Methoxyflavones isolated from the whole plant of Scutellaria rubropunctata Hayata var. rubropunctata promote osteoblast differentiation in MC3T3-E1 cells.

In this study, we isolated two new methoxyflavones (1 and 2) and eight known methoxyflavones (3-10) from the whole plant of Scutellaria rubropunctata Hayata var. rubropunctata (SR). Based on spectroscopic analyses, the methoxyflavones were identified as 5,8,2',6'-tetramethoxy-6,7-methylenedioxyflavone (1) and 5,2',6'-trimethoxy-6,7-methylenedioxyflavone (2). We reported SR might have effects on promoting osteoblast differentiation and stimulating estrogen receptor (ER) in the previous study. Then, the effects of 1-10 on pre-osteoblast MC3T3-E1 cells were investigated, and 1, 2, and 9 were observed to promote alkaline phosphatase activity. To evaluate their effect on osteogenesis-related genes, we performed gene expression analysis using quantitative real-time PCR after treatment of MC3T3-E1 cells with these compounds. Although 2 was only effective at lower concentrations, 1 and 9 upregulated the mRNA levels of Runx2, Osterix, Osteopontin, Osteocalcin, Smad1, and Smad4. These results indicate that 1 and 9 may induce osteoblast differentiation by activating Runx2 via the BMP/Smad pathway and may play a central role in the promotion of osteoblast differentiation by SR. The ER agonist activity of 1-10 were tested using a luciferase reporter assay in HEK293 cells. However, none of the compounds exhibited remarkable activity. Thus, SR may contain other compounds that contribute to its ER agonist activity.

17ß-neriifolin from unripe fruits of Cerbera manghas suppressed cell proliferation via the inhibition of HOXA9-dependent transcription and the induction of apoptosis in the human AML cell line THP-1.

Homeobox A9 (HOXA9) is a transcription factor that is overexpressed in acute myeloid leukemia (AML). It is associated with the pathogenesis and progression of AML, and is a factor responsible for a poor prognosis. Therefore, the development of HOXA9-targeting molecules may contribute to not only better understanding of the mechanism of HOXA9 regulation, but also the development of therapeutic applications. We constructed a reporter assay system using the promoter region of the KBTBD10 gene, to which HOXA9 directly binds and regulates transcription, in the human acute monocytic leukemia cell line THP-1. Using this luciferase gene assay, we screened 1120 plant extracts and a methanol extract of the unripe fruits of Cerbera manghas was found to suppress the reporter gene expression mediated by the KBTBD10 promoter. From the extract, five steroid-type compounds were identified as the active constituents: 7α-neriifolin (1), 17ß-neriifolin (2), 17α-digitoxigenin ß-D-glucosyl-(1 → 4)-α-L-thevetoside (3), 17ß-digitoxigenin ß-D-glucosyl-(1 → 4)-α-L-thevetoside (4), and acetylthevetin B (5). Among the five compounds, 17ß-neriifolin most potently inhibited HOXA9-dependent gene expression without affecting the HOXA9 mRNA levels, and suppressed cell proliferation by inducing apoptosis. The findings on the structure-activity relationships of the compounds from C. manghas may contribute to the development of small molecule inhibitors of HOXA9.

Anchietins A-E: 30-norfriedelane-type triterpenes from Anchietea pyrifolia.

Five undescribed norfriedelane triterpenoids, anchietins A-E, along with three known norfriedelane triterpenoids, 21ß-hydroxycaloncobalactone, and welwitschiilactones B and C, were isolated from the vine of Anchietia pyrifolia. The compounds were characterized by spectroscopic and crystallographic methods, including 2D NMR spectroscopy and single crystal X-ray crystallography. The isolated compounds were evaluated for the cytotoxic activity against HeLa and HL60 cells and for the inhibitory activity against lipopolysaccharide-induced NO production. Further, inhibitory effects on the inducible nitric oxide synthase mRNA expression levels were also assessed.

Effects of a Whole Plant Extract of Scutellaria rubropunctata var. rubropunctata on Bone Metabolism with Estrogen Receptor Activation.

We screened natural resources for estrogen receptor (ER)-activating and bone metabolism-promoting activities with the aim of finding potential treatments for osteoporosis. A screen of 1531 extracts from Ryukyu Arc plants using a luciferase reporter assay identified an 80% MeOH extract of Scutellaria rubropunctata var. rubropunctata (SRE) with dose-dependent ER transcription-promoting activity. Importantly, SRE had no proliferative effect on human breast cancer cells. SRE enhanced the ALP activity of pre-osteoblast MC3T3-E1 cells after 72 h in culture and slightly enhanced mineralization at 14 and 21 d. SRE did not significantly affect the TRAP activity of RAW264.7 cells. Gene expression analysis in MC3T3-E1 cells by quantitative real-time PCR revealed that SRE upregulated the mRNA levels of Runx2, Osterix (Osx), Osteopontin (Opn), Osteocalcin (Ocn), Smad1, Smad4, and Smad5 at 72 h, and those of Runx2, Osx, Smad1, Smad4, and Smad5 at 21 d of osteogenic induction. Analysis of the expression levels of osteogenic markers suggested that SRE may promote osteogenic differentiation by acting at the early stage of differentiation rather than at the late stage of differentiation. These results indicate that SRE activates ER and induces osteoblast differentiation by activating Runx2 and Osx through the BMP/Smad pathway, suggesting that SRE may be useful for the prevention and treatment of postmenopausal osteoporosis.

Inhibitory activity of flavonoids from Ephedrae Herba on hypoxia signaling in PANC-1 cells and the evaluation of their mechanisms.

Pancreatic cancer is a lethal disease with a very poor prognosis. Recent reports indicate that hypoxia signaling mediated by hypoxia-inducible factor (HIF) contributes to the progression of pancreatic cancer. Therefore, elucidating the inhibitor of hypoxia signaling may lead to the development of a candidate for new anticancer agents. During our screening program for HIF inhibitor from crude drug extracts, new acylated kaempferol glycosides, kaempferol 3-O-[4″-(E)-p-coumaroyl-3″-O-dihydroxypalmityl] rhamnoside (1) and kaempferol 3-O-[4″-(E)-p-coumaroyl-2″-O-dihydroxypalmityl] rhamnoside (2), were isolated from an acetone extract of Ephedrae Herba, together with eight known flavonol glycosides (3-10). The structures of novel compounds 1 and 2 were elucidated based on spectroscopic and chemical analyses. Using a cell-based HRE-driven luciferase reporter assay in a PANC-1 pancreatic cancer cell line, we found that these compounds demonstrated potent inhibitory activity on hypoxia signaling with IC50 values of 18.0 ± 0.6 and 13.3 ± 2.2 µM, respectively. Mechanistically, 2 reduced the amount of HIF-1α protein in the nuclear at 30 µM via the ubiquitin-proteasome pathway with no effect on the nuclear translocation of HIF proteins from cytosol and subsequently decreased Glut1 mRNA. These results indicate that 2 inhibits hypoxia signaling through a mechanism involving the reduction of HIF-1α protein levels and Glut1 mRNA and may have anti-pancreatic cancer effects.

Quality evaluation of Pinellia tuber by LC-TOF/MS targeted to ephedrine.

Pinellia tuber (PTE, , , , , , , , ) is derived from the tuber of Pinellia ternata Breitenbach (Araceae), which is a crude drug used in traditional Japanese Kampo medicine for the purpose of antiemesis and expectoration. Since the separation of ephedrine from PTE in 1978, it has been listed as a PTE component in textbooks and internet information. Therefore, there are harmful effects on appropriate use in clinical practice because PTE is dealt with as a crude drug for doping target, and traditional Japanese Kampo medicine containing PTE must be carefully administered to the elderly. However, since the 1978 published report, there has not been any report on the isolation of ephedrine from PTE and the interpretation of biosynthesis remains questionable. In the present study, we analyzed the PTE samples in market distribution products by LC-TOF/MS. From the analysis of the result of ephedrine's m/z 148.113 [M + H-H2O]+, PTE was not detected (n = 55, detection limit: 0.5 ppb). Additionally, the tuber of P. tripartite (PTR, ), the tuber of P. pedatisecta (PPE, ), Arisaema Tuber (ART, ), and the tuber of Typhonium flagelliforme (TFI, ) that have a similar description to PTE were also not detected. Moreover, the genetic analysis of experimental samples showed that PTE is derived from P. ternata. Furthermore, our attempt to isolate ephedrine from PTE based on the past literature was unsuccessful. These results suggest that PTE in market distribution products may not contain ephedrine as a component.

Antiproliferative and Antimigration Activities of Beauvericin Isolated from Isaria sp. on Pancreatic Cancer Cells.

This study describes the antiproliferative and antimigration effects of beauvericin from a culture broth of Isaria sp. in human pancreatic cancer cells (PANC-1). Activity-guided fractionation of the EtOAc extract of cultured broth of Isaria sp. RD055140 afforded beauvericin (1), a new isariotin derivative, 7-O-methylisariotin C (2), together with the known isariotin analogs, TK-57-164A (3) and B (4). As a result of the measurement of the cell viability, 1 inhibited cell growth (IC50 = 4.8 µM) of PANC-1 cells. Furthermore, 1 was found to inhibit the migration activity of PANC-1 cells by upregulating the expression of the E-cadherin gene and reducing N-cadherin and Snail genes in a dose-dependent manner (0.1-1 µM). These activities of 1 had lower concentrations than the cytotoxic activity. These findings suggest that 1 can be used as an anticancer agent against human pancreatic carcinoma.

Marylosides A-G, Norcycloartane Glycosides from Leaves of Cymbidium Great Flower 'Marylaurencin'.

Seven novel norcycloartane glycosides, maryloside A-G (1-7), were isolated from the leaves of Cymbidium Great Flower 'Marylaurencin', along with a known norcycloartane glycoside, cymbidoside (8). These structures were determined on the basis of mainly NMR experiments as well as chemical degradation and X-ray crystallographic analysis. The isolated compounds (1-6 and 8) were evaluated for the inhibitory activity on lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated nitric oxide (NO) production in RAW 264.7 cells. Consequently, 1 and 3 exhibited moderate activity.

Neuroprotective effect of liquiritin as an antioxidant via an increase in glucose-6-phosphate dehydrogenase expression on B65 neuroblastoma cells.

Glycyrrhiza (the roots and rhizomes of licorice) has been used worldwide as both an herbal nutraceutical and herbal medicine. In addition, it is well known that Glycyrrhiza contains various compounds with biological effects, such as anti-viral, anti-inflammatory, immunoregulatory, anti-tumor and neuroprotective effects. Among the various compounds in Glycyrrhiza, the active compounds that show biological activity are thought to include glycyrrhizin, glycyrrhetinic acid, glabridin, licochalcones and liquiritin. In the present study, we investigated the biological effects of three of these compounds (glycyrrhizin, liquiritin and isoliquiritin) on B65 neuroblastoma cells derived from serotonergic neurons. Among these three compounds, only liquiritin enhanced the proliferation of B65 neuroblastoma cells. In contrast, both glycyrrhizin and isoliquiritin, particularly at high concentrations had cytotoxic effects. Cells were treated with various cytotoxic agents and liquiritin could ameliorate the cytotoxicity induced by menadione sodium bisulfate in a dose-dependent manner. We also examined the effect of liquiritin on cell survival by evaluating the expression levels of phospho-p44/42 mitogen-activated protein kinase, cyclin-related proteins and glucose-6-phosphate dehydrogenase, which produces nicotinamide adenine dinucleotide phosphate. Under treatment with liquiritin, the protein expression level of glucose-6-phosphate dehydrogenase increased in a dose-dependent manner. In contrast, the protein expression level of cyclin-related proteins did not change at all under treatment with liquiritin. These results suggest that liquiritin, which is contained in Glycyrrhiza, may enhance cell survival by increasing the protein expression level of glucose-6 phosphate dehydrogenase.

Determination and Identification of a Specific Marker Compound for Discriminating Shrub Chaste Tree Fruit from Agnus Castus Fruit Based on LC/MS Metabolic Analysis.

Shrub Chaste Tree Fruit (SCTF) is defined as the fruits of Vitex rotundifolia L. f. and V. trifolia L. and has been used as a component of some traditional Japanese medicines (Kampo formulations). Agnus Castus Fruit (ACF) is defined as the dried ripe fruits of V. agnus-castus L.; it is used in traditional European medicines, but is becoming popular in Japan as both an over-the-counter drug and as an ingredient in health foods for treating premenstrual syndrome (PMS). To ensure the efficacy and safety of both SCTF and ACF products, it is important to precisely authenticate their botanical origins and to clearly distinguish between SCTF and ACF. Therefore, we tried to identify SCTF-specific marker compounds based on LC/MS metabolic analysis. The multivariate analysis of LC/MS data from SCTF and ACF samples furnished candidate marker compounds of SCTF. An SCTF-specific marker was isolated from SCTF crude drugs and identified as 3-O-trans-feruloyl tormentic acid on the basis of spectroscopic data from NMR and MS. Since avoiding contamination from closely related species is a significant requirement for pharmaceuticals of natural origin, this information will be valuable for the quality control of both SCTF and ACF products from the viewpoint of regulatory science.

Identification of New Diterpenes as Putative Marker Compounds Distinguishing Agnus Castus Fruit (Chaste Tree) from Shrub Chaste Tree Fruit (Viticis Fructus).

Agnus Castus Fruit is defined in the European Pharmacopoeia as the dried ripe fruit of Vitex agnus-castus. In Europe it is used as a medicine targeting premenstrual syndrome and climacteric disorder. In Japan, Agnus Castus Fruit is becoming popular as a raw material for over-the-counter drugs and health food products, though its congenic species, Vitex rotundifolia and Vitex trifolia, have been used as Shrub Chaste Tree Fruit in traditional medicines. Therefore, it is important to discriminate these Vitex plants from the viewpoint of regulatory science. Here we tried to identify putative marker compounds that distinguish between Agnus Castus Fruit and Shrub Chaste Tree Fruit. We analyzed extracts of each crude drug by liquid chromatography-mass spectrometry, and performed differential analysis by comparison of each chromatogram to find one or more peaks characteristic of Agnus Castus Fruit. A peak was isolated and identified as an equilibrium mixture of new compounds named chastol (1) and epichastol (1a). The planar structures of 1 and 1a were determined spectroscopically. Their relative configurations were revealed by nuclear Overhauser effect spectroscopy and differential nuclear Overhauser effect-NMR data. Since avoiding contamination from closely related species is needed for the quality control of natural pharmaceuticals, this information will be valuable to establish a method for the quality control of both, Agnus Castus Fruit and Shrub Chaste Tree Fruit products.

Sinomenine and magnoflorine, major constituents of Sinomeni caulis et rhizoma, show potent protective effects against membrane damage induced by lysophosphatidylcholine in rat erythrocytes.

The effects of the water extract of Sinomeni Caulis et Rhizoma (SCR-WE) and its major constituents, sinomenine (SIN) and magnoflorine (MAG), on moderate hemolysis induced by lysophosphatidylcholine (LPC) were investigated in rat erythrocytes and compared with the anti-hemolytic effects of lidocaine (LID) and propranolol (PRO) as reference drugs. LPC caused hemolysis at concentrations above the critical micelle concentration (CMC), and the concentration of LPC producing moderate hemolysis (60 %) was approximately 10 µM. SCR-WE at 1 ng/mL-100 µg/mL significantly inhibited the hemolysis induced by LPC. SIN and MAG attenuated LPC-induced hemolysis in a concentration-dependent manner from very low to high concentrations (1 nM-100 µM and 10 nM-100 µM, respectively). In contrast, the inhibiting effects of LID and PRO on LPC-induced hemolysis were observed at higher concentrations (1-100 µM) but not at lower concentrations (1-100 nM). Neither SIN nor MAG affected micelle formation of LPC, nor, at concentrations of 1 nM-1 µM, did they attenuate the hemolysis induced by osmotic imbalance (hypotonic hemolysis). Similarly, SCR-WE also did not modify micelle formation or hypotonic hemolysis, except at the highest concentration. These results suggest that SIN and MAG potently protect the erythrocyte membrane from LPC-induced damage and contribute to the beneficial action of SCR-WE. The protective effects of SIN and MAG are mediated by some mechanism other than prevention of micelle formation or protection of the erythrocyte membrane against osmotic imbalance.

Inhibitory effect of chemical constituents from Artemisia scoparia Waldst. et Kit. on triglyceride accumulation in 3T3-L1 cells and nitric oxide production in RAW 264.7 cells.

We investigated the anti-obesity effect of the aerial part of Artemisia scoparia Waldst. et Kit. (Compositae). An 80 % aqueous EtOH extract of the aerial part inhibited triglyceride (TG) accumulation and the nitric oxide (NO) production activity. A new chromane derivative was isolated from the aerial part of A. scoparia Waldst. et Kit. along with 18 known compounds. The structure of the new chromane, scopariachromane (1), was elucidated by spectroscopic analyses. The inhibitory effects of the compounds on TG accumulation activity were examined. Among these, cirsiliol (11) inhibited TG accumulation in 3T3-L1 preadipocytes. Jaceosidin (12) inhibited NO production in a murine macrophage-like cell line (RAW 264.7). These results indicate that the 80 % aqueous EtOH extract and compounds isolated from the aerial part of A. scoparia Waldst. et Kit. may improve obesity-related insulin resistance.

Flavonol acylglycosides from flower of Albizia julibrissin and their inhibitory effects on lipid accumulation in 3T3-L1 cells.

Obesity is a serious health problem worldwide. We investigated the anti-obesity effect of the flower of Albizia julibrissin DURAZZ. (Leguminosae). A 90% EtOH extract of the flower inhibited adipogenesis in 3T3-L1 preadipocytes, as well as the activity of glycerol-3-phosphate dehydrogenase (GPDH) activity. New flavonol acylglycosides (1-4) and eighteen known compounds (5-22) were isolated by bioassay-directed fractionation. These new glycosides were elucidated to be 3″-(E)-p-coumaroylquercitrin (1), 3″-(E)-feruloylquercitrin (2), 3″-(E)-cinnamoylquercitrin (3), and 2″-(E)-cinnamoylquercitrin (4) on the basis of spectroscopic and chemical analysis. These compounds inhibited adipogenesis in 3T3-L1 preadipocytes. In particular, 2 exhibited potent inhibitory effects on triglyceride accumulation. Furthermore, GPDH activity was inhibited by 2. Additionally, 2 inhibited glucose uptake in 3T3-L1 adipocytes. These results indicate that the 90% EtOH extract and compounds isolated from the flower of A. julibrissin inhibit adipogenesis in 3T3-L1 preadipocytes and may have anti-obesity effect through the inhibition of preadipocyte differentiation.

New feruloyl tyramine glycosides from Stephania hispidula YAMAMOTO.

Three new feruloyl tyramine glycosides, N-cis-feruloyl tyramine-4'''-O-beta-D-glucopyranoside (1), N-trans-ferloyl tyramine-4'''-O-beta-D-glucopyranoside (2), and N-trans-feruloyl tyramine-4'-O-beta-D-glucopyranoside (3), along with six known compounds, N-trans-feruloyl-3'''-methoxydopamine-4'-O-beta-D-glucopyranoside (4), haitinosporine (5), tubocurine (6), fuzitine (7), (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside (8), and (-)-lyoniresinol-2alpha-O-beta-D-glucopyranoside (9), were isolated from the stem of Stephania hispidula YAMAMOTO. The structures were elucidated by spectroscopic and chemical analysis.