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Clove, a dried flower buds of Syzygium aromaticum, is used in traditional medicine, for culinary purposes, and in essential oil production. In our preliminary screening of crude drugs used in Japanese Kampo formulas, a methanol (MeOH) extract of clove buds was found to exhibit a melanin induction. To date, the effects of clove buds or their constituents on the activation of melanogenesis remain unclear. Thus, this study aimed to isolate active compounds from the MeOH extract of clove buds associated with melanin synthesis in melanoma cells and to investigate the molecular mechanism involved. The MeOH extract of clove buds increased melanin content in murine B16-F1 melanoma cells. To identify the active compounds responsible for melanin induction, the MeOH extract was suspended in water and successively partitioned using hexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH). Comparative analysis revealed that the EtOAc fraction induced melanin synthesis. Bioassay-guided separation of the EtOAc fraction isolated three compounds including eugenol. The analysis of structure-activity relationships of eugenol and structurally related compounds indicated that eugenol was the most potent melanin inducer among the 11 compounds, and that a hydroxyl group at C-1 and a methoxy group at C-2 may contribute to melanin induction. Eugenol induced melanin synthesis in human HMV-II melanoma cells as well as in B16-F1 cells. Further analysis indicated that eugenol may invoke intracellular tyrosinase activity and expression of tyrosinase, tyrosinaserelated protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). These results suggest that eugenol enhances melanin synthesis by upregulating the expression of MITF and subsequent expression of melanogenic enzymes, and that it may be a potent therapeutic agent for hypopigmentation.

Silibinin is a flavonolignan isolated from milk thistle (Silybum marianum). Silibinin has been reported to possess multiple biological activities; however, its effect on melanogenesis remains unclear. This study investigated the effect of silibinin on melanogenesis in melanoma cells and the associated molecular mechanism. Our findings demonstrated that silibinin markedly increased melanin content in murine B16-F1 and human HMV-II melanoma cells. Silibinin activated intracellular tyrosinase activity and expression of tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). Furthermore, silibinin enhanced the phosphorylation of cyclic AMP-responsive element-binding protein (CREB), protein kinase A (PKA), and p38 mitogen-activated protein kinase (MAPK) but not of Akt and extracellular signal-regulated kinase (ERK). The specific PKA (H-89) and p38 (SB203580) inhibitors significantly attenuated silibinin-mediated melanin synthesis. These results suggest that silibinin is an effective stimulator of melanogenesis through upregulation of the protein expression of melanogenic enzymes activated by the PKA and p38 pathways, leading to CREB phosphorylation and MITF expression. Therefore, silibinin may have potential for use in the treatment of hypopigmentation disorders.

A new high performance liquid chromatography (HPLC) method has been established for quantitative and qualitative analysis of three tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3), which are responsible for anti-trypanosomal and anti-leishmanial activities of Morinda lucida Bentham leaves. Separation of 1–3 from dried 80% aqueous (aq.) ethanol extract was achieved on a reversed-phase cholester column packed with cholesteryl-bonded silica using an acetonitrile-0.1% aq. formic acid mobile phase system. Ultraviolet-visible (UV-VIS) spectroscopy was employed for detection of compounds, and their contents were determined by measuring absorbance at 254 nm. Depending on the above system, several factors potentially affecting the concentration of tetracyclic iridoids were evaluated resulting in several variation on plant organs, seasonality, variation between individual trees, and branch positions within the trees. Moreover, we developed a simple, quick, and effective method for tetracyclic iridoid isolation from M. lucida leaves that consisted of extraction by sonication into 80% aq. ethanol, basic hydrolysis, acid neutralization, liquid-liquid extraction into an organic solvent, and reverse phase open column chromatography. Employing this method, we have succeeded to obtain 1 as a colorless crystal yielding of 0.23%, which was 28 times higher than that of previous isolation method. Setting up methodology in this paper may be important for future in vitro and in vivo studies of tetracyclic iridoids and moreover for their applications in new drug design and development.

Haprin (TRIM36) is a ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin-deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility-related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin-deficient mice having poorer sperm morphology and motility than wild-type mice. Interestingly, haprin-deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.

Our preliminary screening identified an extract from the rhizome of Dioscorea tokoro, which strongly suppressed the proliferation of HepG2 hepatocellular carcinoma cells and inhibited autophagy. This study aimed to isolate active compounds from the rhizome of D. tokoro that exert antiproliferative effects and inhibit autophagy. The bioassay-guided fractionation of the active fraction led to the isolation of two spirostan-type steroidal saponins, dioscin (1) and yamogenin 3-O-α-l-rhamnopyranosyl (1→4)-O-α-l-rhamnopyranosyl(1→2)-β-d-glucopyranoside (2), and the frostane-type steroidal saponin protodioscin (3) from the n-BuOH fraction. Furthermore, acid hydrolysis of 1 and 2 produced the aglycones diosgenin (4) and yamogenin (5), respectively. Compounds 1–5 suppressed proliferation of HepG2 cells. The analysis of structure-activity relationships indicated that the 25(R)-conformation, structures with a sugar moiety, and the spirostan-type aglycone moiety contributed to antiproliferative activity. Analysis of autophagy-related proteins demonstrated that 1–3 clearly increased the levels of both LC3-II and p62, implying that 1–3 deregulate the autophagic pathway by blocking autophagic flux, which results in p62 and LC3-II accumulation. In contrast, 1–3 did not significantly affect caspase-3 activation and PARP cleavage, suggesting that the antiproliferative activity of 1–3 occurred independently of caspase-3-mediated apoptosis. In summary, our study showed that 1–3, active compounds in the rhizome of D. tokoro, suppressed cell proliferation and autophagy, and might be potential agents for autophagy research and cancer chemoprevention.

A coumaric acid analogue with a monoterpene moiety named floraosmanol A (1) was isolated from the flowers of Osmanthisfragrans var. aurantiacus. The chemical structure was elucidated on the basis of chemical and physicochemical evidence. Floraosmanol A (1) significantly inhibited nitric oxide .(NO) production in lipopolysaccharide- (LPS) activated RAW264.7 macrophages and the release of P-hexosaminidase as a marker of degranulation from rat basophile leukemia (RBL-2H3) cells. In addition, several cinnamic acid analogues were conjugated with geraniol and 3-methyl-2-buten-1-ol through an ester linkage. The inhibitory effects on NO production and the release of P-hexosaminidase of the synthesized compounds were examined for structure-activity relationships.

The methanol extract of whole Bacopa monniera plants inhibited pancreatic lipase activity in vitro. From this extract we have reported the isolation of 11 triterpene glycosides and 5 phenylethanoid- and/or phenylpropanoid- glycosides. In this paper, we describe the effects of the methanol extract and/or its constituents on pancreatic lipase activity and the isolation of an active constituent, desrhamnosyl isoacteoside. In addition, the structural requirements for its inhibitory effects were examined. We also examined the effects on the elevation of plasma triglyceride (TG) levels in olive oil loaded mice. The major active constituents, desrhamnosyl isoacteoside and plantainoside B, reduced plasma TG levels in the mice. The inhibitory effects of B. monniera and its constituents on pancreatic lipase activity and plasma TG level are reported for the first time.

Background: The roots of Rehmannia glutinosa (RG) or Rehmanniae Radix are a well-known medicinal material in the Oriental medicine, and its phytochemical profile has been extensively studied with more than 100 individual compounds from Rehmannia species. In contrast, bioactive components of the aerial part of the title plant are largely unknown as only several compounds reported up to date. Objective: The objective was to study on chemical constituents of the aerial parts of the title plant and evaluate the aerial parts as a supplementary source for Rehmanniae radix. Materials and Methods: Solvent extraction, partition, and column chromatography was used to separate individual compounds; spectroscopic data including nuclear magnetic resonance and mass spectrometry were analyzed to determine the chemical structure of the isolates. Results: Eight compounds including five ursane-type triterpenoids for the first time from RG (ursolic acid [1], pomolic acid [2], 2β-hydroxypomolic acid [3], asiatic acid [4] and 7β,24-dihydroxy ursolic acid [5]) and three main glycosides (ajugol [6], aucubin [8], and acteoside [7]) were characterized from the aerial parts of the title plant. Their structures were identified on the basis of spectroscopic data and comparison with those reported in the literature. Conclusion: The current study reveals various ursane triterpenes in the organic portion beside the main hydrophilic glycosides in the RG aerial parts. The occurrence of various ursane triterpenes contributed in part to phytochemical database and evidence of the biological activity associated with potential in use as a medicinal material of the RG leaves.

The dried leaves of Eriobotrya japonica have traditionally been widely used to treat various diseases, such as chronic bronchitis, cough, inflammation, skin disease, and diabetes. Our previous studies have reported several functions and their molecular mechanisms of E. japonica leaf extract and triterpenes contained in the extract. In this review article, we focus on the effects of E. japonica leaf extract and triterpenes on inflammatory mediators, proliferation of cancer cells, and ghrelin production. 1) The leaf extract of E. japonica suppressed inflammatory mediators including nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated RAW264 murine macrophage cells. The anti-inflammatory properties of E. japonica leaf extract resulted from inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions through downregulation of nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinases (MAPK) phosphorylation. 2) E. japonica leaves contain triterpenes such as corosolic acid, ursolic acid, maslinic acid, and oleanolic acid. Corosolic acid exterted the strongest anti-proliferative activity in human leukemia cell lines. Moreover, corosolic acid induced apoptosis mediated by mitochondrial dysfunction and caspase activation. 3) Ghrelin is an appetite-stimulating peptide hormone with an octanoyl modification at serine 3 that is essential for its orexigenic effect. Our investigation of triterpenes from E. japonica, corosolic acid, oleanolic acid, and ursolic acid suppressed octanoylated ghrelin levels in AGS-GHRL8 cell line, which produces octanoylated ghrelin in the presence of octanoic acid, without decreasing transcript expression of ghrelin O-acyltransferase (GOAT) or furin.

Background: Liquiritin (LQ) and its aglycone, liquiritigenin (LQG), are major flavonoids in licorice root (Glycyrrhiza spp.). Our preliminary screening identified LQ and LQG, which promote melanin synthesis in the melanoma cells. In this study, we investigated the molecular mechanism of melanin synthesis activated by LQ and LQG. Methods: Murine (B16-F1) and human (HMVII) melanoma cell lines were treated with LQ or LQG. After incubation, melanin contents, intracellular tyrosinase activity, and cell viability were evaluated. Protein levels were determined using Western blotting. Results: LQ and LQG activated melanin synthesis and intracellular tyrosinase activity. The induction of melanin and intracellular tyrosinase activity by LQG was higher than that by LQ. LQ and LQG induced the expression of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. LQ and LQG also enhanced microphthalmia-associated transcription factor (MITF) expression, and cyclic AMP-responsive element-binding protein (CREB) phosphorylation. The phosphorylation of p38 and extracellular signal-regulated kinase (ERK), but not Akt, was significantly increased by LQ or LQG. Furthermore, LQ- or LQG-mediated melanin synthesis was partially blocked by p38 inhibitor (SB203580) and protein kinase A (PKA) inhibitor (H-89), however, ERK kinase (MEK) inhibitor (U0126) and phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002) had no effect. Conclusions: The results suggest that LQ and LQG enhance melanin synthesis by upregulating the expression of melanogenic enzymes, which were activated by p38 and PKA signaling pathways, leading to MITF expression and CREB phosphorylation.

Background: Autophagy is a catabolic process through which dysfunctional proteins and organelles are degraded, and that is associated with the proliferation of cancer cells. The aim of this study was to screen approximately 130 kinds of crude drugs used in Japanese Kampo formulas to identify crude drugs that would regulate the proliferation through autophagy of human hepatocellular carcinoma HepG2 cells. Methods: Extracts of each crude drug were prepared using methanol. Protein levels were determined using Western blotting. Cell viability was measured by MTT assay. Results: Among the 130 crude extracts, 24 of them increased LC3-II expression. Among these, Goboshi (burdock fruit), Soboku (sappan wood), Mokko (saussurea root), Rengyo (forsythia fruit), and Hikai (dioscorea) notably suppressed the proliferation of HepG2 cells and increased p62 expression levels, which suggested that these five extracts downregulate the autophagic activity resulting in the accumulation of p62. On the other hand, Hishinomi (water chestnut), Biwayo (loquat leaf), and Binroji (areca) induced cell growth and decreased or were uninvolved with p62 expression levels, which implied that these three extracts might induce autophagy modulators for cell growth. Conclusions: The results suggest that the compounds contained in the crude drugs selected for this study could control cell viability by regulating autophagic activity in HepG2 cells. The isolation and identification of the active compounds in these drugs might lead to the development of agents for autophagy research and cancer chemoprevention.

Saffron (Crocus sativus L.) has been used both as a food additive for flavoring and coloring and in traditional medicine. Saffron extract and its main component crocin decrease the growth of several types of human cancer, including colorectal cancer in vitro. Numerous polyps develop in the small intestine in the Adenomatous polyposis coli (Apc) deficiency mice. ApcMin/+ mice are models for human familial adenomatous polyposis and human colon cancer patients. In this study, we examined the efficacy of saffron extract added to diet on reducing the polyp density in ApcMin/+ mice. ApcMin/+ mice were either given a placebo or saffron extract (0.1% and 0.5%) diet for 4 weeks. At 12 weeks of age, intestines were analyzed for polyp number in the small intestine. Our analysis confirmed that crocin (1), crocin-2 (2), and crocin-4 (4) are the major compounds in the saffron extract and the content of 1 in the tested saffron extract was 29.2%. Saffron extract decreased the number of intestinal polyps in a concentration-dependent manner in ApcMin/+ mice. Notably, the number of polyps in the distal small intestine of the mice fed with 0.5% saffron extract was significantly decreased compared with the placebo. These results indicate that saffron extract can reduce the polyp number in the ApcMin/+ mice. Abbreviations Used: FAP: Familial adenomatous polyposis; Apc: Adenomatous polyposis coli.

The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang province, China, showed an inhibitory effect on aldose reductase. From the methanolic extract, five new acylated sucroses, mumeoses F-J, were isolated together with 29 known compounds. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of the isolated compounds on aldose reductase were also investigated. Acylated quinic acid analogs, which are one of the major compounds of the flower buds of P. mume, were shown to substantially inhibit aldose reductase. In particular, mumeic acid-A was found to exhibit a potent inhibitory effect [IC50=0.4 µm].

The methanolic extract and its 1-butanol-soluble fraction from the flower buds of Camellia japonica, cultivated in Yunnan Province, China, showed inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the 1-butanol-soluble fraction, a new 28-nor-oleanane-type and three new oleanane-type triterpene saponins, sanchakasaponins A-D, were isolated together with four known triterpene saponins. Their chemical structures were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells and structure-activity relationships of the saponins were investigated.

A new acylated oleanane-type triterpene oligoglycoside, floratheasaponin K (1), was isolated together with 11 known compounds including floratheasaponins D (2)–G (5), and I (6), chakasaponin V (7), and assamsaponin E (8) from the flower buds of Camellia sinensis cultivated in India. The chemical structure of floratheasaponin K (1) was elucidated on the basis of chemical and physicochemical evidence. In addition, chakasaponins V (7) and I (13) significantly inhibited nitric oxide (NO) production in lipopolysaccharide- (LPS) activated RAW264.7 cells without cytotoxicity.

The methanol extract of whole Bacopa monniera plants inhibited pancreatic lipase activity in vitro. From this extract we have reported the isolation of 11 triterpene glycosides and 5 phenylethanoid- and/or phenylpropanoid- glycosides. In this paper, we describe the effects of the methanol extract and/or its constituents on pancreatic lipase activity and the isolation of an active constituent, desrhamnosyl isoacteoside. In addition, the structural requirements for its inhibitory effects were examined. We also examined the effects on the elevation of plasma triglyceride (TG) levels in olive oil loaded mice. The major active constituents, desrhamnosyl isoacteoside and plantainoside B, reduced plasma TG levels in the mice. The inhibitory effects of B. monniera and its constituents on pancreatic lipase activity and plasma TG level are reported for the first time.

A coumaric acid analogue with a monoterpene moiety named floraosmanol A (1) was isolated from the flowers of Osmanthus fragrans var. aurantiacus. The chemical structure was elucidated on the basis of chemical and physicochemical evidence. Floraosmanol A (1) significantly inhibited nitric oxide (NO) production in lipopolysaccharide- (LPS) activated RAW264.7 macrophages and the release of β-hexosaminidase as a marker of degranulation from rat basophile leukemia (RBL-2H3) cells. In addition, several cinnamic acid analogues were conjugated with geraniol and 3-methyl-2-buten-1-ol through an ester linkage. The inhibitory effects on NO production and the release of β-hexosaminidase of the synthesized compounds were examined for structure-activity relationships.

The methanolic extract and its 1-butanol-soluble fraction from the flower buds of Camellia japonica, cultivated in Yunnan Province, China, showed inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the 1-butanol-soluble fraction, a new 28-nor-oleanane-type and three new oleanane-type triterpene saponins, sanchakasaponins A-D, were isolated together with four known triterpene saponins. Their chemical structures were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells and structure-activity relationships of the saponins were investigated.

温州みかん外果皮に含まれる抗酸化性成分について検討した結果,次の結果が得られた. (1) 温州みかん外果皮の不ケン化物区分にはかなり強い抗酸化作用がある. (2) この抗酸化作胴は温州みかん外果皮に存在するα-およびγ-トコフェロールに基づくものである. (3) 温州みかん外果皮のトコフェロール類はα-トコフェロール,γ-rコフェロールで,β-トコフェロールは存在しない.そしてその含量は,α-トコフェロール100～160γ/g(新鮮物量で),γ-トコフェロール60～70γ/g(新鮮物量で)であった.

2016年度