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Secondary metabolites are produced at low titers by native producers due to tight regulations of their productions in response to environmental conditions. Synthetic biology provides a rational engineering principle for transcriptional optimization of secondary metabolite BGCs (biosynthetic gene clusters). Here, we demonstrate the use of synthetic biology principles for the development of a high-titer strain of the clinically important antibiotic daptomycin. Due to the presence of large NRPS (non-ribosomal peptide synthetase) genes with multiple direct repeats, we employed a top-down approach that allows transcriptional optimization of genes in daptomycin BGC with the minimum inputs of synthetic DNAs. The repeat-free daptomycin BGC was created through partial codon-reprogramming of a NRPS gene and cloned into a shuttle BAC vector, allowing BGC refactoring in a host with a powerful recombination system. Then, transcriptions of functionally divided operons were sequentially optimized through three rounds of DBTL (design-build-test-learn) cycles that resulted in up to ~2300% improvement in total lipopeptide titers compared to the wild-type strain. Upon decanoic acid feeding, daptomycin accounted for ∼ 40% of total lipopeptide production. To the best of our knowledge, this is the highest improvement of daptomycin titer ever achieved through genetic engineering of S. roseosporus. The top-down engineering approach we describe here could be used as a general strategy for the development of high-titer industrial strains of secondary metabolites produced by BGCs containing genes of large multi-modular NRPS and PKS enzymes.

In the search for new natural resources showing plant disease control effects, we found that the methanol extract of Polyalthia longifolia suppressed fungal disease development in plants. To identify the bioactive substances, the methanol extract of P. longifolia was extracted by organic solvents, and consequently, four new 2-oxo-clerodane diterpenes (1-4), a new 4(3 → 2)-abeo-clerodane diterpene (5), together with ten known compounds (6-16) were isolated and identified from the extracts. Of the new compounds, compound 2 showed a broad spectrum of antifungal activity with moderated minimum inhibitory concentration (MIC) values in a range of 50-100 μg/mL against tested fungal pathogens. Considering with the known compounds, compound 6 showed the most potent antifungal activity with an MIC value in the range of 6.3-12.5 μg/mL. When compound 6 was evaluated for an in vivo antifungal activity against rice blast, tomato late blight, and pepper anthracnose, compound 6 reduced the plant disease by at least 60% compared to the untreated control at concentrations of 250 and 500 μg/mL. Together, our results suggested that the methanol extract of twigs and leaves of P. longifolia and its major compound 6 could be used as a source for the development of eco-friendly plant protection agents.

A new bibenzyl, (+)-(1S)-4ʹ,5ʺ-dihydroxy-1,3ʹ,5ʹ,3ʺ-tetramethoxybibenzyl (1), and a new methylflavan, (2S)-7,4′-dihydroxy-5-methoxy-8-methylflavan (2), together with 10 known compounds were isolated from the methanolic extract of the tubers of Bletilla striata. Their chemical structures were elucidated based on spectrometric data interpretation, especially 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) data. All isolates (1 - 12) were evaluated for their inhibitory properties on nitric oxide production induced in RAW 264.7 cells, and dihydrophenantrenes 4 and 6 exhibited the best inhibitory effects with IC50 values of 29.5, and 6.5 μM, respectively. Additionally, significant activity possessed phenantrene 5 and methylflavane 2 with IC50 values of 38.9, and 44.9 μM, respectively.

Carotenoids are not naturally accumulated in the endosperms of rice, which is a cereal crop. In our previous study, biofortified rice seeds accumulated two red color carotenoids, ketocarotenoids including astaxanthin and ketoxanthophylls including capsanthin, via four-step pathway engineering. Their biosynthetic pathways require zeaxanthin as a common precursor and then bifurcated. In this study, cross-fertilization between two astaxanthin (BP and sBP) and two capsanthin (C and CB) rice plants were reciprocally performed to elucidate the rice seed system’s metabolic preference. Filial seeds showed different colors in red hues between mutually interbred lines as well as from parents. The strongest driving force for metabolism was caused by the sBP line for astaxanthin regardless of the parent side and proved to be the most efficacious customized synthetic gene for rice crops. It further resulted in the highest astaxanthin levels when paternal sBP mated with maternal CB line, which has the biggest capacity of total carotenoids. Also, reciprocal crosses between CB and BP lines ascertained their metabolically balanced gene efficacy by showing the pathway preference toward either ketoxanthophylls or ketocarotenoids depending on the paternal parental side. Our study suggests that gene efficacy plays the most decisive role in leading the metabolic path and revealed the unexpectedly paternal parent-of-origin effects in the endosperm. Consequently, we simultaneously biofortified rice crops with two functional red carotenoids and finally generated new red-colored rice varieties.

Eight prenylated xanthones including four new analogues were extracted and purified from the leaves of Garcinia xipshuanbannaensis. Multiple techniques including UV, 1D and 2D NMR, and HRESIMS were used to determine the structures of the isolated xanthones. These xanthones were evaluated for their cytotoxicity toward human cancer cells, and compound 4 exhibited activity against HeLa cells. A cytotoxic mechanism examination revealed the active compound induced cell apoptosis by arresting the cell cycle, increasing the levels of ROS, and inhibiting the expression of p-STAT3 in HeLa cells. In in vivo zebrafish experiments, compound 4 was found to block tumor proliferation and migration and have antiangiogenetic activity, and thus seems worthy of further laboratory evaluation.

Ketocarotenoid–biofortified crops are required as natural additives with respect to their Rubi-like reddish color and strong antioxidative properties for foods and feeds. They can be an alternative means of microalgal and bacterial systems and do not depend on chemical synthesis, providing safe, sustainable, and processing-free platforms. To reach sufficient commercial demands, different metabolic engineering strategies have been used in diverse crop plants. In this study, we summarized the current achievements for de novo production of ketocarotenoids in major crops and described host crop-specific tactics to optimize the ketocarotenoid biosynthetic pathway, considering the following different levels and types of carotenoid being accumulated as precursors: lutein and zeaxanthin in maize seeds; lutein in rice seeds, canola seeds, soybean seeds, and tobacco flowers; zeaxanthin in potato tubers; β–carotene in carrot roots; and lycopene in tomato fruits. This study suggests prospects for the advanced performance of ketocarotenoid biofortification in crops in two crucial points: host crop cultivar and the elaborately designed genetic strategies. The latter includes an increase in the total carotenoid capacity by co-consideration to supply precursors and stabilizers, the prevention of metabolite loss in competing or catabolic pathways or both pathways, and the sensible choice of genes and genetic elements for optimal expression. Furthermore, progresses in synthetic biology and new biotechnology, such as genome editing, could accelerate the development of high-valued “Rubi Crops,” which satisfy the nutritional and functional needs of humans and animals.

Cinnamomum cassia Presl (Cinnamon) has been widely cultivated in the tropical or subtropical areas, such as Yunnan, Fujian, Guandong, and Hainan in China, as well as India, Vietnam, Thailand, and Malaysia. Four new glycosides bearing apiuronic acid (1, 4, 6, and 7) and their sodium or potassium salts (2, 3, and 5), together with 31 known compounds, were isolated from a hot water extract of the bark of C. cassia via repeated chromatography. The structures of the new compounds (1-7) were determined by NMR, IR, MS, and ICP-AES data and by acid hydrolysis and sugar analysis. This is the first report of the presence of apiuronic acid glycosides. Some of the isolates were evaluated for their analgesic effects on a neuropathic pain animal model induced by paclitaxel. Cinnzeylanol (8), cinnacaside (9), kelampayoside A (10), and syringaresinol (11) showed analgesic effects against paclitaxel-induced cold allodynia.

Three new guaianolide lactones (1-3) and four new 9-oxonerolidol glucosides (5-8) together with 20 known compounds were isolated from the MeOH extract of the flowers of Chrysanthemum indicum. Their structures were elucidated based on the interpretation of NMR, HRESIMS, and electronic circular dichroism (ECD) data along with acid hydrolysis. Of the isolates, sesquiterpenoids 1-4 and 15 and flavones 17 and 18 exhibited inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide production in RAW 264.7 cells with IC50 values in the range 0.2-27.0 μM.

Two new tetrahydroprotoberberine N-oxides, 7R,14S-cis-tetrahydrocoptisine N-oxide (1) and 7R,14R-trans-tetrahydrocoptisine N-oxide (2) along with fourteen known compounds (3–16) were isolated from the aerial parts of Chelidonium majus. Their structures were elucidated by spectroscopic and spectrometric methods, such as 1D and 2D-NMR (HSQC, HMBC, NOESY) and HRESIMS. The absolute configurations of 1 and 2 were established by comparison of their experimental and calculated ECD data. All isolates were evaluated for their inhibitory effects on the nitric oxide production in LPS-induced RAW 264.7 macrophages, and compounds 2, 6–10, 13, and 15 showed significant inhibitory effects with the IC50 values ranging from 1.1–31.9 μM.

Bioactivity-guided isolation of a MeOH extract of Aralia cordata led to the isolation of four new ent-pimarane diterpenoids (1-4) and a diacetylene (5) together with 21 known compounds (6-26). Their structures were established based on the interpretation of one- and two-dimensional NMR and HRESIMS data. The absolute configurations of the new isolates were determined by electronic circular dichroism data analysis, single crystal X-ray diffraction, and Mosher's esterification method. All compounds exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages with IC50 values ranging from 1.1 to 69.4 μM.

WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

Rhizopus rot is a serious postharvest disease of various crops caused by Rhizopus spp. and controlled mainly by synthetic fungicides. We detected the antifungal activity of a culture extract of Setosphaeria rostrata F3736 against Rhizopus oryzae. The active ingredient was identified as moriniafungin, a known sordarin derivative, which showed minimum inhibitory concentrations of 1-8 μg/ml against Colletotrichum spp. and 0.03-0.13 μg/ml against Rhizopus spp. in vitro. Moriniafungin showed protective control efficacies against Rhizopus rot on apple and peach fruits. Treatment with 25 μg/ ml moriniafungin delimited the lesion diameter significantly by 100% on R. oryzae-inoculated apple fruits compared with the non-treated control. Treatment with 0.04 μg/ml of moriniafungin reduced the lesion diameter significantly by 56.45%, and treatment with higher concentrations of 0.2-25 μg/ml reduced the lesion diameter by 70-90% on Rhizopus stolonifer var. stolonifer-inoculated peach fruit. These results suggest moriniafungin has potential as a control agent of postharvest diseases caused by Rhizopus spp.

A new phenolic glucoside, (7E,9E)-3-hydroxyavenalumic acid-3-O-[6'-O-(E)-caffeoyl]-β-d-glucopyranoside (1), and three new acetylated flavone glycosides, acacetin-7-O-[β-d-glucopyranosyl(1″″→2″)-4‴-O-acetyl-α-l-rhamnopyranosyl(1‴→6″)]-β-d-glucopyranoside (3), acacetin-7-O-[6″″-O-acetyl-β-d-glucopyranosyl(1″″→2″)-3‴-O-acetyl-α-l-rhamnopyranosyl(1‴→6″)]-β-d-glucopyranoside (5), and acacetin-7-O-[3″″,6″″-di-O-acetyl-β-d-glucopyranosyl(1″″→2″)-4‴-O-acetyl-α-l-rhamnopyranosyl(1‴→6″)]-β-d-glucopyranoside (7), as well as 34 known compounds (2, 4, 6, and 8-38) were isolated from the aerial parts of Elsholtzia ciliata. The chemical structures of the new compounds were determined by spectroscopic/spectrometric data interpretation using NMR and HRESIMS. The neuroprotective effect of the isolated compounds was evaluated by a cell viability assay on HT22 murine hippocampal neuronal cells. Among them, 23 compounds, including new substances 1 and 3, exhibited neuroprotective effects against glutamate-induced HT22 cell death. In particular, compounds 2, 16, 17, 20, 22, 28, 29, and 31 presented potent neuroprotective effects with EC50 values of 1.5-8.3 μM.

Ten new ent-kaurane diterpenoids, including two pairs of epimers 1/2 and 4/5 and a 6,7-seco-ent-kauranoid 10, were obtained from the aerial parts of Isodon serra. The structures of the new compounds were confirmed by extensive spectroscopic methods and electronic circular dichroism (ECD) data analysis. An anti-inflammatory assay was applied to evaluate their nitric oxide (NO) inhibitory activities by using LPS-stimulated BV-2 cells. Compounds 1 and 9 exhibited notable NO production inhibition with IC50 values of 15.6 and 7.3 μM, respectively. Moreover, the interactions of some bioactive diterpenoids with inducible nitric oxide synthase (iNOS) were explored by employing molecular docking studies.

The onion, known as the bulb onion or common onion, is not only a key ingredient in many tasty and healthy vegetarian meals but also many traditional medicines. Nine new flavonoids [cepaflavas A, B (5, 6), cepadials A-D (7-9 and 14), and cepabiflas A-C (10-12)] and six known compounds (1-4, 13, 15) were obtained from the outer skins of Allium cepa L. Among them, compounds 5, 6, and 9 might be artificial products formed during extraction and isolation. New compounds were structurally elucidated using various spectroscopy/spectrometry techniques, including NMR and HRMS, and computational methods. Their absolute configurations were determined using time-dependent density functional theory calculations, combined with ECD spectroscopy, optical rotation calculation, and statistical procedures (CP3 and DP4 analysis). The free radical scavenging assays revealed that the new compounds 10-12 possessed considerable antioxidant activities with IC50 values of 4.25-8.88 and 7.12-8.14 μM against DPPH and ABTS•+, respectively. Compounds 13-15 showed substantial inhibitory activities against both α-glucosidase and protein tyrosine phosphatase 1B (PTP1B), with IC50 values of 0.89-6.80 and 1.13-6.82 μM, respectively. On the basis of molecular docking studies, 13 and 15 were predicted to have high binding capacity and strong affinity toward the active site of PTP1B.

Eight new neo-clerodane diterpenoids (1-8) were acquired from the aerial parts of Ajuga pantantha. Spectroscopic data analysis permitted the definition of their structures, and experimental and calculated electronic circular dichroism data were used to define their absolute configurations. Compounds 2 and 4-8 were found to have NO inhibitory effects with IC50 values of 20.2, 45.5, 34.0, 27.0, 45.0, and 25.8 μM, respectively. The more potent compounds 2, 6, and 8 were analyzed to establish their anti-inflammatory mechanism, including regulation of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins as well as their binding interactions with the two proteins.

A phytochemical survey aiming to acquire pharmacologically active substances has resulted in the isolation of nine new clerodane diterpenoids, named graveospenes A-I (1-9), from the leaves of Casearia graveolens. Spectroscopic methods were employed to establish the structures with their absolute configurations being confirmed by ECD data analysis. A biological evaluation was performed, and compound 1 was found to be cytotoxic to both human lung cancer cells (A549) and human hepatocellular carcinoma cells (HepG2). A mechanism-of-action study on 1 revealed this compound to induce apoptosis of A549 cells and impede them at the G0/G1 stage.

Two new unique angucyclinones (1 and 2) with unprecedented ether bridges connecting carbons 5 and 7 were isolated from the cultures of Streptomyces bulli GJA1, an endophyte of Gardenia jasminoides, together with two known ones (3 and 4). The MS2-based molecular networking system facilitated the isolation of compounds with target functionalities. The stereochemistry of 1 was completely established by ROESY and ECD experiments. Compound 3 showed antivirulence activities by inhibiting the peptide toxin (PSMs) production and the biofilm formation of MRSA. Compounds 3 and 4 showed very potent anti-proliferative effects against OV1 and ES2 ovarian cancer cells.

A new flavone glucoside, acacetin-7-O-(3″-O-acetyl-6″-O-malonyl)-β-d-glucopyranoside (1), two new phenolic glucosides, (3R,7R)-tuberonic acid-12-O-[6'-O-(E)-feruloyl]-β-d-glucopyranoside (14) and salicylic acid-2-O-[6'-O-(E)-feruloyl]-β-d-glucopyranoside (15), and two new phenylpropanoid glucosides, chavicol-1-O-(6'-O-methylmalonyl)-β-d-glucopyranoside (17) and chavicol-1-O-(6'-O-acetyl)-β-d-glucopyranoside(18), as well as 26 known compounds, 2-13, 16, and 19-31, were isolated from the aerial parts of Agastache rugose. The structures of the new compounds were established by spectroscopic/spectrometric methods such as HRESIMS, NMR, and ECD. The anti-inflammatory effect of the isolated compounds was evaluated by measuring their inhibitory activities on prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. New compounds 1, 15, 17, and 18 inhibited LPS-induced PGE2 production with IC50 values of 16.8 ± 0.8, 33.9 ± 4.8, 14.3 ± 2.1, and 48.8 ± 4.4 μM, respectively. Compounds 5, 7, 9-11, 13, 19, 20, 22, and 27-30 showed potent inhibitory activities with IC50 values of 1.7-8.4 μM.

The presence of excessive osteoclasts is a major factor in skeletal diseases. The present study aimed to discover osteoclast differentiation inhibitors from the basidiomycete Xylodon flaviporus. Seven new drimane sesquiterpenoids (1-7) and 7-ketoisodrimenin-5-ene (8) were obtained and characterized by various spectroscopic methods. The isolated compounds were evaluated for their inhibitory effects against receptor activator of nuclear factor-kappa-B ligand-induced osteoclastogenesis in mouse bone marrow macrophages. Compounds 1, 3, and 6 showed potent activities with IC50 values of 1.6, 0.9, and 2.1 μM, respectively, while 4, 5, and 7 exhibited relatively weak activities with IC50 values of 10.7, 10.1, and 8.5 μM, respectively.

Orbitides are plant-derived small cyclic peptides with a wide range of biological activities. Phytochemical investigation of the whole plants of Dianthus chinensis was performed with the aim to discover new bioactive orbitides. Five undescribed proline-containing orbitides, dianthiamides A–E (1–5), were isolated from a methanolic extract of Dianthus chinensis. Their structures were elucidated by extensive analysis of 1D and 2D NMR and HRESI–TOF–MS as well as ESI–MS/MS fragmentation data. The absolute configuration of the amino acid residues of compounds 1–5 was determined by Marfey’s method. All compounds were tested for their cytotoxic activity, and dianthiamide A (1) exhibited weak activity against A549 cell line with IC50 value of 47.9 μM.

The genus Streptomyces is one of the richest sources of secondary metabolite biosynthetic gene clusters (BGCs). Sequencing of a large number of genomes has provided evidence that this well-known bacterial genus still harbors a large number of cryptic BGCs, and their metabolites are yet to be discovered. When taking a gene-first approach for new natural product discovery, BGC prioritization would be the most crucial step for the discovery of novel chemotypes. We hypothesized that strains with a greater number of BGCs would also contain a greater number of silent unique BGCs due to the presence of complex regulatory systems. Based on this hypothesis, we employed a comparative genomics approach to identify a specific Streptomyces phylogenetic lineage with the highest and yet-uncharacterized biosynthetic potential. A comparison of BGC abundance and genome size across 158 phylogenetically diverse Streptomyces type strains identified that members of the phylogenetic group characterized by the formation of rugose-ornamented spores possess the greatest number of BGCs (average, 50 BGCs) and also the largest genomes (average, 11.5 Mb). The study of genetic and biosynthetic diversities using comparative genomics of 11 sequenced genomes and a genetic similarity network analysis of BGCs suggested that members of this group carry a large number of unique BGCs, the majority of which are cryptic and not associated with any known natural product. We believe that members of this Streptomyces phylogenetic group possess a remarkable biosynthetic potential and thus would be a good target for a metabolite characterization study that could lead to the discovery of novel chemotypes. IMPORTANCE It is now well recognized that members of the genus Streptomyces still harbor a large number of cryptic BGCs in their genomes, which are mostly silent under laboratory culture conditions. Activation of transcriptionally silent BGCs is technically challenging and thus forms a bottleneck when taking a gene-first approach for the discovery of new natural products. Thus, it is important to focus activation efforts on strains with BGCs that have the potential to produce novel metabolites. The clade-level analysis of biosynthetic diversity could provide insights into the relationship between phylogenetic lineage and biosynthetic diversity. By exploring BGC abundance in relation to Streptomyces phylogeny, we identified a specific monophyletic lineage associated with the highest BGC abundance. Then, using a combined analysis of comparative genomics and a genetic network, we demonstrated that members of this lineage are genetically and biosynthetically diverse, contain a large number of cryptic BGCs with novel genotypes, and thus would be a good target for metabolite characterization studies.

Five new furanocoumarins, dahuribirin H (1), dahuribirin I (2), (2'S)-(+)-5-(2'-hydroxy-3'-methylbut-3'-enyloxy)-8-(3''-methylbut-2″-enyloxy)psoralen (3), (2'R)-(+)-5-(2',3'-epoxy-3'-methylbutoxy)-8-(3″-methylbut-2″-enyloxy)psoralen (4), and 5-methoxy-8-((Z)-4'-(3″-methylbutanoate)-3'-methylbut-2'-enyloxy)psoralen (5), along with 15 known compounds (6-20), were isolated from the roots of Angelica dahurica. The structures of the new compounds were elucidated by spectroscopic analysis, along with electronic circular dichroism calculations and Mosher ester analysis. Compounds 3, 4, 11, 13, and 16 reduced H2O2-induced cell death in HepG2 cells and attenuated reactive oxygen species (ROS) formation without showing cytotoxicity, suggesting that these compounds might have cytoprotective effects against H2O2-induced oxidative damage via ROS scavenging activities.

Nonhuman primate models are valuable in biomedical research. However, reference data for clinical pathology parameters in cynomolgus and rhesus monkeys are limited. In the present study, we established hematologic and biochemical reference intervals for healthy cynomolgus and rhesus monkeys anesthetized with ketamine hydrochloride. A total of 142 cynomolgus monkeys (28 males and 114 females) and 42 rhesus monkeys (22 males and 20 females) were selected and analyzed in order to examine reference intervals of 20 hematological and 16 biochemical parameters. The effects of sex were also investigated. Reference intervals for hematological and biochemical parameters were separately established by species (cynomolgus and rhesus) and sex (male and female). No sex-related differences were determined in erythrocyte-related parameters for cynomolgus and rhesus monkey housed in indoor laboratory conditions. Alkaline phosphatase and gamma glutamyltransferase were significantly lower in females than males in both cynomolgus and rhesus monkeys aged 48–96 months. The reference values for hematological and biochemical parameters established herein might provide valuable information for researchers using cynomolgus and rhesus monkeys in experimental conditions for biomedical studies.

Eight new geranylated and farnesylated pyranoflavanones (1-8) and two new farnesylated pyranochalcones (9 and 10) were isolated from the methanolic extract of the fruits of Amomum tsao-ko. Their structures were elucidated by extensive analysis of the 1D and 2D NMR spectra and from the HRESIMS, combined with experimental ECD data. All compounds were evaluated for their inhibitory effects on the nitric oxide production against lipopolysaccharide-induced RAW 264.7 macrophages and exhibited IC50 values ranging from 10.6 to 41.5 μM.

A search for bioactive natural products as anticancer lead compounds resulted in the isolation of one previously undescribed and three known clerodane diterpenoids (1–4) from Casearia kurzii. The structures of these compounds were established by analysis of their NMR, MS, and electronic circular dichroism data. The cytotoxic activities of four compounds against three human cancer cell lines were evaluated. Compound 2 was found to be the most active with an IC50 value of 4.1 μM against HeLa cells, and was selected to investigate the possible cytotoxic mechanism.

The ethanolic extract obtained from the stems of Glycosmis pentaphylla was found to suppress antigen-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells. Four new geranylated 2-quinolone alkaloids, named glycopentanolones A–D (1–4), and 12 known metabolites (5–16) were isolated from the ethanolic extract from the stems of G. pentaphylla using bioassay-guided fractionation. Their structures were elucidated by a combination of 1D and 2D NMR, and HRESI-MS. The inhibitory effects of the isolated constituents on β-hexosaminidase release from RBL-2H3 cells were examined, and compounds 1, 5, 8 and 11 exhibited potent inhibitory activity with IC50 values between 0.05 and 4.28 μM.

A phytochemical investigation to obtain bioactive substances as lead compounds or agents for cancer led to the obtainment of six new clerodane diterpenoids, designated as kurzipenes A–F (1–6), from the leaves of Casearia kurzii. Their structures were elucidated on the basis of NMR spectroscopic data analysis and the absolute configurations were confirmed by the time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The cytotoxic activities of compounds 1–6 were evaluated against human lung cancer A549 cell line, human cervical cancer Hela cell line, and human hepatocellular carcinoma HepG2 cell line. Most diterpenoids showed potent cytotoxicities against the three selected cancer cell lines. The preliminary mechanism studies revealed that the most active compound 2, with an IC50 value of 5.3 μM against Hela cells, induced apoptosis and arrested the Hela cell cycle at the G0/G1 stage to exert cytotoxic effects.

Recent studies have revealed that there is a close relationship between neuroinflammation and Alzheimer's disease (AD) and compounds with anti-neuroinflammatory effects are potentially useful for the treatment of AD. A phytochemical investigation to obtain new neuroinflammatory inhibitors resulted in the isolation of four new and three known limonoids from Swietenia mahagoni. The structures of these limonoids were established by NMR, MS, and electronic circular dichroism (ECD) data analysis. Compounds 1–3 feature complicated polycyclic caged structures of limonoid orthoester and represent new examples of phragmalin-type limonoids. All of the isolates showed anti-neuroinflammatory activities by inhibiting nitric oxide (NO) release in LPS-induced murine microglial BV-2 cells with compounds 1 and 3–6 having IC50 values of 26.8, 26.1, 26.0, 37.1, and 16.5 μM, respectively. The possible mechanism of NO inhibition of some bioactive compounds was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the inducible nitric oxide synthase (iNOS) protein.

Two ingenane- (1 and 2), two ent-atisane- (3 and 4), two ent-kaurane- (5 and 6), two ent-abietane- (7 and 8), and one ent-isopimarane-type (9) diterpenoid and 12 known analogues have been isolated from the methanolic extract of the stems of Euphorbia royleana. Their structures, including absolute configurations, were determined by extensive spectroscopic methods and ECD data analysis. The nitric oxide inhibitory activities of those diterpenoids were examined biologically in lipopolysaccharide-stimulated BV-2 cells, with compounds 1, 2, 5–7, 10, and 12 having IC50 values lower than 40 μM. Molecular docking was used to investigated the possible mechanism of compounds 1, 2, 5–7, 10, and 12.

Seven pairs of enantiomeric isoflavones (1a/1b–7a/7b) were obtained from the ethyl acetate extract of the fruits of Maclura tricuspidata (syn. Cudrania tricuspidata), and successfully separated by chiral high-pressure liquid chromatography (HPLC). The structures and absolute configurations of the enantiomeric isoflavones were established on the basic of comprehensive spectroscopic analyses and quantum chemical calculation methods. Compounds 1, 1a, and 1b exhibited neuroprotective activities against oxygen-glucose deprivation/reoxygenation (ODG/R)-induced SH-SY5Y cells death with EC50 values of 5.5 µM, 4.0 µM, and 10.0 µM, respectively. Furthermore, 1, 1a, and 1b inhibited OGD/R-induced reactive oxygen species generation in SH-5Y5Y cells with IC50 values of 6.9 µM, 4.5 µM, and 9.5 µM, respectively.

An undescribed unusual sesquiterpene trimer and three sesquiterpene dimers were isolated from the flowers of Inula japonica. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data as well as HRESIMS data. Inulajaponicolide A has an undescribed carbon skeleton comprising of one xanthanolide and two guaianolide units with the linkage mode of C-11/C-3ʹ and C-11ʹ/C-1ʹʹ via a Diels-Alder cycloaddition reaction. Inulajaponicolides C and D exhibited moderate cytotoxic activity against A 549 and NCI-H460 human cancer cell lines with IC50 values ranging from 8.5 to 17.8 μM. Inulajaponicolides A-D and lineariifolianoid A possessed significant inhibitory potency against nitric oxide production in LPS-induced RAW264.7 cells with IC50 values ranging from 1.0 to 4.1 μM.

As a traditional medicine with potential antioxidant effects, Tenodera angustipennis egg cases (Mantidis ootheca) are a potential source of new bioactive substances. Herein, three new N-acetyldopamine derivatives, namely, (+)-tenoderin A (1a), (−)-tenoderin A (1b), and tenoderin B (2), along with thirteen known compounds (3–15), were isolated from a 70% EtOH extract of T. angustipennis egg cases. Compound 1 was isolated as a racemic mixture, and two enantiomers (1a and 1b) were successfully separated by chiral-phase preparative HPLC. The chemical structures of the new compounds were established by NMR spectroscopy and high-resolution electrospray ionization mass spectrometry, and the absolute configurations of enantiomers 1a and 1b were determined by electronic circular dichroism spectroscopy. All the new compounds exhibited antioxidant activities with IC50 values of 19.45–81.98 μM, as evaluated using free-radical scavenging assays, with the highest activity observed for compound 2. In addition, compounds 1a, 1b, and 2 exhibited inhibitory activities on intracellular reactive oxygen species generation.

The skin is an important organ in the human body that protects the body from environmentally hazardous substances. Reactive oxygen species (ROS) cause inflammatory reactions and degradation of the extracellular matrix leading to skin aging and various cutaneous lesions. This study evaluated the potential of isoflavones isolated from Maclura tricuspidata fruit to prevent TNF-α-induced skin inflammation in normal human dermal fibroblasts (HDFs). It focused on alpinumisoflavone (AIF) that suppressed the accumulation of ROS and nitric oxide (NO) in tumor necrosis factor-alpha (TNF-α)-treated HDFs. AIF inhibited the TNF-α-induced increase in matrix metalloproteinase-1, decreased procollagen I α1, and suppressed pro-inflammatory mediators and pro-inflammatory cytokines, including NO synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6, and IL-8 that trigger inflammatory responses. AIF inhibited nuclear factor-κB and activating protein 1 mitogen-activated protein kinases that were increased by TNF-α stimulation. These results suggest that AIF may protect skin from aging and various cutaneous lesions.

Pleurotus eryngii produces various functional molecules that mediate physiological functions in humans. Recently, we observed that P. eryngii produces molecules that have antidepressant functions. An ethanol extract of the fruiting body of P. eryngii was obtained, and the extract was purified by XAD-16 resin using an open column system. The ethanol eluate was separated by HPLC, and the fraction with an antidepressant function was identified. Using LC-MS, the molecular structure of the HPLC fraction with antidepressant function was identified as that of tryptamine, a functional molecule that is a tryptophan derivative. The antidepressant effect was identified from the ethanol extract, XAD-16 column eluate, and HPLC fraction by a serotonin receptor binding assay and a cell-based binding assay. Furthermore, a forced swimming test (FST) showed that the mice treated with purified fractions of P. eryngii exhibited decreased immobility time compared with nontreated mice. From these results, we suggest that the extract of P. eryngii has an antidepressant function and that it may be employed as an antidepressant health supplement.

Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of “Golden Rice”, a β-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from Thosea asigna virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced β-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition., Highlights • Carotenogenic genes upto four were concatenated via three 2A-like peptides into single cistrons. • Polycistrons stepwisely biosynthesized β-carotene, zeaxanthin and ketocarotenoids in rice. • A Thosea asigna viral 2A peptide is a good alternative to FMDV 2A for plant biotechnology.

Eight new naphtho[1,2-c]furan derivatives (1–8) along with six known analogues (9–14) were isolated from culture medium of the basidiomycete Basidioradulum radula. The structures of these compounds were identified using spectroscopic analysis, and their absolute configurations were resolved using X-ray diffraction, ECD, and VCD. Compounds 7 and 14 inhibited the cell viability of human prostate cancer DU-145 cells with IC50 values of 7.54 ± 0.03 μM and 5.04 ± 0.03 μM, respectively. At 8 μM, compounds 7 and 14 increased the percentage of apoptotic cells and upregulated the protein expression related to the apoptosis caspase pathways in DU-145 cells. Furthermore, the hallmarks of cells undergoing apoptosis, such as chromatin condensation, were also observed at this concentration. However, compound 7 and 14 showed no effect on the proliferation of splenocytes isolated from cyclophosphamide-induce immunosuppressed mice.

Cudratrixanthone U (CTU) is a prenylated xanthone compound isolated from Maclura tricuspidata Bureau (Moraceae). Prenylated xanthones have been reported to exhibit a variety of biological activities. However, the effects of prenylated xanthone on osteoclast differentiation and function are still unclear. Excessive bone resorption by osteoclasts is considered a major cause of diseases such as osteoporosis. Accordingly, suppression of excessive osteoclast formation and function is one of strategies for treating osteoclast related bone diseases. In this study, CTU inhibited osteoclast differentiation and function in RAW264.7 macrophages and BMM cells induced by receptor activator of nuclear factor-κB ligand (RANKL). CTU regulated the formation of TRAF6-TAK1 complex in RANKL-induced RAW264.7 macrophages and BMM cells. Osteoclast-specific genes including those encoding matrix metallopeptidase 9 (MMP-9), dendritic cell-specific transmembrane proteins (DC-STAMP), cathepsin K (CTSK) and chemokine CC motif ligand 4 (CCL4) play an important role in bone resorption and migration, and were effectively regulated by CTU. These results suggest that CTU is a potential therapeutic agent in osteoporosis.

We investigated effects of reblastatins on phenotypic changes in monocytes/macrophages induced by 27-hydroxycholesterol (27OHChol). Treatment of THP-1 monocytic cells with reblastatin derivatives, such as 17-demethoxy-reblastatin (17-DR), 18-dehydroxyl-17-demethoxyreblastatin (WK88-1), 18-hydroxyl-17-demethoxyreblastatin (WK88-2), and 18-hydroxyl-17-demethoxy-4,5-dehydroreblastatin (WK88-3), resulted in blockage of CCL2, CCL3, and CCL4 expression at the transcription and protein levels, which, in turn, impaired migration of monocytes/macrophages and Jurkat T cells expressing CCR5, and almost complete inhibition of transcription of M1 marker cytokines, like CXCL10, CXCL11, and TNF-α. Reblastatins also downregulated surface CD14 as well as soluble CD14 along with inhibition of LPS response and matrix metalloprotease-9 expression. Surface levels of mature dendritic cell (mDC)-specific markers, including CD80, CD83, CD88, CD197, and MHC class I and II molecules, were remarkably down-regulated, and 27OHChol-induced decrease of endocytic activity was recovered following treatment with 17-DR, WK88-1, WK88-2, and WK88-3. However, 15-hydroxyl-17-demethoxyreblastatin (DHQ3) did not affect the molecular or functional changes in monocytic cells induced by 27OHChol. Furthermore, surface levels of CD105, CD137, and CD166 were also down-regulated by 17-DR, WK88-1, WK88-2, and WK88-3, but not by DHQ3. Collectively, results of the current study indicate that, except DHQ3, reblastatins regulate the conversion and differentiation of monocytic cells to an immunostimulatory phenotype and mDCs, respectively, which suggests possible applications of reblastatins for immunomodulation in a milieu rich in oxygenated cholesterol molecules.

Two new compounds, including a nor-pimarane diterpenoid (continentanol, 1) and a phenolic derivative (aralianic acid, 2), along with the known diterpenoids (3–11), polyacetylenes (12–15), phenolic components (16–28), and phytosterols (29 and 30), were isolated from roots of Aralia continentalis. The structures of the new compounds were established by spectroscopic data interpretation, particularly HRESIMS, 1 D and 2 D NMR data including HSQC and HMBC. Also, those of the known compounds were identified by spectral comparison with those of the reported values.

Two new aryltetralin lactone lignans, petasitesins A and B were isolated from the hot water extract of the leaves of butterbur (Petasites japonicus) along with six known compounds. The chemical structures of lignans 1 and 2 were elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data, electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra. Petasitesin A and cimicifugic acid D showed significant inhibitory effects on the production of both prostaglandin E2 (PGE2) and NO in RAW264.7 macrophages. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by compound 1 in RAW264.7 cells. Furthermore, compounds 1 and 3 exhibited strong affinities with both iNOS and COX-2 enzymes in molecular docking studies.

Nine previously undescribed seco-labdane diterpenoids, nudiflopenes A−I, were isolated from the leaves of Callicarpa nudiflora. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of these compounds were established by the modified Mosher's method and experimental and calculated electronic circular dichroism spectra. Nudiflopenes A−I belong to the class of seco-labdane diterpenoids. All of the isolates showed inhibitory activities on lipopolysaccharide-induced nitric oxide (NO) production in murine microglial BV-2 cells. The possible mechanism of NO inhibition of some bioactive compounds was also investigated using molecular docking, which revealed interactions of bioactive compounds with the inducible nitric oxide synthase (iNOS) protein.

Bioassay-guided fractionation of the methanolic extract from the roots of Cynanchum atratum has resulted in the isolation of three new pregnane glycosides (1-3) along with four known compounds (4-7). Their structures were identified by analysis of the spectroscopic data including extensive 2D NMR. All of the isolates were evaluated for their potential to inhibit the melanin production in α-melanocyte stimulating hormone (α-MSH)-activated B16 melanoma cells. Of these, compounds 4-7 dose-dependently inhibited the melanin production with the IC50 values ranging from 4 μM to 33 μM.

Our continuous search for new nitric oxide (NO) inhibitory substances as anti-neuroinflammatory agents for AD resulted in the isolation of one new labdane diterpenoid and three new guaiane sesquiterpenoids, as well as ten known compounds from Blumea balsamifera. Their structures were elucidated by NMR spectroscopic data analysis and the time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The anti-neuroinflammatory effects were examined by inhibiting NO release in LPS-induced murine microglial BV-2 cells. The possible mechanism of NO inhibition of some bioactive compounds was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the iNOS protein.

To improve the utility of A. rugosa as a seasonal edible vegetable, it is necessary to determine the components and contents of reference substances during its different growth stages. Standard markers were characterized as six compounds including a new compound 5 (acacetin 2″-O-acetyl-6″-O-malonyl-β- d -glucopyranoside) by NMR and UPLC-QTof-MS analyses. A method for qualitative/quantitative analysis of the extract was evaluated including compounds (3–5), which are easily converted to tilianin (2) during extraction & isolation process by using UPLC-QTof-MS. The contents of each compound were remarkably changed at different growth stages, especially tilianin group (2–4, and 5), which were the predominant substances (>95%) in all stages. Additionally, more comprehensive chemical information, including the relative retention time, peak area and contents, were obtained simultaneously from the raw material using marker compounds. Therefore, application of this analysis method may relieve the increasing demand for pure standards.

A phytochemical investigation to obtain new NO inhibitors resulted in the isolation of five new spiro diterpenoids (1 −5) from the aerial parts of Scutellaria formosana. The structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of these compounds were established via comparison of experimental and calculated electronic circular dichroism (ECD) spectra. The nitric oxide (NO) inhibitory effects were evaluated and all of the compounds showed inhibitory effects on lipopolysaccharide-induced NO production in murine microglial BV-2 cells. The possible mechanism of NO inhibition of bioactive compounds was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the iNOS protein.

This study evaluated the neuroprotective effects 50% EtOH extract of Maclura tricuspidata fruits (FME50) and isolated compounds, cudraisoflavone I (CFI), and cudraisoflavone H (CFH), on ischemic damage in in vitro model and in vivo model of cerebral ischemia. FME50, CFI, and CFH inhibited OGD/R + glucose-induced neuronal cell death, ROS generation, and Nox4 expression via induction of Nox4-targeting miRNA-25, miRNA-92a, and miRNA-146a in SH-SY5Y cells. Also, FME50 suppressed OGD/R + glucose-induced activation of ASK1-JNK1/p38 MAPK signal cascade. Furthermore, FME50 significantly reduced the MCAO/R-induced brain infarct, Nox4 expression via induction of Nox4-targeting three miRNAs. Additionally, FME50 suppressed MCAO/R-induced MAPK signal pathway. These results demonstrate that FME50, CFI, and CFH exert neuroprotective effects via Nox4 inhibition by the induction of Nox4-targeting miRNAs and inhibition of MAPK signal cascade, suggesting that they might be possible candidates for the treatment of cerebral ischemia.

A phytochemical investigation to obtain new NO inhibitors led to the isolation of nine compounds including one new guaiane-type sesquiterpenoid (1) and two new cleistanthane diterpenoids (2 and 3) from the stems of Trigonostemon howii. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of new compounds 1–3 were established via comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 2 and 3 possess a rare 3,4-seco-cleistanthane diterpenoid skeleton. All of the compounds showed inhibitory effects on lipopolysaccharide-induced NO production in murine microglial BV-2 cells. The further molecular docking studies indicated the strong interactions between some bioactive compounds with the iNOS protein, which revealed the possible and potential mechanism of NO inhibition of bioactive compounds.

Chemical investigation of the Antarctic lichen-derived fungal strain Acremonium sp. SF-7394 yielded a new amphilectane-type diterpene, acrepseudoterin (1), and a new acorane-type sesquiterpene glycoside, isocordycepoloside A (2). In addition, three known fungal metabolites, (−)-ternatin (3), [D-Leu]-ternatin (4), and pseurotin A (5), were isolated from the EtOAc extract of the fungal strain. Their structures were mainly elucidated by analyzing their NMR and MS data. The absolute configuration of 1 was proposed by electronic circular dichroism calculations, and the absolute configuration of the sugar unit in 2 was determined by a chemical method. The inhibitory effects of the isolated compounds on protein tyrosine phosphatase 1B (PTP1B) were evaluated by enzymatic assays, results indicated that acrepseudoterin (1) and [D-Leu]-ternatin (4) dose-dependently inhibited the enzyme activity with IC50 values of 22.8 ± 1.1 μM and 14.8 ± 0.3 μM, respectively. Moreover, compound 1 was identified as a competitive inhibitor of PTP1B.