Promotion of ABCG2 gene expression by neolignans from Piper longum L.

We focused on Piper longum L., a herbal drug produced in Myanmar, which has a renoprotective effect. Thus, we attempted to isolate and identify compounds that enhance the expression of the ABCG2 gene from the aerial parts of the plant except for the fruit. Among the various P. longum extracts, we isolated and identified the components. Using Caco-2 cells, the hABCG2 mRNA expression-enhancing effects of the isolated compounds were compared with the positive reference compound (3-methylcholanthrene [3MC]) using real-time polymerase chain reaction. Six compounds were isolated and identified from the methanol extract of P. longum. Among the isolated compounds, licarin A and neopomatene had lower toxicity and higher hABCG2 mRNA expression-enhancing effects in Caco-2 cells. Suppression of hAhR expression by siRNA reduced the activity of licarin A and neopomatene, as well as the hAhR agonist 3MC, suggesting that these 2 compounds may act as hAhR agonists to promote hABCG2 expression.

Effect of Compounds from Moringa oleifera Lam. on in Vitro Non-Alcoholic Fatty Liver Disease (NAFLD) Model System.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease in the world, with a prevalence of 25 % in many countries. To date, no drug has been approved to treat NAFLD, therefore, the use of phytochemicals to prevent this disease is meaningful. In this study, we focused on the effects of Moringa oleifera Lam. on diabetes, attempted to isolate compounds that regulate NAFLD. Compounds 1 and 2 were isolated from the ethyl acetate fraction of M. oleifera. Spectral data revealed that they were 1-hydroxy-3-phenylpropan-2-yl benzoate (1) and benzyl benzylcarbamate (2), respectively. The three-dimensional structure of compound 1 was determined by single crystal X-ray structural analysis. Neither compound was toxic to HepG2 cells, and compound 1 was found to have a concentration-dependent inhibitory effect on intracellular lipid accumulation induced by stimulation of linoleic acid (LA). As a result of measuring the effects of compound 1 on the intracellular lipid production-related protein, it was found that compound 1 enhanced protein expression that promotes lipolysis. On the other hand, since the action of compound 1 was similar to that of PPARα agonists, it is deduced that compound 1 enhanced the activity of PPARα and further enhanced the expression of lipolytic proteins, which is related to the suppression of intracellular lipid accumulation. Furthermore, as the result of docking simulation, compound 1 had a higher binding affinity to the ligand binding site of PPARα than fenofibrate, which is a PPARα agonist, and thus compound 1 was considered to be promising as an agonist of PPARα.

Inhibitory effect of bofutsushosan (fang feng tong sheng san) on glucose transporter 5 function in vitro.

Bofutsushosan (BTS; fang feng tong sheng san in Chinese) is a formula in traditional Japanese Kampo medicine and Chinese medicine comprising eighteen crude drugs, and is used to treat obesity and metabolic syndrome. Fructose is contained in refreshing beverages as high-fructose corn syrup, and is associated with obesity. Fructose is absorbed via glucose transporter 5 (GLUT5) in the intestine. Therefore, the inhibition of GLUT5 is considered to be a target of obesity drugs. We evaluated the inhibitory effects of BTS extract and its constituents on fructose uptake using Chinese hamster ovary K1 cells, i.e., cells stably expressing GLUT5. Boiled water extract of BTS significantly suppressed GLUT5 function in a concentration-dependent manner without cytotoxicities. Among 18 components of BTS, the boiled water extracts of the rhizome of Zingiber officinale, the root and rhizome of Saposhnikovia divaricata, and the root of Platycodon grandiflorum exhibited significant inhibitory effects on fructose uptake with IC50 values of 314, 119 and 475 µg/ml, respectively. Among the constituents of the rhizome of Z. officinale extract, 6-gingerol significantly inhibited GLUT5 with an IC50 value of 39 µM, while 6-shogaol exhibited a significant but weak inhibition on GLUT5 at 100 µM. One of the mechanisms of action of BTS may be the inhibition of fructose absorption in the intestine, and one of the active components of BTS is the rhizome of Z. officinale and 6-gingerol.

Effect of Anserine/Carnosine Supplementation on Verbal Episodic Memory in Elderly People.

Our goal in this study was to determine whether or not anserine/carnosine supplementation (ACS) is capable of preserving cognitive function of elderly people. In a double-blind randomized controlled trial, volunteers were randomly assigned to an ACS or placebo group at a 1:1 ratio. The ACS group took 1.0 g of an anserine/carnosine (3:1) formula daily for 3 months. Participants were evaluated by psychological tests before and after the 3-month supplementation period. Thirty-nine healthy elderly volunteers (60-78 years old) completed the follow-up tests. Among the tests, delayed recall verbal memory assessed by the Wechsler Memory Scale-Logical Memory showed significant preservation in the ACS group, compared to the placebo group (p = 0.0128). Blood analysis revealed a decreased secretion of inflammatory cytokines, including CCL-2 and IL-8, in the ACS group. MRI analysis using arterial spin labeling showed a suppression in the age-related decline in brain blood flow in the posterior cingulate cortex area in the ACS group, compared to the placebo group (p = 0.0248). In another randomized controlled trial, delayed recall verbal memory showed significant preservation in the ACS group, compared to the placebo group (p = 0.0202). These results collectively suggest that ACS may preserve verbal episodic memory and brain perfusion in elderly people, although further study is needed.

Branched-chain amino acids inhibit the TGF-beta-induced down-regulation of taurine biosynthetic enzyme cysteine dioxygenase in HepG2 cells.

Taurine deficiency has been suggested to contribute to the pathogenesis and complications of advanced hepatic diseases. The molecular basis for a low level of taurine associated with hepatic failure is largely unknown. Using carbon tetrachloride (CCl4)-induced cirrhotic rat model, we found that the activity and expression of cysteine dioxygenase (CDO), a rate-limiting enzyme in taurine synthesis, were significantly decreased in the liver of these rats. To investigate the underlying mechanisms for the suppression, we examined the effects of pathological cytokines on CDO expression in human hepatoma HepG2 cells. Among the several cytokines, transforming growth factor-ß (TGF-ß), one of the key mediators of fibrogenesis, suppressed Cdo1 gene transcription through the MEK/ERK pathway. Finally, we further examined potential effects of branched-chain amino acids (BCAA) on CDO expression, as it has been reported that oral BCAA supplementation increased plasma taurine level in the patients with liver cirrhosis. BCAA, especially leucine, promoted Cdo1 gene transcription, and attenuated TGF-ß-mediated suppression of Cdo1 gene expression. These results indicate that the low plasma level of taurine in advanced hepatic disease is due to decreased hepatic CDO expression, which can be partly attributed to suppressive effect of TGF-ß on Cdo1 gene transcription. Furthermore, our observation that BCAA promotes Cdo1 expression suggests that BCAA may be therapeutically useful to improve hepatic taurine metabolism and further suppress dysfunctions associated with low level of taurine in hepatic diseases.

Effects of oral administration of glucosylceramide on gene expression changes in hairless mouse skin: comparison of whole skin, epidermis, and dermis.

The beneficial effects of dietary glucosylceramide on the barrier function of the skin have been increasingly reported, but the entire mechanism has not been clarified. By DNA microarray, we investigated changes in gene expression in hairless mouse skin when a damage-inducing AD diet and a glucosylceramide diet (GluCer) were imposed. GluCer administration potentially suppressed the upregulation of six genes and the downregulation of four genes in the AD group. Examination of the epidermal and/or dermal expression of Npr3, Cyp17a1, Col1a1, S100a9, Sprr2f, Apol7a, Tppp, and Scd3 revealed responses of various parts of the skin to the diets. In normal hairless mice, GluCer administration induced an increase in the dermal expression of Cyp17a1 and the epidermal expression of Tppp, and a decrease in the epidermal expression of S100a9. Our results provide information on gene expression not only in whole skin but also in the epidermis and dermis that should prove useful in the search for the mechanisms underlying the effects of GluCer on damaged and normal skin.

Suppressive effect of an isoflavone fraction on tumor necrosis factor-alpha-induced interleukin-8 production in human intestinal epithelial Caco-2 cells.

This study demonstrates the effect of soybean components on the tumor necrosis factor-alpha (TNF-alpha)-induced production of interleukin-8 (IL-8), one of the major inflammatory chemokines, in intestinal epithelial-like Caco-2 cells. Among the soybean components, an isoflavone fraction (IFF) suppressed the TNF-alpha-induced IL-8 secretion by Caco-2 cells in a dose-dependent manner, whereas a soyasaponin fraction and soypeptide fraction had no significant effect on TNF-alpha-induced IL-8 secretion. The IL-8 secretion induced by hydrogen peroxide and by IL-1beta was not suppressed by IFF, suggesting that the inhibitory effect of isoflavone was specific for the TNF-alpha-induced regulation of IL-8. The increased expression of IL-8 mRNA by TNF-alpha was almost completely suppressed by IFF. Furthermore, the transcriptional activity of the human IL-8 promoter was increased by the TNF-alpha treatment, and IFF significantly suppressed the IL-8 promoter activity. These results indicate that IFF suppressed TNF-alpha-induced IL-8 production at the transcriptional level in human intestinal Caco-2 cells, suggesting IFF of soybean as a promising food component for preventing intestinal inflammation such as inflammatory bowel disease.

Inhibition of P-glycoprotein enhances the suppressive effect of kaempferol on transformation of the aryl hydrocarbon receptor.

Dioxins enter the body mainly through the diet, bind to the aryl hydrocarbon receptor (AhR), and cause various toxicological effects. In this study, we found that oral administration of kaempferol or ginkgo biloba extract (EGb) containing 24% flavonol at 100 mg/kg body weight suppressed AhR transformation induced by 3-methylcholanthrene at 10 mg/kg body weight in the liver of mice. The suppressive effect of kaempferol was enhanced by verapamil, an inhibitor of P-glycoprotein (P-gp), in ex vivo experiments using a hepatic cytosolic fraction and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enhancement of the suppressive effect by verapamil was also observed in mouse hepatoma Hepa-1c1c7 cells, accompanied by an increase in the uptake of kaempferol into the cells. In conclusion, inhibition of P-gp enhanced the suppressive effect of kaempferol on AhR transformation through an increase in the intracellular kaempferol concentration.

Dietary taurine attenuates dextran sulfate sodium (DSS)-induced experimental colitis in mice.

Effects of dietary taurine on the experimental colitis induced by dextran sulfate sodium (DSS) were studied. C57BL/6 mice administrated taurine or placebo for 5 days were given 3% DSS to induce acute. The colitis was as-sessed using indices such as diarrhea/bleeding scores, colon length change, histological score and tissue myeloperoxidase (MPO) activity. Further, tissue mRNA levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2, were determined by real-time PCR. Taurine supplementation significantly attenuated the severity of diarrhea, colon shortening, histological score, MPO activity elevation and abnormal MIP-2 gene expression, indicating that taurine prevents DSS-induced colitis. Taurine also inhibited the TNF-alpha-induced secretion of IL-8 (a human homologue of MIP-2) from human intestinal epithelial Caco-2 cells. Inhibition of chemokine secretion from intestinal cells may be involved in the mechanisms underlying the cytoprotective function of taurine in the intestinal epithelium.

Activation of pregnane X receptor and induction of MDR1 by dietary phytochemicals.

The pregnane X receptor (PXR) is understood to be the key regulator for gene expression of such drug-metabolizing enzymes and transporters as multidrug-resistant protein 1 (MDR1) and the cytochrome P450 (CYP) family. We examined the effect of dietary phytochemicals on the PXR-dependent transcriptional activity in human intestinal LS180 cells by using a reporter assay. Among approximately 40 kinds of phytochemicals, tangeretin and ginkgolides A and B markedly induced the PXR-dependent transcriptional activity and also the activity of the human MDR1 promoter. The expression levels of MDR1 mRNA as well as of CYP3A4 mRNA, another gene regulated by PXR, were significantly increased by these phytochemicals. Furthermore, an increase was observed of the MDR1 protein and its functional activity by tangeretin and by ginkgolides A and B. These findings strongly suggest that tangeretin and ginkgolides A and B activated PXR, thereby regulating detoxification enzymes and transporters in the intestines.

Transepithelial transport of alpha-lipoic acid across human intestinal Caco-2 cell monolayers.

Alpha-lipoic acid (LA) is used in dietary supplements or food with antioxidative functions. The mechanism for the intestinal absorption of alpha-lipoic acid was investigated in this study by using human intestinal Caco-2 cell monolayers. LA was rapidly transported across the Caco-2 cell monolayers, this transport being energy-dependent, suggesting transporter-mediated transport to be the mechanism involved. The LA transport was strongly dependent on the pH value, being accelerated in the acidic pH range. Furthermore, such monocarboxylic acids as benzoic acid and medium-chain fatty acids significantly inhibited LA transport, suggesting that a proton-linked monocarboxylic acid transporter (MCT) was involved in the intestinal transport of LA. The conversion of LA to the more antioxidative dihydrolipoic acid was also apparent during the transport process.

Inhibitory effect of a bitter melon extract on the P-glycoprotein activity in intestinal Caco-2 cells.

Extracts of bitter melon, soybean, dokudami and welsh onion by 40% methanol increased the accumulation of rhodamine-123 by Caco-2 cells, suggesting that these extracts inhibited P-glycoprotein (P-gp). The extract of bitter melon was separated in a tC18 cartridge column and the eluate from 80% acetonitrile most markedly increased the [(3)H]-daunomycin accumulation by Caco-2 cells. The inhibitory compounds in the bitter melon fraction were isolated by HPLC with Pegasil C4 and Pegasil ODS columns. The HPLC fraction having the highest activity was analyzed by (1)H-NMR and FAB-MS, and the active compound was identified as 1-monopalmitin. The inhibitory activities of 1-monopalmitin and its related compounds suggested that the inhibition of P-gp activity was not dependent on the degree of unsaturation of fatty acid in the monoglyceride, but on the chain length. It was also suggested that the monoglyceride structure played an important role in the inhibition of P-gp activity. Monoglycerides could therefore alter the pharmacokinetics of drugs by inhibiting the P-gp-mediated efflux.

A bitter melon extract inhibits the P-glycoprotein activity in intestinal Caco-2 cells: monoglyceride as an active compound.

P-glycoprotein (P-gp) is a 170 kDa membrane protein that belongs to the ATP-binding cassette (ABC) transporter superfamily. In normal tissues, P-gp functions as an ATP-dependent efflux pump that excretes highly hydrophobic xenobiotic compounds, playing an important role in protecting the cells/tissues from xenobiotics. In the present study, chemical substances that could directly modulate the intestinal P-gp activity were searched in vegetables and fruits. By using human intestinal epithelial Caco-2 cells as a model of the small intestinal cells, we observed that a bitter melon fraction extracted from 40% methanol showed the greatest increase of the rhodamine-123 accumulation by Caco-2 cells. Inhibitory compounds in the bitter melon fraction were then isolated by HPLC using Pegasil C4 and Pegasil ODS columns. The HPLC fraction having the highest activity was analyzed by (1)H-NMR and FAB-MS, and the active compound was identified as 1-monopalmitin. It is interesting that certain types of monoglyceride might be involved in the drug bioavailability by specifically inhibiting the efflux mediated by P-gp.