基于川芎内生枯草杆菌初步研究川芎中川芎嗪的来源

A preliminary study on origin of ligustrazine in Chuanxiong Rhizoma based on endogenetic Bacillus subtilis

川芎嗪是中药川芎的重要的活性成分,其含量一直存在争议。药用植物内生菌可代谢产生与宿主相同的活性物质,该研究从川芎内生枯草杆菌这一途径,体外将分离的内生菌接种至川芎基质进行固态发酵,初步研究川芎中川芎嗪的来源。采用组织研磨法分离川芎内生枯草杆菌,结合形态学特征、常规生理生化反应及16S rRNA分子技术对内生菌株进行鉴定;筛选出在川芎基质培养基中生长良好的菌株,进行固态发酵,采用川芎发酵基质(40 g川芎细粉,100 mL无菌水,121℃,灭菌30 min)37℃发酵培养30 d;UPLC检测发酵基质、川芎药材中川芎嗪、乙偶姻的含量。结果显示,从川芎中分离出5株内生枯草芽孢杆菌Bacillus subtilis编号为LB3,LB3-2-1,LB4,LB5,LB6-2。UPLC检测结果显示,川芎药材、空白川芎发酵基质中未检测到川芎嗪,接种川芎内生菌的川芎发酵基质中均检测到川芎嗪及其前体物质乙偶姻。该研究表明川芎内生枯草杆菌可利用川芎发酵基质产生川芎嗪,内生枯草杆菌与川芎中川芎嗪的积累具有一定相关性,川芎嗪可能来源于川芎中内生枯草杆菌的分解代谢。

Ligustrazine is an important active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma, but its content is a controversial topic. The endophytes of medicinal plants have the ability to produce the same active substances as the host, so this report focused on the endophytic Bacillus subtilis, to study the origin of ligustrazine in Chuanxiong Rhizoma preliminarily by inoculating the isolated endophytic B. subtilis to the Chuanxiong Rhizoma medium in vitro for solid state fermentation. Tissue grinding method was used to isolate the endogenetic B. subtilis. The morphological features, conventional physiological and biochemical reactions and 16 S rRNA molecular techniques were combined to identify the endogenetic strains. Then, the strains that grew well in the medicinal matrix of Chuanxiong Rhizoma were screened out for further fermentation studies. The solid-state fermentation was performed at 37 ℃ for 30 d using Chuanxiong Rhizoma fermentation medium(40 g Chuanxiong Rhizoma powder, 100 mL sterile water, 121 ℃, sterilization for 25 minutes). UPLC was used to detect the contents of ligustrazine, acetoin in the Chuanxiong Rhizoma fermentation medium and Chuanxiong Rhizoma. All the five strains were Gram-positive and had spores. Phylogenetic analysis of the 16 S rRNA sequence showed that the endophytes were B. subtilis. The results of UPLC showed that ligustrazine was detected in the Chuanxiong Rhizoma fermentation medium inoculated with endogenetic B. subtilis LB3, LB3-2-1, LB4, LB5 and LB6-2, while not detected neither in blank Chuanxiong Rhizoma fermentation medium nor in Chuanxiong Rhizoma. This study showed that the endogenetic B. subtilis of Ligusticum chuanxiong Hort. can make use of Chuanxiong Rhizoma fermentation medium to produce ligustrazine. Endogenetic B. subtilis has a certain correlation with the accumulation of ligustrazine in Rhizoma Chuanxiong. We speculate that the ligustrazine may be derived from the catabolism of endogenetic B. subtilis in Ligusticum chuanxiong.