Inhibition of protein tyrosine phosphatase 1B by serratane triterpenes from Huperzia serrata and their molecular docking study.

During the search for protein tyrosine phosphatase 1B (PTP1B) inhibitory compounds from the natural resources, two new serratane triterpenes, 3-O-dihydro-p-coumaroyltohogenol (1) and 21-O-acetyltohogenol (2), along with four known serratane triterpenes (3-6), were isolated from the whole plant of Huperzia serrata. The chemical structures of compounds 1 and 2 were determined by NMR study, HRMS analysis, and chemical modification. All isolates were evaluated for their PTP1B inhibitory activities. Among the isolates, compounds 1, 3, 5 and 6 exhibit moderate inhibitory activities against PTP1B. Kinetic studies demonstrated that they are competitive inhibitors. Molecular docking studies support these experimental results by showing that compounds 1, 3, 5 and 6 interact with the active site of PTP1B, clarifying the structure-activity relationship. This study suggests that serratane triterpenes from H. serrata have potential as starting skeletons for anti-diabetes or anti-obesity agents.

New insights into the composition and diversity of endophytic bacteria in cultivated Huperzia serrata.

Endophytic bacteria play crucial roles in the growth and bioactive compound synthesis of host plants. In this study, the composition and diversity of endophytic bacteria in the roots, stems, and leaves from 3-year-old artificially cultivated Huperzia serrata were investigated using Illumina HiSeq sequencing technology. Total effective reads were assigned to 936 operational taxonomic units (OTUs), belonging to 12 phyla and 289 genera. A total of 28, 3, and 2 OTUs were exclusive to the roots, stems, and leaves, respectively. The bacterial richness and diversity in the roots were significantly lower than those in the leaves and stems. The dominant genera with significant distribution differences among these plant tissue samples were Burkholderia-Caballeronia-Paraburkholderia, Sphingomonas, Acidibacter, Bradyrhizobium, Bryobacter, Methylocella, Nocardioides, Acidothermus, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium. Furthermore, the differences in the bacterial communities associated with these plant tissue samples were visualized using principal coordinate analysis and cluster pedigree diagrams. Linear discriminant analysis effect size explained statistically significant differences among the endophytic bacterial microbiota in these plant tissue samples. Overall, this study provides new insights into the diversity and distribution patterns of endophytic bacteria in the different tissues of H. serrata.

Neuroprotective effects of total alkaloids fraction of Huperzia serrata on scopolamine-induced neurodegenerative animals.

Huperzia serrata contains Huperzine A (HupA)-an alkaloid used to treat cognitive dysfunction. In this study, we used the total alkaloids (HsAE) to investigate their potential in managing cognitive impairment in comparison with HupA. The antioxidant activity was measured by DPPH assay. In the cellular study, the cell viability and level of ACh of SH-SY5Y cells were evaluated after pretreated with HsAE and scopolamine. For in vivo assay, mice were pre-treated with HsAE, and HupA and undergone scopolamine injection for cognitive impairment. The behavioral tests including the Y-maze and Morris water maze test and the AChE activity, the SOD, CAT, MDA level in the hippocampus and cortex were evaluated. HsAE showed significant scavenging properties on DPPH radicals. HsAE was not toxic to SH-SY5Y cells, and can rescue these cells upon scopolamine treatment. Intriguingly, HsAE showed the neuroprotection against scopolamine-induced amnesia in mice. Moreover, HsAE decreased AChE activity, MDA level, increased antioxidative enzyme activity in the hippocampus as well as cortex of mice, which was relatively better than that of HupA. These findings suggested that HsAE may significantly protect the neurons of mice with scopolamine-induced memory impairment connected to AChE depletion and oxidative stress.

Lycopodium Alkaloids from Huperzia serrata and Their Anti-acetylcholinesterase Activities.

One new fawcettimine-type alkaloid (1), one new miscellaneous-type alkaloid (2), four new lycodine-type alkaloids (3-6), and eight known ones (7-14) were isolated from the whole plants of Huperzia serrata. Their structures and absolute configurations were elucidated based on spectroscopic data, X-ray diffraction, ECD calculation and Mosher's method. Compound 1 was a rare C18 N2 -type Lycopodium alkaloid, possessing serratinine skeleton with an amide side chain in C-5. The absolute configuration of the 18-OH of compounds 4-6 were first determined by Mosher's method. Moreover, compounds 1-14 were assayed anti-acetylcholinesterase effect in vitro, and compound 7 showed significant anti-acetylcholinesterase activity with an IC50 value of 16.18±1.64 µM.

Diversity of endophytic fungal community in Huperzia serrata from different ecological areas and their correlation with Hup A content.

BACKGROUND: Huperzine A (Hup A) has attracted considerable attention as an effective therapeutic candidate drug used to treat Alzheimer's disease. Whereas, the production of Hup A from wild plants faced a major challenge, which is the wild Huperzia Serrata harbor a low Hup A content, has a long-life cycle, and has a small yield. At present, several reports showed that Hup A is produced by various endophytic fungal strains isolated from H. serrata, thereby providing an alternative method to produce the compound and reduce the consumption of this rare and endangered plant. However, till now, very few comprehensive studies are available on the biological diversity and structural composition of endophytic fungi and the effects of endophytic fungi on the Hup A accumulation in H. serrata. RESULTS: In this research, the composition and diversity of fungal communities in H. serrata were deciphered based on high-throughput sequencing technology of fungal internal transcribed spacer regions2 (ITS2). The correlation between endophytic fungal community and Hup A content was also investigated. Results revealed that the richness and the diversity of endophytic fungi in H. serrata was various according to different tissues and different ecological areas. The endophytic fungal communities of H. serrata exhibit species-specific, ecological-specific, and tissue-specific characteristics. There are 6 genera (Ascomycota_unclassified, Cyphellophora, Fungi_unclassified, Sporobolomyces, and Trichomeriaceae_unclassified) were significantly positively correlated with Hup A content in all two areas, whereas, there are 6 genera (Auricularia, Cladophialophora, Cryptococcus, Mortierella, and Mycena) were significantly negatively correlated with Hup A content of in all two areas. CONCLUSIONS: This study indicated a different composition and diverse endophytic fungal communities in H. serrata from different organs and ecological areas. The current study will provide the realistic basis and theoretical significance for understanding the biological diversity and structural composition of endophytic fungal communities in H. serrata, as well as providing novel insights into the interaction between endophytic fungi and Hup A content.

The temporal and spatial endophytic fungal community of Huperzia serrata: diversity and relevance to huperzine A production by the host.

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.

Hupertimines A-E, Fawcettimine-Type Lycopodium Alkaloids from Huperzia serrata.

Five new fawcettimine-type Lycopodium alkaloids, hupertimines A-E (1-5), were discovered from the whole plant of Huperzia serrata, along with two known alkaloids, 8α-hydroxyphlegmariurine B (6) and 8ß-hydroxyphlegmariurine B (7). The structures of 1-7 were identified through HR-MS, IR, 1 H, 13 C, and 2D NMR, and single-crystal X-ray diffraction analysis. Structurally, compound 1 was the fourth example of Lycopodium alkaloid with an ether linkage between C-5 and C-13 and 2 was the third example of Lycopodium alkaloid with a 5/5/5/5/6 pentacyclic ring system and featuring a 1-aza-7-oxabicyclo[2.2.1]heptane unit. Compounds 1-7 were tested for their BACE1 inhibitory activity. In addition, the correct 1 H- and 13 C-NMR data for 7 were reported in current study.

Endophytic Fungal Community of Huperzia serrata: Diversity and Relevance to the Production of Huperzine A by the Plant Host.

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.

Five novel and highly efficient endophytic fungi isolated from Huperzia serrata expressing huperzine A for the treatment of Alzheimer's disease.

Huperzine A (Hup A) is an important drug for treating Alzheimer's disease (AD) and mainly extracted from the Huperzia serrata (Thunb.) Trevis. (Lycopodiaceae) (HS). Nevertheless, the content of Hup A in HS is very low of 0.007% with growing circle of 8 to 10 years, and the chemical synthesis of Hup A still has some insurmountable limitations in the industrialized production. So, the available resources of Hup A for clinical treatment of AD are scarce. The purpose of this work was to construct a biosynthesis platform based on the endophytic fungi from HS. In this work, five endophytic fungi Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V were firstly found and isolated from the Chinese folk medicine HS, which were identified according to their morphological characteristics and nuclear ribosomal DNA ITS sequences. The highest efficient fungus could effectively biosynthesize Hup A in a liquid culture of 319.8 ± 0.17 mg/L which were 112 times higher than that of other reported conventional endophytic fungi. Moreover, these fungi with higher hereditary stability could possess the initial expressing ability of Hup A after 40 generations, and the expressed Hup A from these biosynthesis systems has prior physicochemical properties, a better inhibition activity of acetylcholinesterase and a lower cytotoxicity compared with the listed active pharmaceutical ingredients (APIs) of Hup A. These results provide promising alternative resources for producing Hup A at an industrial scale by biosynthesis, and it may also shed light on millions of AD patients. KEY POINTS: • Five novel endophytic fungi with high stability could highly express prior Hup A Graphical abstract.

A novel huperzine A-producing endophytic fungus Fusarium sp. Rsp5.2 isolated from Huperzia serrate.

The isolation, identification and characterization of a novel huperzine A (HupA)-producing fungal strain Rsp5.2 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in Vietnam. The fifty-eight endophytic fungi were recovered from roots of natural H. serrata in Lao Cai province of northern Vietnam and screened for HupA-producing by thin-layer chromatography (TLC). The only one of the 58 strains, Rsp5.2, could produce HupA. The amount of HupA produced by Rsp5.2 was quantified to be 19.45 µg g-1 dried mycelium by high-performance liquid chromatography (HPLC). Acetylcholine esterase (AchE) inhibition (IC50) of the crude HupA extract from Rsp5.2 fermentation broth was 2.849 ± 0.0026 µg mL-1. The fungus was identified as Fusarium sp. Rsp5.2 by morphological characteristics and Internal Transcribed Spacer (ITS) sequences. This is the first report of Fusarium sp. as a HupA-producing endophyte isolated from H. serrata.

Biotransformation of Huperzine B by a Fungal Endophyte of Huperzia serrata.

The biotransformation of huperzine B (hupB), one of the characteristic bioactive constituents of the medicinal plant Huperzia serrata, by a fungal endophyte of the host plant was studied. One new compound, 8α,15α-epoxyhuperzine B (1), along with two known oxygenated hupB analogs, 16-hydroxyhuperzine B (2) and carinatumin B (3), was isolated and identified. The structures of all the isolates were deduced by spectroscopic methods including NMR, MS, IR, and UV spectra. The known compounds 2 and 3 were obtained from a microbial source for the first time. To the best of our knowledge, it is the first report on the microbial transformation of hupB and would facilitate further structural modification of hupB by chemo-enzymatic method. In the LPS-induced neuro-inflammation injury assay, 8α,15α-epoxyhuperzine B (1) exhibited moderate neuroprotective activity by increasing the viability of U251 cell lines with an EC50 of 40.1 nm.

Global transcriptome analysis of Huperzia serrata and identification of critical genes involved in the biosynthesis of huperzine A.

BACKGROUND: Huperzia serrata (H. serrata) is an economically important traditional Chinese herb with the notably medicinal value. As a representative member of the Lycopodiaceae family, the H. serrata produces various types of effectively bioactive lycopodium alkaloids, especially the huperzine A (HupA) which is a promising drug for Alzheimer's disease. Despite their medicinal importance, the public genomic and transcriptomic resources are very limited and the biosynthesis of HupA is largely unknown. Previous studies on comparison of 454-ESTs from H. serrata and Phlegmariurus carinatus predicted putative genes involved in lycopodium alkaloid biosynthesis, such as lysine decarboxylase like (LDC-like) protein and some CYP450s. However, these gene annotations were not carried out with further biochemical characterizations. To understand the biosynthesis of HupA and its regulation in H. serrata, a global transcriptome analysis on H. Serrata tissues was performed. RESULTS: In this study, we used the Illumina Highseq4000 platform to generate a substantial RNA sequencing dataset of H. serrata. A total of 40.1 Gb clean data was generated from four different tissues: root, stem, leaf, and sporangia and assembled into 181,141 unigenes. The total length, average length, N50 and GC content of unigenes were 219,520,611 bp, 1,211 bp, 2,488 bp and 42.51%, respectively. Among them, 105,516 unigenes (58.25%) were annotated by seven public databases (NR, NT, Swiss-Prot, KEGG, COG, Interpro, GO), and 54 GO terms and 3,391 transcription factors (TFs) were functionally classified, respectively. KEGG pathway analysis revealed that 72,230 unigenes were classified into 21 functional pathways. Three types of candidate enzymes, LDC, CAO and PKS, responsible for the biosynthesis of precursors of HupA were all identified in the transcripts. Four hundred and fifty-seven CYP450 genes in H. serrata were also analyzed and compared with tissue-specific gene expression. Moreover, two key classes of CYP450 genes BBE and SLS, with 23 members in total, for modification of the lycopodium alkaloid scaffold in the late two stages of biosynthesis of HupA were further evaluated. CONCLUSION: This study is the first report of global transcriptome analysis on all tissues of H. serrata, and critical genes involved in the biosynthesis of precursors and scaffold modifications of HupA were discovered and predicted. The transcriptome data from this work not only could provide an important resource for further investigating on metabolic pathways in H. serrata, but also shed light on synthetic biology study of HupA.

Isolation, diversity and acetylcholinesterase inhibitory activity of the culturable endophytic fungi harboured in Huperzia serrata from Jinggang Mountain, China.

Huperzia serrata has many important medicinal properties with proven pharmacological potential. Some of these properties may be mediated by its endophytic fungi. To test this hypothesis, in the present study, we provided a first insights into evaluating the species composition and acetylcholinesterase (AChE) inhibitory activity of the culturable endophytic fungi of H. serrata from the regional at Jinggang Mountain in southeastern China. A total number of 885 fungal isolates distributed across 44 genera and 118 putative species were obtained from 1422 fragments of fine H. serrata roots, stems and leaves base on ITS-rDNA sequences BLAST analysis. The endophytic fungi were phylogenetically diverse and species-rich, with high rate of colonization and isolation. The assemble of endophytic fungi consisted mainly of Ascomycota (97.15%), followed by Basidiomycota (1.92%) and unknown fungal species (0.90%). Colletotrichum (64.29%), Phyllosticta (3.39%), Hypoxylon (2.81%), Xylaria (2.25%) and Nigrospora (2.04%) were the most abundant genera, whereas the remaining genera were infrequent groups. Although, roots yielded low abundance strains, the diverse and species-rich were both higher than that of stems and leaves. In addition, out of the 247 endophytic fungi strains determinated, 221 fungal extracts showed AChE inhibition activities in vitro. Among them, 22 endophytic fungi strains achieved high inhibitory activity (≥50%) on AChE which belongs to 13 genera and five incertae sedis strains. Four endophytic fungi designated as JS4 (Colletotrichum spp.), FL14 (Ascomycota spp.), FL9 (Sarcosomataceae spp.) and FL7 (Dothideomycetes spp.) were displayed highly active (≥80%) against AChE, which the inhibition effects were even more intense than the positive control. Our findings highlight that H. serrata grown in Jinggang Mountain harbors a rich and fascinating endophytic fungus community with potential AChE inhibitory activity, which could further broaden the natural acetylcholinesterase inhibitors resources used for Alzheimer's disease treatment.

Japanese Huperzia serrata extract and the constituent, huperzine A, ameliorate the scopolamine-induced cognitive impairment in mice.

Huperzia serrata has been used as a Chinese folk medicine for many years. It contains huperzine A, which has a protective effect against memory deficits in animal models; however, it is unclear if H. serrata extract exerts any effects in Alzheimer's disease (AD) models. We used H. serrata collected in Japan and determined its huperzine A content using HPLC. We determined its inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. H. serrata extract (30 mg/kg/day) and donepezil (10 mg/kg/day) were orally administrated for 7 days. After repeated administration, we performed the Y-maze and passive avoidance tests. H. serrata extract contained 0.5% huperzine A; H. serrata extract inhibited AChE, but not BuChE. H. serrata extract ameliorated cognitive function in mice. These results indicate that Japanese H. serrata extract ameliorates cognitive function deficits by inhibiting AChE. Therefore, H. serrata extract may be valuable for the prevention or treatment of dementia in AD.

Lycopodium alkaloids from Huperzia serrata and their cholinesterase inhibitory activities.

Huperzia serrata, belonging to the Lycopodiaceae family, has been traditionally utilized for the management of treating rheumatic numbness, arthritic pain, dysmenorrhea, and contusions. This plant is a rich source of lycopodium alkaloids, some of which have demonstrated notable cholinesterase inhibitory activity. The objective of this study was to identify lycopodium alkaloids with cholinesterase inhibitory properties from H. serrata. The structures of these alkaloids were elucidated by HRESIMS, NMR (including a 1H-15N HMBC experiment), ECD methods and single-crystal X-ray diffraction. The inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were assessed using a modified Ellman's method. Consequently, sixteen lycopodium alkaloids (1-16), including ten previously undescribed ones named huperradines A-G and huperradines I-K (1-7 and 9-11), along with one previously undescribed naturally occurring compound, huperradine H (8), were isolated from H. serrata. Among these, compounds 7 and 1 exhibited potent and moderate AChE inhibition, with IC50 values of 0.876 ± 0.039 µM and 13.125 ± 0.521 µM, respectively. Our results suggest that huperradine G (7) may be a promising lead compound for the development of new AChE inhibitors for Alzheimer's disease.

Secondary metabolites from Penicillium sp. HS-11, a fungal endophyte of Huperzia serrata.

Three new sorbicillinoids sorbicatechols E-G (1-3), along with seven known compounds 4-10, were obtained from the ethanol extract of Penicillium sp. HS-11, a fungal endophyte of the medicinal plant Huperzia serrata. The structures of 1-3 were established by detailed interpretation of the spectroscopic data and their absolute configurations were established by comparative analyses of the ECD spectra. Sorbicatechol G (3) represented the first hybrid sorbicillinoid bearing a tetralone skeleton. In the in-vitro bioassay, trichodimerol (5) exhibited moderate inhibitory activity against the Escherichia coli ß-glucuronidase (EcGUS) with an IC50 value of 92.0 ± 9.4 µM.

Combined transcriptome and widely targeted metabolome analysis reveals the potential mechanism of HupA biosynthesis and antioxidant activity in Huperzia serrata.

Introduction: Huperzia serrata is a traditional Chinese herb that has gained much attention for its production of Huperzine A (HupA). HupA has shown promise on treating Alzheimer's disease (AD). However, the biosynthetic pathway and molecular mechanism of HupA in H. serrata are still not well understood. Methods: Integrated transcriptome and metabolome analysis was performed to reveal the molecular mechanisms related to HupA biosynthesis and antioxidant activity in Huperzia serrata. Results: HT (in vitro H. serrata thallus) exhibits higher antioxidant activity and lower cytotoxicity than WH (wild H. serrata). Through hierarchical clustering analysis and qRT-PCR verification, 7 important enzyme genes and 13 transcription factors (TFs) related to HupA biosynthesis were detected. Among them, the average |log2FC| value of CYP (Cytochrome P450) and CAO (Copper amine oxidase) was the largest. Metabolomic analysis identified 12 metabolites involved in the HupA biosynthesis and 29 metabolites related to antioxidant activity. KEGG co-enrichment analysis revealed that tropane, piperidine and pyridine alkaloid biosynthesis were involved in the HupA biosynthesis pathway. Furthermore, the phenylpropanoid, phenylalanine, and flavonoid biosynthesis pathway were found to regulate the antioxidant activity of H. serrata. The study also identified seven important genes related to the regulation of antioxidant activity, including PrAO (primary-amine oxidase). Based on the above joint analysis, the biosynthetic pathway of HupA and potential mechanisms of antioxidant in H. serrata was constructed. Discussion: Through differential transcriptome and metabolome analysis, DEGs and DAMs involved in HupA biosynthesis and antioxidant-related were identified, and the potential metabolic pathway related to HupA biosynthesis and antioxidant in Huperzia serrata were constructed. This study would provide valuable insights into the HupA biosynthesis mechanism and the H. serrata thallus medicinal value.

[Mining and identification of a biosynthetic gene cluster producing xanthocillin analogues from Penicillium chrysogenum MT-40, an endophytic fungus of Huperzia serrata].

Xanthocillin is a unique natural product with an isonitrile group and shows remarkable antibacterial activity. In this study, the genome of an endophytic fungus Penicillium chrysogenum MT-40 isolated from Huperzia serrata was sequenced, and the gene clusters with the potential to synthesize xanthocillin analogues were mined by local BLAST and various bioinformatics analysis tools. As a result, a biosynthetic gene cluster (named for) responsible for the biosynthesis of xanthocillin analogues was identified by further heterologous expression of the key genes in Aspergillus oryzae NSAR1. Specifically, the ForB catalyzes the synthesis of 2-formamido-3-(4-hydroxyphenyl) acrylic acid, and the ForG catalyzes the dimerization of 2-formamido-3-(4-hydroxyphenyl) acrylic acid to produce the xanthocillin analogue N, N'-(1, 4-bis (4-hydroxyphenyl) buta-1, 3-diene-2, 3-diyl) diformamide. The results reported here provide a reference for further discovery of xanthocillin analogues from fungi.

Two new diterpenoids from Penicillium chrysogenum MT-12, an endophytic fungus isolated from Huperzia serrata.

Two new diterpenoids, penicichrysogene A (1) and penicichrysogene B (2), were isolated from the solid substrate fermentation cultures of Penicillium chrysogenum MT-12, an endophytic fungus isolated from the medicinal plant of Huperzia serrata. Their structures were elucidated on the basis of extensive spectroscopic and spectrometric data (1D and 2D NMR, UV, IR, and HRESIMS). The absolute configurations of 1 and 2 were assigned on the basis of experimental and calculated electronic circular dichroism spectra. Compound 1 exhibited inhibitory activity on ATP release of thrombin-activated platelets with IC50 = 42.7 ± 3.5 µM.

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

Huperzia serrata is a traditional herb and endangered Chinese medicinal material, which has attracted much attention due to its production of Huperzine A (HupA). In vitro propagation of H. serrata is considered a new way to relieve the resource pressure of H. serrata. In this study, three different genotypic wild H. serrata were used for in vitro propagation. Then, the antioxidant activity and the content of HupA in the regenerated H. serrata were investigated. The results showed the survival rate of the explant was increased to 25.37% when using multiple sterilization processes. The best induction medium for H. serrata was the Schenk and Hildebrandt (SH) medium supplemented with 0.5 mg·L-1 Naphthalene acetic acid (NAA) and 0.1 mg·L-1 2,4-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 vitro H. serrata had higher antioxidant activity than that of wild H. serrata. This study provides a reliable in vitro H. serrata culture protocol and laid an important foundation for the antioxidant capacity of the thallus and the content of HupA.