目的 探究人體腸道菌群體外生物轉(zhuǎn)化大黃過程條件及成分變化。方法 采集3名健康兒童（1～3歲）新鮮糞便制備菌群懸液；以活菌數(shù)保持最多為依據(jù)進行初始培養(yǎng)基的選擇；按照初始培養(yǎng)基中原有的0.5%碳源和0.5%氮源的添加量，進行不同碳源/氮源組合，依據(jù)活菌計數(shù)結(jié)果判斷最優(yōu)組合；通過指紋圖譜技術(shù)篩選菌群體外培養(yǎng)條件，包括大黃添加方式（大黃粉或大黃水浸液）、轉(zhuǎn)化時間（24或48 h）、氧需求（有氧或無氧）；基于LC-MS代謝組學(xué)進行轉(zhuǎn)化前后成分分析，采用SIMCA軟件（V16.0.2）進行主成分分析（PCA），差異代謝物分析采用MetaboAnalyst 5.0。結(jié)果 菌落計數(shù)和指紋圖譜結(jié)果顯示，蔗糖/大豆胨為改良YCFA培養(yǎng)基最適組合，粉狀大黃為添加方式，培養(yǎng)基與大黃粉最適添加體積質(zhì)量比為32∶1，有氧培養(yǎng)24 h為最優(yōu)生物轉(zhuǎn)化條件。代謝組學(xué)結(jié)果表明，主要代謝產(chǎn)物共計763種，數(shù)目較多的代謝物類別分別為脂質(zhì)和類脂質(zhì)分子（244種）、苯丙烷和聚酮化合物（138種）、有機雜環(huán)化合物（101種）、有機酸及其衍生物（92種）和苯類（59種）；轉(zhuǎn)化前后代謝物PCA圖能良好區(qū)分，確定18種關(guān)鍵差異代謝物，山柰酚-3-O-刺槐二糖苷和薯蕷皂苷上調(diào)最明顯。結(jié)論 采用指紋圖譜技術(shù)聯(lián)合代謝組學(xué)方法揭示了大黃在菌群生物轉(zhuǎn)化前后存在的成分差異，通過菌群生物轉(zhuǎn)化可提高大黃活性成分的溶出和轉(zhuǎn)變。

Objective To explore the conditions and composition changes in the process of Rhei Radix et Rhizoma biotransformation in vitro by human gut microbiota. Methods Fresh feces from three healthy children (1-3 years old) were collected to prepare bacterial flora suspension. The initial medium was selected based on the maximum number of viable bacteria. According to the amount of 0.5% carbon source and 0.5% nitrogen source added in the initial medium, different carbon source/nitrogen source combinations were performed, and the optimal combination was determined according to the viable count results. The out-ofpopulation culture conditions of bacteria were screened by fingerprint technology, including Rhei Radix et Rhizoma addition method (Rhei Radix et Rhizoma powder or Rhei Radix et Rhizoma water immersion), transformation time (24 or 48 h), oxygen requirement (aerobic or anaerobic). Component analysis before and after transformation was performed based on LC-MS metabolomics, principal component analysis (PCA) was performed using SIMCA software (V16.0.2), and MetaboAnalyst 5.0 was used for differential metabolite analysis. Results The results of colony count and fingerprinting technology showed that the optimal combination of sucrose/soy peptone as the modified YCFA medium, and powdered Rhei Radix et Rhizoma as the addition method, the optimal addition ratio (v/w) of the medium and Rhei Radix et Rhizoma powder was 32:1. Culturing in oxygen for 24 h was the optimal conditions for biotransformation. The results of metabolomics showed that there were 763 main metabolites in total, the most abundant metabolite groups were lipids and lipid-like molecules (244 species), phenylpropanes and polyketides (138 species), organic heterocyclic compounds (101 species), organic acids and their derivatives (92 species), and benzenes (59 species). The PCA diagrams of metabolites before and after biotransformation were well distinguished, also, 18 key different metabolites were identified. Biorobin and dioscin were up-regulated most obviously. Conclusion The compositional differences of Rhei Radix et Rhizoma before and after biotransformation were revealed by fingerprint technology and metabolomics. The dissolution and transformation of Rhei Radix et Rhizoma active components could be improved by biotransformation of the flora.

R283.1;R284.1

天津市教委科研計劃任務(wù)（2018KJ032）