Screening of growth inhibitors for epithelial-mesenchymal transition-induced cells by TGF-ß from plant-based sources identified the active compound hydroxychavicol from Piper bitle.

Epithelial-mesenchymal transition (EMT) has recently been associated with cancer invasion, metastasis, and resistance. In our previous study, we discovered nanaomycin K, a natural growth inhibitor for EMT-induced Madin Darby canine kidney (MDCK) cells, from the cultured broth of actinomycetes. However, the screening method was undeveloped, because the activity of nanaomycin K was discovered accidentally. In this study, we established a screening method by analyzing the characteristics of nanaomycin K in MDCK cells. Nanaomycin K showed the characteristic growth inhibitory activity on MDCK cells cultured under four conditions: medium containing dimethyl sulfoxide, SB431542, TGF-ß, and a mixture of SB431542 and TGF-ß. The activity was stronger in TGF-ß-treated cells than in DMSO-treated cells. In the mixture of SB431542 and TGF-ß-treated cells, the activity of nanaomycin K was suppressed. The anti-cancer agents, mitomycin C, cisplatin, and staurosporine, lacked the characteristics as that of nanaomycin K for these four treatment conditions. Since these four conditions distinguish between the effects of nanaomycin K and other anti-cancer agents in EMT-induced cells, the screening method was established. Among the 13,427 plant extracts tested, Piper betle leaf extract displayed growth inhibitory activity against EMT-induced cells. Through the purification of the extract via bio-guided fractionation, hydroxychavicol was isolated as an active compound. The cytotoxic activity of hydroxychavicol was stronger in EMT-induced MDCK cells than in control cells. However, its cytotoxic activity was suppressed in EMT-inhibited cells. Furthermore, hydroxychavicol exhibited same activity against SAS cells (human squamous cell carcinoma of the tongue). Thus, we have successfully established a screening method for growth inhibitors of EMT-induced cells and have discovered an inhibitor from plant-based sources.

Identification of an Oligostilbene, Vaticanol B, from Dryobalanops aromatica Leaves as an Antiviral Compound against the Hepatitis C Virus.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Although current medications using direct-acting antivirals (DAAs) are highly effective and well-tolerated for treating patients with chronic HCV, high prices and the existence of DAA-resistant variants hamper treatment. There is thus a need for easily accessible antivirals with different mechanisms of action. During the screening of Indonesian medicinal plants for anti-HCV activity, we found that a crude extract of Dryobalanops aromatica leaves possessed strong antiviral activity against HCV. Bioassay-guided purification identified an oligostilbene, vaticanol B, as an active compound responsible for the anti-HCV activity. Vaticanol B inhibited HCV infection in a dose-dependent manner with 50% effective and cytotoxic concentrations of 3.6 and 559.5 µg/mL, respectively (Selectivity Index: 155.4). A time-of-addition study revealed that the infectivity of HCV virions was largely lost upon vaticanol B pretreatment. Also, the addition of vaticanol B following viral entry slightly but significantly suppressed HCV replication and HCV protein expression in HCV-infected and a subgenomic HCV replicon cells. Thus, the results clearly demonstrated that vaticanol B acted mainly on the viral entry step, while acting weakly on the post-entry step as well. Furthermore, co-treatment of the HCV NS5A inhibitor daclatasvir with vaticanol B increased the anti-HCV effect. Collectively, the present study has identified a plant-derived oligostilbene, vaticanol B, as a novel anti-HCV compound.

Retusone A, a Guaiane-Type Sesquiterpene Dimer from Wikstroemia retusa and Its Inhibitory Effects on Histone Acetyltransferase HBO1 Expression.

Retusone A (1), a new sesquiterpene dimer consisting of two guaiane-type sesquiterpenoids, and oleodaphnal (2) were isolated from heartwood of Wikstroemia retusa (Thymelaeaceae). The planar structure of 1 was elucidated on the basis of HRESIMS and NMR spectroscopic data, and the relative stereochemistry was established by X-ray diffraction analysis. The absolute configuration of 1 was determined by electronic circular dichroism. Compound 1 suppressed luciferase reporter gene expression driven by the HBO1 (histone acetyltransferase binding to ORC1) gene promoter in human breast cancer MCF7 cells. Compound 1 also decreased the expression of endogenous HBO1 mRNA and protein, and inhibited proliferation of the cells. These results suggest that retusone A (1), which has a unique dimeric sesquiterpenoid structure with inhibitory activity against HBO1 expression, may contribute to the development of a novel therapeutic candidate for the treatment of breast cancer.

Phenanthroindolizine alkaloids from Boehmeria sieboldiana leaves exhibit cytotoxicity against human cancer cell lines.

To explore useful natural compounds from indigenous medicinal plants, the cytotoxic properties from a methanolic extract of Boehmeria sieboldiana leaves against human cancer cell lines were isolated in the present study. After purification of the extract, seco-dehydroantofine B (1) together with two known phenanthroindolizine alkaloids, seco-dehydroantofine A (2) and septicine (3), were isolated. The structure of seco-dehydroantofine B was elucidated by performing comprehensive one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The cytotoxicity of these compounds against five human tumor cell lines was evaluated. Compound 3 exhibited anti-tumor activity at IC50 values of 50.0, 66.9, 50.0, and 153.7 µM against MKN1, SAS, HL-60, and THP-1 cells, respectively.

Diversity of Adenostemma lavenia, multi-potential herbs, and its kaurenoic acid composition between Japan and Taiwan.

Adenostemma lavenia (L.) Kuntze (Asteraceae) is widely distributed in tropical regions of East Asia, and both A. lavenia and A. madurense (DC) are distributed in Japan. In China and Taiwan, A. lavenia is used as a folk medicine for treating lung congestion, pneumonia, and hepatitis. However, neither phylogenic nor biochemical analysis of this plants has been performed to date. We have reported that the aqueous extract of Japanese A. lavenia contained high levels of ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11αOH-KA; a kaurenoic acid), which is a potent anti-melanogenic compound. Comparison of chloroplast DNA sequences suggested that A. lavenia is originated from A. madurense. Analyses of kaurenoic acids revealed that Japanese A. lavenia and A. madurense contained high levels of 11αOH-KA and moderate levels of 11α,15OH-KA, while Taiwanese A. lavenia mainly contained 9,11αOH-KA. The diverse biological activities (downregulation of Tyr, tyrosinase, gene expression [anti-melanogenic] and iNOS, inducible nitric oxide synthase, gene expression [anti-inflammatory], and upregulation of HO-1, heme-oxygenase, gene expression [anti-oxidative]) were associated with 11αOH-KA and 9,11αOH-KA but not with 11α,15OH-KA. Additionally, 11αOH-KA and 9,11αOH-KA decreased Keap1 (Kelch-like ECH-associated protein 1) protein levels, which was accompanied by upregulation of protein level and transcriptional activity of Nrf2 (NF-E2-related factor-2) followed by HO-1 gene expression. 11αOH-KA and 9,11αOH-KA differ from 11α,15OH-KA in terms of the presence of a ketone (αß-unsaturated carbonyl group, a thiol modulator) at the 15th position; therefore, thiol moieties on the target proteins, including Keap1, may be important for the biological activities of 11αOH-KA and 9,11αOH-KA and A. lavenia extract.

Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability.

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 µM, and 20.2 µM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.

Endotoxin Contamination and Reaction Interfering Substances in the Plant Extract Library.

Endotoxin is an unintentional contaminant that has numerous activities and can affect various biological experiments using cells. In this study, we measured the endotoxin activity of samples from a plant extract library (PEL) and determined their degrees of contamination. Endotoxin was detected in approx. 48% (n = 139) and approx. 4% (n = 5) of field-collected and crude drug samples, respectively, and in concentrations >5.0 EU/mL in some samples. The concentrations of endotoxin that affect cells in vitro vary depending on the target cell type. Although the degree of contamination varied in the present study, it was considered to have little effect on the cell experiments. More than 150 PEL samples had problems with reaction courses or recovery rates of Limulus amoebocyte lysate (LAL) tests. In the LAL tests, using three plant extracts [Sanguisorba officinalis L. (Rosaceae), Oenothera biennis L. (Onagraceae), and Lythrum salicaria L. (Lythraceae)], the polyphenolic compounds in the plant extracts affected LAL test and their effects differed depending on the plant species. When the 16 single polyphenol compounds were added to the LAL tests, the compounds with caffeoyl and pyrogallol moieties were found to affect the LAL reaction course and recovery rate. Furthermore, none of the compounds had any effects at concentrations of 1 µM. Because the plant extracts contained analogs of various polyphenolic compounds, they were presumed to actually act synergistically. Our findings demonstrated that attention must be paid to the recovery rate and reaction process of LAL tests with samples containing polyphenolic compounds.

Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

The Importance of 11α-OH, 15-oxo, and 16-en Moieties of 11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Its Inhibitory Activity on Melanogenesis.

Cosmetic industries have an interest in exploring and developing materials that have the potential to regulate melanin synthesis in human skin. Although melanin protects the skin from ultraviolet irradiation, excess melanin can be undesirable, particularly on the face where spots or freckles are associated with an appearance of aging. In this study, we found that ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11α-OH KA) in Pteris dispar Kunze strongly inhibited melanin synthesis by suppressing tyrosinase gene expression. The melanogenic transcription factor microphthalmia-associated transcription factor (MITF) is required for this suppression. However, 11α-OH KA did not modulate the expression level or activity of MITF. Structure-activity relationship analyses suggested that the 11α-OH, 15-oxo, and 16-en moieties of 11α-OH KA are essential for the suppression of melanin synthesis. On the other hand, the 19-COOH moiety is important for preventing cellular toxicity associated with 11α-OH KA and its related compounds. These results suggest that 11α-OH KA is an attractive target for potential use in the production of cosmetic items.

Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3.

Osteoarthritis is a common debilitating joint disorder. Risk factors for osteoarthritis include age, which is associated with thinning of articular cartilage. Here we generate chondrocyte-specific salt-inducible kinase 3 (Sik3) conditional knockout mice that are resistant to osteoarthritis with thickened articular cartilage owing to a larger chondrocyte population. We also identify an edible Pteridium aquilinum compound, pterosin B, as a Sik3 pathway inhibitor. We show that either Sik3 deletion or intraarticular injection of mice with pterosin B inhibits chondrocyte hypertrophy and protects cartilage from osteoarthritis. Collectively, our results suggest Sik3 regulates the homeostasis of articular cartilage and is a target for the treatment of osteoarthritis, with pterosin B as a candidate therapeutic.

Callicarpa longissima extract, carnosol-rich, potently inhibits melanogenesis in B16F10 melanoma cells.

Cosmetic industries focus on developing materials and resources that regulate skin pigmentation. Melanin, the major pigment in human skin, protects the skin against damage from ultraviolet light. An ethanolic extract of the leaves of Callicarpa longissima inhibits melanin production in B16F10 mouse melanoma cells by suppressing microphthalmia-associated transcription factor (MITF) gene expression. Following purification and analysis using liquid chromatography-mass spectrometry (LC-MS), NMR, and biochemical assays, carnosol was determined to be responsible for the major inhibitory effect of the C. longissima extract on melanin production. Carnosol is an oxidative product of carnosic acid, whose presence in the extract was also confirmed by an authentic reference. The carnosol and carnosic acid content in the extract was approximately 16% (w/w). These results suggest that C. longissima is a novel, useful, and attractive source of skin-whitening agents.

Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes.

Salt-inducible kinases (SIKs), members of the 5'-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.

Salt-inducible kinase 3 deficiency exacerbates lipopolysaccharide-induced endotoxin shock accompanied by increased levels of pro-inflammatory molecules in mice.

Macrophages play important roles in the innate immune system during infection and systemic inflammation. When bacterial lipopolysaccharide (LPS) binds to Toll-like receptor 4 on macrophages, several signalling cascades co-operatively up-regulate gene expression of inflammatory molecules. The present study aimed to examine whether salt-inducible kinase [SIK, a member of the AMP-activated protein kinase (AMPK) family] could contribute to the regulation of immune signal not only in cultured macrophages, but also in vivo. LPS up-regulated SIK3 expression in murine RAW264.7 macrophages and exogenously over-expressed SIK3 negatively regulated the expression of inflammatory molecules [interleukin-6 (IL-6), nitric oxide (NO) and IL-12p40] in RAW264.7 macrophages. Conversely, these inflammatory molecule levels were up-regulated in SIK3-deficient thioglycollate-elicited peritoneal macrophages (TEPM), despite no impairment of the classical signalling cascades. Forced expression of SIK3 in SIK3-deficient TEPM suppressed the levels of the above-mentioned inflammatory molecules. LPS injection (10 mg/kg) led to the death of all SIK3-knockout (KO) mice within 48 hr after treatment, whereas only one mouse died in the SIK1-KO (n = 8), SIK2-KO (n = 9) and wild-type (n = 8 or 9) groups. In addition, SIK3-KO bone marrow transplantation increased LPS sensitivity of the recipient wild-type mice, which was accompanied by an increased level of circulating IL-6. These results suggest that SIK3 is a unique negative regulator that suppresses inflammatory molecule gene expression in LPS-stimulated macrophages.

Inhibition of hepatitis C virus replication by chalepin and pseudane IX isolated from Ruta angustifolia leaves.

Hepatitis C virus (HCV) infection is highly prevalent among global populations, with an estimated number of infected patients being 170 million. Approximately 70-80% of patients acutely infected with HCV will progress to chronic liver disease, such as liver cirrhosis and hepatocellular carcinoma, which is a substantial cause of morbidity and mortality worldwide. New therapies for HCV infection have been developed, however, the therapeutic efficacies still need to be improved. Medicinal plants are promising sources for antivirals against HCV. A variety of plants have been tested and proven to be beneficial as antiviral drug candidates against HCV. In this study, we examined extracts, their subfractions and isolated compounds of Ruta angustifolia leaves for antiviral activities against HCV in cell culture. We isolated six compounds, chalepin, scopoletin, γ-fagarine, arborinine, kokusaginine and pseudane IX. Among them, chalepin and pseudane IX showed strong anti-HCV activities with 50% inhibitory concentration (IC50) of 1.7 ± 0.5 and 1.4 ± 0.2 µg/ml, respectively, without apparent cytotoxicity. Their anti-HCV activities were stronger than that of ribavirin (2.8 ± 0.4 µg/ml), which has been widely used for the treatment of HCV infection. Mode-of-action analyses revealed that chalepin and pseudane IX inhibited HCV at the post-entry step and decreased the levels of HCV RNA replication and viral protein synthesis. We also observed that arborinine, kokusaginine and γ-fagarine possessed moderate levels of anti-HCV activities with IC50 values being 6.4 ± 0.7, 6.4 ± 1.6 and 20.4 ± 0.4 µg/ml, respectively, whereas scopoletin did not exert significant anti-HCV activities at 30 µg/ml.

Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps.

Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH(2)) and EMP-EF (FDVMGIIKKIAGAL-NH(2)), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

In vitro screening of leishmanicidal activity in myanmar timber extracts.

Seventy-five Myanmar timber extracts belonging to 27 families were examined for their leishmanicidal activities. Some timber extracts had significant leishmanicidal activity, especially extracts of Millettia pendula, which exhibited the most potent activity (MLC 3.1 microg/ml, MIC 1.6 microg/ml). Other timber extracts showing potent activity included those from Cedrela serrata, Cedrela toona, Cordia fragrantissima, Calophyllum kunstleri, Dalbergia cultrate, Grevillea robusta, Haplophragma adenophyllum, Michelia champaca, and Tectona grandis. From a literature search for reports on the chemical constituents of these plants, most constituents were found to be quinone derivatives or other compounds with unsaturated carbonyl groups.