离体培养滇重楼根茎中甾体皂苷类化学成分的研究

Chemical constituents of steroidal saponins in rhizome of Paris polyphylla var. yunnanensis cultured in vitro

该研究采用LC-MS与HPLC技术对离体培养条件下3种不同产地滇重楼根茎中甾体皂苷类化学成分进行定性、定量分析,探究在离体培养条件下滇重楼根茎中能否合成包含2020年版《中国药典》中质量控制指标性成分重楼皂苷Ⅰ、Ⅱ和Ⅶ在内的甾体皂苷类化学成分。从3种不同产地云龙长新(S1)、大理凤仪(S2)和洱源牛街(S3)离体培养滇重楼根茎中鉴定出滇重楼皂苷A、D、E,原重楼皂苷Ⅱ,重楼皂苷G、H,重楼皂苷Ⅰ、Ⅱ、Ⅴ、Ⅵ、Ⅶ,薯蓣皂苷,纤细薯蓣皂苷,薯蓣次皂苷A,Tg, isomer of Th, saponin Th, reclinatoside, proto-paris D,pseudoproto-dioscin, 23-O-glc-(23S,25R)-spirost-5-en-3β,23α-diol-3-O-rha-(1→2)-[ara(1→4)]-glc或27-O-glc-(23S,25R)-spirost-5-en-3β,27α-diol-3-O-rha-(1→2)-[ara(1→4)]-glc共20个甾体皂苷类化学成分。其中,从离体培养S1滇重楼根茎中检测到重楼皂苷Ⅰ、Ⅱ、Ⅶ,质量分数分别为0.109 1%、0.165 2%、0.051 03%,总量为0.325 3%;从离体培养S2滇重楼根茎中检测到重楼皂苷Ⅱ、Ⅶ,质量分数分别为0.192 2%、0.074 23%,总量为0.266 5%;从离体培养S3滇重楼根茎中检测到重楼皂苷Ⅱ、Ⅶ,质量分数分别为0.207 7%、0.186 9%,总量为0.394 6%。上述结果表明在离体培养条件下滇重楼根茎中可以合成包含2020年版《中国药典》中质量控制指标性成分重楼皂苷Ⅰ、Ⅱ、Ⅶ在内的甾体皂苷类化学成分,但总量尚未达到2020年版《中国药典》不得少于0.60%的规定。因此,该研究滇重楼离体培养方案可行,但离体培养条件还需进一步改善。

In this study, liquid chromatography-mass spectrometry(LC-MS) and high performance liquid chromatography(HPLC) were employed for qualitative and quantitative analysis of the steroidal saponins in rhizomes of Paris polyphylla var. yunnanensis from three different habitats cultured in vitro, in an attempt to explore whether the rhizomes of the medicinal herb cultured in vitro can synthesize the steroidal saponins, including polyphyllinsⅠ, Ⅱ, and Ⅶ, the quality markers specified in Chinese Pharmacopoeia(2020 edition). A total of 20 steroidal saponins were identified in the rhizomes from Changxin, Yunlong(S1), Fengyi, Dali(S2), and Niujie, Eryuan(S3): parisyunnanoside A and parisyunnanoside D or E, proto-polyphyllin Ⅱ, polyphyllins G and H, polyphyllinsⅠ, Ⅱ, Ⅴ, Ⅵ, and Ⅶ, dioscin, gracillin, prosapogenin A, Tg, isomer of Th, saponin Th, reclinatoside, proto-pairs D, pseudoproto-dioscin, and 23-O-glc-(23S,25R)-spirost-5-en-3β,23α,27-triol-3-O-rha-(1→2)-[ara(1→4)]-glc or 27-O-glc-(23S,25R)-spirost-5-en-3β,27α-diol-3-O-rha-(1→2)-[ara(1→4)]-glc. Among them, polyphyllinsⅠ, Ⅱ, and Ⅶ were detected in the rhizomes from S1, with the mass fraction of 0.109 1%, 0.165 2%, and 0.051 03%, respectively(total 0.325 3%). Polyphyllins Ⅱ and Ⅶ were identified in the rhizomes from S2 with the respective mass fraction of 0.192 2% and 0.074 23% and total content of 0.266 5%. Moreover, polyphyllins Ⅱ and Ⅶ were also found in the rhizomes from S3, which had the mass fraction of 0.207 7% and 0.186 9%, separately, with the total content of 0.394 6%. Thus, steroidal saponins, including the quality makers polyphyllins Ⅰ, Ⅱ, and Ⅶ recorded in Chinese Pharmacopoeia(2020 edition) can be synthesized in rhizomes of Paris polyphylla var. yunnanensis cultured in vitro, but their total content fails to meet the standard(0.60% in Chinese Pharmacopoeia). Therefore, in vitro culture of the Paris polyphylla var. yunnanensis is feasible, but the culture conditions need to be further improved.