目的 建立靈芝菌與何首烏雙向發(fā)酵的最佳發(fā)酵體系，并探討靈芝菌生物轉(zhuǎn)化對(duì)何首烏特異質(zhì)肝毒性的影響。方法 70%乙醇回流提取生何首烏粉末制備何首烏醇提物（PMEE，二苯乙烯苷質(zhì)量分?jǐn)?shù)為7.85%）；HPLC法檢測(cè)二苯乙烯苷轉(zhuǎn)化率，考察底物濃度、發(fā)酵溫度、轉(zhuǎn)速、瓶裝量、接種量、發(fā)酵時(shí)間6個(gè)因素對(duì)靈芝菌與PMEE雙向發(fā)酵體系的影響，并制備不同二苯乙烯苷轉(zhuǎn)化率的發(fā)酵物。采用脂多糖 （LPS） 制備 SD 大鼠特異質(zhì)肝毒性模型，考察 PMEE 高、低劑量 （以生首烏計(jì)2.16、1.08 g·kg^-1，分別為4、2倍臨床等效劑量）及靈芝菌轉(zhuǎn)化后PMEE發(fā)酵物（二苯乙烯苷的轉(zhuǎn)化率分別為0、50%、75%、100%，以生首烏計(jì)2.16 g·kg^-1）的特異質(zhì)肝毒性，ELISA法檢測(cè)血清丙氨酸氨基轉(zhuǎn)氨酶（ALT）、天冬氨酸氨基轉(zhuǎn)氨酶（AST）、乳酸脫氫酶（LDH）的釋放量，HE染色法觀察肝臟病理學(xué)改變。結(jié)果 靈芝菌轉(zhuǎn)化PMEE的最佳發(fā)酵條件為發(fā)酵溫度 28 ℃、搖床轉(zhuǎn)速 180 r·min^-1、菌種接種量 2.5%、培養(yǎng)基瓶裝量 25%、底物質(zhì)量濃度 19.11 mg·mL^-1，轉(zhuǎn)化時(shí)間 88～104 h，二苯乙烯苷轉(zhuǎn)化率與轉(zhuǎn)化時(shí)間呈一定的線(xiàn)性關(guān)系，線(xiàn)性方程為Y＝20.657X－12.959，R²＝0.988。與對(duì)照組比較，模型組的ALT、AST、LDH無(wú)顯著性差異，提示LPS特異質(zhì)肝毒性模型造模成功；與模型組比較，PMEE靈芝菌轉(zhuǎn)化后二苯乙烯苷轉(zhuǎn)化率為 75%、100% 組 ALT、AST、LDH 的釋放量無(wú)顯著差異，其他給 藥 組 ALT、AST、LDH 釋 放 量 均 顯著升高（P＜0.01），且ALT、AST、LDH的釋放量隨二苯乙烯苷含量的減少而減少。與對(duì)照組相比，模型組有少量炎癥因子增加，但無(wú)顯著性病理學(xué)改變，未見(jiàn)肝損傷現(xiàn)象；PMEE高劑量組出現(xiàn)肝細(xì)胞廣泛性壞死，胞核消失，有許多大小不一的脂滴空泡，偶有灶性炎癥；PMEE低劑量組可見(jiàn)多數(shù)匯管區(qū)有大量庫(kù)普弗細(xì)胞浸潤(rùn)，多數(shù)肝細(xì)胞水腫，有少量肝細(xì)胞胞質(zhì)內(nèi)可見(jiàn)小泡性或細(xì)顆粒狀脂質(zhì)空泡；PMEE靈芝菌轉(zhuǎn)化組隨著二苯乙烯苷含量降低，肝臟損傷程度也減輕，二苯乙烯苷轉(zhuǎn)化率為100%組基本上未見(jiàn)肝細(xì)胞損傷。結(jié)論 靈芝菌生物轉(zhuǎn)化能降低何首烏特異質(zhì)肝毒性，減毒作用與減少何首烏中二苯乙烯苷含量相關(guān)。

Objective To establish of a fermentation system for Polygonum multiflorum Thunb. by Ganoderma lucidum, and effects of biotransformation of G. lucidum on idiosyncratic hepatotoxicity of P. multiflorum was investigated. Methods The ethanol extract of P. multiflorum (PMEE, mass fraction of stilbene glycoside 7.85%) was prepare by 70% ethanol reflux extraction of raw P. multiflorum powder. HPLC method was used to detect the stilbene glycoside conversion rate, and the effects of six factors, including substrate concentration, fermentation temperature, rotational speed, bottled volume, inoculation amount, and fermentation time, on the bidirectional fermentation system of G. lucidum and PMEE, and to prepare fermentation products with different stilbene glycoside conversion rates. Using lipopolysaccharide (LPS) to prepare a specific hepatotoxicity model in SD rats, the specific hepatotoxicity of high and low doses of PMEE (calculated as 2.16, 1.08 g·kg^-1, respectively, 4 and 2 times the clinical equivalent dose) and PMEE fermented products after transformation by G. lucidum (the conversion rates of stilbene glycosides are 0%, 50%, 75%, and 100%, respectively, 2.16 g·kg^-1 based on raw P. multiflorum) was investigated. The release of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were detected by ELISA, and the pathological changes of the liver were observed by HE staining. Results The optimal fermentation conditions for the transformation of PMEE by G. lucidum were fermentation temperature of 28 ℃ , rotating speed of the shaking table of 180 r·min^-1, inoculum inoculation amount of 2.5%, bottled amount of culture medium of 25%, and substrate mass concentration of 19.11 mg·mL^-1. There was a linear relationship between the conversion rate of stilbene glycoside and the conversion time within 88 to 104 hours, and the linear equation was Y=20.657 X－12.959, with a square of correlation coefficient R²=0.988. Compared with control group, there was no significant difference in ALT, AST, and LDH in the model group, indicating that the LPS specific hepatotoxicity model was successfully established. Compared with the model group, there was no significant difference in the release of ALT, AST, and LDH of PMEE fermented products after transformation by G. lucidum groups (the conversion rates of stilbene glycosides are 0%, 50%, 75%, and 100%), while the release of ALT, AST, and LDH in other treatment groups were significantly increased (P＜0.01), and the release of ALT, AST, and LDH decreased with the decrease of stilbene glycoside content. Compared with the control group, there was a small increase in inflammatory factors in the model group, but no significant pathological changes were observed, and no liver injury was observed. In the high dose group of PMEE, extensive necrosis of hepatocytes, disappearance of nuclei, many lipid droplet vacuoles of varying sizes, and occasional focal inflammation were observed. In the low dose group of PMEE, there were a large number of Kupffer cells infiltrating most portal areas, most liver cells were edema, and a small amount of vesicular or granular lipid vacuoles were visible in the cytoplasm of liver cells. With the decrease of the content of stilbene glycoside in the PMEE G. lucidum transformation group, the degree of liver damage was also reduced. The group with a stilbene glycoside conversion rate of 100% basically had no liver cell damage. Conclusion Biotransformation of G. lucidum can reduce heterologous hepatotoxicity of P. multiflorum. The toxicity reduction effect is related to reducing the content of stilbene glycosides in P. multiflorum.

湖南省衛(wèi)生健康委科研計(jì)劃項(xiàng)目（D202313058583）；湖南省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目（湘教通【2022】174號(hào)：2924）；湖南中醫(yī)藥大學(xué)校級(jí)大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目（X202010541027）；湖南中醫(yī)藥大學(xué)校級(jí)科研基金項(xiàng)目（2020XJJJ020）