Hiroyuki Fuchino

Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species

Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti‐HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti‐HCV activity with 50%‐inhibitory concentrations (IC50) of 20.0 and 8.0u2009μg/mL, respectively. Through bioactivity‐guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti‐HCV compounds, their IC50 being 8.8, 7.2, 4.6 and 16.4u2009μg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti‐HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti‐HCV activity, their IC50 being 2.5 and 6.2u2009μg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti‐HCV activity, with an IC50 of 3.7u2009μg/mL. Time‐of‐addition analysis revealed that all Glycyrrhiza‐derived anti‐HCV compounds tested in this study act at the post‐entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.

Antiviral activities of Indonesian medicinal plants in the East Java region against hepatitis C virus

BackgroundHepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities.MethodsEthanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b).ResultsFour of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 μg/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 μg/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 μg/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 μg/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes.ConclusionsEthanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.

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

Background: Salt-inducible kinases (SIKs) are capable of suppressing gluconeogenic gene expression in hepatocytes when they are overexpressed. Results: However, enhanced gluconeogenic programs are observed only in SIK3-defective hepatocytes. Conclusion: SIK3 is the major kinase that down-regulates gluconeogenesis. Significance: The present study proposes that SIK3 could be a new target of diabetic care. 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.

Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites, pheophorbide a and pyropheophorbide a, against hepatitis C virus

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo‐Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n‐hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti‐HCV activities with 50%‐inhibitory concentrations (IC50) of 20.6, 6.1, and 6.6u2009μg/mL, respectively. Bioactivity‐guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti‐HCV compound present in the extracts (IC50 u2009=u20090.3u2009μg/mL). It was also found that pyropheophorbide a possesses anti‐HCV activity (IC50u2009=u20090.2u2009μg/mL). The 50%‐cytotoxic concentrations (CC50) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2u2009μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti‐HCV activities. Time‐of‐addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post‐entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.

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.

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.

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 16xa0% (w/w). These results suggest that C. longissima is a novel, useful, and attractive source of skin-whitening agents.

Pterosin B has multiple targets in gluconeogenic programs, including coenzyme Q in RORα–SRC2 signaling

Hepatic gluconeogenic programs are regulated by a variety of signaling cascades. Glucagon-cAMP signaling is the main initiator of the gluconeogenic programs, including glucose-6-phosphatase catalytic subunit (G6pc) gene expression. Pterosin B, an ingredient in Pteridium aquilinum, inhibits salt-inducible kinase 3 signaling that represses cAMP-response element-binding protein regulated transcription coactivator 2, an inducer of gluconeogenic programs. As the results, pterosin B promotes G6pc expression even in the absence of cAMP. In this work, however, we noticed that once cAMP signaling was initiated, pterosin B became a strong repressor of G6pc expression. The search for associated transcription factors for pterosin B actions revealed that retinoic acid receptor-related orphan receptor alpha-steroid receptor coactivator 2 (RORα-SRC2) complex on the G6pc promoter was the target. Meanwhile, pterosin B impaired the oxidation-reduction cycle of coenzyme Q in mitochondrial oxidative phosphorylation (OXPHOS); and antimycin A, an inhibitor of coenzyme Q: cytochrome c-oxidoreductase (termed mitochondrial complex III), also mimicked pterosin B actions on RORα-SRC2 signaling. Although other respiratory toxins (rotenone and oligomycin) also suppressed G6pc expression accompanied by lowered ATP levels following the activation of AMP-activated kinase, minimal or no effect of these other toxins on RORα-SRC2 activity was observed. These results suggested that individual components in OXPHOS differentially linked to different transcriptional machineries for hepatic gluconeogenic programs, and the RORα-SRC2 complex acted as a sensor for oxidation-reduction cycle of coenzyme Q and regulated G6Pc expression. This was a site disrupted by pterosin B in gluconeogenic programs.

Determination of (E)-ferulic acid content in the root of Angelica acutiloba: a simple chemical evaluation method for crude drug quality control

The root of Angelica acutiloba Kitagawa is an important crude drug in Kampo medicines (traditional Japanese medicine). Chemical evaluation of crude drugs is crucial to ensuring the safety and efficacy of herbal medicine; however, there is currently no chemical standard for the A. acutiloba crude drug in Japanese pharmacopoeia. (E)-ferulic acid (FA) is an important active ingredient of Angelica spp., including A. sinensis (Oliv.) Diels, and has been suggested as a marker for quality evaluation of those crude drugs. However, it has been controversial whether FA is a reliable marker constituent of A. acutiloba. To achieve effective extraction of FA from A. acutiloba, we compared three different extraction methods: alkaline hydrolysis, ethanol extraction, and hexane extraction. FA levels in these extracts were assessed using high performance liquid chromatography (HPLC), and alkaline hydrolysis was found to be the most effective. Furthermore, in the hydrolysate, FA was distinctly identified by thin layer chromatography (TLC) analysis. These results provide useful information for the quality control of the A. acutiloba crude drug.