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.

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.

Antimalarial activity of borrelidin and fumagilin in Plasmodium berghei-infected mice.

Background: Malaria remains a significant global health burden, with drug resistance posing a major challenge to its control. The emergence of resistance to antimalarial drugs represents a critical issue in malaria management, as it heightens the likelihood of morbidity and mortality associated with the disease. There is an urgent requirement for a novel candidate drug with a distinct mechanism of action. Aim: In light of the ongoing challenges in malaria management, particularly the emergence of drug resistance, this study aimed to investigate the efficacy of a novel combination therapy of borrelidin and fumagilin against Plasmodium berghei infection on Swiss Webster mice. The findings of this study could contribute to developing new and effective antimalarial treatments. Methods: This study employed a unique approach, using Swiss Webster mice aged 6-8 weeks and dividing them into five groups, each with five mice. The therapeutic efficacy of the combination treatment was evaluated through a comprehensive assessment of parasitemia levels, survival rates, and histological changes in the liver and spleen. This rigorous methodology ensures the reliability and validity of our findings. Results: The combination of borrelidin and fumagilin led to the lowest parasitemia at 5%, contrasting with the control group reaching 15%. Moreover, the combination group exhibited the highest inhibition rate of 69.6% on day nine post-infection. Histopathological alterations were limited to sinusoid dilation, hepatocyte ballooning, and the presence of hemozoin. Conclusion: These findings suggest that the combination of borrelidin and fumagilin holds promise as a potential antimalarial therapy.

Exploring Indonesian actinomycete extracts for anti-tubercular compounds: Integrating inhibition assessment, genomic analysis, and prediction of its target by molecular docking.

Tuberculosis (TB) is the foremost cause of infectious fatality globally. The primary global challenge in combatting TB lies in addressing the emergence of drug-resistant variants of the disease. However, the number of newly approved agents for treating TB has remained remarkably low over recent decades. Hence, research endeavors for discovering novel anti-TB agents are always needed. In the present study, we screened over 1,500 culture extracts from actinomycetes isolated in Indonesia for their inhibitory activity against Mycobacterium smegmatis used as a surrogate in the primary screening. The initial screening yielded approximately 6.2 % hit extracts, with a selection criterion of >80 % growth inhibition. The confirmed hit extracts were subsequently subjected to growth inhibition assay against Mycobacterium bovis and Mycobacterium tuberculosis. Approximately 20 % of the hit extracts that showed growth inhibition also exhibited efficacy against M. bovis BCG and M. tuberculosis H37Rv pathogenic strain. An active compound was successfully purified from a large-scale culture of the most potent representative extract by high-performance liquid chromatography and thin-layer chromatography. The structure of the active compound was elucidated by mass spectrometry and nuclear magnetic resonance. This compound displayed structural similarities to actinomycin group and exhibited robust inhibition, with IC50 values of 0.74, 0.02, and 0.07 µg/mL against M. smegmatis, M. bovis, and M. tuberculosis, respectively. The Actinomycetes strain A612, which produced the active compound, was taxonomically classified by phylogenetic analysis of 16s rRNA gene and whole genome sequencing data as Streptomyces parvus. Computational genome analysis utilizing anti-SMASH 7.0 unveiled that S. parvus A612 strain harbors 40 biosynthetic gene clusters with the potential to produce 16 known (with >70 % similarity) and 24 unknown compounds. A non-ribosomal peptide synthesis (NRPS) gene cluster associated with actinomycin D biosynthesis was also identified, boasting an 85 % similarity. Molecular docking analysis of actinomycin D and 21 potential M. tuberculosis targets revealed possible interactions with multiple targets. The purified active compound inhibited recombinant M. tuberculosis shikimate kinase (MtSK), which validated the results obtained from the docking analysis.

Agro-Physiological Traits of Kaffir Lime in Response to Pruning and Nitrogen Fertilizer under Mild Shading.

Mild shading has been reported to increase leaf production in kaffir lime (Citrus hystrix) through the improvement of agro-physiological variables, such as growth, photosynthesis, and water-use efficiency; however, there is still a knowledge gap concerning its growth and yield after experiencing severe pruning in harvest season. Additionally, a specific nitrogen (N) recommendation for leaf-oriented kaffir lime is still unavailable due to its lesser popularity compared to fruit-oriented citrus. The present study determined the best pruning level and N dose based on agronomy and the physiology of kaffir lime under mild shading. Nine-month-old kaffir lime seedlings grafted to rangpur lime (C. limonia) were arranged in a split-plot design, i.e., N dose as a main plot and pruning as a subplot. Comparative analysis resulted in 20% higher growth and a 22% higher yield in the high-pruned plants by leaving 30 cm of main stem above the ground rather than short ones with a 10 cm main stem. Both correlation and regression analysis strongly highlighted the importance of N for leaf numbers. Plants treated with 0 and 10 g N plant-1 experienced severe leaf chlorosis due to N deficiency, while those treated with 20 and 40 g N plant-1 showed N sufficiency; thus, the efficient recommendation for kaffir lime leaf production is 20 g N plant-1.

Anti-SARS-CoV-2 Agents in Artemisia Endophytic Fungi and Their Abundance in Artemisia vulgaris Tissue.

Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therapeutic agents for the disease are being developed. Endophytes are diverse and produce various secondary metabolites and bioactive substances. We isolated 13 endophytes from the leaves and stems of Artemisia vulgaris. Antiviral testing using the culture extracts of these endophytic fungi revealed that five isolates effectively inhibited the replication of SARS-CoV-2. These extracts were used to study the inhibitory effect of SARS-CoV-2 on 3C-like protease, and two isolates proved useful. Both isolates were from the genus Colletotrichum; therefore, the percentage of Artemisia endophytic fungi in the plant tissue was observed to be an important factor in plant site selection. Thus, we conducted a macroanalysis using next-generation sequencing to analyze the percentage of endophytes in the stems (whole, skin, and inner), leaves, roots, and cultivating soil, as well as to determine the location of each genus. To the best of our knowledge, this study is the first to report that Colletotrichum spp. are abundant in stems and that stem-based methods are the most efficient for isolating endophytes targeting Colletotrichum spp.

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.

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.

Relationship among Agroclimatic Variables, Soil and Leaves Nutrient Status with the Yield and Main Composition of Kaffir Lime (Citrus hystrix DC) Leaves Essential Oil.

Previous studies revealed the impact growing location has on the quantity and quality of essential oils derived from numerous Citrus spp., except on the kaffir lime. This study aims to analyze the relationship shared by agroclimatic variables and soil-plant nutrient status to kaffir lime leaves essential oil yield and main composition. The experiment was conducted between February and April 2019 in four growing locations, namely Bogor (6°36'36″ S, 106°46'47″ E), West Bandung (6°48'12″ S, 107°39'16″ E), Pasuruan (7°45'5″ S, 112°40'6″ E) and Tulungagung (8°6'27″ S, 112°0'35″ E). The highest essential oil yield was obtained from Bogor (1.5%), while the lowest one was from Tulungagung (0.78%). The yield was positively and significantly correlated with the rainfall, soil organic carbon, soil pH, and macronutrient levels, i.e., nitrogen, phosphorus, and magnesium. Citronellal, the major component in metabolites' profile of kaffir lime leaves essential oils, was significantly affected by the growing location. The absolute content of citronellal was positively and significantly correlated with the actual soil pH and leaf Ca content; furthermore, it negatively correlated with the leaf content of Fe, Mn, Zn, Cu. Pearson correlation analysis also showed (i) a negative significant correlation between the relative percentage of citronellol and annual rainfall intensity; (ii) a negative significant correlation between altitude and relative percentage of caryophyllene, and (iii) a positive significant correlation between the relative percentage of linalool and leaf K content.

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.

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.

Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant.

The microbial transformation of five flavans (1-5) by endophytic fungi isolated from the tea plant Camellia sinensis was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. oxidized stereoselectively at C-4 position of (+)-catechin (1) and (-)-epicatechin (2) to give the correspondent 3,4-cis-dihydroxyflavan derivatives (6, 10), respectively. (-)-Epicatechin 3-O-gallate (3) and (-)-epigallocatechin 3-O-gallate (4) were also oxidized by the fungus into 3,4-dihydroxyflavan derivatives (10, 12) via (-)-epicatechin (2) and (-)-epigallocatechin (11), respectively. Meanwhile, (-)-gallocatechin 3-O-gallate (5), (-)-catechin (ent-1) and (+)-epicatechin (ent-2), which possess a 2S-phenyl substitution, resisted the biotransformation.