Diversity and epimedium biotransformation potential of cultivable endophytic fungi associated with Epimedium brevicornum Maxim in the Qinling Mountains, China

Abstract

Background: The use of biocatalysis technology to manufacture rare natural products can solve the contradiction between the market demand for rare natural products in large health industry fields and the protection and sustainable development of wildlife resources. However, the currently available research on fungal endophytes, which are great potential resources for glycoside hydrolase biocatalysts, is still insufficient. In this study, endophytic fungi from Epimedium brevicornum Maxim. were isolated in the Qinling Mountains, identified and tested for their potential to biotransform epimedium extracts into minor epimedium flavonoids.

Results: A total of 84 representative morphotype strains were isolated and identified via ITS rDNA sequence analyses and were grouped into 32 taxa. The Shannon‒Wiener index (H', 3.089) indicated that E. brevicornum Maxim. harboured abundant fungal resources. Ten strains showed strong β-glucosidase activity and exhibited the ability to biotransform major epimedium flavonoids into deglycosylated minor epimedium flavonoids, such as baohuoside I and icaritin, via various glycoside-hydrolysing pathways. Among these strains, strains 8509 and F8889, which were initially characterized as Aspergillus ochraceus and A. protuberus, have the potential for further development in the biotransformation of epimedium extracts into minor epimedium flavonoids because of their excellent biosafety, enzyme activity, and enzymatic characteristics. The enzyme activity of the crude enzyme obtained by freeze-drying from the F8509 fermentation broth supernatant reached 78.24 ± 2.48 U/g. Further research revealed that major glycosylated flavonoids from 100 g/L epimedium extracts were bio-transformed completely into minor deglycosylated flavonoids in 90 min after the addition of 1 g/L crude enzyme. In addition, the liquid phase separation conditions were optimized, and ethyl alcohol and water were ultimately used as the mobile phase for efficient separation of the conversion products at equal flow degrees.

Conclusions: This study not only identified a series of candidates for the biotransformation of minor epimedium flavonoids but also provided an efficient purification method. More importantly, this study also demonstrated the important value of endophytes in the biotransformation of rare natural products.

Keywords: Epimedium brevicornum Maxim; Biotransformation; Diversity; Endophytic fungi.

Fig. 1

Isolation and diversity analysis of the culturable endophytic fungi from E. brevicornum Maxim. A The isolates were subsequently grown on PDA plates. B Species richness and Shannon–Wiener diversity index analysis. C Phylogenetic tree of culturable endophytic fungi from E. brevicornum Maxim. in the Qinling Mountains, China

Fig. 2

The relative abundance (RA, %) of endophytic fungi at the genus (A), family (B), order (C), and class (D) levels

Fig. 3

Screening of β-glucosidase-producing endophytic fungi and analysis of their properties for the bioconversion of the major flavonol glycosides from the extracts of epimedium. A Screening of β-glucosidase produced on esculin-R2A agar. B The characteristics of the β-glycosidase-producing isolates and their taxonomic status. C HPLC spectra of the flavonol glycoside standards and bioconversion products from 7 strains with β-glucosidase activity. D Comparison of the activity of the pNPG enzyme in different strains

Fig. 4

Effects of reaction temperature (A), pH (B) and organic solvents (C methanol, D ethanol, E DMSO) on pNPG enzyme activity

Fig. 5

Enzyme preparation and transformation process. A Enzyme activity of fermentation broth and freeze-dried crude enzyme. B HPLC analysis of the bioconversion products at different biotransformation times. The composition of flavonoids in the initial epimedium extracts (C) and the converted product (D)

Fig. 6

Optimization of the bioconversion product separation and purification process and identification of the conversion product structure. Gradient HPLC was used for the detection of flavonol glycoside standards (A) and bioconversion products (B). Separation of the conversion products (C) and separation process optimization (D) by equi-HPLC. Stability verification of the purification process (E) and mass spectrum analysis of the purification products (F)

Fig. 7

Schematic diagram of the direct biotransformation of high-glycosylation flavonoids (epimedin A, epimedin B, and epimedin C) into low-glycosylation flavonoids (icariin, baohuoside I, 2``-O-icariside II) by glucosidase