硫磺熏蒸药材特征标志物快速发现和专属性检控方法研究

Screening the sulfur-fumigated herbs is critical in guarantee the quality of herbs. The residue of sulfur dioxide is currently used as marker in Chinese Pharmacopeia to screen the sulfur-fumigated herbs. Many studies found that sulfur-fumigation could induce ingredients in those herbs such as Radix Paeoniae Alba transformed into their sulfur-containing derivatives, and that the false negative result might obtain when sulfur dioxide determination method was used to screen the sulfur-fumigated herbs such as Radix Paeoniae Alba owing to the uncompleted transformation of sulfur-containing derivatives into sulfur dioxide. It is urgent to establish specifical methods for screening these sulfur-fumigated herbs with ingredients transformed into sulfur-containing derivatives. In present proposal, six medicinal herbs, including Radix Angelicae Sinensis, Radix paeoniae Alba, Cortex Moutan, Radix Platycodonis, Rhizoma Zingiberis, Radix Codonopsis, which containing ingredients with different kinds of chemical structures and often being sulfur-fumigated, will be selected as representative herbs, and analyzed by LC-MS based plant metabolomics and chemical profiling methods to rapidly discover and identify characteristic sulfur-containing compounds newly-generated in sulfur-fumigated herbs, and then the sulfur-containing compounds will be utilized as reference compounds together with the reference sulfur-fumigated herbs to establish quality control method of sulfur-fumigated herbs. The following hypothesis will be verified: the newly-generated sulfur-containing compounds are characteristic chemical markers in sulfur-fumigated herbs, which can be used as reference compounds to establish specific methods for the quality control of sulfur-fumigated herbs, and that this specific method might be the supplemental method to sulfur dioxide determination method in screening sulfur-fumigated herbs. From this study, the scientific basis and research example for revising the current quality control method of sulfur-fumigated herbs will be provided.

硫熏药材的检控对保证药材质量具有重要意义。现版《中国药典》采用SO2残留限量检控硫熏药材。研究证明硫熏可导致白芍等药材产生新的含硫衍生物，我们研究发现应用SO2残留限量法检控硫熏白芍时，其中含硫衍生物不能全部转化成SO2，检测结果易出现假阴性。针对这些硫熏新生含硫衍生物的药材，急需创建专属性检控方法。本项目拟在对白芍研究的基础上，再以含不同结构类型化合物且常被硫熏的当归、党参、桔梗、干姜和牡丹皮为例，采用基于LC-MS技术的植物代谢组学研究策略和化学成分表征方法，快速发现和鉴定硫熏新生含硫衍生物，并用作参照物，结合硫熏对照药材建立检控方法，验证如下假说：硫熏新生含硫衍生物是硫熏药材特征标志物，具有专属性，用作参照物可以创建硫熏药材专属性检控方法，作为SO2残留限量法的补充。通过该假说的验证，阐明以硫熏特征标志物为参照物创建检控方法的专属性，为国家相关标准的修订提供科学依据和研究范例。

本项目针对当前硫熏中药材二氧化硫残留限量检控方法的不足，提出了基于LC-MS技术的靶标和非靶标代谢组学和化学成分表征方法，快速发现和鉴定硫熏中药中特征含硫衍生物，通过证明硫熏特征含硫衍生物的专属性，建立以硫熏特征含硫衍生物为参照的硫熏药材检控方法的研究策略。项目以六种含不同结构类型的中药材为研究对象，取了如下研究结果。1. 白芍含硫衍生物Paeoniflorin sulfonate含量与硫熏程度呈对应关系，Paeoniflorin sulfonate作为参照物用于硫熏白芍的定性及定量鉴别具有可行性；通过分离、制备Paeoniflorin sulfonate，建立起以Paeoniflorin sulfonate为参照物的UPLC-QTOF-MS/MS硫熏白芍专属性检控方法。2. 发现硫熏当归中藁本内酯类含硫衍生物(3Z)-6-sulfite-ligustilide和 (3E)-6-sulfite-ligustilide等，针对含硫衍生物的特征性离子m/z 273.08 (ESI+)和m/z 271.06 (ESI-)，建立以特征离子结合硫熏当归对照药材的UPLC-QTOF-MS/MS硫熏当归专属性检控方法。3. 发现和鉴定硫熏干姜中1个姜烯酚含硫衍生物，建立以姜烯酚含硫衍生物特征性离子m/z 357.13为诊断离子的硫熏干姜专属性检控方法。4. 发现硫熏牡丹皮中10个蒎烷单萜苷含硫衍生物，优选了单萜苷含硫衍生物质谱响应较高的离子m/z 543.12，m/z 559.11和m/z 647.14作为特征离子，建立以特征离子为参照的硫熏牡丹皮专属性检控方法。5. 发现和鉴定硫熏党参中白术内酯Ⅲ含硫衍生物和白术内酯Ⅱ含硫衍生物，利用白术内酯Ⅲ含硫衍生物的特征离子，建立以m/z 327.09作为提取离子的硫熏党参筛查方法。6. 桔梗和硫熏桔梗中共鉴定出34个化合物，其中15种桔梗皂苷的含量发生了显著变化，未发现桔梗中含硫衍生物。通过项目的实施，阐明以硫熏特征标志物为参照物创建检控方法的专属性，为完善和修订国家相关标准提供科学依据和研究范例。