Environmental differences between Japan and Indonesia provide endophyte diversity associated with Artemisia plant and variety of artemisinin derivatives in microbial conversion.

We compared the endophytic compositions of Artemisia plant from different environments (Japan and Indonesia) to demonstrate that the endophytic filamentous fungi in both species differed based on their environments. To prove that the species were identical, both Artemisia plants were identified by comparing the scanning electron micrographs of their pollens, as well as the nucleotide sequences (ribosomal internal transcribed spacer and mitochondrial maturase K) of the two gene regions. After isolating the endophytic filamentous fungi from each plant, we observed that those from Japan and Indonesia comprised 14 and 6 genera, respectively. We assumed that the genera, Arthrinium and Colletotrichum, which exist in both Artemisia species, were species-specific filamentous fungi, while the other genera were environment-dependent. In the microbial-conversion reaction with artemisinin as a substrate using Colletotrichum sp., the peroxy bridge of artemisinin, which is an active site for achieving antimalarial effect, was converted into an ether bond. However, the reaction using the environment-dependent endophyte did not eliminate the peroxy bridge. These endophytic reactions indicated the different roles of endophytes within Artemisia plants.

Challenging Structure Elucidation of Lumnitzeralactone, an Ellagic Acid Derivative from the Mangrove Lumnitzera racemosa.

The previously undescribed natural product lumnitzeralactone (1), which represents a derivative of ellagic acid, was isolated from the anti-bacterial extract of the Indonesian mangrove species Lumnitzera racemosa Willd. The structure of lumnitzeralactone (1), a proton-deficient and highly challenging condensed aromatic ring system, was unambiguously elucidated by extensive spectroscopic analyses involving high-resolution mass spectrometry (HRMS), 1D 1H and 13C nuclear magnetic resonance spectroscopy (NMR), and 2D NMR (including 1,1-ADEQUATE and 1,n-ADEQUATE). Determination of the structure was supported by computer-assisted structure elucidation (CASE system applying ACD-SE), density functional theory (DFT) calculations, and a two-step chemical synthesis. Possible biosynthetic pathways involving mangrove-associated fungi have been suggested.

Endophytic fungi inhabiting Physalis angulata L. plant: diversity, antioxidant, and antibacterial activities of their ethyl acetate extracts.

OBJECTIVES: Endophytic fungi are an essential source of biologically active compounds. They have the ability to synthesize secondary metabolites which are the same or have a high degree of similarity to their host plants. In this study, we aimed to explore the biodiversity and the bioactivities of active metabolites produced by 14 endophytic fungi isolated from the medicinal plant Physalis angulata L. (PA). METHODS: Fourteen endophytic fungi were isolated from the flowers, stems, leaves, and fruit husks of PA. The endophytic fungi were cultured and incubated in the PDB medium at room temperature. After three weeks, the cultures were extracted using ethyl acetate and dried using a rotary evaporator. The antioxidant activity was evaluated against DPPH while antibacterial activity was evaluated against Escherichia coli and Staphylococcus aureus using microdilution technique. TLC analysis was also done to profile the active compounds within the extract. RESULTS: Hyphomycetes fungus isolated from the flower of PA exhibited a moderate antioxidant activity with an antioxidant index value of 0.59 (IC50 = 52.43 µg/mL). Six isolates have strong antibacterial activity against E. coli and S. aureus with minimum inhibitory concentration (MIC) value ranging from 8-64 µg/mL. These endophytic fungi are one Hyphomycetes fungus isolated from the flower, one Fusarium sp. isolated from the stem, and four Colletotrichum sp. isolated from leaf and fruit husk of PA. CONCLUSIONS: Endophytic fungi isolated from PA are potential novel sources of active metabolites especially for antibacterial compounds.

Endophyte composition and Cinchona alkaloid production abilities of Cinchona ledgeriana cultivated in Japan.

New eight endophytic filamentous fungi were isolated from the young stems of Cinchona ledgeriana (Rubiaceae) cultivated in Japan. They were classified into four genera based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2), including the 5.8S ribosomal DNA region. Of the eight fungi isolated, there were five genera Cladosporium, one Meira sp., one Diaporthe sp. and one Penicillium sp. Genus of Cladosporium and Meira were first isolated fungi from Cinchona plant. In a previous study, we applied the same process to the same plant cultivated in Indonesia. The endophyte compositions for the two cultivation regions were found to differ at the genera level. The ability of Cinchona endophytes cultivated in Japan to produce Cinchona alkaloids was also assessed. We found that three isolates have producing ability of Cinchona alkaloids. However, the amount produced was very small compared to that produced by the endophytes of Indonesian Cinchona ledgeriana. In addition, the total content amount of Cinchona alkaloids, especially quinine, produced by the extract of Cinchona cultivated in Japan was much smaller than that from Indonesia. These finding indicate that endophyte composition has an influence on the Cinchona alkaloid content amount in the Cinchona ledgeriana host.

Two-dimensional NMR analysis of Angiopteris evecta rhizome and improved extraction method for angiopteroside.

INTRODUCTION: The rhizome of Angiopteris evecta is of academic interest in Kalimantan, Indonesia, from an ethnobotanical perspective. Angiopteroside is a substance of pharmaceutical importance that is found in the rhizome of A. evecta. OBJECTIVE: The aims of this research are to improve the extraction method for angiopteroside from the rhizome, compared to that in a previous report, and to determine the yield of angiopteroside from the rhizome of A. evecta, as well as to obtain precise data for extractives from the rhizome of A. evecta by using two-dimensional NMR spectroscopy and liquid chromatography-mass spectrometry (LC-MS). METHODOLOGY: We investigated the chemical constituents of the whole rhizome by means of two-dimensional NMR (heteronuclear single quantum coherence or HSQC) spectroscopy, neutral sugar analysis using the alditol acetate method, and lignin analysis using alkaline nitrobenzene oxidation and Klason lignin methods. LC-MS revealed the purity of the angiopteroside. Antimicrobial assays were also performed for the purified angiopteroside by using a broth microdilution method. RESULTS: Angiopteroside was isolated by Soxhlet extraction with aqueous acetone followed by preparative thin-layer chromatography (eluent: 20% methanol/dichloromethane). LC-MS revealed that angiopteroside can be found in the rhizome of A. evecta in 9.9% yield, which is an extremely high yield for a plant extractive. CONCLUSION: HSQC analysis is a powerful tool for surveying compounds in plant materials, such as the whole rhizome of A. evecta. Soxhlet extraction with aqueous acetone is an effective method for extracting glycosides from plant materials.

Composition of the endophytic filamentous fungi associated with Cinchona ledgeriana seeds and production of Cinchona alkaloids.

Four kinds of endophytic filamentous fungi (code names: CLS-1, CLS-2, CLS-3, and CLS-4) associated with the seeds of Cinchona ledgeriana (Rubiaceae) from West Java, Indonesia, were isolated. All of the isolates were classified into Diaporthe spp. based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) including the 5.8S ribosomal DNA region. All four of these endophytic fungi produce Cinchona alkaloids, mainly quinine and quinidine, in synthetic liquid medium.

Bisanthraquinone metabolites produced by the endophytic fungus Diaporthe sp.

Two bisanthraquinones named (+)-epicytoskyrin (1) and (+)-1,1'-bislunatin (2) were produced by the endophytic fungus from a tea plant, which is a species closely related to Diaporthe phaseolorum strain sw-93-13. The chemical structures of the metabolites were elucidated on the basis of the physicochemical properties including the circular dichroism (CD) spectrum.

Composition of the endophytic filamentous fungi isolated from the tea plant Camellia sinensis.

It has been found by ribosomal DNA analysis that the endophytic filamentous fungi isolated from the tea plant Camellia sinensis (Theaceae) are composed of six groups; one Fusarium sp., one Penicillium sp., two Schizophyllum sp., and two Diaporthe sp..

Biooxidation of (+)-catechin and (-)-epicatechin into 3,4-dihydroxyflavan derivatives by the endophytic fungus Diaporthe sp. isolated from a tea plant.

The microbial transformation of (+)-catechin (1) and (-)-epicatechin (2) by endophytic fungi isolated from a tea plant was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. transformed them (1, 2) into the 3,4-cis-dihydroxyflavan derivatives, (+)-(2R,3S,4S)-3,4,5,7,3',4'-hexahydroxyflavan (3) and (-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavan (7), respectively, whereas (-)-catechin (ent-1) and (+)-epicatechin (ent-2) with a 2S-phenyl group resisted the biooxidation.