大叶紫金牛活性成分的研究
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摘要
本论文利用多种现代色谱手段从紫金牛科紫金牛属植物大叶紫金牛(Ardisia gigantifoliaStapf)干燥根茎60％乙醇提取物中分离得到了17个化合物。通过化学和光谱学(IR、MS、NMR)方法鉴定了它们的结构，分别为：没食子酸(1)，(2R，3S，4S，4aR，10bS)-(—)-岩白菜素(2)，(2R，3S，4S，4aR，10bS)-(+)-8-O-(3′，5′)-二甲基没食子酰基岩白菜素(3)，(—)-10b-羟基岩白菜素(4)，(+)-5-(1′，2′-二羟基戊基)-苯-1，3-二醇(5)，(—)-5-(1′，2′-二羟基戊基)-苯-1，3-二醇(6)，(—)-表儿茶素(7)，(—)-4′-羟基-3′，5′-二甲氧基苯基-β-D-[6-O-(4″-羟基-3″，5″-二甲氧基苯甲酰基)]-葡萄糖苷(8)，(—)-4′-羟基-3′-甲氧基苯基-β-D-[6-O-(4″-羟基-3″，5″-二甲氧基苯甲酰基)]-葡萄糖苷(9)，(—)-4′-羟基-2′，6′-二甲氧基苯基-β-D-[6-O-(4″-羟基-3″-甲氧基苯甲酰基)]-葡萄糖苷(10)，(—)-3′-羟基-4′-甲氧基苯基-β-D-[6-O-(4″-羟基-3″，5″-二甲氧基苯甲酰基)]-葡萄糖苷(11)，β-谷甾醇(12)，3β-O-{α-L-吡喃鼠李糖基-(1→3)-[β-D-吡喃木糖基-(1→2)]-β-D-吡喃半乳糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→2)]-α-L-吡喃阿拉伯糖基}-16α-羟基-13，28-环氧齐墩果烷(13)，3β-O-{α-L-吡喃鼠李糖基-(1→3)-[β-D-吡喃葡萄糖基-(1→3)-β-D-吡喃木糖基-(1→2)]-β-D-吡喃半乳糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→2)]-α-L-吡喃阿拉伯糖基}-16α-羟基-13，28-环氧齐墩果烷(14)，3β-O-{α-L-吡喃鼠李糖基-(1→3)-[β-D-吡喃木糖基-(1→2)]-β-D-吡喃半乳糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→2)]-α-L-吡喃阿拉伯糖基}-西克拉敏A(15)，3β-O-{α-L-吡喃鼠李糖基-(1→3)-[β-D-吡喃葡萄糖基-(1→3)-β-D-吡喃木糖基-(1→2)]-β-D-吡喃半乳糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→2)]-α-L-吡喃阿拉伯糖基}-西克拉敏A(16)，3β-O-{α-L-吡喃鼠李糖基-(1→3)-[β-D-吡喃木糖基-(1→2)]-β-D-吡喃半乳糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→2)]-α-L-吡喃阿拉伯糖基}-16α-羟基-13，28-环氧-30-乙酰氧基齐墩果烷(17)。
17个化合物中，有1个小分子酚酸类化合物(化合物1)，3个香豆素类化合物(化合物2—4)，2个酚类化合物(化合物5，6)，1个黄烷类化合物(化合物7)，4个酚苷类化合物(化合物8—11)，1个甾醇类化合物(化合物12)以及5个三萜皂苷类化合物(化合物13—17)。新化合物共6个，分别为化合物3—6，10，11，化合物8和9为首次从该属植物中分离得到，化合物1和7为首次从该种植物中分离得到。
采用抑制大鼠巨噬细胞NO释放的活性测试方法，对非皂苷类化合物进行了活性评价，发现酚苷类化合物8，10显示出了很强的NO释放抑制作用，并具有初步的构效关系。
采用清除1，1-二苯基-2-苦基苯肼(DPPH)自由基的活性测试方法，考察了非皂苷类化合物的体外抗氧化能力。结果表明化合物1，5，7，8，11具有很好的清除DPPH自由基的作用，并具有初步的构效关系。
本课题组张晓明硕士曾经从大叶紫金牛中分离得到过11个三萜皂苷类化合物，初步体外细胞毒活性测试结果显示，部分三萜皂苷类化合物对NCI-H460(人非小细胞肺癌)、SF-268(人神经胶质瘤)、MCF-7(人乳腺癌)以及HepG2(人肝癌)细胞具有很强的细胞毒活性。本论文对大叶紫金牛中含量较大、活性显示较好的三萜皂苷类化合物进行累计，得到化合物13—17。
本论文发现，该植物中大量含有的没食子酸、岩白菜素以及含有其片段的衍生物具有抑制大鼠巨噬细胞NO释放、清除DPPH自由基的生物活性，部分三萜皂苷类化合物具有抗癌活性，结合其民间药用我们推测大叶紫金牛的疗效是由于该植物中所含有的酚酸类和酚苷类成分具有潜在的iNOS选择性抑制、抗氧化以及三萜皂苷类成分抗癌作用的综合体现。在上述工作的基础上，建立了活性部位群乙酸乙酯萃取物的指纹图谱。本文为今后对该种中草药的开发和利用提供了初步的理论和实验依据。
In this dissertation, the dried rhizome of Ardisia gigantifolia Stapf was extracted with 60% ethanol, and seventeen compounds were isolated using various chromatographic techniques. The structures of the compounds were elucidated on the basis of physical-chemical evidence and spectral analysis. The compounds were established as: gallic acid (1),(2R, 3S, 4S, 4aR, 10bS)-(-)-bergenin (2),(2R, 3S, 4S, 4aR, 10bS)-(+)-3, 4, 10-trihydroxy-2-(hydroxymethyl)-9-methoxy-6-oxo-2,3,4,4a,6,10b-h exahydropyrano[3,2-c]isochromen-8-yl 4-hydroxy-3,5-dimethoxybenzoate (3),(-)-3,4,8,10,10b-pentahydroxy-2-(hydroxymethyl)-9-methoxy-2,3,4,4a-tetrahydropyrano [3,2-c]iso chromen-6(10bH)-one (4), (+)-5-(1,2-dihydroxypentyl)benzene-1,3-diol(5),(-)-5-(1,2-dihydroxypentyl)benzene-1, 3-diol (6), (-)-epicatechin (7),(-)-4'-hydroxy-3', 5'-dimethoxyphenyl-β-D-[6-O-(4"-hydroxy-3", 5"-dimethoxybenzoyl)]-glucopyra noside(8),(-)-4'-hydroxy-3'-methoxyphenyl-β-D-[6-O-(4"-hydroxy-3", 5"-dimethoxybenzoyl)]-glucopyranosi de(9),(-)-4'-hydroxy-2', 6'-dimethoxyphenyl-β-D-[6-O-(4"-hydroxy-3"-methoxybenzoyl)]-glucopyranosi de(10),(-)-3'-hydroxy-4'-methoxyphenyl-β-D-[6-O-(4"-hydroxy-3", 5"-dimethoxybenzoyl)]-glucopyranosi de (11),β-sitosterol (12), 3β-O-{α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside}-16α-hydroxy-13, 28-epoxy-oleanane(13), 3β-O-{α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside}-16α-hydroxy-13, 28-epoxy-oleanane(14), 3β-O-α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside-cyclamiretin A (15), 3β-O-α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→2)]-β-D-g alactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside-cyclamiretin A(16), 3β-O-{α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside}-16α-hydroxy-13, 28-epoxy-30-acetoxyoleane (17), respectively. All the compounds include one phenolic acid compound(1), three coumarins (2-4), two phenolic compounds(5, 6), one flavan(7), four phenolic glycoside compounds(8-11), one sterol(12) and five triterpenoid saponins(13-17). Compounds 3-6, 10, 11 are new compounds, compounds 8 and 9 were isolated from Ardisia for the first time, compounds 1 and 7 were isolated from Ardisia gigantifolia for the first time.
The inhibitory effects and their IC_(50) values of the non-saponin compounds on NO production in murine macrophage activated by LPS and IFN-γwere estimated, and phenolic glycoside compounds 8 and 10 showed strong inhibitory effects. The primary structure-activity relationship was discussed.
We also tested the DPPH free radical scavenging activity of the non-saponin compounds. Partial phenolic acid compounds and phenolic glycoside compounds were found to have strong antioxidant activity and primary structure-activity relationship.
In our laboratory, eleven triterpenoid saponins, with four kinds of aglycone, were isolated from Ardisia gigantifolia before, and some of them showed potent cytotoxic activity against NCI-H460, SF-268, MCF-7 and HepG2 tumor cell lines in the preliminary tests. In my works, five triterpenoid saponins(13-17) were accumulated.
The results indicated that the anti-inflammation components and the potent free radical scavenger are gallic acid, bergenin and their derivates. In summary, the phenolic acid compounds and phenolic glycoside compounds as the potent NO inhibitors, free radical scavengers play the important role in the anti-inflammatory effects of Ardisia gigantifolia, and the anti-cancer components are triterpenoid saponins. Furthermore, the finger printing of the acetic ether part of the Ardisia gigantifolia was tried to established. It provided a reasonable ground for continuous study of this traditional Chinese medicine.
17个化合物中，有1个小分子酚酸类化合物(化合物1)，3个香豆素类化合物(化合物2—4)，2个酚类化合物(化合物5，6)，1个黄烷类化合物(化合物7)，4个酚苷类化合物(化合物8—11)，1个甾醇类化合物(化合物12)以及5个三萜皂苷类化合物(化合物13—17)。新化合物共6个，分别为化合物3—6，10，11，化合物8和9为首次从该属植物中分离得到，化合物1和7为首次从该种植物中分离得到。
采用抑制大鼠巨噬细胞NO释放的活性测试方法，对非皂苷类化合物进行了活性评价，发现酚苷类化合物8，10显示出了很强的NO释放抑制作用，并具有初步的构效关系。
采用清除1，1-二苯基-2-苦基苯肼(DPPH)自由基的活性测试方法，考察了非皂苷类化合物的体外抗氧化能力。结果表明化合物1，5，7，8，11具有很好的清除DPPH自由基的作用，并具有初步的构效关系。
本课题组张晓明硕士曾经从大叶紫金牛中分离得到过11个三萜皂苷类化合物，初步体外细胞毒活性测试结果显示，部分三萜皂苷类化合物对NCI-H460(人非小细胞肺癌)、SF-268(人神经胶质瘤)、MCF-7(人乳腺癌)以及HepG2(人肝癌)细胞具有很强的细胞毒活性。本论文对大叶紫金牛中含量较大、活性显示较好的三萜皂苷类化合物进行累计，得到化合物13—17。
本论文发现，该植物中大量含有的没食子酸、岩白菜素以及含有其片段的衍生物具有抑制大鼠巨噬细胞NO释放、清除DPPH自由基的生物活性，部分三萜皂苷类化合物具有抗癌活性，结合其民间药用我们推测大叶紫金牛的疗效是由于该植物中所含有的酚酸类和酚苷类成分具有潜在的iNOS选择性抑制、抗氧化以及三萜皂苷类成分抗癌作用的综合体现。在上述工作的基础上，建立了活性部位群乙酸乙酯萃取物的指纹图谱。本文为今后对该种中草药的开发和利用提供了初步的理论和实验依据。
In this dissertation, the dried rhizome of Ardisia gigantifolia Stapf was extracted with 60% ethanol, and seventeen compounds were isolated using various chromatographic techniques. The structures of the compounds were elucidated on the basis of physical-chemical evidence and spectral analysis. The compounds were established as: gallic acid (1),(2R, 3S, 4S, 4aR, 10bS)-(-)-bergenin (2),(2R, 3S, 4S, 4aR, 10bS)-(+)-3, 4, 10-trihydroxy-2-(hydroxymethyl)-9-methoxy-6-oxo-2,3,4,4a,6,10b-h exahydropyrano[3,2-c]isochromen-8-yl 4-hydroxy-3,5-dimethoxybenzoate (3),(-)-3,4,8,10,10b-pentahydroxy-2-(hydroxymethyl)-9-methoxy-2,3,4,4a-tetrahydropyrano [3,2-c]iso chromen-6(10bH)-one (4), (+)-5-(1,2-dihydroxypentyl)benzene-1,3-diol(5),(-)-5-(1,2-dihydroxypentyl)benzene-1, 3-diol (6), (-)-epicatechin (7),(-)-4'-hydroxy-3', 5'-dimethoxyphenyl-β-D-[6-O-(4"-hydroxy-3", 5"-dimethoxybenzoyl)]-glucopyra noside(8),(-)-4'-hydroxy-3'-methoxyphenyl-β-D-[6-O-(4"-hydroxy-3", 5"-dimethoxybenzoyl)]-glucopyranosi de(9),(-)-4'-hydroxy-2', 6'-dimethoxyphenyl-β-D-[6-O-(4"-hydroxy-3"-methoxybenzoyl)]-glucopyranosi de(10),(-)-3'-hydroxy-4'-methoxyphenyl-β-D-[6-O-(4"-hydroxy-3", 5"-dimethoxybenzoyl)]-glucopyranosi de (11),β-sitosterol (12), 3β-O-{α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside}-16α-hydroxy-13, 28-epoxy-oleanane(13), 3β-O-{α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside}-16α-hydroxy-13, 28-epoxy-oleanane(14), 3β-O-α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside-cyclamiretin A (15), 3β-O-α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→2)]-β-D-g alactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside-cyclamiretin A(16), 3β-O-{α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside}-16α-hydroxy-13, 28-epoxy-30-acetoxyoleane (17), respectively. All the compounds include one phenolic acid compound(1), three coumarins (2-4), two phenolic compounds(5, 6), one flavan(7), four phenolic glycoside compounds(8-11), one sterol(12) and five triterpenoid saponins(13-17). Compounds 3-6, 10, 11 are new compounds, compounds 8 and 9 were isolated from Ardisia for the first time, compounds 1 and 7 were isolated from Ardisia gigantifolia for the first time.
The inhibitory effects and their IC_(50) values of the non-saponin compounds on NO production in murine macrophage activated by LPS and IFN-γwere estimated, and phenolic glycoside compounds 8 and 10 showed strong inhibitory effects. The primary structure-activity relationship was discussed.
We also tested the DPPH free radical scavenging activity of the non-saponin compounds. Partial phenolic acid compounds and phenolic glycoside compounds were found to have strong antioxidant activity and primary structure-activity relationship.
In our laboratory, eleven triterpenoid saponins, with four kinds of aglycone, were isolated from Ardisia gigantifolia before, and some of them showed potent cytotoxic activity against NCI-H460, SF-268, MCF-7 and HepG2 tumor cell lines in the preliminary tests. In my works, five triterpenoid saponins(13-17) were accumulated.
The results indicated that the anti-inflammation components and the potent free radical scavenger are gallic acid, bergenin and their derivates. In summary, the phenolic acid compounds and phenolic glycoside compounds as the potent NO inhibitors, free radical scavengers play the important role in the anti-inflammatory effects of Ardisia gigantifolia, and the anti-cancer components are triterpenoid saponins. Furthermore, the finger printing of the acetic ether part of the Ardisia gigantifolia was tried to established. It provided a reasonable ground for continuous study of this traditional Chinese medicine.
引文
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