Solid state fermentation process with Aspergillus kawachii enhances the cancer-suppressive potential of silkworm larva in hepatocellular carcinoma cells.

BACKGROUND: Mulberry silkworm larvae (Bombyx mori) are known as the oldest resource of food and traditional medicine. Although silkworm larvae have been reported to treat various chronic diseases, the effect of fermentation by microorganisms improving the biological activities of silkworm larvae was not reported. In the present study, fermented silkworm larvae was developed via solid-state fermentation with Aspergillus kawachii and investigated its anti-cancer activity in human hepatocellular carcinoma cells. METHODS: We investigated the anti-cancer effects of unfermented (SEE) and fermented silkworm larva ethanol extract (FSEE) on HepG2 human hepatocellular carcinoma cells as well as compared changes in free amino acid, fatty acid, and mineral contents. Anti-cancer activities were evaluated by SRB staining, cell cycle analysis, Annexin V staining, Hoechst staining, DNA fragmentation analysis and western blot analysis. Fatty acid, free amino acid and mineral contents of SEE and FSEE were determined by gas chromatography, amino acid analyzer and flame atomic absorption spectrophotometer, respectively. RESULTS: Compared with SEE, treatment with FSEE resulted in apoptotic cell death in HepG2 cells characterized by G0/G1 phase cell cycle arrest, DNA fragmentation, and formation of apoptotic bodies. Furthermore, FSEE significantly up-regulated pro-apoptotic as well as down-regulated anti-apoptotic proteins in HepG2 cells. However, an equivalent concentration of SEE did not induce cell cycle arrest or apoptosis in HepG2 cells. Moreover, fermentation process by Aspergillus kawachii resulted in enhancement of fatty acid contents in silkworm larvae, whereas amino acid and mineral contents were decreased. CONCLUSION: Collectively, this study demonstrates that silkworm larvae solid state-fermented by Aspergillus kawachii strongly potentiates caspase-dependent and -independent apoptosis pathways in human hepatocellular carcinoma cells by regulating secondary metabolites.

Anti-Pigmentation Effects of Eight Phellinus linteus-Fermented Traditional Crude Herbal Extracts on Brown Guinea Pigs of Ultraviolet B-Induced Hyperpigmentation.

We have previously found that mycelia culture broth of eight kinds of traditional herbal extracts fermented with Phellinus linteus (previously named as 8-HsPLCB) not only inhibited melanin and tyrosinase activity, but also reduced the contents of melanogenesis-related proteins, including tyrosinase and microphthalmia-associated transcription factor, in 3-isobutyl-1-methylxanthine-stimulated B16F0 melanoma cells. For a further study, the effect of 8-HsPLCB against skin pigmentation in brown guinea pigs with ultraviolet B (UVB)-induced hyperpigmentation was investigated. 8-HsPLCB (3%) and arbutin (2%) as positive controls were applied topically twice daily for 4 weeks to the hyperpigmented areas. 8-HsPLCB showed skin-lightening effect as effective as arbutin, one of the most widely used in whitening cosmetics. Melanin index values as the degree of pigmentation showed a significant reduction week by week post 8-HsPLCB treatment and then substantially reduced by 4 weeks. The degree of depigmentation after 4 weeks of topical application with 8-HsPLCB was 32.2% as compared with before treatment (0 week). Moreover, using Fontana-Masson staining and hematoxylin-eosin staining, 8-HsPLCB reduced melanin pigmentation in the basal layer of the epidermis and epidermal thickness changes exposed to the UV-B irradiation as compared with non-treatment and vehicle treatment. The intensity of the skin-lightening effect of 8-HsPLCB was similar to arbutin. These results suggest that the skin-lightening effect of 8-HsPLCB might be resulted from inhibition of melanin synthesis by tyrosinase in melanocytes. To conclude, 8-HsPLCB treatment showed reduction of the melanin pigment and histological changes induced by UV irradiation in brown guinea pigs.

Triptolide-Mediated Apoptosis by Suppression of Focal Adhesion Kinase through Extrinsic and Intrinsic Pathways in Human Melanoma Cells.

Triptolide (TPL) has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis.

Identification of 5-hydroxy-3,6,7,8,3´,4´-hexamethoxyflavone from Hizikia fusiforme involved in the induction of the apoptosis mediators in human AGS carcinoma cells.

An 80% ethanol extract of Hizikia fusiforme was obtained and followed by successive fractionation using the organic solvents n-hexane, ethyl acetate, and n-butanol to identify the antioxidative substance. The aqueous part of the nbutanol fractionation step, showing high antioxidative activity, was subjected to reverse-phase liquid chromatography. As a result, a substance purified from a BB-2 fraction showed high antioxidative activity. The m/z 419 [M+H] molecular ion peak in the fraction was observed by the analysis of the ESI-LC/MS spectrum. By the analysis of 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) spectra, a unique compound of the fraction was biochemically identified as a 5-hydroxy-3,6,7,8,3´,4´- hexamethoxyflavone (5HHMF). We also investigated the effect of 5HHMF on human gastric AGS carcinoma cells. Western blot analysis suggested that the flavone substantially increased the levels of the death receptor-associated apoptosis mediators Fas, Fas L, FADD, TRADD, and DR4 in a concentration-dependent manner. The levels of Fas, Fas L, TRADD, and DR4 in the cells treated with 5HHMF (5 microgram/ml) were approximately 26.4-, 12.8-, 6.7-, and 9.8- times higher than those of non-treated cells, respectively. Of note, the level of FADD protein in the cells exposed to 5HHMF (1 microgram/ml) increased approximately 9.6-times. In addition, the cleavage of caspase-3, -8, and -9 in cultured AGS cells treated with 5HHMF was significantly confirmed. Therefore, our results suggest that 5HHMF from H. fusiforme is involved in the induction of death receptor-associated apoptosis mediators in human gastric AGS carcinoma cells.

Cordycepin induces apoptosis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells.

In this study, the effect of cordycepin (3'-deoxyadenosine), a major component of Cordyceps militaris, an ingredient of traditional Chinese medicine was investigated for the first time on apoptotsis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells. Cordycepin significantly inhibited the proliferation of human neuroblastoma SK-N-BE(2)-C and human melanoma SK-MEL-2 cells with IC50 values of 120 microM and 80 microM, respectively. Cordycepin treatment at 120 microM and 80 microM, respectively, induced apoptosis in both cells and caused the increase of cell accumulation in a time-dependent manner at the apoptotic sub-G1 phase, as evidenced by the flow cytometry (FCM) and annexin V-fluorescein isothiocyanate (FITC) analyses. Western blot analysis revealed the induction of active caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage by cordycepin treatment. These results suggest that cordycepin is a potential candidate for cancer therapy of neuroblastoma and melanoma.

Effect of fermented Angelicae gigantis Radix on carbon tetrachloride-induced hepatotoxicity and oxidative stress in rats.

This study is aimed to evaluate the protective effect of fermented Angelicae gigantis Radix (AGR) with Monascus purpureus strain on carbon tetrachloride (CCl(4))-induced hepatotoxicity and oxidative stress in rats. The activities of liver marker enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and the levels of lipid peroxidation were increased when CCl(4) was treated but these parameters were significantly decreased by fermented AGR treatment. CCl(4) treatment exhibited decrease in serum concentrations of triglyceride, total cholesterol, HDL-cholesterol, and free fatty acids, and these were also decreased by fermented AGR administration. The level of serum leptin was significantly lower in fermented AGR administration than that in normal control group. CCl(4) treatment significantly increased the concentration of liver triglyceride. The current study observed significant elevations of the thiobarbituric acid-reactive substances (TBARS) levels in the liver homogenate, mitochondrial, and microsomal fractions of CCl(4) control group compared with normal control group. CCl(4) treatment resulted in a significant decrease in the levels of plasma and hepatic glutathione, but these reductions were significantly increased by fermented AGR administration. CCl(4) induced the marked hepatocytes necrosis and fatty accumulation around the central veins. Accordingly, fermented AGR may be an ideal candidate for the hepatoprotective effect in animal model.

Effect of betaine on the hepatic damage from orotic acid-induced fatty liver development in rats.

Betaine prevents hepatic damage caused by ethanol and carbone tetrachloride (CCl4) in rats. Present study was to investigate the effect of betaine on the hepatic microsomal triglyceride transfer protein (MTP) mRNA expression in orotic acid (OA)-induced fatty liver in rats. OA feeding was attributed to the significant increase in the hepatic levels of triglyceride and the serum levels of ALT and AST and resulted in typical histology of fatty liver contained numerous largely fat droplets. While concomitant supplementation of betaine to OA diet was slightly reduced the hepatic triglyceride concentrations and was significantly decreased ALT activity. Hepatic MTP mRNA expression by OA treatment increased by 14% despite triglyceride accumulation in the liver in OA treatment rats relative to rats fed a normal diet without OA supplemented, but MTP expression by simultaneous supplementation of OA and betaine was slightly decreased by 7.9% as compared to the OA-feeding rats. A significant elevation of TBARS contents in the liver homogenate, microsome, and mitochondrial fractions of the OA-feeding rats compared with the normal rats, however, these increases were significantly or slightly decreased by simultaneous addition of OA and betaine. The increases of hepatic OA and betaine levels in OA feeding rats was also found when compared to the normal rats, but these increases were significantly lowered in the concomitant supplementation OA and betaine. The content of Fe was significantly increased in the OA feeding rats, but this elevation showed significantly recovered as low as the normal level by concomitant with OA and betaine. Zinc content was also significantly decreased in the OA feeding rats compared with the normal rats, but this reduction was more significantly elevated by concomitant with OA and betaine. Hepatic glutathione content in the OA feeding rats was similar to that of the normal rats, but this content was slightly reduced without statistically significant differences. But, a significant elevation in the hepatic glutathione content was found in the simultaneously administration OA and betaine. The hepatocytes contained numerous largely fat droplets induced by OA administration and was slightly reduced by simultaneous supplementation of OA and betaine. Present study demonstrated that betaine has a weak preventive action on the OA-induced triglyceride accumulation.

Effect of fermented sea tangle on the alcohol dehydrogenase and acetaldehyde dehydrogenase in Saccharomyces cerevisiae.

Sea tangle, a kind of brown seaweed, was fermented with Lactobacillus brevis BJ-20. The gamma-aminobutyric acid (GABA) content in fermented sea tangle (FST) was 5.56% (w/w) and GABA in total free amino acid of FST was 49.5%. The effect of FST on the enzyme activities and mRNA protein expression of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) involved in alcohol metabolism in Saccharomyces cerevisiae was investigated. Yeast was cultured in YPD medium supplemented with different concentrations of FST powder [0, 0.4, 0.8, and 1.0% (w/v)] for 18 h. FST had no cytotoxic effect on the yeast growth. The highest activities and protein expressions of ADH and ALDH from the cell-free extracts of S. cerevisiae were evident with the 0.4% and 0.8% (w/v) FST-supplemented concentrations, respectively. The highest concentrations of GABA as well as minerals (Zn, Ca, and Mg) were found in the cell-free extracts of S. cerevisiae cultured in medium supplemented with 0.4% (w/v) FST. The levels of GABA, Zn, Ca, and Mg in S. cerevisiae were strongly correlated with the enzyme activities of ADH and ALDH in yeast. These results indicate that FST can enhance the enzyme activities and protein expression of ADH and ALDH in S. cerevisiae.

Supplementation of SK1 from Platycodi radix ameliorates obesity and glucose intolerance in mice fed a high-fat diet.

This study investigated the beneficial effects of SK1 on obesity and insulin resistance in C57BL/6 mice, which were fed a high-fat diet (37% calories from fat). SK1 is an edible saponin-rich compound from Platycodi radix. The mice were supplemented with two doses of SK1 (0.5% and 1.0%, wt/wt) for 9 weeks. The body weight, visceral fat mass, and adipocyte area were significantly decreased in the SK1 supplemented-groups in a dose-dependent manner compared to the high-fat group. The SK1 supplement significantly lowered plasma triglycerides, total cholesterol, and free fatty acid levels, whereas it significantly elevated the fecal excretion of lipids in the diet-induced obese mice. Supplementation of SK1 decreased the triglyceride and cholesterol levels and the accumulation of lipid droplets in the liver compared to the high-fat control group. High-fat diet induced glucose intolerance and insulin resistance with the elevation of blood glucose levels compared to the normal group; however, the SK1 supplement significantly improved postprandial glucose levels and insulin resistance index. After 9 weeks of being fed a high-fat diet, the mice presented with significantly increased activities of hepatic fatty acid synthase, fatty acid beta-oxidation, and glucokinase; however, both 0.5% and 1.0% SK1 supplementation normalized these activities. Notably, SK1 supplementation effectively diminished the ratio of fatty acid biosynthesis to fatty acid oxidation compared to the high-fat group. These results indicate that SK1 exhibits a potential anti-obesity effect and may prevent glucose intolerance by reducing body weight and fat accumulation, increasing fecal lipid excretions, and regulating hepatic lipid and glucose metabolism in high-fat fed mice.

Antihyperglycemic effect of stem bark powder from paper mulberry (Broussonetia kazinoki Sieb.) in type 2 diabetic Otsuka Long-Evans Tokushima fatty rats.

The effect of stem bark powder from paper mulberry (PMSB) on serum glucose, insulin, fructosamine, and lipid concentrations, as well as enzyme activities that serve as liver injury markers, was investigated in genetically diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats. Both nondiabetic Long-Evans Tokushima Otsuka (LETO) rats and diabetic OLETF rats (30 weeks old) were fed a semisynthetic diet with or without 50 g/kg PMSB for 8 weeks and then compared. The OLETF control rats showed a high amount of daily water intake in comparison to those in the LETO group. The concentrations of glucose, fructosamine, total lipid, triglyceride, total cholesterol, and high-density lipoprotein-cholesterol and the activities of aspartate aminotransferase and alanine aminotransferase (ALT) in serum were higher in the OLETF control rats than those in the LETO control rats. However, PMSB ingestion decreased the serum levels of glucose, fructosamine, triglyceride, and total cholesterol and the activity of ALT in the OLETF rats, but not in the LETO rats. The concentration of serum insulin was also significantly increased by PMSB consumption in the OLETF rats compared to the OLETF control rats. These results suggest that PMSB might have an antihyperglycemic effect in the OLETF rat and that the increased blood insulin level would be an important regulatory factor for improving hyperglycemia in the current animal model.