Analysis of Chuanxiong Rhizoma substrate on production of ligustrazine in endophytic Bacillus subtilis by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Abstract

Ligustrazine was the active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma. However, the content of ligustrazine is very low. We proposed a hypothesis that ligustrazine was produced by the mutual effects between endophytic Bacillus subtilis and the Ligusticum chuanxiong Hort. This study aimed to explore whether the endophytic B. subtilis LB5 could make use of Chuanxiong Rhizoma fermentation matrix to produce ligustrazine and clarify the mechanisms of action preliminarily. Ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry analysis showed the content of ligustrazine in Chuanxiong Rhizoma was below the detection limit (0.1 ng/mL), while B. subtilis LB5 produced ligustrazine at the yield of 1.0268 mg/mL in the Chuanxiong Rhizoma‐ammonium sulfate fermentation medium. In the fermented matrix, the reducing sugar had a significant reduction from 12.034 to 2.424 mg/mL, and rough protein content increased from 2.239 to 4.361 mg/mL. Acetoin, the biosynthetic precursor of ligustrazine, was generated in the Chuanxiong Rhizoma‐Ammonium sulfate (151.2 mg/mL) fermentation medium. This result showed that the endophytic bacteria B. subtilis LB5 metabolized Chuanxiong Rhizoma via secreted protein to consume the sugar in Chuanxiong Rhizoma to produce a considerable amount of ligustrazine. Collectively, our preliminary research suggested that ligustrazine was the interaction product of endophyte, but not the secondary metabolite of Chuanxiong Rhizoma itself.

Key Phytochemicals and Biological Functions of Chuanxiong Rhizoma Against Ischemic Stroke: A Network Pharmacology and Experimental Assessment

“…Obviously, compounds that are widely distributed in a variety of herbs and have good pharmacological properties are more likely to be identified as active phytochemicals. Unlike the conventional approach, the present study obtained data on active CR component, including aromatic acids, phthalides and tetramethylpyrazine from recent UHPLC-MS/MS-based studies (Yin et al, 2019;Wang et al, 2020). In stark contrast, butylidenephthalide, neocnidilide, senkyunolide A and z-ligustilide are only present in 3, 6, 10, and 13 herbs, respectively, including CR (data from HERB, date of search: October 20, 2021).…”

“…This study included 21 main bioactive phytochemicals of CR, namely 12 aromatic acids, eight phthalides, and tetramethylpyrazine (Wang et al, 2020;Yin et al, 2019). According to the UHPLC-MS/MS data (Wang et al, 2020), the component with the highest content is z-ligustilide (7.16 ± 0.36 mg/g CR), followed by senkyunolide A (5.2 ± 0.36 mg/g CR) and coniferyl ferulate (1.98 ± 0.08 mg/g CR) (Figure 1).…”

“Breeding on Mountains” Resulted in the Reorganization of Endophytic Fungi in Asexually Propagated Plants (Ligusticum chuanxiong Hort.)

“…It has been found that the endophytic fungi of L. chuanxiong are often significantly related to their active ingredients. For example, the content of ibutilide is significantly related to endophytes, such as Penicillium and Wardell , and Bacillus subtilis can secrete Ligustrazine ( Yin et al, 2018a , b , 2019 ). Therefore, endophytic fungi can affect the quality of the rhizome of L. chuanxiong by regulating the production of its secondary metabolites.…”

Influence of tissue and geographic locality on culturable endophytic bacteria of Atractylodes macrocephala

“…Interestingly, recent studies have reported Bacillus’ s ability to produce several bioactive compounds, including alkaloids, terpenoids, flavonoids and steroids [25, 26, 27]. Bacillus subtilis LB5 metabolized Chuanxiong Rhizoma and produced ligustrazine [28]. B.…”

“…Interestingly, recent studies have reported Bacillus's ability to produce several bioactive compounds, including alkaloids, terpenoids, flavonoids and steroids [25,26,27]. Bacillus subtilis LB5 metabolized Chuanxiong Rhizoma and produced ligustrazine [28]. B. altitudinis (LB 5-3) isolated from Panax ginseng, an elicitor, increased ginsenoside accumulation in ginseng adventitious root culture [29].…”