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Diversity of Endophytic Fungi Isolated From Huperzia serrata and Research on the Secondary Metabolites of Penicillium panissanguineum SZSS 4-2-2

Ya-Shuai Kang,Hao-Wen Zhu,Xue Zhao,Hao Ma,Yu Yang,Rui Xu,Fu-Yao Luo,Er-Wei Li,Qian Luo,You-Zhi Zhang,Chang-Wei Li

A total of 46 endophytic fungi were isolated from Huperzia serrata and then cultured in a standard liquid medium and PDB medium. Cytotoxicity and antimicrobial activity assays revealed that seven crude extracts exhibited significant inhibitory effects on MCF-7 and SH-SY5Y cell lines; four crude extracts effectively inhibited the growth of Candida albicans , and 12 crude extracts demonstrated potent inhibition effects on Staphylococcus aureus . Strains displaying antimicrobial activity or potential for producing novel metabolites were selected for identification. In total, 14 strains representing 7 genera were identified, 5 of which had not been previously documented regarding their secondary metabolites. A fungus, Penicillium panissanguineum SZSS 4-2-2 was preliminarily selected for further chemical investigation. From the fermentation extract of P. panissanguineum SZSS 4-2-2, one new compound ( 1 ), one new natural product ( 2 ), and two known compounds ( 3 and 4 ) were isolated. The structures of these compounds were elucidated through spectral analysis and calculated nuclear magnetic resonance and electronic circular dichroism data. Compound 1 demonstrated a significantly stronger inhibitory effect on lipopolysaccharide-induced nitric oxide (NO) production in RAW 264.7 macrophages, with an IC 50 value of 64.91 µM. By contrast, compounds 2 , 3 , and 4 exhibited only moderate inhibitory effects on NO production. Graphical In this study, a total of 46 endophytic fungi were isolated from H. serrata. Strains displaying antimicrobial activity or potential for producing novel metabolites were selected for identification. And, from the fermentation extract of one endophytic fungus, P. panissanguineum SZSS 4-2-2, one new compound (1), one new natural product (2), and two known compounds (3 and 4) were isolated.

In Vitro Propagation, Huperzine A Content and Antioxidant Activity of Three Genotypic Huperzia serrata

Yan Yang,Liangfang Dai,Decai Wu,Limin Dong,Yisheng Tu,Jiankun Xie,Xiangdong Luo

Huperzia serratais a traditional herb and endangered Chinese medicinal material, which has attracted much attention due to its production of Huperzine A (HupA). In vitro propagation ofH. serratais considered a new way to relieve the resource pressure ofH. serrata. In this study, three different genotypic wildH. serratawere used for in vitro propagation. Then, the antioxidant activity and the content of HupA in the regeneratedH. serratawere investigated. The results showed the survival rate of the explant was increased to 25.37% when using multiple sterilization processes. The best induction medium forH. serratawas the Schenk and Hildebrandt (SH) medium supplemented with 0.5 mg·L−1Naphthalene acetic acid (NAA) and 0.1 mg·L−12,4-Dichlorophenoxyacetic acid (2,4-D), where the regeneration rate of the explant was to 57.04%. The best proliferation medium was the SH medium with NAA (1.0 mg·L−1), as the biomass of in vitro tissue increased 164.17 ± 0.41 times. High-performance liquid chromatography analysis showed that the in vitro culture of three genotypes could produce HupA and the content of HupA was 53.90–87.17 µg·g−1. The antioxidant experiment showed that the methanol extract of in vitroH. serratahad higher antioxidant activity than that of wildH. serrata. This study provides a reliable in vitroH. serrataculture protocol and laid an important foundation for the antioxidant capacity of the thallus and the content of HupA.Keywords:genotypes;Huperzia serrata;Huperzine A;micropropagation;sterilization;antioxidant

Huperzia serrata Thallus as a Host-Derived Elicitor Enhances Alkaloid and Terpenoid Biosynthesis in a Sporothrix Endophyte: Insights from Multi-Omics

Endophytic fungi fromHuperzia serrata(H. serrata) are promising sources for exploring novel acetylcholinesterase (AChE) inhibitors. In this study, 111 endophytic fungal strains were isolated from wildH. serrata, of which 18 exhibited AChE inhibition rates exceeding 90%. Among them, an endophyticSporothrixisolate (HS2-S-26) showed a 98.37% AChE inhibition rate and could produce a range of AChE inhibitors, including dauricine. Comparative metabolomics and transcriptomics betweenH. serratathallus (HT)-treated and untreated cultures revealed that the HT elicitor reprogrammed HS2-S-26 metabolism, affecting 6708 genes and 49 secondary metabolites. Alkaloids and terpenoids were the two largest classes, containing 10 and 11 compounds, respectively. Differential metabolites and differentially expressed genes were co-enriched in 59 KEGG pathways related to the biosynthesis and metabolism of alkaloids, terpenoids, and tyrosine metabolism, etc. HT likely promoted alkaloid accumulation by upregulating the expression of tyrosine biosynthesis genes (AROF,TYR1,GOT2, andARO8) and may also enhance the supply of dopamine, potentially contributing to dauricine accumulation. Meanwhile, HT may promote terpenoid biosynthesis via activation of core pathway genes (MVAK2,GGPS1,FDFT1) and inhibition of branch pathway genes (COQ2,WRBA). Through multi-omics, this study demonstrates that host thallus acts as an effective elicitor of endophytic secondary metabolism, offering insights into host–endophyte regulatory crosstalk.

Simultaneous separation and determination of three huperzine alkaloids in Huperzia serrata and its preparations by cyclodextrin-modified mixed micellar electrokinetic capillary chromatography

Yuhang Yang,Hua Wang,Haixia Yu,Hongfen Zhang,Guangbin Zhang,Ruimiao Chang,Anjia Chen

In this study, a simple and sensitive cyclodextrin-modified mixed micellar electrokinetic capillary chromatography (CD-MEKC) method has been developed for the simultaneous separation and determination of Huperzine A (HupA), Huperzine B (HupB) and Huperzine C (HupC) in Huperzia serrata (H. serrata). The optimal conditions (pH 9.3) were composed of 10 mM sodium tetraborate solution, 40 mM sodium dodecyl sulfate (SDS), 50 mM sodium cholate (SC) and 3.0 mM mono-(6-ethylenediamine-6-deoxy)-β-cyclodextrin (ED-β-CD). The separation and determination process were performed on a P/ACE MDQ capillary electrophoresis system, the separation voltage was 15 kV, the temperature was 25 °C and the detection wavelength was 308 nm. Under the optimum conditions, the migration time was less than 9 min. The LOD and LOQ were between 0.38 and 0.80 μg/mL and 1.2–2.3 μg/mL, respectively. The developed method, with excellent precision and accuracy, was applied for the determination of three alkaloids in H. serrata and its formulations.