Metabolomic analysis of hairy roots of endangered Mongolian medicine Ophiopogon japonicus
Ammopiptanthus mongolicus (Maxim. ex kon) Cheng f, also known as Mongolian holly, belongs to the subfamily Fabaceae and the genus Ammopiptanthus. It is the only super drought tolerant evergreen broad-leaved shrub species in desert areas of China and has been listed in the first batch of rare and endangered protected plants by the state. Sha Dongqing is a traditional ethnic medicinal herb (Mongolian medicine) called “Menghe Harigane”. Its branches and leaves can be used as medicine, with the effects of dispelling wind and dampness, promoting blood circulation, dispersing blood stasis, and relieving pain. It can also be used topically to treat frostbite, especially for the treatment of rheumatoid arthritis, with significant effects. It contains abundant bioactive substances in its body, including alkaloids, flavonoids and isoflavones, organic acids, stilbenes, sterols and monoterpenes, etc., which have the effects of lowering blood sugar, anti-tumor, antiviral, bactericidal, antioxidant, insecticidal, etc.
Large scale propagation and cultivation of hairy roots can obtain a large amount of ideal medicinal substances. So far, secondary metabolites produced through hairy root cultivation have covered various substances such as alkaloids, glycosides, flavonoids, quinones, polysaccharides, etc. Liu et al. established a ginseng hairy root culture system, and the total saponin content in the hairy roots reached 2.468%, which is nearly twice the content of the original ginseng medicinal material. Another study found that infection of licorice explants with Agrobacterium tumefaciens LBA9402 and R1601 resulted in hairy roots. The total amount of five flavonoids was 1.5 times higher than that in callus tissue, and the content of licorice chalcone was 15.5 times higher than that in callus tissue. Yang et al. induced the cultivation of Eucommia ulmoides hairy roots, and the mass fraction of the secondary metabolite aucubin was higher than that of natural roots and bark, reaching up to 30.105 mg/g. At present, domestic and foreign scholars’ research on Mongolian holly mainly focuses on its above ground medicinal parts and chemical components and pharmacological activities in seeds, and there have been no reports on the metabolic accumulation of its hairy root tissues. This study selected the hairy root system of Mongolian holly with good growth and compared it with its natural roots. The UHPLC-QTOF-MS experimental technique was used to conduct non targeted metabolomics analysis on the samples, and the differential accumulation of metabolites and metabolic pathways in the hairy root tissues were preliminarily understood. This provides a theoretical basis for the accumulation and pathway analysis of metabolites in Mongolian holly hairy roots, and provides a reference for the production of active metabolites in Mongolian holly through the hairy root pathway in the later stage.

 

This study used UHPLC-Q-TOF-MS technology to perform metabolomics analysis on the hairy roots and natural roots of Mongolian holly. 1706 and 1639 differentially expressed metabolites were screened in positive and negative ion modes, respectively. Among them, 1455 and 1095 metabolites were up-regulated. The differential metabolites between the two are mainly enriched in amino acid metabolism and secondary metabolite biosynthesis pathways. Among the secondary metabolite biosynthesis pathways, there are 11 metabolic pathways including flavonoid/flavonoid biosynthesis, alkaloid biosynthesis, β – lactam biosynthesis, and glucosinolate biosynthesis. The flavonoid/flavonoid biosynthesis pathway involves the most differential metabolites, accounting for 34%, of which 88.2% show upregulation. Flavonoids are a class of 2-phenylchromenone compounds, which can be divided into chalcones, flavanones, flavonoids, flavonols, isoflavones, anthocyanins, and rosewood pigments according to their skeletons. Glycosylation, malonylation, hydroxylation, isopentenyl, acetylation modifications, and polymerization reactions ultimately lead to the diversity of this compound family. These chemical modifications have important effects on the solubility, mobility, and degradability of flavonoids. The diversity of the structure and molecular properties of flavonoids enables them to interact with various subcellular targets, thereby affecting the biological activity of plants, microorganisms, and animals. They are one of the important carriers for plants to exert their chemical ecological functions. Research has found that total flavonoids extracted from Mongolian holly seeds have various biological activities and good immune enhancement effects. They have significant promoting effects on the proliferation of immune active cells, the production and release of related immune cytokines and antibodies in mice. The main active component of total flavonoids in Mongolian holly seeds is mangiferin, which accounts for 52.48% of the total flavonoid content. Through the analysis of this study, it is preliminarily judged that the hairy roots of Mongolian holly have a strong ability to synthesize and accumulate flavonoids/flavonoid substances. This is similar to the research results of scholars on the functional components of Mongolian holly, indicating that its hairy root tissue has potential development value. On the basis of this metabolomics analysis, targeted metabolomics analysis of its flavonoid/flavonoid biosynthesis pathway is needed in the future, and related studies such as chemical component isolation, extraction, and activity identification of hairy root tissues need to be carried out to provide reference for whether Mongolian holly hairy roots can be used as active metabolite production tissues for Mongolian medicine holly.