,400 MHz)δ:6.70(1H,s,H2′),6.67(1H,d,J=8.0 Hz,H6′),6.57(1H,d,J=8.0 Hz,H5′),5.88(1H,s,H8),5.67(1H,s,H6),4.46(1H,d,J=7.6 Hz,H2),3.80(1H,m,H3),2.64(1H,dd,J=5.2,16.0 Hz,H4a),2.33(1H,dd,J=8.0,16.0 Hz,H4b);13CNMR(DMSOd6,125 MHz)δ:81.0(C2),66.3(C3),27.8(C4),156.1(C5),95.1(C6),156.4(C7),93.6(C8),155.3(C9),99.0(C10),130.6(C1′),114.5(C2′),144.8(C3′,4′),115.0(C5′),118.4(C6′)。以上波谱数据与文献[11]数据基本一致,鉴定化合物4为(+)儿茶素。

化合物5 灰白色粉末,mp 242~243 ℃,[α]20D +7.1(c 0.166,CH3OH)。ESIMS m/z 343[M+Na]+;1HNMR(DMSOd6,400 MHz)δ:6.37(2H,s,H2′,6′),5.86(1H,s,H6),5.82(1H,s,H8),4.87(1H,d,J=10.8 Hz,H2),4.38(1H,d,J=10.8 Hz,H3);13CNMR(DMSOd6,125 MHz)δ:83.2(C2),71.6(C3),197.4(C4),163.3(C5),96.0(C6),167.1(C7),95.0(C8),162.5(C9),100.3(C10),127.1(C1′),106.9(C2′,6′),145.7(C3′,5′),133.4(C4′)。以上波谱数据与文献[12]数据基本一致,鉴定化合物5为蛇葡萄素。

化合物6 灰白色粉末,mp 235~237 ℃,[α]20D-35.5(c 0.251,CH3OH)。ESIMS m/z 289[M-H]-;1HNMR(DMSOd6,400 MHz)δ:6.88(1H,s,H2′),6.64(2H,s,H5′,6′),5.88(1H,s,H6),5.70(1H,s,H8),4.71(1H,br s,H2),3.98(1H,m,H3),2.64(1H,dd,J=16.0,5.2 Hz,H4a),2.45( 1H,dd,J=16.0,2.4 Hz,H4b);13CNMR(DMSOd6,125 MHz)δ:78.1(C2),64.9(C3),28.2(C4),155.8(C5),95.1(C6),156.5(C7),94.1(C8),156.2(C9),98.5(C10),130.6(C1′),114.8(C2′),144.5(C3′),144.4(C4′),114.9(C5′),118.0(C6′)。以上波谱数据与文献[11]数据基本一致,鉴定化合物6为(-)表儿茶素。

化合物7 白色粉末,mp 185~186 ℃,[α]20D-59.7(c 0.250,CH3OH)。ESIMS m/z 273[M-H]-;1HNMR(DMSOd6,400 MHz)δ:7.21(2H,d,J=8.8 Hz,H2′,6′),6.70(2H,d,J=8.8 Hz,H3′,5′),5.88(1H,d,J=2.0 Hz,H6),5.70(1H,d,J=2.0 Hz,H8),4.78(1H,br s,H2),4.00(1H,m,H3),2.67(1H,dd,J=4.4,16.4 Hz,H4a),2.48(1H,dd,J=3.6,16.4 Hz,H4b);13CNMR(DMSOd6,125 MHz)δ:78.0(C2),64.8(C3),28.2(C4),155.7(C5),95.1(C6),156.5(C7),94.1(C8),156.2(C9),98.4(C10),129.9(C1′),128.2(C2′,6′),114.4(C3′,5′),156.5(C4′)。以上波谱数据与文献[13]数据基本一致,故鉴定化合物7为(-)表阿福豆素。

化合物8 灰白色粉末,mp 178~179 ℃,[α]20D-173.3(c 0.107,CH3OH)。ESIMS m/z 465[M+Na]+;1HNMR(DMSOd6,400 MHz)δ:6.87(1H,s,H2′),6.79(2H,s,H2″,6″),6.71(1H,d,J=8.0 Hz,H6′),6.62(1H,d,J=8.0 Hz,H5′),5.92(1H,s,H6),5.81(1H,s,H8),5.28(1H,br s,H2),5.00(1H,m,H3),2.91(1H,dd,J=17.2,3.6 Hz,H4a),2.66(1H,dd,J=17.2,4.0 Hz,H4b);13CNMR(DMSOd6,125 MHz)δ:76.5(C2),68.1(C3),25.6(C4),155.6(C5),95.5(C6),156.5(C7),94.3(C8),156.4(C9),97.2(C10),129.3(C1′),114.3(C2′),144.8(C3′),144.7(C4′),115.0(C5′),117.4(C6′),165.3(C-O),108.5(C2″,6″),145.6(C3″,5″),139.4(C4″)。以上数据与文献[11]的数据基本一致,故鉴定化合物8为(-)表儿茶素3O没食子酸酯。

化合物9 灰白色粉末,mp 173~174 ℃,[α]20D -171.9(c 0.253,CH3OH)。ESIMS m/z 427[M+H]+;1HNMR(DMSOd6,400 MHz)δ:7.27(2H,d,J=8.4 Hz,H2′,6′),6.80(2H,s,H2″,6″),6.67(2H,d,J=8.4 Hz,H3′,5′),5.93(1H,d,J=2.0 Hz,H6),5.82(1H,d,J=2.0 Hz,H8),5.31(1H,br s,H2),5.09(1H,m,H3),2.92(1H,dd,J=17.2,4.4 Hz,H4a),2.69(1H,dd,J=17.2,2.4 Hz,H4b);13CNMR(DMSOd6,125 MHz)δ:76.5(C2),68.2(C3),25.5(C4),155.6(C5),95.5(C6),156.5(C7),94.3(C8),156.4(C9),97.2(C10),128.7(C1′),127.8(C2′,6′),114.7(C3′,5′),156.8(C4′),165.1(C-O),118.9(C1″),108.5(C2″,6″),145.5(C3″,5″),138.8(C4″)。以上光谱数据与文献[11]数据基本一致,故鉴定化合物9为(-)表阿福豆素3O没食子酸酯。


化合物10 灰白色粉末,mp 180~181 ℃,[α]20D+ 20.4(c 0.251,CH3OH)。ESIMS m/z 443[M+H]+;1HNMR(DMSOd6,400 MHz)δ:6.83(2H,s,H2″,6″),6.73(1H,d,J=1.6 Hz,H2′),6.66(1H,d,J=8.4 Hz,H5′),6.60(1H,dd,J=1.6,8.4 Hz,H6′),5.92(1H,d,J=2.0 Hz,H6),5.80(1H,d,J=2.0 Hz,H8),5.23(1H,m,H3),5.06(1H,d,J=6.0 Hz,H2),2.65(1H,dd,J=4.8,16.4 Hz,H4a),2.57(1H,dd,J=5.6,16.4 Hz,H4b);13CNMR(DMSOd6,125 MHz)δ:77.1(C2),69.0(C3),23.1(C4),156.2(C5),95.4(C6),153.7(C7),94.0(C8),156.8(C9),97.3(C10),129.2(C1′),113.5(C2′),145.1(C3′),145.0(C4′),117.5(C5′),115.3(C6′),165.1(C-O),119.0(C1″),108.6(C2″,6″),145.5(C3″,5″),138.8(C4″)。以上光谱数据与文献[14]数据基本一致,故鉴定该化合物为(+)儿茶素3O没食子酸酯。

化合物11 灰白色粉末,mp 173~174 ℃,[α]20D+11.5(c 0.090,CH3COCH3)。 ESIMS m/z 449[M+Na]+;1HNMR(DMSOd6,400 MHz)δ:7.16(2H,d,J=8.8 Hz,H2′,6′),6.81(2H,s,H2″,6″),6.70(2H,d,J=8.8 Hz,H3′,5′),5.93(1H,d,J=2.0 Hz,H6),5.79(1H,d,J=2.0 Hz,H8),5.23(1H,m,H3),5.08(1H,d,J=6.8 Hz,H2),2.73(1H,dd,J=5.2,16.4 Hz,H4a),2.57(1H,dd,J=6.8,16.4 Hz,H4b);13CNMR(DMSOd6,125 MHz)δ:77.3(C2),69.1(C3),23.7(C4),154.7(C5),95.6(C6),156.8(C7),94.1(C8),156.2(C9),97.5(C10),128.5(C1′),127.7(C2′,6′),115.1(C3′,5′),157.0(C4′),165.0(C-O),119.0(C1″),108.5(C2″,6″),145.5(C3″,5″),138.9(C4″)。该化合物与化合物9相对分子质量相同,仅在1HNMR中发现H2与H3裂分明显,与文献[15]数据比较基本一致,故鉴定该化合物为(+)阿福豆素3O没食子酸酯。

化合物12 黄色粉末,mp 207~208 ℃。ESIMS m/z 457[M+Na]+;1HNMR(DMSOd6,400 MHz)δ:7.64(1H,d,J=8.4 Hz,H6′),7.49(1H,s,H2),6.82(1H,d,J=8.4 Hz,H5′),6.37(1H,s,H6),6.16(1H,s,H8),5.26(1H,d,J=5.2 Hz,H1″),3.50~3.74(4H,m,H2″,3″,4″,5″a),3.20(1H,J=10.4 Hz,H5″b);13CNMR(DMSOd6,125 MHz)δ:156.1(C2),133.7(C3),177.4(C4),161.1(C5),98.8(C6),164.8(C7),93.5(C8),156.3(C9),103.7(C10),122.0(C1′),115.7(C2′),145.0(C3′),148.6(C4′),115.3(C5′),120.8(C6′),101.4(C1″),71.6(C2″),70.7(C3″),66.0(C4″),64.2(C5″)。以上光谱数据与文献[16]报道一致,故鉴定该化合物为槲皮素3OαL吡喃阿拉伯糖苷。

化合物13 白色粉末,mp 174~175 ℃,[α]20D-151.6(c 0.249,MeOH)。ESIMS m/z 433[M+Na]+;1HNMR(DMSOd6,400 MHz)δ:7.63(2H,d,J=8.8 Hz,H2″,6″),6.89(1H,d,J=1.6 Hz,H2′),6.77(2H,d,J=8.8 Hz,H3″,5″),6.69(1H,dd,J=1.6,8.4 Hz,H6′),6.62(1H,d,J=8.4 Hz,H5′),5.92(1H,d,J=2.4 Hz,H6),5.82(1H,d,J=2.4 Hz,H8),5.33(1H,m,H3),5.04(1H,d,J=2.4 Hz,H2),2.93(1H,dd,J=16.0,4.4 Hz,H4a),2.70(1H,dd,J=16.0,2.4 HZ,H4b);13CNMR(DMSOd6,125 MHz)δ:76.3(C2),68.4(C3),25.5(C4),155.5(C5),95.5(C6),156.5(C7),94.2(C8),156.4(C9),97.1(C10),129.3(C1′),114.2(C2′),144.8(C3′),144.7(C4′),115.1(C5′),117.3(C6′),164.9(C-O),119.9(C1″),131.3(C2″,6″),115.3(C3″,5″),162.3(C4″)。以上数据与文献[17]报道数据基本一致,故鉴定该化合物为(-)epicatechin3Ophydroxybenzoate。

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Chemical constituents of Camellia sinensis var. assamica

ZHU Hongbo, LI Baomin, LIU Chao, CHEN Ruoyun*

(State Key Laboratory of Bioactive Substance and function of Natural Medicines, Ministry of Education, Institute of

Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China)

[Abstract] To study the chemical constituents of Camellia sinensis var. assamica. The compounds were isolated by NKA Macroporous resin silica gel, Sephadex LH20, RPCl8 column chromatographies and semipreparative HPLC,and their structures were elucidated by physicochemical properties and spectral analysis. Thirteen compounds were isolated and identified as caffeine (1), theobromine (2), gallic acid (3), (+)catechin (4), ampelopsin (5), (-)epicatechin (6), (-)epiafzelechin (7), (-)epicatechin3Ogallate (8), (-)epiafzelechin3Ogallate (9), (+)catechin3Ogallate (10), (+)afzelechin3Ogallate (11), quemefin3OαLarabinopyranosid (12), and (-)epicatechin3Ophydroxybenzoate (13). Compounds 2, 5, 1013 were isolated from this plant for the first time, and compound 11 is a new natural product.

[Key words] Puerh tea; flavane; ampelopsin

doi:10.4268/cjcmm20130925

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普洱茶百科
普洱茶百科
普洱茶制作工艺详解 含高清视频教程 熟知普洱茶是中国黑茶代表之一,其口感醇厚,功效作用丰富,常饮对于自身健康有积极的作用。如何选购适合饮用的普洱茶,首先要了解
普洱茶