硫磺熏蒸对人参中皂苷类成分组成、吸收和代谢的影响

Sulfur-fumigation is commonly used to protect herbs from worms, insects and molds. However, the influence of sulfur-fumigation on the quality and efficacy of herbs need to be systematically investigated. Our previous studies showed that after sulfur-fumigation, the immune adjustment function of Panax ginseng was weakened and some of ginsenosides had transformed into their sulfur-containing derivatives, among which ginsenoside Re sulfate and ginsenoside Rg1 sulfite, whose structure were assigned by their quasi-molecular ions and fragments, were detected as the main sulfur-containing ginsenoside derivatives in sulfur-fumigated ginseng by LC-MS analysis. In this proposal, the influence of sulfur-fumigation on the chemical composition, absorption and metabolism of ginsenosides will be investigated. The chemical profiles of ginsenosides and their metabolites in plasma after oral administration of total ginsenosides of sulfur-fumigated and non-fumigated ginseng will be compared by LC-MS based chemical profiling approach. Ginsenoside Re sulfate and ginsenoside Rg1 sulfite will be isolated and their chemical structures will be further confirmed by MS and NMR analysis. The absorption and metabolism of ginsenoside Re sulfate and ginsenoside Rg1 sulfite will be studied by Caco-2 cellular monolayer model, in situ intestinal absorption model, liver microsomes-cocktail probe method and intestinal microbiota incubation method. All results obtained would provide more detailed scientific evidence to evaluate the influence of sulfur-fumigation on the quality and efficacy of Panax ginseng.

硫磺熏蒸是当前常用的中药材杀虫防霉方法，但硫磺熏蒸对药材品质和功效的影响仍需系统研究和评价。我们前期研究发现，硫磺熏蒸可降低人参的免疫调节作用，使部分人参皂苷转化成人参皂苷含硫衍生物，其中人参皂苷Re和Rg1的硫酸酯化物为硫熏人参中主要的含硫衍生物。本项目拟应用基于LC-MS的化学轮廓表征方法比较硫熏前后人参总皂苷及口服血中移行成分化学轮廓差异，应用植物化学手段分离、纯化和确证人参皂苷Re和Rg1的硫酸酯化物，应用Caco-2细胞模型、原位肠吸收模型、肝微粒体-cocktail药物探针和肠道菌温孵代谢方法比较人参皂苷Re和Rg1及相应硫酸酯化物的吸收和代谢情况，从人参皂苷组成、吸收和代谢三个层次研究硫磺熏蒸对人参内在品质和功效的影响，为全面评价硫磺熏蒸作为人参杀虫防霉方法的合理性提供科学依据。

人参经硫熏生成一系列人参皂苷含硫衍生物，其体内吸收、代谢过程不明。阐明该类衍生物结构、理化性质、体内吸收代谢行为是保证用药安全的首要问题。本项目考察硫磺熏蒸对原有人参皂苷类成分组成的影响；进行硫熏前后人参皂苷入血成分及代谢产物轮廓表征；最后以人参皂苷Re 25-羟基硫酸酯及Rg1 25-羟基亚硫酸酯为代表，通过单向在体肠灌流、Caco-2细胞转运、体外肠道菌、肝S9代谢、探针底物法等研究其吸收、代谢特性和过程，探讨硫熏对人参主要活性成分的影响规律，为合理控制硫熏人参药材提供科学依据。.主要研究结果：.(1)硫熏人参醇提液中9种皂苷总量下降54 %；水煎液中下降64%，严重影响人参质量。.(2)对硫熏前后人参皂苷类成分、入血成分及粪便代谢物进行轮廓表征，发现药材新增20个人参皂苷含硫衍生物；粪便中有44个，血中有6个，说明硫磺熏蒸改变人参入血成分。.(3)硫熏人参以70%乙醇提取，浓缩液用75%乙醇醇沉，上清液经D101大孔树脂分离富集皂苷部位，经制备高效液相分离纯化皂苷含硫衍生物，分别为Re的25-羟基硫酸酯及Rg1的25-羟基亚硫酸酯。.(4)经人/大鼠肠道菌体外代谢，Re、Rg1及其25-羟基硫化物共生成25个代谢产物，其中18个来自硫化物，以水解脱糖反应为主，硫酸酯键不易断裂，保持含硫形式。人参皂苷Re及Rg1 Peff值为0.4×10-4左右，吸收差；其25-OH硫/亚硫酸酯在各肠段有效吸收系数和常数较原人参皂苷均更差，生物利用度更低；四者均非P-gp底物。Caco-2单层细胞膜转运研究结果显示，硫化物表观渗透系数Papp低于原型，外排系数小于1，属被动转运模式。对肝药酶影响研究表明，Re、Rg1 25-OH 硫/亚硫酸酯对人肝微粒体CYP3A4和CYP1A2有诱导作用，对CYP2C9和CYP2E1有抑制作用，与原型一致，且对CYP2C19亦有诱导作用。Re、Rg1及其硫化物在人/大鼠肝脏中均不发生Ⅰ、Ⅱ相代谢。.综上，硫熏人参中含硫衍生物经肠道菌代谢生成更多含硫代谢物并吸收入血；以Re 25-羟基硫酸酯及Rg1 25-羟基亚硫酸酯为例，发现其吸收较原型差，生物利用度更低，对肝药酶影响情况与原人参皂苷有差异。为确保用药安全，必须全面评价其安全性后，才能判断是否可用硫磺熏蒸方法对人参药材进行杀虫、防霉、防蛀，本项目为全面合理控制硫磺熏蒸人参药材提供科学依据。