A new secondary metabolite from the marine-derived fungus Phomopsis lithocarpus FS508.

Based on the One Strain-Many Compounds (OSMAC) strategy, the secondary metabolites of Phomopsis lithocarpus FS508 were investigated. As a result, a new secondary metabolite, 4-methoxy-3-[4-(acetyloxy)-3-methyl-2-butenyl]benzoic acid (1) as well as eleven known compounds were isolated from the fermentation product of the strain FS508. Their structures were determined by NMR, IR, UV, and MS spectroscopic data analyses. All the isolated compounds were evaluated for cytotoxic and anti-inflammatory activities. Among them, compounds 3 and 9 displayed potent cytotoxicity against HepG-2 cell line, and compounds 2, 3 and 12 showed significant anti-inflammatory activities.

Cytotoxic pyrone derivatives from the deep-sea-derived fungus Cladosporium halotolerans FS702.

Two new compounds (R)-6-((8S)-hydroxypropyl)-2-methyl-5,6-dihydro-4H-pyran-4-one (1) and (R)-6-((8R)-hydroxypropyl)-2-methyl-5,6-dihydro-4H-pyran-4-one (2), together with four known compounds were isolated from the marine-derived fungus Cladosporium halotolerans FS702. The structures of these compounds were determined on the basis of extensive spectroscopic analysis including 1D/2D NMR, IR, UV, HRESIMS, ECD calculations as well as the modified Mosher's method. Cytotoxic assay results showed that compound 2 had significant cytotoxic activity against SF-268, MCF-7, HepG-2, and A549 cells lines with IC50 values of 0.16, 0.47, 0.33 and 0.23 µM, respectively.

3-Carboxy-indole derivatives from the deep-sea-derived fungus Phomopsis tersa FS441.

Three new compounds phomtersines A-C (1-3) together with nine known compounds were isolated from the marine-derived fungus Phomopsis tersa FS441. Their structures were sufficiently established by spectroscopic methods, including extensive 1D and 2D NMR techniques and modified Snatzke's method. Moreover, compounds 1-12 were evaluated for cytotoxic and anti-inflammatory activities. As a result, phomtersine B (2) and the known compound 10 showed moderate cytotoxic activity against the four tested cell lines with IC50 values ranging from 20.21 to 36.53 µM, and phomtersine A (1) exhibited moderate inhibitory activity against LPS-induced NO production.

Comparative physicochemical, hormonal, transcriptomic and proteomic analyses provide new insights into the formation mechanism of two chemotypes of Pogostemon cablin.

Patchouli (Pogostemon cablin) is an aromatic plant, and its oil has diverse applications in medicine, food, and cosmetics. Patchouli alcohol is the principal bioactive constituent of its volatile oil. In China, patchouli is typically categorized into two types: patchoulol-type (PA-type) and pogostone-type (PO-type). The study evaluated physiological and biochemical indicators, phytohormone metabolites and conducted transcriptome and proteome analyses on both two chemotypes. The PA-type exhibited higher levels of chlorophyll a, b, and carotenoids than the PO-type. In total, 35 phytohormone metabolites representing cytokinin, abscisic acid, gibberellin, jasmonic acid, and their derivatives were identified using UPLC-MS/MS, 10 of which displayed significant differences, mainly belong to cytokinins and jasmonates. Transcriptome analysis identified 4,799 differentially expressed genes (DEGs), while proteome analysis identified 150 differentially expressed proteins (DEPs). Regarding the transcriptome results, the DEGs of the PO-type showed significant downregulation in the pathways of photosynthesis, photosynthesis-antenna protein, porphyrin and chlorophyll metabolism, carotenoid biosynthesis, sesquiterpene and triterpenoid biosynthesis, and starch and sucrose metabolism, but upregulation in the pathway of zeatin synthesis. A combination of transcriptome and proteome analyses revealed that the DEGs and DEPs of lipoxygenase (LOX2), ß-glucosidase, and patchouli synthase (PTS) were collectively downregulated, while the DEGs and DEPs of Zeatin O-xylosyltransferase (ZOX1) and α-amylase (AMY) were jointly upregulated in the PO-type compared to the PA-type. Differential levels of phytohormones, variations in photosynthetic efficiency, and differential expression of genes in the sesquiterpene synthesis pathway may account for the morphological and major active component differences between the two chemotypes of patchouli. The findings of this study offer novel perspectives on the underlying mechanisms contributing to the formation of the two patchouli chemotypes.

Plastid Phylogenomic Insights into the Inter-Tribal Relationships of Plantaginaceae.

Plantaginaceae, consisting of 12 tribes, is a diverse, cosmopolitan family. To date, the inter-tribal relationships of this family have been unresolved, and the plastome structure and composition within Plantaginaceae have seldom been comprehensively investigated. In this study, we compared the plastomes from 41 Plantaginaceae species (including 6 newly sequenced samples and 35 publicly representative species) representing 11 tribes. To clarify the inter-tribal relationships of Plantaginaceae, we inferred phylogenic relationships based on the concatenated and coalescent analyses of 68 plastid protein-coding genes. PhyParts analysis was performed to assess the level of concordance and conflict among gene trees across the species tree. The results indicate that most plastomes of Plantaginaceae are largely conserved in terms of genome structure and gene content. In contrast to most previous studies, a robust phylogeny was recovered using plastome data, providing new insights for better understanding the inter-tribal relationships of Plantaginaceae. Both concatenated and coalescent phylogenies favored the sister relationship between Plantagineae and Digitalideae, as well as between Veroniceae and Hemiphragmeae. Sibthorpieae diverged into a separate branch which was sister to a clade comprising the four tribes mentioned above. Furthermore, the sister relationship between Russelieae and Cheloneae is strongly supported. The results of PhyParts showed gene tree congruence and conflict to varying degrees, but most plastid genes were uninformative for phylogenetic nodes, revealing the defects of previous studies using single or multiple plastid DNA sequences to infer the phylogeny of Plantaginaceae.

SMRT sequencing of full-length transcriptome and gene expression analysis in two chemical types of Pogostemon cablin (Blanco) Benth.

BACKGROUND: Pogostemon cablin (Blanco) Benth. also called patchouli, is a traditional medicinal and aromatic plant that grows mainly in Southeast Asia and China. In China, P. cablin is divided into two chemical types: the patchouliol-type and the pogostone-type. Patchouliol-type patchouli usually grow taller, with thicker stems and bigger leaves, and produce more aromatic oil. METHODS: To better understand the genetic differences between the two chemical types that contribute to their differences in morphology and biosynthetic capabilities, we constructed de novo transcriptomes from both chemical types using the Pacific Biosciences (PacBio) Sequel platform and performed differential expression analysis of multiple tissues using Illumina short reads. RESULTS: In this study, using single-molecule real-time (SMRT) long-read sequencing, we obtained 22.07 GB of clean data and 134,647 nonredundant transcripts from two chemical types. Additionally, we identified 126,576 open reading frames (ORFs), 100,638 coding sequences (CDSs), 4,106 long noncoding RNAs (lncRNAs) and 6,829 transcription factors (TFs) from two chemical types of P. cablin. We adopted PacBio and Illumina sequencing to identify differentially expressed transcripts (DEGs) in three tissues of the two chemical types. More DEGs were observed in comparisons of different tissues collected from the same chemical type relative to comparisons of the same tissue collected from different chemical types. Furthormore, using KEGG enrichment analysis of DEGs, we found that the most enriched biosynthetic pathways of secondary metabolites of the two chemical types were "terpenoid backbone biosynthesis", "phenylpropanoid biosynthesis", "plant hormone signal transduction", "sesquiterpenoid and triterpenoid biosynthesis", "ubiquinone and other terpenoid-quinone biosynthesis", "flavonoid biosynthesis", and "flavone and flavonol biosynthesis". However, the main pathways of the patchouliol-type also included "diterpene biosynthesis" and "monoterpene biosynthesis". Additionally, by comparing the expression levels of the three tissues verified by qRT-PCR, more DEGs in the roots were upregulated in the mevalonate (MVA) pathway in the cytoplasm, but more DEGs in the leaves were upregulated in the methylerythritol phosphate (MEP) pathway in the plastid, both of which are important pathways for terpenoids biosynthesis. These findings promote the study of further genome annotation and transcriptome research in P. cablin.

[Biosynthesis and metabolism regulation of terpenoids in Pogostemon cablin: a review].

The terpenoids in Pogostemon cablin have complex structures and abundant pharmacological effects. Patchouli alcohol(PA) and pogostone(PO) have a high medicinal value by virtue of anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other biological activities. Due to the low content of terpenoid metabolites in P. cablin, the study of biosynthesis and metabolism regulation can provide a biosynthetic basis for obtaining high-content terpenoids. In this study, key enzyme genes in biosynthesis, transcription factors in metabolism regulation, spatio-temporal expression of terpene synthase were reviewed, aiming to provide a reference for the development, protection, and utilization of P. cablin resources.

[Effects of 5-azacytidine on DNA methylation and index components in patchouliol-type Pogostemon cablin].

This study aims to explore the relationship of DNA methylation with the contents of the index components as well as the growth and development of Pogostemon cablin. The demethylation reagent 5-azacytidine(5-azaC) was used to treat the tissue culture seedlings of patchouliol-type P. cablin. High performance liquid chromatography was employed to evaluate the changes of DNA methy-lation in P. cablin, and GC-MS to detect the contents of index components in P.cablin. The agronomic characters of P.cablin were measured using the common methods. The results showcased that DNA methylation of P.cablin was significantly reduced by 5-azaC in a concentration-dependent manner. Thirty days after treatment with 5-azaC at different concentrations, the content of patchouli alcohol changed slightly; compared with that in the control group, the content of pogostone in 50 µmol·L~(-1) and 100 µmol·L~(-1) 5-azaC groups was significantly up-regulated. The 100 µmol·L~(-1) 5-azaC group had the largest differences in contents of pogostone and patchouli alcohol compared with the control group, followed by the 50 µmol·L~(-1) 5-azaC group. Ninety days after disinhibition, the content of pogostone in the treatment group was significantly increased and the content of patchouli alcohol was significantly decreased. In addition, 5-azaC significantly inhibited the growth and development of P.cablin in a dose-dependent manner. These results indicate that DNA methylation regulates the biosynthesis of the index components in patchouliol-type P.cablin and proper demethylation can directly promote the synthesis of pogostone and indirectly affect the accumulation of patchouli alcohol.

The complete plastomes of two flowering epiparasites (Phacellaria glomerata and P. compressa): Gene content, organization, and plastome degradation.

A plant parasite obligately parasitizing another plant parasite is referred to as epiparasite, which is extremely rare in angiosperms, and their complete plastome sequences have not been characterized to date. In this study, the complete plastomes of two flowering epiparasites: Phacellaria compressa and P. glomerata (Amphorogynaceae, Santalales) were sequenced. The plastomes of both species are of similar size, structure, gene content, and arrangement of genes to other hemiparasites in Santalales. Their plastomes were characterized by the functional loss of plastid-encoded NAD(P)H-dehydrogenase and infA genes, which strongly coincides with the general pattern of plastome degradation observed in Santalales hemiparasites. Our study demonstrates that the relatively higher level of nutritional reliance on the host plants and the reduced vegetative bodies of P. compressa and P. glomerata do not appear to cause any unique plastome degradation compared with their closely related hemiparasites.

Intercropping With Turmeric or Ginger Reduce the Continuous Cropping Obstacles That Affect Pogostemon cablin (Patchouli).

Continuous cropping (CC) restricts the development of the medicinal plant cultivation industry because it alters soil properties and the soil microbial micro-ecological environment. It can also lead to reductions in the chemical contents of medicinal plants. In this study, we intercropped continuously cropped Pogostemon cablin (patchouli) with turmeric or ginger. High-throughput sequencing was used to study the soil bacteria and fungi. Community composition, diversity, colony structure, and colony differences were also analyzed. A redundancy analysis (RDA) was used to study the interactions between soil physical and chemical factors, and the bacteria and fungi. The correlations between the soil community and the soil physical and chemical properties were also investigated. The results showed that intercropping turmeric and ginger with patchouli can improve soil microbial abundance, diversity, and community structure by boosting the number of dominant bacteria, and by improving soil bacterial metabolism and the activities of soil enzymes. They also modify the soil physical and chemical properties through changes in enzyme activity, soil pH, and soil exchangeable Ca (Ca). In summary, turmeric and ginger affect the distribution of dominant bacteria, and increase the contents of the active ingredient in patchouli. The results from this study suggested that the problems associated with continuously cropping patchouli can be ameliorated by intercropping it with turmeric and ginger.

The complete plastome of Gymnotheca chinensis (Saururaceae) and its phylogenetic analysis.

The complete plastome of Gymnotheca chinensis, an important medicinal herb, was firstly elucidated and analyzed in this study. The plastome is 161,621 bp in size, which comprises of one large single-copy (LSC) region and one small single-copy (SSC) region of 89,291 bp and 18,592 bp, respectively, separated by a pair of IR regions of 26,869 bp each. It encodes a total of 132 genes, including 87 protein-coding genes, 37 tRNA, and 8 rRNA. The phylogeny robustly supports that G. chinensis is sister to the clade including Piper kadsura, Piper cenocladum, Saruma henryi, Asarum sieboldii.

Characterization of the complete plastome of medicinal plant Saururus chinensis (Saururaceae).

Saururus chinensis is an important medicinal plant in Southeast Asia. Here, we determined the first complete plastome of S. chinensis using high throughput Illumina sequencing technology. The S. chinensis plastome is 161,494 bp in length and presents a typical quadripartite structure consisting of one large single-copy region (LSC, 88,863 bp), one small single-copy region (SSC, 18,679 bp), and a pair of inverted repeat regions (IRs, 26,976 bp each). The phylogenetic analysis robustly supports that S. chinensis is sister to the group including the Saruma henryi, Asarum sieboldii, Piper kadsura, Piper cenocladum.

Complete plastome of Houttuynia cordata (Saururaceae), a medicinal and edible plant.

The complete plastome of Houttuynia cordata, an important medicinal and edible plant, was identified and sequenced in this study. The circular plastome is 160,217 bp in length and consists of a pair of inverted repeats (IRs 26,854 bp each), which is separated by a large single-copy region (LSC, 88,189 bp) and a small single-copy region (SSC, 18,320 bp). It encodes 132 genes, of which 114 are unique genes (80 protein-coding genes, 30 tRNAs, and 4 rRNAs). The phylogenetic analysis strongly reveals the sister group between H. cordata and the clade including Piper kadsura, Piper cenocladum, Saruma henryi, and Asarum sieboldii.

Molecular Identification of Three Aquilaria (Thymelaeaceae) Species through DNA Barcoding.

Aquilaria LAM. is an endangered tropical tree that produces agarwood, a common ingredient in medicine, perfumes and incense. The species endemic to China, Aquilaria yunnanensis, is often misidentified as the two valuable species, Aquilaria sinensis and Aquilaria crassna. In present study, three DNA barcodes (internal transcribed spacer (ITS), maturase K gene (matK) and trnL-trnF) were used to evaluate whether these genes can be used to discriminate the three species, and evaluate the phylogenetic relationship between the three Aquilaria species. For accurate identification of the three Aquilaria species, a total of 26 nucleotide variations were detected when comparing the three DNA barcodes. We found that A. sinensis is closely related to A. crassna based on combination of nuclear and chloroplast DNA barcodes, and is closely related to A. yunnanensis based on chloroplast DNA barcodes. Taken together, we suggest that the combination of ITS+matK and ITS+trnL-trnF are suitable for identifying these three Aquilaria species.

In vitro induction and morphological characteristics of octoploid plants in Pogostemon cablin.

This paper describes an efficient colchicine-mediated technique for in vitro induction of octoploids in Pogostemon cablin and its confirmation by flow cytometry and chromosome numbers. The highest octoploid induction ratio was obtained by 0.05% colchicine treatment for 72 h. The chromosome number of octoploid seedlings was 2n = 8x = 128. Colchicine-induced tetraploids and octoploids planted in soil remained stable after 6 months. There were 31 lines of octoploid plants obtained. The leaf characteristics of P. cablin tetraploids and octoploids were compared. The larger leaves and stomata of transplants can be used to identify putative octoploids in P. cablin. Most octoploid lines exhibited higher patchoulic alcohol contents than the controls after 6 months of cultivation. Our results demonstrated that polyploidy induction can be beneficial in improving the medicinal value of P. cablin.

[Study on cytotoxic secondary metabolites of endophytic fungus Diaporthe longicolla A616 from Pogostemon cablin].

To study active secondary metabolites of endophytic fungus Diaporthe longicolla A616 isolated from Pogostemon cablin. Ten compounds were isolated from fermentation product of the strain 616 by silica gel, reverse phase silica gel, Sephadex-LH20, HPLC and so on. Their structures were identified as 1,3-diamino-1,3-dimethylurea(1),(7R,9R)-7-hydroxy-9-propyl-5-nonen-9-olide(2), Ergosta-5,7,22-trien-3ß-ol(3),(22E,24R)-ergosta-4,6,8(14)-22-tetraen-3-one(4),(22E,24R)-3ß,5α-dihydroxy-6ß-ergosta-7,22-diene(5), citreoisocoumarin(6), glycerol monolinoleate(7), 1-(2-hydroxyethoxy)ethyl(E)-octadec-9-enoate(8), cyclo-(L-Pro-L-Ala)(9), cyclo(L)-Pro-(L)-Val(10), respectively, based on extensive spectroscopic analysis and literature comparisons. Compounds 6-10 were isolated from the genus Diaporthe for the first time. All isolated compounds were evaluated for in vitro cytotoxic activities against SF-268, MCF-7, NCI-H460 and HepG-2 tumor cell lines. Compounds 4 and 5 showed potent growth inhibitory activities against the four cell lines with IC50 values of 5.3, 6.5, 12.2, 6.1µmol•L⁻¹ and 8.2, 5.2, 6.1, 9.4µmol•L⁻¹, respectively.

[Study on Chemical Constituents of Petroleum Ether Fraction from Rubus alceaefolius].

OBJECTIVE: To investigate the chemical constituents of Rubus alceaefolius. METHODS: Nine compounds were isolated and purified from the petroleum ether extract of 95% alcohol extract of Rubus alceaefolius by repeated column chromatography on silica, Sephadex LH-20 and structurally identified by spectral analysis. RESULTS: The compounds were identified as chrysophanol(1), physcion (2), ß-sitosterol(3), 3-oxotirucalla-7, 24-dien-21-oic acid(4), myricadiol(5), 19-α-hydroxy-3-acetyl-ursolic acid(6), N-benzoylphenylalaninyl-N-benzoylphenylalaninate(7), aurantiamide acetate(8) and euscaphic acid(9). CONCLUSION: Compounds land 4~8 are isolated from this plant for the first time, and compounds 4 - 8 are found in plants of Rubus genus for the first time.

[Cultural regionalization for Notopterygium incisum based on 3S technology platform. I. Evaluation for growth suitability for N. incisum based on ecological factors analysis by Maxent and ArcGIS model].

Growth suitability as assessment indicators for medicinal plants cultivation was proposed based on chemical quality determination and ecological factors analysis by Maxent and ArcGIS model. Notopterygium incisum, an endangered Chinese medicinal plant, was analyzed as a case, its potential distribution areas at different suitability grade and regionalization map were formulated based on growth suitability theory. The results showed that the most suitable habitats is Sichuan province, and more than 60% of the most suitable areawas located in the western Sichuan such as Aba and Ganzi prefectures for N. incisum. The results indicated that habitat altitude, average air temperature in September, and vegetation types were the dominant factors contributing to the grade of plant growth, precipitation and slope were the major factors contributing to notopterol accumulation in its underground parts, while isoimperatorin in its underground parts was negatively corelated with precipitation and slope of its habitat. However, slope as a factor influencing chemical components seemed to be a pseudo corelationship. Therefore, there were distinguishing differences between growth suitability and quality suitability for medicinal plants, which was helpful to further research and practice of cultivation regionalization, wild resource monitoring and large-scale cultivation of traditional Chinese medicine plants.

[Determination of anthraquinones from different medicinal parts of Berchemia lineata by HPLC].

OBJECTIVE: To develop an HPLC method for determination of emodin,chrysophanol and physcion from different medicinal parts of Berchemia lineata. METHODS: Samples were analyzed on Diamonsil ODS C18 (250 mm x 4. 6 mm,5 µm), with the mobile phase consisted of methanol-0. 20% phosphoric acid solution(74: 26). The flow rate was 1.0 mL/min,column temperature was set at 35 °C ,and detection UV wavelength was 254 nm. RESULTS: The linear range of emodin, chrysophanol and physcion was 0. 00201~ 0. 0804 µg,0. 0066~0. 264 µg and 0. 0124 ~0. 496 µg,with the average recovery was 100. 43% ,101. 29% and 98. 36% ,respectively. The content of total anthraquinones in root was higher than that in taten of Berchemia lineata. CONCLUSION: The method is simple,accurate and reliable for quality control of Berchemia lineata.

[Effects of agarwood formation induced by Fusarium sp. A2 on distribution and community composition of endophytic fungi in leaves of Aquilaria sinensis].

The aim of the paper is to observe the distribution of the endophytic fungi in leaves of Aquilaria sinensis by using permanent paraffin-cut section, optical microscope photography and histochemistry. Total DNA was extracted with modified CTAB method and rDNA ITS regions of plant and endophytic fungi were amplified with eukaryotic universal primers. The rDNA ITS amplicon was characterized by RFLP analysis, sequencing of rDNA ITS library and phylogenetic analyses using PAUP by maximum parsimony. Fusarium sp. A2 was used to induce the formation of resinous in A. sinensis trees. As a result, endophytic fungi mainly distributed in spongy and phloem in leaf. Endophytic fungi distributed in the phloem in agarwood-producing tree and had a relatively high abundance. Phoma sp. and Collectrotrichum sp. were the absolute advantage species in the leaf tissues of non-resinous and agarwood-producing tree, respectively. Collectrotrichum sp. was the only fungal species detected both in the two types of A. sinensis with different levels of abundance. The culture-independent molecular method can be used to identify fungal species directly and rapidly from the plant tissues. Endophytic fungal communities in non-resinous and agarwood-producing A. sinensis leaf tissues were quite different.