Chemical constituents from Morus alba with proprotein convertase subtilisin/kexin type 9 expression and secretion inhibitory activity.

Six new flavanones, including sanggenol W (1), morusalnol D-F (2-4) and neovanone A and B (5 and6), and fourteen known compounds were isolated from the methanol extract of the dried root bark of Morus alba using various column chromatographic methods. Their structures were elucidated using spectroscopic methods. The isolated compounds were tested in vitro for LDLR, PCSK9 and IDOL mRNA regulatory activity, and it was found that betulinic acid (13) showed the most potent effect on downregulation of PCSK9 and upregulation of LDLR at both mRNA and protein levels, showing comparable results to berberine, the positive control. In addition, betulinic acid (13) inhibited PCSK9 secretion, indicating its role as a future PCSK9 synthesis inhibitor.

Computational Prediction of the Phenotypic Effect of Flavonoids on Adiponectin Biosynthesis.

In silico machine learning applications for phenotype-based screening have primarily been limited due to the lack of machine-readable data related to disease phenotypes. Adiponectin, a nuclear receptor (NR)-regulated adipocytokine, is relatively downregulated in human metabolic diseases. Here, we present a machine-learning model to predict the adiponectin-secretion-promoting activity of flavonoid-associated phytochemicals (FAPs). We modeled a structure-activity relationship between the chemical similarity of FAPs and their bioactivities using a random forest-based classifier, which provided the NR activity of each FAP as a probability. To link the classifier-predicted NR activity to the phenotype, we next designed a single-cell transcriptomics-based multiple linear regression model to generate the relative adiponectin score (RAS) of FAPs. In experimental validation, estimated RAS values of FAPs isolated from Scutellaria baicalensis exhibited a significant correlation with their adiponectin-secretion-promoting activity. The combined cheminformatics and bioinformatics approach enables the computational reconstruction of phenotype-based screening systems.

Alkaloids and Coumarins with Adiponectin-Secretion-Promoting Activities from the Leaves of Orixa japonica.

Fractionation of a methanol extract of Orixa japonica leaves led to the identification of five new quinoline alkaloids (1, 2, 4, 8, and 9), three new coumarins (15, 17, and 19), and 20 known compounds. The structures were determined by analysis of 1D and 2D NMR spectroscopic data. The absolute configuration of 19 was proposed by electronic circular dichroism calculation. Among the compounds tested in the phenotypic screening to measure adiponectin secretion in human bone marrow mesenchymal stem cells, metabolites 4 and 12 stimulated adiponectin secretions with EC50 values of 13.8 and 25.8 µM, respectively. Further PPARγ binding assay and molecular modeling suggested that compounds 4 and 12 are selective PPARγ agonists.

No Interference of H9 Extract on Trastuzumab Pharmacokinetics in Their Combinations.

Trastuzumab is used to treat breast cancer patients overexpressing human epidermal growth factor receptor 2, but resistance and toxicity limit its uses, leading to attention to trastuzumab combinations. Recently, the synergistic effect of trastuzumab and H9 extract (H9) combination against breast cancer has been reported. Because drug exposure determines its efficacy and toxicity, the question of whether H9 changes trastuzumab exposure in the body has been raised. Therefore, this study aimed to characterize trastuzumab pharmacokinetics and elucidate the effect of H9 on trastuzumab pharmacokinetics at a combination dose that shows synergism in mice. As a result, trastuzumab showed linear pharmacokinetics after its intravenous administration from 1 to 10 mg/kg. In the combination of trastuzumab and H9, single and 2-week treatments of oral H9 (500 mg/kg) did not influence trastuzumab pharmacokinetics. In the multiple-combination treatments of trastuzumab and H9 showing their synergistic effect (3 weeks of trastuzumab with 2 weeks of H9), the pharmacokinetic profile of trastuzumab was comparable to that of 3 weeks of trastuzumab alone. In tissue distribution, the tissue to plasma ratios of trastuzumab below 1.0 indicated its limited distributions within the tissues, and these patterns were unaffected by H9. These results suggest that the systemic and local exposures of trastuzumab are unchanged by single and multiple-combination treatments of H9.

Flavonostilbenes Isolated from the Stems of Rhamnoneuron balansae as Potential SIRT1 Activators.

The cumulative effects of cell damage result in aging, which gradually decreases human function in various aspects and leads to multiple age-related chronic diseases. To overcome the adverse effects of aging, silent mating type information regulation 2 homologue (SIRT1) activators are promising bioactive compounds that mimic calorie restriction to improve quality of life and prevent aging. In this study, 11 new flavonostilbenes (1-11) and three known compounds (12-14) were purified from stems of Rhamnoneuron balansae. The structures of the new compounds were determined using extensive data from spectroscopic methods, including NMR and HRESIMS. Their absolute configurations were deduced by ECD calculations with coupling constant analysis. All of the isolated new compounds (1-11) were evaluated for their effects on SIRT1 deacetylase activity, the NAD+/NADH ratio, and the AMP-activated protein kinase activation level in cell-based assays. The results showed that rhamnoneuronal D (1) exhibits promising biological activity in several in vitro models related to SIRT1 and suggest it is a potential natural-product-based antiaging agent.

Selaginellin Derivatives from Selaginella tamariscina and Their Upregulating Effects on Low-Density Lipoprotein Receptor Expression.

Two new dimeric selaginellins, diselaginellins C and D (1 and 2), a new unusual derivative, selapiginellin A (4), a new selaginpulvilin U (5), and a known derivative, diselaginellin A (3), were isolated from Selaginella tamariscina (P. Beauv.) Spring. Among these compounds, selapiginellin A (4) is the first naturally occurring compound comprising an ether-linked dimer of a selaginellin and a selaginpulvilin. The absolute configurations of 1, 2, and 4 were elucidated by spectroscopic data analyses. Compound 5 was found to regulate mRNA expression of the low-density lipoprotein receptor (LDLR) gene and LDLR-related genes.

Salicinoyl Quinic Acids and Their Prostaglandin E2 Production Inhibitory Activities from the Fruits of Casearia grewiifolia.

Phytochemical investigation on the dried fruits of Casearia grewiifolia led to the identification of 10 new salicinoyl quinic acid derivatives (1-10), a new benzoyl quinic acid (11), and two known compounds (12 and 13). The structures of the new compounds were elucidated by interpreting 1D and 2D NMR spectroscopic data including HMBC and EXSIDE along with a chemical method for sugar unit analysis. All isolates were evaluated for their inhibitory activities against prostaglandin E2 (PGE2) production in ultraviolet B (UVB)-irradiated HaCat keratinocytes. Of the isolates tested, compounds 6 and 12 were found to inhibit PGE2 production with IC50 values of 20.5 and 28.8 µM, respectively.

Sesquiterpenoids from the Aerial Parts of Salvia plebeia with Inhibitory Activities on Proprotein Convertase Subtilisin/Kexin Type 9 Expression.

Phytochemical investigation of the methanol extract of the aerial parts of Salvia plebeia aided by a proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression screening assay in HepG2 cells led to the identification of 19 compounds including one new norsesquiterpene (1), six new eudesmane sesquiterpenoids (2-5, 8, and 11), and 12 known compounds. The structures of all compounds were elucidated by interpretation of their 1D and 2D NMR spectroscopic and MS data. Furthermore, computational prediction of ECD or chemical shifts was used to propose the absolute configurations of the new structures. All isolates were assessed for their inhibitory activities against PCSK9 mRNA expression and PCSK9-low-density lipoprotein receptor (LDLR) interactions. None of the isolated compounds inhibited PCSK9 and LDLR interactions. However, compounds 1, 9, and 10 downregulated PCSK9 mRNA expression.

Pharmacokinetic Properties of Moracin C in Mice.

Moracin C from Morus alba fruits, also known as the mulberry, has been proven to exhibit inhibitory activities against lipoxygenase enzymes, TNF-α and interleukin-1ß secretion, and proprotein convertase subtilisin/kexin type 9 expression. Despite the various pharmacological activities of moracin C, its pharmacokinetic characteristics have yet to be reported. Here, the pharmacokinetic parameters and tissue distribution of moracin C have been investigated in mice, and the plasma concentration of moracin C with multiple dosage regimens was simulated via pharmacokinetic modeling. Our results showed that moracin C was rapidly and well absorbed in the intestinal tract, and was highly distributed in the gastrointestinal tract, liver, kidneys, and lungs. Moracin C was distributed in the ileum, cecum, colon, and liver at a relatively high concentration compared with its plasma concentration. It was extensively metabolized in the liver and intestine, and its glucuronidated metabolites were proposed. In addition, the simulated plasma concentrations of moracin C upon multiple treatments (i.e., every 12 and 24 h) were suggested. We suggest that the pharmacokinetic characteristics of moracin C would be helpful to select a disease model for in vivo evaluation. The simulated moracin C concentrations under various dosage regimens also provide helpful knowledge to support its pharmacological effect.

Prenylated Flavonoid Glycosides with PCSK9 mRNA Expression Inhibitory Activity from the Aerial Parts of Epimedium koreanum.

Phytochemical investigation on the n-BuOH-soluble fraction of the aerial parts of Epimedium koreanum using the PCSK9 mRNA monitoring assay led to the identification of four previously undescribed acylated flavonoid glycosides and 18 known compounds. The structures of new compounds were elucidated by NMR, MS, and other chemical methods. All isolated compounds were tested for their inhibitory activity against PCSK9 mRNA expression in HepG2 cells. Of the isolates, compounds 6, 7, 10, 15, and 17-22 were found to significantly inhibit PCSK9 mRNA expression. In particular, compound 7 was shown to increase LDLR mRNA expression. Thus, compound 7 may potentially increase LDL uptake and lower cholesterol levels in the blood.

Effect of Water Extract of Mangosteen Pericarp on Donepezil Pharmacokinetics in Mice.

The pharmacokinetic (PK) change in a drug by co-administered herbal products can alter the efficacy and toxicity. In the circumstances that herb-drug combinations have been increasingly attempted to alleviate Alzheimer's disease (AD), the PK evaluation of herb-drug interaction (HDI) is necessary. The change in systemic exposure as well as target tissue distribution of the drug have been issued in HDIs. Recently, the memory-enhancing effects of water extract of mangosteen pericarp (WMP) has been reported, suggesting a potential for the combination of WMP and donepezil (DNP) for AD treatment. Thus, it was evaluated how WMP affects the PK change of donepezil, including systemic exposure and tissue distribution in mice after simultaneous oral administration of DNP with WMP. Firstly, co-treatment of WMP and donepezil showed a stronger inhibitory effect (by 23.0%) on the neurotoxicity induced by Aß(25-35) in SH-SY5Y neuroblastoma cells than donepezil alone, suggesting that the combination of WMP and donepezil may be more effective in moderating neurotoxicity than donepezil alone. In PK interaction, WMP increased donepezil concentration in the brain at 4 h (by 63.6%) after administration without affecting systemic exposure of donepezil. Taken together, our results suggest that WMP might be used in combination with DNP as a therapy for AD.

Glucose Uptake-Stimulating Galloyl Ester Triterpenoids from Castanopsis sieboldii.

Using molecular networking-guided isolation, three new galloyl ester triterpenoids (1-3), two new hexahydroxydiphenic acid-conjugated triterpenoids (6 and 7), and four known compounds (4, 5, 8, and 9) were isolated from the fruits and leaves of Castanopsis sieboldii. The chemical structures of 1-3, 6, and 7 were elucidated on the basis of interpreting their NMR, HRESIMS, and ECD spectra. All compounds (1-9) were evaluated for their glucose uptake-stimulating activities in differentiated adipocytes using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose as a fluorescent-tagged glucose probe. Compounds 2 and 9 resulted in a 1.5-fold increase in glucose uptake. Among them, compound 2 from the fruits showed an upregulation of p-AMPK/AMPK ratio in differentiated C2C12 myoblasts to support the mechanism proposed of glucose uptake stimulation.

A stilbene dimer and flavonoids from the aerial parts of Chromolaena odorata with proprotein convertase subtilisin/kexin type 9 expression inhibitory activity.

Investigation of components of the chloroform-soluble and ethyl acetate-soluble extracts of the aerial parts of Chromolaena odorata L. selected by PCSK9 mRNA expression monitoring assay in HepG2 cells led to the isolation of a new stilbene dimer, (+)-8b-epi-ampelopsin A (1), and 30 known compounds (2-31). The structures of the isolates were established by interpretation of NMR spectroscopic data and the stereochemistry of the new stilbene (1) was proposed based on ECD and NMR calculations. Among the isolates, 1, 5,6,7,4'-tetramethoxyflavanone (6), 5,6,7,3',4'-pentamethoxyflavanone (7), acacetin (18), and uridine (21) were found to inhibit PCSK9 mRNA expression with IC50 values of 20.6, 21.4, 31.7, 15.0, and 13.7 µM, respectively. Furthermore, the most abundant isolate among the selected compounds, 6, suppressed PCSK9 and low-density lipoprotein receptor protein expression in addition to downregulating the mRNA expression of HNF-1α.

Differential Effects of Saikosaponins A, B2, B4, C and D on Alcohol and Chocolate Self-Administration in Rats.

AIMS: Treatment with saikosaponin A (SSA)-an ingredient of the medicinal herb, Bupleurum falcatum-has been reported to suppress several addictive-like behaviors, including morphine, cocaine, alcohol and chocolate self-administration in male rats. The aim of this investigation was to investigate whether saikosaponins of B. falcatum other than SSA affect alcohol and chocolate self-administration in rats. METHODS: Ovariectomized female Sardinian alcohol-preferring (sP) and Wistar rats were trained to self-administer alcohol (15%, v/v) and a chocolate solution [5% (w/v) Nesquik® in water], respectively, under fixed ratio schedules of reinforcement. The following saikosaponins were compared to SSA: saikosaponin D (SSD; epimer of SSA), saikosaponin C (SSC), saikosaponin B2 (SSB2) and saikosaponin B4 (SSB4). All saikosaponins were tested acutely at the doses of 0, 0.25, 0.5 and 1 mg/kg (i.p.). RESULTS: Treatment with SSA and SSD resulted in highly similar, marked reductions in alcohol self-administration; SSC failed to alter lever-responding for alcohol, while SSB2 and SSB4 produced intermediate reductions. Only SSA and SSD reduced chocolate self-administration, with SSC, SSB2 and SSB4 being ineffective. CONCLUSIONS: The wide spectrum of efficacy of saikosaponins in reducing alcohol and chocolate self-administration suggests that even relatively small structural differences are sufficient to produce remarkable changes in their in vivo pharmacological profile. Together, these results confirm that roots of B. falcatum may be an interesting source of compounds with anti-addictive potential.

Effect of treatment period with LC478, a disubstituted adamantayl derivative, on P-glycoprotein inhibition: its application to increase docetaxel absorption in rats.

1. Treatment periods of P-glycoprotein (P-gp) inhibitors have revealed different efficacies. We have previously reported dose-dependent inhibition of P-gp in single-treatment with LC478. However, whether repeated treatment with LC478 can inhibit P-gp even at its ineffective single-treatment dose remains unknown. 2. Therefore, the purpose of this study was to assess the effect of repeated treatment (i.e., 7-day treatment) with LC478 on P-gp known to affect docetaxel bioavailability in rats. Effects of LC478 on P-gp mediated efflux and expression in MDCK-MDR1 cells, P-gp ATPase activity, and binding site with P-gp were evaluated.3. The 7-day treatment with LC478 increased docetaxel absorption via intestinal P-gp inhibition in rats. Intestinal concentrations of LC478 were 8.31-10.3 µM in rats after 7-day treatment of LC478. These concentrations were close to 10 µM that reduced P-gp mediated docetaxel efflux and P-gp expression in MDCK-MDR1 cells. Considering that intestinal LC478 concentrations after 1-day treatment were 2.68-4.19 µM, higher LC478 concentrations after 7-day treatment might have driven P-gp inhibition and increased docetaxel absorption. LC478 might competitively inhibit P-gp considering its stimulated ATPase activity and its binding site with nucleotide binding domain of P-gp. 4. Therefore, repeated treatment with LC478 can determine its feasibility for P-gp inhibition and changing docetaxel bioavailability.

Standardized Extract of Atractylodis Rhizoma Alba and Fructus Schisandrae Ameliorates Coughing and Increases Expectoration of Phlegm.

Cough and phlegm frequently occur in respiratory diseases like upper respiratory tract infections, acute bronchitis, and chronic obstructive pulmonary diseases. To relieve these symptoms and diseases, various ingredients are being used despite the debates on their clinical efficacy. We aimed to investigate the effects of the extract CKD-497, composed of Atractylodis Rhizoma Alba and Fructus Schisandrae, in relieving cough and facilitating expectoration of phlegm. CKD-497 was found to inhibit inflammatory mediators such as interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) in lipopolysaccharide (LPS)-treated mouse macrophages and transient receptor potential cation channel 1 (TRPV-1)-overexpressed human bronchial epithelial cells stimulated by capsaicin. CKD-497 decreased the viscosity of the mucin solution. During in vivo experiments, CKD-497 reduced coughing numbers and increased expectoration of phlegm via mucociliary clearance enhancement. Collectively, these data suggest that CKD-497 possesses potential for cough and phlegm expectoration treatment.

Combined MS/MS-NMR Annotation Guided Discovery of Iris lactea var. chinensis Seed as a Source of Viral Neuraminidase Inhibitory Polyphenols.

In this study, the chemical diversity of polyphenols in Iris lactea var. chinensis seeds was identified by combined MS/MS-NMR analysis. Based on the annotated chemical profile, the isolation of stilbene oligomers was conducted, and consequently, stilbene oligomers (1-10) were characterized. Of these, compounds 1 and 2 are previously undescribed stilbene dimer glycoside (1) and tetramer glycoside (2), respectively. Besides, to evaluate this plant seed as a rich source of stilbene oligomers, we quantified three stilbene oligomers of I. lactea var. chinensis seeds. The contents of three major stilbene oligomers-trans-ε-viniferin (3), vitisin A (6), and vitisin B (9)-in I. lactea var. chinensis seeds were quantified as 2.32 (3), 4.95 (6), and 1.64 (9) mg/g dry weight (DW). All the isolated compounds were tested for their inhibitory activities against influenza neuraminidase. Compound 10 was found to be active with the half maximal inhibitory concentration (IC50) values at 4.76 µM. Taken together, it is concluded that I. lactea var. chinensis seed is a valuable source of stilbene oligomers with a human health benefit.

Prenylated Flavonoids from the Roots and Rhizomes of Sophora tonkinensis and Their Effects on the Expression of Inflammatory Mediators and Proprotein Convertase Subtilisin/Kexin Type 9.

Seven new prenylated flavonoids (1-7) and one new prenylated phenylpropiophenone (8) were isolated from roots and rhizomes of Sophora tonkinensis, along with nine known compounds (9-17). The structures 1-8 were elucidated by spectroscopic data analysis and comparison with reported values. Compounds 8 and 12 (7-methoxyebenosin) showed inhibitory activities against nitric oxide production in lipopolysaccharide-induced RAW264.7 cells, with IC50 values of 8.1 and 6.2 µM, respectively. They also significantly lowered expression of CSF2, TNF, and IL-1ß. Lonchocarpol A (10) and erybraedin D (16) at concentrations of 20 µM downregulated proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression in HepG2 cells. Moreover, erybraedin D (16) inhibited PCSK9 protein synthesis (IC50 7.8 µM), while simultaneously activating AMP-activated protein kinase and acetyl-CoA carboxylase.

7-O-Methylwogonin from Scutellaria baicalensis Disturbs Mitotic Progression by Inhibiting Plk1 Activity in Hep3B Cells.

Polo-like kinase 1, a mitotic Ser/Thr kinase, has emerged as a molecular target for the development of anticancer drugs. In this study, we found that polo-like kinase 1 activity was inhibited by 7-O-methylwogonin and related flavones, including baicalein, dihydrobaicalein, and viscidulin II, isolated from Scutellaria baicalensis. Although dihydrobaicalein exhibited the highest polo-like kinase 1 inhibitory activity among the four compounds, it also inhibited other kinases, such as vaccinia-related kinase 2 and polo-like kinase 2. Baicalein and viscidulin II also showed low selectivity to polo-like kinase 1 since they inhibited polo-like kinase 3 and polo-like kinase 2, respectively. However, 7-O-methylwogonin exhibited selective polo-like kinase 1 inhibitory activity, as evidenced from in vitro kinase assays based on fluorescence resonance energy transfer assays and ADP-Glo kinase assays. In addition, examination of mitotic morphology and immunostaining using specific antibodies for the mitotic markers, p-histone H3 and mitotic protein monoclonal 2, in Hep3B cells showed that 7-O-methylwogonin treatment increased mitotic cell populations due to inhibition of mitotic progression as a result of polo-like kinase 1 inhibition. The pattern of 7-O-methylwogonin-induced mitotic arrest was similar to that of BI 2536, a specific polo-like kinase 1 inhibitor. Thus, it was suggested that 7-O-methylwogonin disturbed mitotic progression by inhibiting polo-like kinase 1 activity. These data suggest that 7-O-methylwogonin, a polo-like kinase 1 inhibitor, may be a useful anticancer agent because of its polo-like kinase 1 selectivity and effectiveness.

Molecular Targets of Genistein and Its Related Flavonoids to Exert Anticancer Effects.

Increased health awareness among the public has highlighted the health benefits of dietary supplements including flavonoids. As flavonoids target several critical factors to exert a variety of biological effects, studies to identify their target-specific effects have been conducted. Herein, we discuss the basic structures of flavonoids and their anticancer activities in relation to the specific biological targets acted upon by these flavonoids. Flavonoids target several signaling pathways involved in apoptosis, cell cycle arrest, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/AKT kinase, and metastasis. Polo-like kinase 1 (PLK1) has been recognized as a valuable target in cancer treatment due to the prognostic implication of PLK1 in cancer patients and its clinical relevance between the overexpression of PLK1 and the reduced survival rates of several carcinoma patients. Recent studies suggest that several flavonoids, including genistein directly inhibit PLK1 inhibitory activity. Later, we focus on the anticancer effects of genistein through inhibition of PLK1.