目的 優(yōu)化奶制手參Gymnadenia conopsea的炮制工藝并采用HPLC法建立其指紋圖譜及指標(biāo)成分含量測(cè)定方法。方法 基于單因素實(shí)驗(yàn)結(jié)合Box-Behnken設(shè)計(jì)-響應(yīng)面法（Box-Behnken design-response surface methodology，BBD-RSM），以料液比、炮制溫度和炮制時(shí)間為考察因素，結(jié)合層次分析法（analytic hierarchy process，AHP）和基于指標(biāo)相關(guān)性的權(quán)重分配法（criteria importance through intercriteria correlation，CRITIC）綜合賦權(quán)，以綜合評(píng)分作為工藝優(yōu)化評(píng)價(jià)標(biāo)準(zhǔn)。采用HPLC法建立指紋圖譜，應(yīng)用中藥色譜指紋圖譜相似度評(píng)價(jià)系統(tǒng)（2012.130723版本）、IBM SPSS Statistics 27.0、SIMCA 14.1軟件，結(jié)合層次聚類(lèi)分析（hierarchical cluster analysis，HCA）、主成分分析（principal component analysis，PCA）和正交偏最小二乘法-判別分析（orthogonal partial least squares-discrimination analysis，OPLS-DA）對(duì)不同產(chǎn)地的奶制手參質(zhì)量進(jìn)行評(píng)價(jià)。結(jié)果 確定最佳炮制工藝參數(shù)為料液比1∶2、溫度85 ℃、時(shí)間15 min，經(jīng)3批驗(yàn)證試驗(yàn)綜合評(píng)分為83.446（RSD為0.544%）。奶制手參中天麻素、對(duì)羥基苯甲醇、dactylorhin B、loroglossin、dactylorhin A和militarine的平均質(zhì)量分?jǐn)?shù)分別為0.143%、0.300%、0.600%、0.490%、0.870%和0.880%。指紋圖譜確定了8個(gè)共有峰，13批樣品相似度為0.897～0.996，HCA將13批奶制手參分為3類(lèi)；OPLS-DA篩選出militarine和對(duì)羥基苯甲醇為關(guān)鍵差異性成分。結(jié)論 建立的炮制工藝穩(wěn)定可靠，所構(gòu)建的HPLC指紋圖譜結(jié)合多成分含量測(cè)定的質(zhì)量控制方法科學(xué)合理，可為奶制手參質(zhì)量控制提供依據(jù)。

Objective To optimize the processing technology of milk-processed Shoushen (Gymnadenia conopsea, GC) and establish its fingerprint and determination method for index components by HPLC. Methods Based on single-factor experiments combined with Box-Behnken design-response surface methodology (BBD-RSM), the solid-liquid ratio, processing temperature, and processing time were investigated. The comprehensive weighting method combining analytic hierarchy process (AHP) and criteria importance through intercriteria correlation (CRITIC) was used, with the comprehensive score as the evaluation standard for process optimization. The HPLC method was applied to establish the fingerprint, and the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (Version 2012.130723), IBM SPSS Statistics 27.0, and SIMCA 14.1 software were used. Quality evaluation of milk-processed GC from different producing areas was conducted by combining hierarchical cluster analysis (HCA) and orthogonal partial least squares-discrimination analysis (OPLS-DA). Results The optimal processing parameters were determined as solid-liquid ratio 1:2, temperature 85 ℃, and time 15 min. The comprehensive score of three batches of verification tests was 83.446 (RSD is 0.542%). The average contents of gastrodin, p-hydroxybenzyl alcohol, dactylorhin B, loroglossin, dactylorhin A, and militarine in milk-processed GC were 0.143%, 0.300%, 0.600%, 0.490%, 0.870%, and 0.880%, respectively. Eight common peaks were identified in the fingerprint, and the similarity of 13 batches of samples was 0.897—0.996. HCA classified the 13 batches of milk-processed GC into three categories; OPLS-DA screened out militarine and p-hydroxybenzyl alcohol as key differential components. Conclusion The established processing technology is stable and reliable, and the quality control method combining HPLC fingerprint and multi-component content determination is scientific and reasonable, which can provide a basis for the quality control of milk-processed GC.

R283.6

國(guó)家重點(diǎn)研發(fā)計(jì)劃資助項(xiàng)目（2023YFC3504403）