Antcin A, a phytosterol regulates SARS-CoV-2 spike protein-mediated metabolic alteration in THP-1 cells explored by the 1 H-NMR-based metabolomics approach.

The mechanism of SARS-CoV-2 spike protein-mediated perturbations of metabolic pathways and modulation of antcin A, a steroid-like compound isolated from Taiwanofungus camphoratus, are not studied. Here, we investigated the metabolic alteration by SARS-CoV-2 spike protein and the regulatory effect of antcin A on SARS-CoV-2 spike protein-induced metabolic changes in the Phorbol 12-myristate 13-acetate (PMA)-induced human monocytes (THP-1) using proton nuclear magnetic resonance (1 H-NMR) and MetaboAnalyst 5.0 software. The cytotoxic potential of SARS-CoV-2 spike protein, antcin A, and dexamethasone was assessed by MTT assay. The metabolomic perturbations and their relation to human coronaviruses' receptors were evaluated by qPCR. This study indicated that the altered metabolites mediated by SARS-CoV-2 protein, such as methionine, phosphoenolpyruvic acid, canadine, glutamine, ethanolamine, and phenylalanine, were significantly reversed by antcin A. In addition, antcin A significantly inhibited SARS-CoV-2 spike protein-mediated up-regulation of TLR-4 and ACE2 receptors, while GRP78 inhibition was not statistically significant. This is the first study to use 1 H-NMR to investigate SARS-CoV-2 spike protein-induced metabolomic changes in PMA-induced THP-1 cells. Antcin A significantly reversed metabolomic alters while dexamethasone failed to fix them. Therefore, we believe that antcin A could be a potential candidate for therapeutic agents for viral infections related to a metabolic abnormality.

Lucidone inhibits autophagy and MDR1 via HMGB1/RAGE/PI3K/Akt signaling pathway in pancreatic cancer cells.

Gemcitabine (GEM) drug resistance remains a difficult challenge in pancreatic ductal adenocarcinoma (PDAC) treatment. Therefore, identifying a safe and effective treatment strategy for PDAC is urgent. Lucidone is a natural compound extracted from the fruits of Lindera erythrocarpa Makino. However, the role of lucidone in PDAC inhibition remains unclear. In addition, high-mobility group box 1 (HMGB1) and receptor for advanced glycation end products (RAGE) are involved in multidrug resistance protein 1 (MDR1) regulation and GEM resistance. Thus, this study aimed to explore the function of lucidone in tumor cytotoxicity and chemosensitivity through the suppression of RAGE-initiated signaling in PDAC cells. The data showed that lucidone significantly promoted apoptotic cell death and inhibited the expression of autophagic proteins (Atg5, Beclin-1, LC3-II, and Vps34) and MDR1 by inhibiting the HMGB1/RAGE/PI3K/Akt axis in both MIA Paca-2 cells and MIA Paca-2GEMR cells (GEM-resistant cells). Notably, convincing data were also obtained in experiments involving RAGE-specific siRNA transfection. In addition, remarkable cell proliferation was observed after treatment with lucidone combined with GEM, particularly in MIA Paca-2GEMR cells, indicating that lucidone treatment enhanced chemosensitivity. Collectively, this study provided the underlying mechanism by which lucidone treatment inhibited HMGB1/RAGE-initiated PI3K/Akt/MDR1 signaling and consequently enhanced chemosensitivity in PDAC.

Limonene protects human skin keratinocytes against UVB-induced photodamage and photoaging by activating the Nrf2-dependent antioxidant defense system.

Long term exposure to solar ultraviolet B (UVB) radiation is one of the primary factors of premature skin aging and is referred to as photoaging. Also, mammalian skin exposed to UVB triggers an increase in production of α-melanocyte-stimulating hormone (α-MSH), which is critically involved in the pathogenesis of hyperpigmentary skin diseases. This study investigated the protective effect of limonene on UVB-induced photodamage and photoaging in immortalized human skin keratinocytes (HaCaT) in vitro. Initially, we determined cell viability and levels of reactive oxygen species (ROS) in UVB-irradiated HaCaT cells. Pretreatment with limonene increased cell viability followed by inhibition of intracellular ROS generation in UVB-irradiated HaCaT cells. Interestingly, the antioxidative activity of limonene was directly correlated with an increase in expression of endogenous antioxidants, including heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO-1), and γ-glutamylcysteine synthetase (γ-GCLC), which was associated with enhanced nuclear translocation and activation of NF-E2-related factor-2 (Nrf2). Indeed, Nrf2 knockdown reduced limonene's protective effects. Additionally, we observed that limonene treatment inhibited UVB-induced α-MSH secretion followed by inhibition of proopiomelanocortin (POMC) via suppression of p53 transcriptional activation. Moreover, limonene prevented UVB-mediated depletion of tight junction regulatory proteins, including occludin and zonula occludens-1. On the other hand, limonene treatment significantly decreased matrix metalloproteinase-2 levels in UVB-irradiated HaCaT cells. Based on these results, limonene may have a dermato-protective effect in skin cells by activating the Nrf2-dependent cellular antioxidant defense system.

Characterization of Virus-Inducible Orchid Argonaute 5b Promoter and Its Functional Characterization in Nicotiana benthamiana during Virus Infection.

Plant ARGONAUTES (AGOs) play a significant role in the defense against viral infection. Previously, we have demonstrated that AGO5s encoded in Phalaenopsis aphrodite subsp. formosana (PaAGO5s) took an indispensable part in defense against major viruses. To understand the underlying defense mechanism, we cloned PaAGO5s promoters (pPaAGO5s) and analyzed their activity in transgenic Nicotiana benthamiana using ß-glucuronidase (GUS) as a reporter gene. GUS activity analyses revealed that during Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) infections, pPaAGO5b activity was significantly increased compared to pPaAGO5a and pPaAGO5c. Analysis of pPaAGO5b 5'-deletion revealed that pPaAGO5b_941 has higher activity during virus infection. Further, yeast one-hybrid analysis showed that the transcription factor NbMYB30 physically interacted with pPaAGO5b_941 to enhance its activity. Overexpression and silencing of NbMYB30 resulted in up- and downregulation of GUS expression, respectively. Exogenous application and endogenous measurement of phytohormones have shown that methyl jasmonate and salicylic acid respond to viral infections. NbMYB30 overexpression and its closest related protein, PaMYB30, in P. aphrodite subsp. formosana reduced CymMV accumulation in P. aphrodite subsp. formosana. Based on these discoveries, this study uncovers the interaction between virus-responsive promoter and the corresponding transcription factor in plants.

Color variation in young and senescent leaves of Formosan sweet gum (Liquidambar formosana) by the gene regulation of anthocyanidin biosynthesis.

In certain plants, leaf coloration occurs in young and senescent leaves; however, it is unclear whether these two developmental stages are controlled by the same regulatory mechanisms. Formosan sweet gum (Liquidambar formosana Hance) is a subtropical deciduous tree species that possesses attractive autumnal leaf coloration. The color of young leaves is closer to purplish red, while senescent leaves are more orange-red to dark red. It was confirmed that delphinidin and cyanidin are the two anthocyanidins that contribute to the color of Formosan sweet gum leaves, and the content of different anthocyanins influences the appearance of color. To elucidate the regulation of anthocyanidin biosynthesis, recombinant DIHYDROFLAVONOL-4-REDUCTASEs (LfDFR1 and LfDFR2) (EC 1.1.1.234) were produced, and their substrate acceptability was investigated both in vitro and in planta. The functions of flavanones and dihydroflavonols modification by FLAVONOID 3' HYDROXYLASE (LfF3'H1) (EC 1.14.14.82) and FLAVONOID 3'5' HYDROXYLASE (LfF3'5'H) (EC 1.14.14.81) were verified using a transient overexpression experiment in Nicotiana benthamiana. The results showed that LfMYB5 induced LfF3'5'H and LfMYB123 induced both LfF3'H1 and LfDFR1 in spring when the leaves were expanding, whereas LfMYB113 induced LfF3'H1, LfDFR1, and LfDFR2 in late autumn to winter when the leaves were undergoing leaf senescence. In conclusion, the color variation of Formosan sweet gum in young and senescent leaves was attributed to the composition of anthocyanidins through the transcriptional regulation of LfF3'H1 and LfF3'5'H by LfMYB5, LfMYB113, and LfMYB123.

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms.

Taiwania cryptomerioides is a monotypic gymnosperm species, valued for the high decay resistance of its wood. This durability has been attributed to the abundance of terpenoids, especially the major diterpenoid metabolite ferruginol, with antifungal and antitermite activity. Specialized diterpenoid metabolism in gymnosperms primarily recruits bifunctional class-I/II diterpene synthases (diTPSs), whereas monofunctional class-II and class-I enzymes operate in angiosperms. In this study, we identified a previously unrecognized group of monofunctional diTPSs in T. cryptomerioides, which suggests a distinct evolutionary divergence of the diTPS family in this species. Specifically, five monofunctional diTPS functions not previously observed in gymnosperms were characterized, including monofunctional class-II enzymes forming labda-13-en-8-ol diphosphate (LPP, TcCPS2) and (+)-copalyl diphosphate (CPP, TcCPS4), and three class-I diTPSs producing biformene (TcKSL1), levopimaradiene (TcKSL3) and phyllocladanol (TcKSL5), respectively. Methyl jasmonate (MeJA) elicited the accumulation of levopimaradiene and the corresponding biosynthetic diTPS genes, TcCPS4 and TcKSL3, is consistent with a possible role in plant defense. Furthermore, TcCPS4 and TcKSL3 are likely to contribute to abietatriene biosynthesis via levopimaradiene as an intermediate in ferruginol biosynthesis in Taiwania. In conclusion, this study provides deeper insight into the functional landscape and molecular evolution of specialized diterpenoid metabolism in gymnosperms as a basis to better understand the role of these metabolites in tree chemical defense.

Sinensol-C Isolated from Spiranthes sinensis Inhibits Adipogenesis in 3T3-L1 Cells through the Regulation of Adipogenic Transcription Factors and AMPK Activation.

Obesity is an abnormal medical condition caused by accumulation of body fat that presents negative health impacts. Adipocyte hyperplasia, also known as adipogenesis, is one of the major manifestations of obesity. In the present study, we isolated six phenanthrene derivatives (compounds 1-6) from the ethyl acetate fraction of Spiranthes sinensis and investigated their anti-adipogenic activity. We found that among the six phenanthrene derivatives, compound 6 (sinensol-C) exhibited strong inhibitory activity against intracellular lipid accumulation in 3T3-L1 adipocytes, with an IC50 value of 12.67 µM. Sinensol-C remarkably suppressed the accumulation of lipid droplets and adipogenesis, via down-regulation of adipogenic transcription factors, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), sterol regulatory element binding protein-1 (SREBP-1c), fatty acid synthase (FAS), and fatty acid binding protein 4 (FABP4), during adipocyte differentiation in 3T3-L1 cells. In addition, treatment with sinensol-C significantly increased the adenosine monophosphate-activated protein kinase (AMPK) activity in 3T3-L1 cells. Taken together, these data strongly suggest that sinensol-C regulates adiogenesis via down-regulation of adipogenic transcription factors and up-regulation of AMPK. Furthermore, this is the first study that demonstrates that sinensol-C has the capacity to modulate adipogenesis.

MicroRNA-708 activation by glucocorticoid receptor agonists regulate breast cancer tumorigenesis and metastasis via downregulation of NF-κB signaling.

Therapeutic administration of glucocorticoids (GCs) is frequently used as add-on chemotherapy for palliative purposes during breast cancer treatment. Recent studies have shown that GC treatment induces microRNA-708 in ovarian cancer cells, resulting in impaired tumor cell proliferation and metastasis. However, the regulatory functions of GCs on miR-708 and its downstream target genes in human breast cancer cells (BCCs) are poorly understood. In this study, we found that treatment with either the synthetic GC dexamethasone (DEX) or the natural GC mimic, antcin A (ATA) significantly increased miR-708 expression by transactivation of glucocorticoid receptor alpha (GRα) in MCF-7 and MDA-MB-231 human BCCs. Induction of miR-708 by GR agonists resulted in inhibition of cell proliferation, cell-cycle progression, cancer stem cell (CSC)-like phenotype and metastasis of BCCs. In addition, GR agonist treatment or miR-708 mimic transfection remarkably inhibited IKKß expression and suppressed nuclear factor-kappaB (NF-κB) activity and its downstream target genes, including COX-2, cMYC, cyclin D1, Matrix metalloproteinase (MMP)-2, MMP-9, CD24, CD44 and increased p21CIP1 and p27KIP1 that are known to be involved in proliferation, cell-cycle progression, metastasis and CSC marker protein. BCCs xenograft models indicate that treatment with GR agonists significantly reduced tumor growth, weight and volume. Overall, our data strongly suggest that GR agonists induced miR-708 and downstream suppression of NF-κB signaling, which may be applicable as a novel therapeutic intervention in breast cancer treatment.

Antcin-A Modulates Epithelial-to-Mesenchymal Transition and Inhibits Migratory and Invasive Potentials of Human Breast Cancer Cells via p53-Mediated miR-200c Activation.

Antcin-A (ATA) is a steroid-like phytochemical isolated from the fruiting bodies of a precious edible mushroom Antrodia cinnamomea. We previously showed that ATA has strong anti-inflammatory and anti-tumor effects; however, other possible bioactivities of this unique compound remain unexplored. In the present study, we aimed to investigate the modulation of epithelial-to-mesenchymal transition (EMT), anti-migration, and anti-invasive potential of ATA against human breast cancer cells in vitro. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were incubated with ATA for 24 h. Wound healing, trans-well invasion, western blot, q-PCR, F-actin staining, and immunofluorescence assays were performed. We found that treatment with ATA significantly blocked EMT processes, as evidenced by upregulation of epithelial markers (E-cadherin and occludin) and downregulation of mesenchymal markers (N-cadherin and vimentin) via suppression of their transcriptional repressor ZEB1. Next, we found that ATA could induce miR-200c, which is a known player of ZEB1 repression. Further investigations revealed that ATA-mediated induction of miR-200c is associated with transcriptional activation of p53, as confirmed by the fact that ATA failed to induce miR-200c or suppress ZEB1 activity in p53 inhibited cells. Further in vitro wound healing and trans-well invasion assays support that ATA could inhibit migratory and invasive potentials of breast cancer cells, and the effect was likely associated with induced phenotypic modulation. Taken together, the present study suggests that antcin-A could be a lead phyto-agent for the development of anti-metastatic drug for breast cancer treatment.

Identification, Functional Characterization, and Seasonal Expression Patterns of Five Sesquiterpene Synthases in Liquidambar formosana.

Terpenoids are a large group of important secondary metabolites that are involved in a variety of physiological mechanisms, and many are used commercially in the cosmetics and pharmaceutical industries. During the past decade, the topic of seasonal variation in terpenoid biosynthesis has garnered increasing attention. Formosan sweet gum ( Liquidambar formosana Hance) is a deciduous tree species. The expression of terpene synthase and accumulation of terpenoids in leaves may vary in different seasons. Here, four sesquiterpene synthases (i.e., LfTPS01, LfTPS02, LfTPS03, and LfTPS04) and a bifunctional mono/sesquiterpene synthase ( LfTPS05) were identified from Formosan sweet gum. The gene expression of LfTPS01, LfTPS02, and LfTPS03 showed seasonal diversification, and, in addition, expression of LfTPS04 and LfTPS05 was induced by methyl jasmonate treatment. The major products LfTPS01, LfTPS02, LfTPS04, and LfTPS05 are hedycaryol, α-selinene, trans-ß-caryophyllene, α-copaene/δ-cadinene, and nerolidol/linalool, respectively. The data indicated that the sesquiterpenoid content in the essential oil of Formosan sweet gum leaves shows seasonal differences that were correlated to the sesquiterpene synthase gene expression.

Three New Iridoid Derivatives Have Been Isolated from the Stems of Neonauclea reticulata (Havil.) Merr. with Cytotoxic Activity on Hepatocellular Carcinoma Cells.

Three new iridoids, namely neonanin A (1), neonanin B (2) and neoretinin A (3), as well as twelve known compounds, 6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyran (4), 4-epi-alyxialactone (5), loganetin (6), loganin (7), phenylcoumaran-α'-aldehyde (8), cleomiscosin A (9), ficusal (10), balanophonin (11), vanillic acid (12), p-coumaric acid (13), cis,trans-abscisic acid (14), and trans,trans-abscisic acid (15) were isolated from the stems of Neonauclea reticulata (Havil.) Merr. These new structures were determined by the detailed analysis of spectroscopic data and comparison with the data of known analogues. Compounds 1⁻13 were evaluated using an in-vitro MTT cytotoxic assay for hepatocellular carcinoma (HCC) cells, and the preliminary results showed that ficusal (10), balanophonin (11), and p-coumaric acid (13) exhibited moderate cytotoxic activity, with EC50 values of 85.36 ± 4.36, 92.63 ± 1.41, and 29.18 ± 3.48 µg/mL against Hep3B cells, respectively.

Klyflaccicembranols A-I, New Cembranoids from the Soft Coral Klyxum flaccidum.

New cembranoids klyflaccicembranols A-I (1-9), along with gibberosene D (10), have been isolated from the organic extract of a Formosan soft coral Klyxum flaccidum. Their structures were established by extensive spectroscopic analyses, including 2D NMR spectroscopy, and spectral data comparison with related structures. The cytotoxicity of the isolated metabolites, as well as their nitric oxide (NO) inhibitory activity, were evaluated and reported. Metabolites 2, 4, 6, 8 and 9 were found to exhibit variable activities against a limited panel of cancer cell lines in a range of IC50 16.5-49.4 µM. Among the tested cembranoids, compounds 4, 5, 6, and 9 significantly inhibited NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages at a dose of 50 µg/mL.

Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development.

Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long been used as a remedy for cancer, hypertension, and hangover, with an annual market of over $100 million (US) in Taiwan. We obtained a 32.15-Mb genome draft containing 9,254 genes. Genome ontology enrichment and pathway analyses shed light on sexual development and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, antroquinonol, and antrocamphin. We identified genes differentially expressed between mycelium and fruiting body and 242 proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to the production of medicinal secondary metabolites. Genes of secondary metabolite biosynthetic pathways showed expression enrichment for tissue-specific compounds, including 14-α-demethylase (CYP51F1) in fruiting body for converting lanostane to ergostane triterpenoids, coenzymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting body. Our data will be useful for developing a strategy to increase the production of useful metabolites.

New Benzenoid Derivatives and Other Constituents from Lawsonia inermis with Inhibitory Activity against NO Production.

Three new benzenoid derivatives, lawsoinermone (1), inermidioic acid (2), and inermic acid (3) have been isolated from the aerial part of Lawsonia inermis, together with 11 known compounds (4-14). The structures of three new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4-6, 13 and 14 were evaluated for inhibition of nitric oxide production in LPS-stimulated product of nitrite in RAW 264.7 cells with IC50 values of 6.12, 16.43, 18.98, 9.30, 9.30 and 14.90 µg/mL, respectively.

Phytoplasma SAP11 alters 3-isobutyl-2-methoxypyrazine biosynthesis in Nicotiana benthamiana by suppressing NbOMT1.

Phytoplasmas are bacterial phytopathogens that release virulence effectors into sieve cells and act systemically to affect the physiological and morphological state of host plants to promote successful pathogenesis. We show here that transgenic Nicotiana benthamiana lines expressing the secreted effector SAP11 from Candidatus Phytoplasma mali exhibit an altered aroma phenotype. This phenomenon is correlated with defects in the development of glandular trichomes and the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP). IBMP is a volatile organic compound (VOC) synthesized by an O-methyltransferase, via a methylation step, from a non-volatile precursor, 3-isobutyl-2-hydroxypyrazine (IBHP). Based on comparative and functional genomics analyses, NbOMT1, which encodes an O-methyltransferase, was found to be highly suppressed in SAP11-transgenic plants. We further silenced NbOMT1 through virus-induced gene silencing and demonstrated that this enzyme influenced the accumulation of IBMP in N. benthamiana In vitro biochemical analyses also showed that NbOMT1 can catalyse IBHP O-methylation in the presence of S-adenosyl-L-methionine. Our study suggests that the phytoplasma effector SAP11 has the ability to modulate host VOC emissions. In addition, we also demonstrated that SAP11 destabilized TCP transcription factors and suppressed jasmonic acid responses in N. benthamiana These findings provide valuable insights into understanding how phytoplasma effectors influence plant volatiles.

Metabolite Profiling and Comparison of Bioactivity in Antrodia cinnamomea and Antrodia salmonea Fruiting Bodies.

Antrodia cinnamomea is a precious edible mushroom endemic to Taiwan that has been claimed to have significant health promotion activities. Antrodia salmonea is a new species of the genus Antrodia. In this study, we compared the metabolites and bioactivity of A. cinnamomea and A. salmonea fruiting bodies. The volatiles of A. cinnamomea and A. salmonea were characterized and 3,4,5-trimethoxybenzaldehyde was found to be the most abundant compound in A. cinnamomea; the other abundant compounds were δ-guaiene, isolongifolene, 1-octen-3-ol, 4-terpinenol, α-guaiene, and p-cymene. In A. salmonea, the main volatiles were α-cedrene, 1-octen-3-ol, D-limonene, cadinadiene, germacrene D, isolongifolene, and α-muurolene. Furthermore, five ergostane-type triterpenoids and two lanostane-type triterpenoids were selected as index compounds characterizing A. cinnamomea and A. salmonea extracts. The content of each compound varied between the two species. (R,S)-antcin B was the most abundant compound in A. cinnamomea fruiting bodies (75.18 ± 0.11 µg/mg). However, (R,S)-antcin C (184.85 ± 0.96 µg/mg) was the major triterpenoid in the A. salmonea fruiting body. Furthermore, two compounds, antcin M and methyl antcinate K, were only present in the A. salmonea fingerprint; therefore, antcin M and methyl antcinate K may be important for distinguishing between A. cinnamomea and A. salmonea fruiting bodies. Finally, examination of anti-inflammation activity and cytotoxicity showed that A. salmonea had more anti-inflammatory activity than A. cinnamomea; however, A. salmonea was more cytotoxic than A. cinnamomea. In conclusion, the composition and bioactivity of the fruiting bodies of A. cinnamomea and A. salmonea varies. Therefore, it is recommended that further toxicological evaluation and investigation of the biological activity of A. salmonea is carried out to ensure its safe and efficacious use as an alternative to A. cinnamomea.

Sesquiterpenoids and Diterpenoids from the Wood of Cunninghamia konishii and Their Inhibitory Activities against NO Production.

Three new sesquiterpenoids, 2α-hydroxy-3,3,6α,9ß-tetramethyltricyclo[4,3,2(1,4)]undecane (1), 11-acetoxyeudesman-4ß-ol (4), and 2α,3ß-dihydroxy-4ß-methyl-6,8,10-cadinatriene (6), four known sesquiterpenoids (2, 3, 5, and 7), together with eight known diterpenoids (8-15), were isolated from the wood of Cunninghamia konishii. Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. Four sesquiterpenoids (1, 4, 5, and 6) and all the diterpenoids (8-15) were evaluated for inhibition of nitric oxide production in lipopolysaccharides (LPS)-activated RAW 264.7 macrophages and the results showed that compounds 10 and 15 exhibited moderate inhibitory activities against nitric oxide production.

New Diphenol and Isocoumarins from the Aerial Part of Lawsonia inermis and Their Inhibitory Activities against NO Production.

Lawsonia inermis Linn (Lythraceae), also known as henna, is a small shrub or tree distributed throughout Taiwan's Lanyu Island, in North Africa, and in Australia. Its leaves are used as a folk medicine for the treatment of external hemorrhage and fingernail abscesses. Investigation of the ethyl acetate (EtOAc)-soluble fractions from methanol extract of the aerial part of Lawsonia inermis has led to the isolation of a new diphenol, (Z)-4,4'-(prop-1-ene-1,3-diyl)diphenol (1), two new isocoumarin carbonates, inermiscarbonates A (2) and B (3), and six known compounds, 4'-hydroxyflavanone (4), apigenine (5), kampferol (6), luteolin (7), quercetin (8), and (-)-catechin (9). Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. Compounds 1 and 4-9 were evaluated for the inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated product of nitrite in RAW 264.7 cells with IC50 values of 5.63, 15.72, 8.67, 6.67, 6.17, 7.61, and 14.52 µg/mL, respectively.

Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

Bornyl cinnamate inhibits inflammation-associated gene expression in macrophage cells through suppression of nuclear factor-κB signaling pathway.

Formosan sweetgum (Liquidamber formosana) is an endemic tree species. Various parts of this tree are used as a traditional Chinese medicine for treating pain, inflammation, and rheumatic disorders. In this study, we investigated the anti-inflammatory potential of bornyl cinnamate, a cinnamic acid derivative from the essential oil of L. formosana. Pretreatment with bornyl cinnamate significantly inhibited lipopolysaccharide-induced proinflammatory molecules, including nitric oxide, prostaglandin-E2, tumor necrosis factor α, and interleukin-1ß production, in murine macrophage RAW 264.7 cells. RT-PCR and immunoblotting analysis revealed that the inhibition of the proinflammatory molecules occurred through the downregulation of their corresponding mediator genes. Immunofluorescence and luciferase reporter assays revealed that the inhibition of proinflammatory genes by bornyl cinnamate was caused by the suppression of nuclear translocation and transcriptional activation of the redox-sensitive transcription factor nuclear factor κB. In addition, bornyl cinnamate increased the protein stability of the inhibitor of nuclear factor κB, an endogenous repressor of nuclear factor κB, through inhibition of its phosphorylation and proteasomal degradation. Furthermore, bornyl cinnamate significantly blocked the lipopolysaccharide-induced activation of I-κB kinase α, an upstream kinase of the inhibitor of nuclear factor κB α. Taken together, these results suggest that bornyl cinnamate could inhibit proinflammatory molecules through the suppression of the redox-sensitive nuclear factor κB signaling pathway.