灰树花化学成分及抗肿瘤细胞增殖作用研究
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摘要
灰树花(Grifola frondosa)是一种珍贵的食药两用菌,属担子菌亚门,层菌纲,非褶菌目,多孔菌科,树花菌属真菌。有抗肿瘤、提高免疫力、降血糖、抗病毒、抗辐射、清除自由基等功效。灰树花中已知的化学成分主要包括多糖、核酸、多酚、氨基酸、微量元素等,对灰树花中的小分子化合物仅报道过粗多酚,由于未进行充分的纯化,无法全面了解化合物的类型、含量及它们与灰树花药理作用的关系。
本文对灰树花小分子化合物进行了深入的分离、纯化并对获得的化合物进行化学结构研究和抑制肿瘤细胞增殖作用的测定。值得指出的是,我们发现5,8-位存在过氧键对抑制肿瘤细胞增殖作用很重要,该结果为抗癌先导化合物的修饰积累了经验,以加快抗癌新药研发的速度。
本论文运用一种新型高效的天然产物检识方法对灰树花依次对水相、乙醇相、石油醚相进行了系统检识,结果表明,水相中含有三萜皂苷；乙醇相含有生物碱、萜类内酯、香豆素、有机酸、黄酮、甾体、三萜类物质；石油醚相含有甾体和三萜类物质。
灰树花分别用95%乙醇提取、乙酸乙酯萃取、硅胶柱层析,得到13个组分。运用MTT比色法,以人乳腺癌细胞MCF-7模型进行筛选,逐级活性追踪,最终追踪到隐藏在上千种化合物中的活性目标物。使用新型微量重结晶法纯化,得到了三个化合物。用1HNMR、13CNMR核磁共振等波谱学方法鉴定了3个甾醇类化合物的结构,分别为：5α,8β-过氧麦角甾-6,22-二烯-3β-醇、麦角甾醇3β,5α,6β-三羟基麦角甾醇。
运用MTT比色法、流式细胞术研究它们对MCF-7细胞增殖的影响,发现三种化合物具有不同程度的抑制作用。5α,8β-过氧麦角甾-6,22E-二烯-3β-醇能明显MCF-7增殖,且有明显的促凋亡作用；麦角甾醇和3β,5α,6β-三羟基麦角甾醇轻微抑制MCF-7增殖。
化合物Ⅰ、Ⅱ、Ⅲ的结构极相似,因此进行构效分析,寻找起抗癌作用的关键基团,结果表明,5,8-位含有过氧键对麦角甾醇类物质具有抑制MCF-7增殖作用是极其重要的。
综上所述,本文的主要结果如下
1首次提出灰树花中含有香豆素、萜类内酯、黄酮、有机酸、生物碱、甾体、三萜等多种天然物质,对灰树花中含有的小分子物质有了全面系统的认识,为深度开发利用灰树花打下了基础。
2首次从灰树花中纯化得到以下三种化合物：5α,8β-过氧麦角甾-6,22E-二烯-3β-醇,麦角甾醇和3β,5α,6β-三羟基麦角甾醇,对灰树花的化学成分有了更深入的了解。
3 5α,8β-过氧麦角甾-6,22-二烯-3β-醇有明显抑制MCF-7增殖的作用,并有明显的促凋亡作用,麦角甾醇和3β,5α,6β-三羟基麦角甾醇有轻微抑制其增殖的作用。
4通过效分析,5,8-位存在过氧键对麦角甾醇类物质具有抑制MCF-7增殖作用重要,为抗癌药研发过程中的关键步骤即抗癌先导化合物的化学修饰积累经验。
5改进了天然产物的检识方法,大幅降低了检识过程中的假阳性和假阴性率；改进了传统重结晶法,可实现毫克级样品的纯化。
Grifola frondosa is a kind of precious edible and medical fungi, which belongs to layer of bacteria Basidiomycotina Class of non-Aphyllophorales Polyporaceae tree flowers sp. It can suppress tumor growth, enhance immunity, lower blood glucose, prevent virus, resist radiation, and eliminate free radical. The chemical constituents of Grifola frondosa included polysaccharide, nuclear acid, polyphenol, amino acid and trace elements. It was reported that the compounds of Grifola frondosa with low molecule contained raw polyphenol, however, due to insufficient analysis on Grifola frondosa, it was difficult to know the types, contents of compounds of Grifola frondosa, as well as their pharmacological mechanism.
Our study focus on the isolation and purification of compounds with low molecule from Grifola frondosa. And we also investigate their chemical structure and anti-cancer effects. It was found that peroxy bond at 5,8-sites in compounds played an important role in suppressing the proliferation of cancer cells, and it was significant for modification of compounds to suppress tumor growth and accelerating the research on anti-cancer drugs.
Isolated with water, ethanol and petroleum ether, which was new and efficient, it was found that water extract of Grifola frondosa contained triterpenoid saponin, ethanol extract contained alkaloid, terpene lactone, coumarin, organic acid, flavonoid, steroid and triterpenoid; petroleum ether extract contained steroid and triterpenoid.
Thirteen kinds of constituents were obtained through extraction with 95% ethanol and ethyl acetate, then by silica gel column chromatography. MTT assays were used to determine the activity of various constituents on MCF-7 cell, and the active compounds were obtained finally. Three compounds were obtained by purification with micro recrystallization. And the structures of these compounds were identified by 1HNMR,13CNMR NMR spectroscopy:compound I (5α,8β-epidioxyergosta-6,22diene-3β-ol), compoundsⅡ(ergosterol) and compoundsⅢ(3β, 5α,6β-trihydroxyergosta-7,22-diene).
MTT assay and flow cytometry were used to determined the inhibition of these compounds on MCF-7 cells. It was found that these three compounds could suppress the proliferation of cancer cells variously.5α,8β-epidioxyergosta-6,22diene-3β-ol significantly inhibited the proliferation of MCF-7 and led to apoptosis of cells; while ergosterol sterol 3β,5α,6β-trihydroxy ergosterol slightly inhibited proliferation of MCF-7.
CompoundsⅠ,Ⅱ,Ⅲhad very similar structure, therefore it was necessary to conduct dynamic structure-activity analysis to find the key groups for anti-cancer. It was showed that peroxy bond at 5,8-site of the ergot sterols played an important part in inhibiting the proliferation of MCF-7.
In summary, the main results are as follows:
1 For the first time it was report that Grifola frondosa contained coumarin, terpene lactones, flavonoids, organic acids, alkaloids, steroids, triterpenoids and other natural substances. The overall and systemic comprehension over small molecules laid the foundation for the development of complete utilization of Grifola frondosa.
2 Three compounds were purified from Grifola frondosa for the first time: 5α,8β-epidioxyergosta-6,22diene-3β-ol; ergosterol and 3β,5α,6β-trihydroxyergosta-7,22-diene. Providing a further undstanding of chemical constituents of Grifola frondosa.
3 5α,8β-epidioxyergosta-6,22diene-3β-ol could significant inhibition on MCF-7 and led to apoptosis of cells, providing a profound understanding that the types and contents of compounds in Grifola frondosa, and their relationship with the pharmacological effects of Grifola frondosa.
4 Peroxy bond at 5,8-site of the ergot sterols played an important part in inhibiting the proliferation on MCF-7, providing informations for modification of compounds which was the key step during the development of anti-cancer research.
5 It was important to improve the traditional method for natural products detction, dramatically reducing the false positive and negetive results. It was also significant to improve a method for recrystallization, enabling the purification of microgram samples.
本文对灰树花小分子化合物进行了深入的分离、纯化并对获得的化合物进行化学结构研究和抑制肿瘤细胞增殖作用的测定。值得指出的是,我们发现5,8-位存在过氧键对抑制肿瘤细胞增殖作用很重要,该结果为抗癌先导化合物的修饰积累了经验,以加快抗癌新药研发的速度。
本论文运用一种新型高效的天然产物检识方法对灰树花依次对水相、乙醇相、石油醚相进行了系统检识,结果表明,水相中含有三萜皂苷；乙醇相含有生物碱、萜类内酯、香豆素、有机酸、黄酮、甾体、三萜类物质；石油醚相含有甾体和三萜类物质。
灰树花分别用95%乙醇提取、乙酸乙酯萃取、硅胶柱层析,得到13个组分。运用MTT比色法,以人乳腺癌细胞MCF-7模型进行筛选,逐级活性追踪,最终追踪到隐藏在上千种化合物中的活性目标物。使用新型微量重结晶法纯化,得到了三个化合物。用1HNMR、13CNMR核磁共振等波谱学方法鉴定了3个甾醇类化合物的结构,分别为：5α,8β-过氧麦角甾-6,22-二烯-3β-醇、麦角甾醇3β,5α,6β-三羟基麦角甾醇。
运用MTT比色法、流式细胞术研究它们对MCF-7细胞增殖的影响,发现三种化合物具有不同程度的抑制作用。5α,8β-过氧麦角甾-6,22E-二烯-3β-醇能明显MCF-7增殖,且有明显的促凋亡作用；麦角甾醇和3β,5α,6β-三羟基麦角甾醇轻微抑制MCF-7增殖。
化合物Ⅰ、Ⅱ、Ⅲ的结构极相似,因此进行构效分析,寻找起抗癌作用的关键基团,结果表明,5,8-位含有过氧键对麦角甾醇类物质具有抑制MCF-7增殖作用是极其重要的。
综上所述,本文的主要结果如下
1首次提出灰树花中含有香豆素、萜类内酯、黄酮、有机酸、生物碱、甾体、三萜等多种天然物质,对灰树花中含有的小分子物质有了全面系统的认识,为深度开发利用灰树花打下了基础。
2首次从灰树花中纯化得到以下三种化合物：5α,8β-过氧麦角甾-6,22E-二烯-3β-醇,麦角甾醇和3β,5α,6β-三羟基麦角甾醇,对灰树花的化学成分有了更深入的了解。
3 5α,8β-过氧麦角甾-6,22-二烯-3β-醇有明显抑制MCF-7增殖的作用,并有明显的促凋亡作用,麦角甾醇和3β,5α,6β-三羟基麦角甾醇有轻微抑制其增殖的作用。
4通过效分析,5,8-位存在过氧键对麦角甾醇类物质具有抑制MCF-7增殖作用重要,为抗癌药研发过程中的关键步骤即抗癌先导化合物的化学修饰积累经验。
5改进了天然产物的检识方法,大幅降低了检识过程中的假阳性和假阴性率；改进了传统重结晶法,可实现毫克级样品的纯化。
Grifola frondosa is a kind of precious edible and medical fungi, which belongs to layer of bacteria Basidiomycotina Class of non-Aphyllophorales Polyporaceae tree flowers sp. It can suppress tumor growth, enhance immunity, lower blood glucose, prevent virus, resist radiation, and eliminate free radical. The chemical constituents of Grifola frondosa included polysaccharide, nuclear acid, polyphenol, amino acid and trace elements. It was reported that the compounds of Grifola frondosa with low molecule contained raw polyphenol, however, due to insufficient analysis on Grifola frondosa, it was difficult to know the types, contents of compounds of Grifola frondosa, as well as their pharmacological mechanism.
Our study focus on the isolation and purification of compounds with low molecule from Grifola frondosa. And we also investigate their chemical structure and anti-cancer effects. It was found that peroxy bond at 5,8-sites in compounds played an important role in suppressing the proliferation of cancer cells, and it was significant for modification of compounds to suppress tumor growth and accelerating the research on anti-cancer drugs.
Isolated with water, ethanol and petroleum ether, which was new and efficient, it was found that water extract of Grifola frondosa contained triterpenoid saponin, ethanol extract contained alkaloid, terpene lactone, coumarin, organic acid, flavonoid, steroid and triterpenoid; petroleum ether extract contained steroid and triterpenoid.
Thirteen kinds of constituents were obtained through extraction with 95% ethanol and ethyl acetate, then by silica gel column chromatography. MTT assays were used to determine the activity of various constituents on MCF-7 cell, and the active compounds were obtained finally. Three compounds were obtained by purification with micro recrystallization. And the structures of these compounds were identified by 1HNMR,13CNMR NMR spectroscopy:compound I (5α,8β-epidioxyergosta-6,22diene-3β-ol), compoundsⅡ(ergosterol) and compoundsⅢ(3β, 5α,6β-trihydroxyergosta-7,22-diene).
MTT assay and flow cytometry were used to determined the inhibition of these compounds on MCF-7 cells. It was found that these three compounds could suppress the proliferation of cancer cells variously.5α,8β-epidioxyergosta-6,22diene-3β-ol significantly inhibited the proliferation of MCF-7 and led to apoptosis of cells; while ergosterol sterol 3β,5α,6β-trihydroxy ergosterol slightly inhibited proliferation of MCF-7.
CompoundsⅠ,Ⅱ,Ⅲhad very similar structure, therefore it was necessary to conduct dynamic structure-activity analysis to find the key groups for anti-cancer. It was showed that peroxy bond at 5,8-site of the ergot sterols played an important part in inhibiting the proliferation of MCF-7.
In summary, the main results are as follows:
1 For the first time it was report that Grifola frondosa contained coumarin, terpene lactones, flavonoids, organic acids, alkaloids, steroids, triterpenoids and other natural substances. The overall and systemic comprehension over small molecules laid the foundation for the development of complete utilization of Grifola frondosa.
2 Three compounds were purified from Grifola frondosa for the first time: 5α,8β-epidioxyergosta-6,22diene-3β-ol; ergosterol and 3β,5α,6β-trihydroxyergosta-7,22-diene. Providing a further undstanding of chemical constituents of Grifola frondosa.
3 5α,8β-epidioxyergosta-6,22diene-3β-ol could significant inhibition on MCF-7 and led to apoptosis of cells, providing a profound understanding that the types and contents of compounds in Grifola frondosa, and their relationship with the pharmacological effects of Grifola frondosa.
4 Peroxy bond at 5,8-site of the ergot sterols played an important part in inhibiting the proliferation on MCF-7, providing informations for modification of compounds which was the key step during the development of anti-cancer research.
5 It was important to improve the traditional method for natural products detction, dramatically reducing the false positive and negetive results. It was also significant to improve a method for recrystallization, enabling the purification of microgram samples.
引文
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