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Huperzine A (HupA), a natural Lycopodium alkaloid derived from Huperzia serrata (Thunb. ex Murray) Trev. plants, is a highly active acetylcholinesterase inhibitor and a key compound used for treating Alzheimer’s disease (AD). Recently, HupA has been reported in various endophytic fungi isolated from H. serrata . In the present study, 153 endophytic fungi were isolated from healthy tissues of H. serrata collected from natural populations in Lam Dong province of Central Vietnam. The endophytic fungi were identified based on morphological characteristics and Internal Transcribed Spacer sequences. Among them, 34 strains were classified into seven genera belonging to Ascomycota , including Alternaria , Fusarium , Trichoderma , Penicillium , Paecilomyces , and Phoma , and eight strains belonging to the genus Mucor ( Zygomycota ). The other strains remained unidentified. According to the results of thin-layer chromatography and high-performance liquid chromatography, only one of the 153 strains, Penicillium sp. LDL4.4, could produce HupA, with a yield 1.38 mg l −1 (168.9 µg g −1 dried mycelium) when cultured in potato dextrose broth, which was considerably higher than that of other reported endophytic fungi. Such a fungus is a promising candidate and alternative to presently available HupA production techniques for treating AD and preventing further memory decline.

Huperzine A is a preferred treatment option for Alzheimer’s disease. Huperzia serrata (Thunb. ex Murray) Trev. (H. serrata) has garnered significant attention for its ability to produce Huperzine A (HupA). However, natural populations of wild H. serrata (WH) are rapidly declining. Fortunately, our group obtained two types of H. serrata thalli (OT and ST) capable of stably producing Huperzine A, which have the potential to serve as an alternative resource to WH. To evaluate the feasibility of this strategy, we conducted a comprehensive assessment of both WH and H. serrata thallus. The results indicated that compared to WH, ST and OT exhibited stronger anti-inflammatory and antioxidant activities, with lower cytotoxicity. Notably, ST demonstrated a strong radical scavenging activity, reaching 93.23% (DPPH at 0.2 μg/mL) and 99.87% (ABTS at 4 μg/mL), and reduced nitrite production from 10.29 μM to 6.51 μM at 50 µg/mL. GC-MS and widely targeted metabolomics analyses revealed that the higher antioxidant and anti-inflammatory activities for ST and OT were due to higher concentrations of phenolic acids and flavonoids compared to WH. In addition, the HupA content in ST reached 36.56% of that found in WH. KEGG enrichment analysis revealed that the flavonoid, phenylalanine, and phenylpropanoid biosynthesis pathways may be involved in regulating the antioxidant activity. P-coumaroyl quinic acid and caffeoyl quinic acid are the crucial metabolites for antioxidant activity. These findings suggested that the H. serrata thallus could serve as a sustainable alternative to WH.

Huperzine A (HupA) can alleviate Alzheimer’s disease due to its reversible inhibition of acetylcholinesterase (AChE). The chemical synthesis and plant extraction of HupA is plagued by route complexity and resource scarcity, respectively. Although some endophytic fungi from Huperzia serrata can independently biosynthesize HupA, their yields are trivial. After a comprehensive investigation of HupA-producing H. serrata across China, we focused on the endophytic fungi from Hunan and Hubei provinces, which demonstrated high-level HupA. Morphological characteristics and internal transcriptional sequence (ITS) analysis revealed their diversity. Among the four HupA-producing endophytic fungi, Colletotrichum kahawae is the best-performing and was thus subjected to fermentation optimization. When its fermentation medium was supplemented with H. serrata flavonoids daidzein and apigenin, HupA yields reached 58.38 μg/g (dry cell weight, dcw) and 72.21 μg/g dcw, respectively. In contrast, the addition of L-lysine and H. serrata extracts led to yields of 50.17 μg/g dcw and 255.32 μg/g dcw, respectively. Transcriptomic analysis revealed that H. serrata extracts substantially upregulated the expression of HupA biosynthesis genes in C. kahawae. Overall, H. serrata extracts outperformed L-lysine, daidzein, and apigenin in boosting HupA production, as they encompass all the necessary nutrients for C. kahawae growth. This study not only connotes a nutritional exchange between H. serrata and C. kahawae during long-term coevolution but also offers insights for harnessing plant extracts for the overproduction of desired metabolites in endophytic fungi.

Background Plants maintain the steady-state balance of the mutually beneficial symbiosis relationship with their endophytic fungi through secondary metabolites. Meanwhile endophytic fungi can serve as biological inducers to promote the biosynthesis and accumulation of valuable secondary metabolites in host plants through a variety of ways. The composition and structure of endophytic fungal community are affected by many factors, including tissues, seasons and so on. In this work, we studied the community diversity, temporal and spatial pattern of endophytic fungi detected from the roots, stems and leaves of Huperzia serrata in different seasons. The correlation between endophytic fungi and huperzine A (HupA) content in plants was analyzed. Results A total of 7005 operational taxonomic units were detected, and all strains were identified as 14 phyla, 54 classes, 140 orders, 351 families and 742 genera. Alpha diversity analysis showed that the diversity of endophytic fungi in stem and leaf was higher than that in root, and the diversity in summer (August) was lower than that in other months. NMDS analysis showed that the endophytic fungal communities of leaves, stems and roots were significantly different, and the root and leaf communities were also different between four seasons. Through correlation analysis, it was found that 33 genera of the endophytic fungi of H. serrata showed a significant positive correlation with the content of HupA ( p  < 0.05), of which 13 genera ( Strelitziana, Devriesia, Articulospora, Derxomyces, Cyphellophora, Trechispora, Kurtzmanomyces, Capnobotryella, Erythrobasidium, Camptophora, Stagonospora, Lachnum, Golubevia ) showed a highly significant positive correlation with the content of HupA ( p  < 0.01). These endophytic fungi may have the potential to promote the biosynthesis and accumulation of HupA in plant. Conclusions This report is the first time to analyze the diversity of endophytic fungi in tissues of H. serrata in different seasons, which proves that there is variability in different tissues and seasonal distribution patterns. These findings provide references to the study of endophytic fungi of H. serrata.

Plant-derived (−)-Huperzine A (HupA), the bioactive enantiomer of the acetylcholinesterase inhibitor used for Alzheimer’s disease (AD) therapy, is limited by the scarcity of Huperzia serrata ( HS ), while chemically synthesized (+)-HupA is clinically unviable due to high toxicity and low activity (1/30 that of (−)-HupA), creating a critical bottleneck for HupA pharmaceutical development that necessitates microbial biosynthesis solutions. In this study, we isolated the endophytic bacterium Serratia marcescens HL-1 from HS (identified via morphological characterization and 16S rRNA sequencing) and developed a novel co-cultivation strategy with Trichoderma harzianum NSW-V in modified PDA medium (26 °C, 2 days): This co-cultivation system sustained the stable HupA biosynthetic capacity of both Serratia marcescens HL-1 and Trichoderma harzianum NSW-V, synergistically enhancing the (−)-HupA yield of the endophytic bacterium to 32.976 ± 0.21 mg/L (biosynthetic HupA, BHA) and concurrently boosting the HupA production of the fungal strain; this revealed a positive upward trend in HupA yield with co-cultivation intervention, but no statistically significant differences were observed between groups ( p  > 0.05), and notably co-cultivation restored the strain’s HupA-synthesizing capacity when its native production potential declined. BHA exhibited physicochemical properties and crystal structure identical to plant-derived (−)-HupA (PHA), as validated by NMR spectroscopy and molecular docking analyses. Furthermore, we identified a novel pharmacological role for HupA: BHA protected pancreatic islet β-cells in a palmitic acid-induced injury model, where cell viability increased from 58.2% to 71.2% (one-way ANOVA followed by Dunnett’s test, p  < 0.01, n  = 3 independent experiments), revealing an unprecedented role of HupA in pancreatic β-cell protection beyond its well-established anti-AD activity. Key points •  Co-cultivation of endophytic fungi and bacteria could highly express prior HupA.

As the population ages globally, there seem to be more people with Alzheimer’s disease. Unfortunately, there is currently no specific treatment for the disease. At present, Huperzine A (HupA) is one of the best drugs used for the treatment of Alzheimer’s disease and has been used in clinical trials for several years in China. HupA was first separated from Huperzia serrata, a traditional medicinal herb that is used to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China, and has been confirmed to have acetylcholinesterase inhibitory activity. With the very slow growth of H. serrata, resources are becoming too scarce to meet the need for clinical treatment. Some endophytic fungal strains that produce HupA were isolated from H. serrate in previous studies. In this article, the diversity of the endophytic fungal community within H. serrata was observed and the relevance to the production of HupA by the host plant was further analyzed. A total of 1167 strains were obtained from the leaves of H. serrata followed by the stems (1045) and roots (824). The richness as well as diversity of endophytic fungi within the leaf and stem were higher than in the root. The endophytic fungal community was similar within stems as well as in leaves at all taxonomic levels. The 11 genera (Derxomyces, Lophiostoma, Cyphellophora, Devriesia, Serendipita, Kurtzmanomyces, Mycosphaerella, Conoideocrella, Brevicellicium, Piskurozyma, and Trichomerium) were positively correlated with HupA content. The correlation index of Derxomyces with HupA contents displayed the highest value (CI = 0.92), whereas Trichomerium showed the lowest value (CI = 0.02). Through electrospray ionization mass spectrometry (ESI-MS), it was confirmed that the HS7-1 strain could produce HupA and the total alkaloid concentration was 3.7 ug/g. This study will enable us to screen and isolate the strain that can produce HupA and to figure out the correlation between endophytic fungal diversity with HupA content in different plant organs. This can provide new insights into the screening of strains that can produce HupA more effectively.

Huperzine A (HupA) is a potent acetylcholinesterase (AChE) inhibitor of a great consideration as a prospective drug candidate for Alzheimer’s disease treatment. Production of HupA by endophytes offers an alternative challenge to reduce the massive plant harvest needed to meet the increasing demand of HupA. In the current study, some endophytic fungal and actinobacterial isolates from the Chinese herb, Huperzia serrata , underwent liquid fermentation, alkaloid extraction, and screening for AChE inhibition and HupA production. Among these isolates, Alternaria brassicae AGF041 strain was the only positive strain for HupA production with the maximum AChE inhibition of 75.5%. Chromatographic analyses verified the identity of the produced HupA. The HupA production was efficiently maximized up to 42.89 μg/g of dry mycelia, after optimization of thirteen process parameters using multifactorial statistical approaches, Plackett–Burman and central composite designs. The statistical optimization resulted in a 40.8% increase in HupA production. This is the first report to isolate endophytic actinobacteria with anti-AChE activity from H. serrata , and to identify an endophytic fungus A. brassicae as a new promising start strain for a higher HupA yield.

Colletotrichum gloeosporioides is a major agricultural pathogen of crops that has also been identified as an endophyte of the medicinal plant Huperzia serrata. Both H. serrata and C. gloeosporioides produce huperzine A, a potential treatment for Alzheimer's disease. In this study, a nonpathogenic C. gloeosporioides strain (NWUHS001) was isolated and its genome sequenced. Gene structure prediction identified 15,413 protein‐coding genes and 879 noncoding RNAs. Through PHI‐base database prediction, we found that NWUHS001 lacks two key pathogenicity genes CgDN3 and cap20, which may be the cause of its nonpathogenicity. Comparative genomic analysis showed that the number of genes encoding pectin lyase B (pelB), pectin lyase (pnl) and polygalacturonase (pg) in NWUHS001 was significantly lower than that in pathogenic strains during the expansion of mycelium into host tissues. This caused slow growth and incapability to penetrate host cells. In contrast, in NWUHS001, genes involved in carbon acquisition such as ribose and amino sugar metabolic pathways were enriched, indicating active metabolite exchange with the host. In addition, by comparing the genome of NWUHS001 with that of the host H. serrata, we found that polyketosynthetase (pksIII), a key gene in the host huperzine A biosynthetic pathway, may possibly have been acquired from the fungus by horizontal gene transfer (HGT). This study explained the possible genetic evolution mechanism of C. gloeosporioides from pathogenicity to nonpathogenicity, which is of value for studying the interaction between microorganisms and plants. It also provided clues to the genetic evolution of the biosynthetic pathway of huperzine A. A Colletotrichum gloeosporioides strain from Huperzia serrata lacks key pathogenicity genes CgDN3 and cap20 and has fewer pelB, pnl and pg genes. pksIII, for synthesis of huperzine A, was probably acquired by the host plant from the fungus.

Alkaloid-enriched extract of Huperzia serrata (Thunb.) Trevis (Lycopodiaceae) (HsAE) can potentially be used to manage neuronal disorders. This study determines the anti-neuroinflammatory effects of HsAE on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and the underlying mechanisms. BV-2 cells were pre- or post-treated with different concentrations of HsAE (25-150 µg/mL) for 30 min before or after LPS induction. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and no cytotoxicity was found. Nitric oxide (NO) concentration was determined using Griess reagent. The levels of prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were determined using enzyme-linked immunosorbent assay. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 and the phosphorylation of mitogen-activated protein kinase (MAPK) were analyzed using western blotting. HsAE reduced LPS-induced NO production with half-maximal inhibitory concentration values of 99.79 and 92.40 µg/mL at pre- and post-treatment, respectively. Pre-treatment with HsAE at concentrations of 50, 100, and 150 µg/mL completely inhibited the secretion of PGE2, TNF-α, IL-6, and IL-1β compared to post-treatment with HsAE. This suggests that prophylactic treatment is better than post-inflammation treatment. HsAE decreased the expression levels of iNOS and COX-2 and attenuated the secretion of pro-inflammatory factors by downregulating the phosphorylation of p38 and extracellular signal-regulated protein kinase in the MAPK signaling pathway. HsAE exerts anti-neuroinflammatory effects on LPS-stimulated BV-2 cells, suggesting that it may be a potential candidate for the treatment of neuroinflammation in neurodegenerative diseases.

Endophytic fungi play an important role in plant growth. The composition and structure of endophytes vary in different plant tissues, which are specific habitats for endophyte colonization. To analyze the diversity and structural composition of endophytic fungi from toothed clubmoss ( Huperzia serrata ) that was artificially cultivated for 3 years, we investigated endophytic fungi from the roots, stems and leaves using comparative sequence analysis of the ITS2 region of the fungal rRNA genes sequenced with high-throughput sequencing technology. Seven fungal phyla were identified, and fungal diversity and structure varied across different tissues, with the most distinctive community features found in the roots. A total of 555 operational taxonomic units (OTUs) were detected, and 198 were common to all samples, and 43, 16, 16 OTUs were unique to the root, stem, leaf samples, respectively. Taxonomic classification showed that Ascomycota and Basidiomycota were dominant phyla, and Cladosporium , Oidiodendron , Phyllosticta , Sebacina and Ilyonectria were dominant genera. The relative abundance heat map at the genus level suggested that H . serrata had characteristic endophytic fungal microbiomes. Line discriminant analysis effect size analysis and principal coordinate analysis demonstrated that fungal communities were tissue-type and tissue-site specific. Overall, our study provides new insights into the complex composition of endophytic fungi in H . serrata .