Potential natural product 3,4-seco-schitriterpenoids from Kadsura japonica L. as anti-neuroinflammatory agents.

In the present study, the undescribed schitriterpenoids, kadsujanonols A-I (1-9), and eleven reported compounds (10-20) were isolated from K. japonica L. vines. Their structures of 3,4-seco-schitriterpenoids were elucidated mainly by spectroscopic analyses including 1H-, 13C-, and 2D-NMR, IR, HRESIMS spectra. The spatial configurations were determined by the single-crystal X-ray diffraction analysis of kadsujapnonol A (1), 15, 17, and 18, CD data and computational analysis. Furthermore, all isolates were evaluated for the anti-neuroinflammatory activity on LPS-stimulated NO production in BV2 microglial cells and compounds 2, 4, 5, 7, 9, 11, 13-16, and 18 exposed better or comparable suppression abilities than PDTC. Among them, kadlongilactone B (14) showed the best significant inhibiting ability (IC50 = 0.87 µg/mL) and the effect is through the attenuation of the inflammatory transcription factor p65NF-κB. Preliminary structure-activity relationship revealed that δ-lactone at the side chain and 7-member lactone at C-3/C-4, and 3,4:9,10 ring opening are important.

Genetic and Biochemical Characterization of Halogenation and Drug Transportation Genes Encoded in the Albofungin Biosynthetic Gene Cluster.

Albofungin, a hexacyclic aromatic natural product, exhibits broad-spectrum antimicrobial activity. Its biosynthesis, regulation, and resistance remain elusive. Here, we report the albofungin (abf) biosynthetic gene cluster (BGC) from its producing strain Streptomyces tumemacerans JCM5050. The nascent abf BGC encodes 70 putative genes, including regulators, transporters, type II polyketide synthases (PKSs), oxidoreductase, and tailoring enzymes. To validate the intactness and functionality of the BGC, we developed an Escherichia coli-Streptomyces shuttle bacterial artificial chromosome system, whereby the abf BGC was integrated into the genome of a nonproducing host via heterologous conjugation, wherefrom albofungin can be produced, confirming that the BGC is in effect. We then delimited the boundaries of the BGC by means of in vitro CRISPR-Cas9 DNA editing, concluding a minimal but essential 60-kb abf BGC ranging from orfL to abf58. The orfA gene encoding a reduced flavin adenine dinucleotide (FADH2)-dependent halogenase was examined and is capable of transforming albofungin to halogen-substituted congeners in vivo and in vitro. The orfL gene encoding a transporter was examined in vivo. The presence/absence of orfA or orfL demonstrated that the MIC of albofungin is subject to alteration when an extracellular polysaccharide intercellular adhesin was formed. Despite that halogenation of albofungin somewhat increases binding affinity to transglycosylase (TGase), albofungin with/without a halogen substituent manifests similar in vitro antimicrobial activity. Halogenation, however, limits overall dissemination and effectiveness given a high secretion rate, weak membrane permeability, and high hydrophobicity of the resulting products, whereby the functions of orfA and orfL are correlated with drug detoxification/resistance for the first time. IMPORTANCE Albofungin, a natural product produced from Streptomycetes, exhibits bioactivities against bacteria, fungi, and tumor cells. The biosynthetic logic, regulations, and resistance of albofungin remain yet to be addressed. Herein, the minimal albofungin (abf) biosynthetic gene cluster (BGC) from the producing strain Streptomyces tumemacerans JCM5050 was precisely delimited using the Escherichia coli-Streptomyces shuttle bacterial artificial chromosome system, of which the gene essentiality was established in vivo and in vitro. Next, we characterized two genes orfA and orfL encoded in the abf BGC, which act as a reduced flavin adenine dinucleotide (FADH2)-dependent halogenase and an albofungin-congeners transporter, respectively. While each testing microorganism exhibited different sensitivities to albofungins, the MIC values of albofungins against testing strains with/without orfA and/or orfL were subject to considerable changes. Halogen-substituted albofungins mediated by OrfA manifested overall compromised dissemination and effectiveness, revealing for the first time that two functionally distinct proteins OrfA and OrfL are associated together, exerting a novel "belt and braces" mechanism in antimicrobial detoxification/resistance.

Novel 11-norbetaenone isolated from an entomopathogenic fungus Lecanicillium antillanum.

A novel nor-betaenone compound, 11-norbetaenone (1), was isolated from the culture broth of an entomopathogenic fungus Lecanicillium antillanum. The structure was determined on the basis of 1D and 2D NMR spectroscopic data. The absolute stereochemistry of 1 was further confirmed by X-ray single crystallography analysis. It is the first secondary metabolite reported from the species Lecanicillium antillanum. And it is also the first time that a betaenone-type compound was isolated from the genus Lecanicillium. Furthermore, 11-norbetaenone (1) displayed significant anti-angiogenic effect by suppressing tube formation.

Inflammation Modulatory Phorbol Esters from the Seeds of Aquilaria malaccensis.

The tree Aquilaria malaccensis is a valuable source of agarwood, which is used in herbal medicinal preparations. Phytochemical research on A. malaccensis seeds has led to the isolation of four new phorbol esters (1-4), two known phorbol esters (5, isolated from Nature for the first time, and 6), and two known glycerides (7 and 8). The structures of these isolates were elucidated by means of spectroscopic data interpretation. The inflammation-modulatory activities of the isolates on elastase release and superoxide anion generation in human neutrophils were evaluated. Interestingly, phorbol esters 1, 5, and 6 showed potent inhibitory activity on elastase release in human neutrophils, with IC50 values of 2.7, 0.8, and 2.1 µM, respectively. All isolated phorbol esters exerted enhancing activity on superoxide anion generation. The results indicated that phorbol esters may play a bilateral modulatory role in the processes of inflammation. In addition, the compounds were evaluated for their cytotoxic properties against HepG2 (hepatoma), MDA-MB-231 (breast), and A549 (lung) cancer cells, but all compounds were inactive for all cell lines used (IC50 > 10 µM).

Biosynthesis of Complex Indole Alkaloids: Elucidation of the Concise Pathway of Okaramines.

The okaramines are a class of complex indole alkaloids isolated from Penicillium and Aspergillus species. Their potent insecticidal activity arises from selectively activating glutamate-gated chloride channels (GluCls) in invertebrates, not affecting human ligand-gated anion channels. Okaramines B (1) and D (2) contain a polycyclic skeleton, including an azocine ring and an unprecedented 2-dimethyl-3-methyl-azetidine ring. Owing to their complex scaffold, okaramines have inspired many total synthesis efforts, but the enzymology of the okaramine biosynthetic pathway remains unexplored. Here, we identified and characterized the biosynthetic gene cluster (oka) of 1 and 2, then elucidated the pathway with target gene inactivation, heterologous reconstitution, and biochemical characterization. Notably, we characterized an α-ketoglutarate-dependent non-heme FeII dioxygenase that forged the azetidine ring on the okaramine skeleton.

Ecdysones from Zoanthus spp. with inhibitory activity against dengue virus 2.

Bioassay-guided fractionation of an ethanolic extract of Zoanthus spp. collected in Taiwan has resulted in the isolation of one new ecdysone, zoanthone A (1), along with thirteen known compounds (2-14). The structures of these compounds were determined by spectroscopic methods, especially 2D NMR analyses. The in vitro antiviral activities of all isolated ecdysones (1-14) against dengue virus type 2 (DENV-2) were evaluated using DENV infectious system. New compound (1) exhibited potent antiviral activity (EC50=19.61 ± 2.46 µM) with a selectivity index (CC50/EC50) value of 36.7. The structure-activity relationships of isolated ecdysones against DENV-2 were concluded. Molecular docking information of 3 and NS5 polymerase was performed either.

Zoanthamine-Type Alkaloids from the Zoanthid Zoanthus kuroshio Collected in Taiwan and Their Effects on Inflammation.

Zoanthus kuroshio is a colorful zoanthid with a fluorescent pink oral disc and brown tentacles, which dominates certain parts of the Taiwanese and Japanese coasts. This sea anemone is a rich source of biologically active alkaloids. In the current investigation, two novel halogenated zoanthamines [5α-iodozoanthenamine (1) and 11ß-chloro-11-deoxykuroshine A (2)], along with four new zoanthamines [18-epi-kuroshine A (3), 7α-hydroxykuroshine E (4), 5α-methoxykuroshine E (5), and 18-epi-kuroshine E (6)], and six known compounds were isolated from Z. kuroshio. Compounds 1 and 2 are the first examples of halogenated zoanthamine-type alkaloids isolated from nature. Compounds 3 and 6 are the first zoanthamine stereoisomers with a cis-junction of the A/B rings. All isolated compounds were evaluated for their anti-inflammatory activities by measuring their effects on superoxide anion generation and elastase release by human neutrophils in response to fMLP.

Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis.

The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia against diseases such as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC50 value less than 1 µg/mL. Therefore, the present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structure of 1 was assigned by means of 1D and 2D NMR data and high-resolution mass spectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulation assay with IC50 values of 1.7 and 11 nM, respectively, with a therapeutic index up to 71,000. The antiallergic activities of A. malaccensis seeds and aquimavitalin (1) have never been revealed before. The results indicated that A. malaccensis seeds and the pure compound have the potential for use in the treatment of allergy.

Tetranortriterpenes and Limonoids from the Roots of Aphanamixis polystachya.

Phytochemical investigation of the acetone extract from the roots of Aphanamixis polystachya resulted in isolation of four new tetranortriterpenes (1-4) in addition to one protolimonoid (methyl-1ξ,7R-diacetoxy-23R,25-dihydroxy-20S,24R-21,24-epoxy-3,4-seco-apotirucall-4(28),14(15)-diene-3-oate (5)), five known limonoids (rohituka 3 (6), rohituka 7 (7), nymania 1 (8), rubrin G (9), prieurianin (10)) and a steroid (2,3-dihydroxy-5-pregnan-16-one (11)). Their structures were determined by spectroscopic analyses, including 2D-NMR (COSY, HMQC, HMBC, and NOESY) and high-resolution electrospray ionization mass spectrometry (HRESIMS). Cytotoxic and anti-inflammatory activities of these compounds were evaluated. Compounds 4 and 5 showed significant inhibition against superoxide generation and elastase release by human neutrophils in response to (formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B) (FMLP/CB).

Pandalisines A and B, novel indolizidine alkaloids from the leaves of Pandanus utilis.

Two novel alkaloids named pandalisines A (1) and B (2), constituting a new class of C8-substituted indolizidine moiety, were isolated from the leaves of Pandanus utilis. The structures of these new compounds were established by their mass and spectroscopic data. The absolute configuration was determined by the comparison of experimental CD and calculated ECD spectra. A plausible biosynthetic pathway for compounds 1 and 2 is advanced. The cytotoxic activities of the isolated alkaloids against A-549, Hep-G2, and MDA-MB-231 cancer cell lines were evaluated. The result showed that 1 and 2 are the first non-cytotoxic indolizidine alkaloids.

Alkylamides of Acmella oleracea.

Phytochemical investigation of the flowers of Acmella oleracea had resulted in the isolation of one new alkylamide, (2E,5Z)-N-isobutylundeca-2,5-diene-8,10-diynamide (1), together with four known analogues (2-5). The structures of these compounds were determined by the interpretation of spectroscopic methods, especially NMR technologies (COSY, HSQC, HMBC, and NOESY). In addition, a convenient method for concentrating the alkylamide-rich fraction and analyzing fingerprint profile of A. oleracea was established.

Limonoids from the seeds of Swietenia macrophylla with inhibitory activity against dengue virus 2.

Fractionation of an ethanol-soluble extract of the seeds of Swietenia macrophylla yielded six new limonoids, swielimonoids A-F (1-6), along with 20 known compounds. Compounds 1 and 2, mexicanolide-type limonoids, were assigned with an α,ß-unsaturated δ-lactone moiety (ring D) and a C═C bond between C-8 and C-30. Compounds 3-6 could be categorized as highly oxygenated phragmalin-type limonoids. The structures of these new compounds were elucidated through the interpretation of spectroscopic data. The antidengue virus 2 activities of the isolated components from S. macrophylla were investigated, and of 12 compounds subjected to bioassay, compounds 2 and 7-10 were found to show inhibitory activity in the range 3.5 to 12.5 µM. Among these, the new limonoid 2 exhibited significant antiviral activity (EC50 = 7.2 ± 1.33 µM) with a selectivity index (CC50/EC50) value of >27.7.

Seven new sesquiterpenoids from the fruits of Schisandra sphenanthera.

Fractionation of the EtOH extract from the fruits of Schisandra sphenanthera resulted in the isolation of seven new sesquiterpenoids, 1-7, in addition to the known metabolites 8-23. Among them, schiscupatetralin A (1) possesses an unprecedented structure with a CC bond between cuparenol and tetralin. The isolated new compounds were evaluated for their anti-HSV-1 and anti-inflammatory activities. The results revealed that compound 4 exhibited anti-HSV-1 activity, while compound 6 showed a significant anti-inflammatory activity.

New Lignans from the Leaves and Stems of Kadsura philippinensis.

Three novel C19 homolignans, taiwankadsurins D (1), E (2) and F (4), and two new C18 lignans kadsuphilins N (3) and O (5) were isolated from the aerial parts of Taiwanese medicinal plant Kadsura philippinensis. The structures of compounds 1-5 were determined by spectroscopic analyses, especially 2D NMR techniques. The structure of compound 5 was further confirmed by X-ray crystallographic analysis. Compounds 1 and 2 have a 3,4-{1'-[(Z)-2''-methoxy-2''-oxoethylidene]}-pentano(2,3-dihydrobenzo[b]furano)-3-(2'''-methoxycarbonyl-2'''-hydroxy-2''',3'-epoxide) skeleton.

Kaguacidine A: a novel spirohydantoin-containing cucurbitane glycoside from vines of Momordica charantia L.

The spirohydantoin-containing cucurbitane-type triterpenoid, kaguacidine A (1), was isolated and purified from 95% ethanol extract of vines of Momordica charantia L. (Cucurbitaceae). Its unprecedented chemical structure, a spirohydantoin substituent at C-23 of cucurbitane, was elucidated by extensive spectroscopic analyses, including HRESIMS, IR, optical rotation, 1 D- and 2 D-NMR spectra. The possible biosynthetic pathway is deduced and may be attributed to the metabolic activity of microbial symbionts in M. charantia L. Compound 1 was evaluated for anti-inflammatory activity against LPS-induced NO production in RAW 264.7 cells and anti-proliferative activity against four cancer cell lines, including HEp-2, MCF-7, Hep-G2, and WiDr. Compound 1 showed moderate anti-inflammatory activity with an IC50 value of 18.5 ± 0.4 µg/mL and weak anti-proliferative activity against MCF-7, HEp-2, Hep-G2, and WiDr with IC50 values of >40, 33.8 ± 0.6, 31.0 ± 0.7, and 27.0 ± 0.7 µM, respectively.

N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry.

Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

Anti-inflammatory effect of euphane- and tirucallane-type triterpenes isolated from the traditional herb Euphorbia neriifolia L.

The Euphorbiaceae plant Euphorbia neriifolia L. is distributed widely in India, Thailand, Southeastern China, and Taiwan and used as a carminative and expectorant to treat several inflammation-related diseases, such as gonorrhoea, asthma, and cancer. In the course of our search for potential anti-inflammatory agents from the titled plant, 11 triterpenes from the stem of E. neriifolia were isolated and reported in our previous endeavor. Given its rich abundance in triterpenoids, the ethanolic extract in this follow-up exploration has led to the isolation of additional eight triterpenes, including six new euphanes-neritriterpenols H and J-N (1 and 3-7)-one new tirucallane, neritriterpenol I (2), and a known compound, 11-oxo-kansenonol (8). Their chemical structures were elucidated on the basis of spectroscopic data, including 1D- and 2D NMR, and HRESIMS spectra. The absolute stereochemistry of neritriterpenols was determined by single-crystal X-ray diffraction analysis, ICD spectra, and DP4+ NMR data calculations. Compounds 1-8 were also evaluated for their anti-inflammatory activity by using lipopolysaccharide (LPS)-stimulated IL-6 and TNF-α on RAW 264.7 macrophage cells. Intriguingly, the euphane-type triterpenes (1 and 3-8) showed an inhibitory effect on LPS-induced IL-6 but not on TNF-α, while tirucallane-type triterpene 2 showed strong inhibition on both IL-6 and TNF-α.

Four cucurbitane glycosides taimordisins A-D with novel furopyranone skeletons isolated from the fruits of Momordica charantia.

Four novel triterpene glycosides, taimordisins A-D (1-4), were discovered from fresh fruits of Taiwanese Momordica charantia. The chemical framework and relative stereochemistry of these four natural products were isolated, purified, and determined by using various separation and spectroscopy techniques. Each of them features a unique bicyclic-fused or trifuso-centro-fused ring system. Notably, 1 and 2 are cucurbitane-based compounds possessing a new C-24 and C-2″ carbon-carbon linkage with 5-hydroxy-2-(hydroxymethyl)tetrahydro-4H-pyran-4-one and 6-(hydroxymethyl)tetrahydro-4H-pyran-3,4,4-triol units, respectively, and represented an unprecedented molecular skeleton. In terms of biosynthesis, they all originate from a common precursor 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-ß-glucopyranoside. Of two sugar moieties, the one at 23-O-ß-glucopyranoside grants each individual congener uniqueness likely through microbial symbiont-mediated intramolecular transformation into two major types of furo[2,3-b]pyranone and furo[3,2-c]pyranone derivatives. These new products possess desirable anti-inflammatory biological activities in addition to being generally regarded as safe.

Anti-inflammatory sesquiterpene and triterpene acids from Mesona procumbens Hemsley.

Mesona procumbens Hemsley is a plant conventionally processed to provide popular food materials and herbal medicines in Asia. In this study, six triterpene acids, including five new ones (mesonaic acids D-H, 1-5), and one proximadiol-type sesquiterpene (7) were isolated from the methanolic extract of the air-dried M. procumbens. Chemical structures of 1‒7 were established by spectroscopic methods, especially 2D NMR techniques (1H-1H COSY, HSQC, HMBC, and NOESY) and HRESIMS. Concerning their biological activities, compounds 1, 2, 6, and 7 were examined manifesting high inhibition toward the pro-inflammatory NO production with EC50 values ranging from 12.88 to 21.21 µM, outrunning the positive control quercetin (24.12 µM). The mesoeudesmol B (7) identified from M. procumbens is the very first example, which exhibited high anti-inflammatory activity diminishing the level of the lipopolysaccharide-induced NO in RAW264.7 macrophage cells, thereby suppressing the secretion of pro-inflammatory cytokines TNF-α and IL-6 and the level of two critical downstream inflammatory mediators iNOS and COX-2.

ß-Hydroxylation of α-amino-ß-hydroxylbutanoyl-glycyluridine catalyzed by a nonheme hydroxylase ensures the maturation of caprazamycin.

Caprazamycin is a nucleoside antibiotic that inhibits phospho-N-acetylmuramyl-pentapeptide translocase (MraY). The biosynthesis of nucleoside antibiotics has been studied but is still far from completion. The present study characterized enzymes Cpz10, Cpz15, Cpz27, Mur17, Mur23 out of caprazamycin/muraymycin biosynthetic gene cluster, particularly the nonheme αKG-dependent enzyme Cpz10. Cpz15 is a ß-hydroxylase converting uridine mono-phosphate to uridine 5' aldehyde, then incorporating with threonine by Mur17 (Cpz14) to form 5'-C-glycyluridine. Cpz10 hydroxylates synthetic 11 to 12 in vitro. Major product 13 derived from mutant Δcpz10 is phosphorylated by Cpz27. ß-Hydroxylation of 11 by Cpz10 permits the maturation of caprazamycin, but decarboxylation of 11 by Mur23 oriented to muraymycin formation. Cpz10 recruits two iron atoms to activate dioxygen with regio-/stereo-specificity and commit electron/charge transfer, respectively. The chemo-physical interrogations should greatly advance our understanding of caprazamycin biosynthesis, which is conducive to pathway/protein engineering for developing more effective nucleoside antibiotics.