Fermentation of mulberry leaves with Cordyceps militaris enhanced anti-adipogenesis activity in 3T3-L1 cells through down-regulation of PPAR-? pathway signaling

Objective: To establish an efficacious and efficient fermentation method of enhancing the anti-adipogenesis effect of mulberry (Morus alba) leaves using Cordyceps militais. Methods: Dried mulberry leaves, dried mulberry leaves with 50% raw silkworm pupa and raw silkworm pupa were fermented with Cordyceps militais for 4 weeks at 25 °C, after which the dried mulberry leaves and fermented product were extracted with 70% ethanol and subjected to high performance liquid chromatography (HPLC). The contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid were determined. We then used the 3T3-L1 cells to investigate whether extracts of fermentation enhanced anti-adipogenesis activity in vitro. Results: HPLC showed that fermentation changed the contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid. Furthermore, fermented dried mulberry leaves with 50% raw silkworm pupa had a better efficacy of anti-adipogenesis than dried mulberry leaves, fermented dried mulberry leaves and fermented silkworm pupa and inhibited triglycerides accumulation and glucose consumption. Additionally, fermented dried mulberry leaves with 50% raw silkworm pupa inhibited PPAR-? signaling. Conclusions: Fermentation with Cordyceps militaris enhanced anti-adipogenesis efficacy of mulberry leaves.

Inhibition of endoplasmic reticulum stress in high-fat-diet-induced obese C57BL/6 mice: Efficacy of a novel extract from mulberry (Morus alba) leaves fermented with Cordyceps militaris / 한국실험동물학회지

A few clues about correlation between endoplasmic reticulum (ER) stress and mulberry (Morus alba) leaves were investigated in only the experimental autoimmune myocarditis and streptozotocin-induced diabetes. To investigate whether a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) could suppress ER in fatty liver, alterations in the key parameters for ER stress response were measured in high fat diet (HFD)-induced obese C57L/6 mice treated with EMfC for 12 weeks. The area of adipocytes in the liver section were significantly decreased in the HFD+EMfC treated group as compared to the HFD+Vehicle treated group, while their level was higher in HFD+Vehicle treated group than No treated group. The level of the eukaryotic initiation factor 2 alpha (eIF2α) and inositol-requiring enzyme 1 beta (IRE1α) phosphorylation and CCAAT-enhancer-binding protein homologous protein (CHOP) expression were remarkably enhanced in the HFD+Vehicle treated group. However, their levels were restored in the HFD+EMfC treated group, although some differences were detected in the decrease rate. Similar recovery was observed on the ER stress-induced apoptosis. The level of Caspase-3, Bcl-2 and Bax were decreased in the HFD+EMfC and HFD+orlistat (OT) treated group compared to the HFD+Vehicle treated group. The results of the present study therefore provide first evidence that EMfC with the anti-obesity effects can be suppressed ER stress and ER stress-induced apoptosis in the hepatic steatosis of HFD-induced obesity model.

α-Isocubebene modulates vascular tone by inhibiting myosin light chain phosphorylation in murine thoracic aorta

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms, but the mechanism responsible for this activity has not been determined. To determine the role played by ICB on the regulation of vascular tone, we investigated the inhibitory effects of ICB on vascular contractile responses by adrenergic α-receptor agonists. In addition, we investigated the role on myosin light chain (MLC) phosphorylation and cytosolic calcium concentration in vascular smooth muscle cells (VSMC). In aortic rings isolated from C57BL/6J mice, ICB significantly attenuated the contraction induced by phenylephrine (PE) and norepinephrine (NE), whereas ICB had no effects on KCl (60 mM)-induced contraction. In vasculatures precontracted with PE, ICB caused marked relaxation of aortic rings with or without endothelium, suggesting a direct effect on VSMC. In cultured rat VSMC, PE or NE increased MLC phosphorylation and increased cytosolic calcium levels. Both of these effects were significantly suppressed by ICB. In conclusion, our results showed that ICB regulated vascular tone by inhibiting MLC phosphorylation and calcium flux into VSMC, and suggest that ICB has anti-hypertensive properties and therapeutic potential for cardiovascular disorders related to vascular hypertension.

Lipolytic effect of novel extracts from mulberry (Morus alba) leaves fermented with Cordyceps militaris in the primary adipocytes derived from SD rats / 한국실험동물학회지

Mulberry (Morus alba) leaves are known to have therapeutic effects on lipid metabolism including lipogenesis, lipolysis and hyperlipidemia. However, novel compounds with strong lipolytic ability among 27 extracts of the mulberry leaves fermented with Cordyceps militaris (EMfCs) have not yet been identified. Therefore, the cAMP concentration and cell viability were measured in the primary adipocytes of SD (Sprague Dawley) rats and 3T3-L1 cells after treatment of 27 EMfCs. Briefly, mulberry leaves powders amended with three different concentrations (0, 25 and 50%) of silkworm pupae (SWP) powder were fermented with 10% C. militaris (v/w) during three different periods (3, 4 and 6 weeks). A total of 27 extracts were obtained from the fermented mulberry leaves powders using three different solvents (dH2O, 50% EtOH and 95% EtOH). Among the 27 EMfCs treated groups, a significant increase in the concentration of cAMP was detected in primary adipocytes treated with 10 extracts when compared with the Vehicle treated group. However, their cAMP concentration did not agree completely with the non-toxicity, although most extracts showed non-toxicity. Furthermore, the concentration of cAMP and level of free glycerol gradually increased in a dose dependent manner (100, 200 and 400 µg/mL) of 4M3-95 contained cordycepin without any significant toxicity. Overall, the results of this study provide strong evidence that 4M3-95 extract derived from EMfCs can stimulate the lipolysis of primary adipocytes at an appropriate concentration and therefore have the potential for use as lipolytic agents to treat obesity.

Therapeutic Effects of S-Petasin on Disease Models of Asthma and Peritonitis

To explore the anti-allergic and anti-inflammatory effects of extracts of Petasites genus, we studied the effects of s-petasin, a major sesquiterpene from Petasites formosanus (a butterbur species) on asthma and peritonitis models. In an ovalbumin-induced mouse asthma model, s-petasin significantly inhibited the accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar fluids. S-petasin inhibited the antigen-induced degranulation of beta-hexosamidase but did not inhibit intracellular Ca2+ increase in RBL-2H3 mast cells. S-petasin inhibited the LPS induction of iNOS at the RNA and protein levels in mouse peritoneal macrophages. Furthermore, s-petasin inhibited the production of NO (the product of iNOS) in a concentration-dependent manner in the macrophages. Furthermore, in an LPS-induced mouse model of peritonitis, s-petasin significantly inhibited the accumulation of polymorpho nuclear and mononuclear leukocytes in peritoneal cavity. This study shows that s-petasin in Petasites genus has therapeutic effects on allergic and inflammatory diseases, such as, asthma and peritonitis through degranulation inhibition in mast cells, suppression of iNOS induction and production of NO in macrophages, and suppression of inflammatory cell accumulation.

Induction of apoptosis by a hexane extract of aged black garlic in the human leukemic U937 cells

BACKGROUND/OBJECTIVES: In this study, the apoptogenic activity and mechanisms of cell death induced by hexane extract of aged black garlic (HEABG) were investigated in human leukemic U937 cells. MATERIALS/METHODS: Cytotoxicity was evaluated by MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide) assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay. RESULTS: Exposure to HEABG was found to result in a concentration- and time-dependent growth inhibition by induction of apoptosis, which was associated with an up-regulation of death receptor 4 and Fas legend, and an increase in the ratio of Bax/Bcl-2 protein expression. Apoptosis-inducing concentrations of HEABG induced the activation of caspase-9, an initiator caspase of the mitochodrial mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. HEABG also induced apoptosis via a death receptor mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid, and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. However, pre-treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, significantly blocked the HEABG-induced apoptosis of these cells, and increased the survival rate of HEABG-treated cells, confirming that HEABG-induced apoptosis is mediated through activation of caspase cascade. CONCLUSIONS: Based on the overall results, we suggest that HEABG reduces leukemic cell growth by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways, implying its potential therapeutic value in the treatment of leukemia.

Transcriptome Analysis of the Effects of Gomisin A on the Recovery of Carbon Tetrachloride-Induced Damage in Rat Liver / 한국실험동물학회지

Gomisin A possesses a hepatic function-facilitating property in liver-injured rats. Its preventive action on carbon tetrachloride-induced cholestasis is due to maintenance of the function of the bile acids-independent fraction. To investigate alterations in gene expression after gomisin A treatment on injured rat liver, DNA microarray analyses were performed on a Rat 44K 4-Plex Gene Expression platform with duplicated reactions after gomisin A treatment. We identified 255 up-regulated and 230 down-regulated genes due to the effects of gomisin A on recovery of carbon tetrachloride-induced rat liver damage. For functional characterization of these genes, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes biochemical pathways analyses were performed. Many up-regulated or down-regulated genes were related to cell cycle or focal adhesion and cell death genes, respectively. Our microarray experiment indicated that the liver repair mechanism induced by gomisin A was strongly associated with increased gene expressions related to cell cycle and suppression of the gene expression related in cell death.

Hypertension, Polydipsia, Polyuria, and Hypokalemia Associated with Renin-Secreting Wilms' Tumor in a Child

A 3-year-old boy with Wilms' tumor had unusual severe hypertension, polydipsia, polyuria, and hypokalemia. Physical examination on admission was unremarkable except for the presence of a smooth, firm mass in the right abdomen. Computerized tomography showed a tumor occupying upper two thirds of the right kidney. Plasma renin activity and aldosterone concentration were markedly elevated, 37.7 mg/ml/hour (normal supine 0.15-2.33 mg/ml/hour) and 120.1 ng/dl (normal supine 1 to 16 ng/dl), respectively. His hypertension, varied from 150/90 mmHg as high as 240/180 mmHg, was not effectively controlled by antihyperensive drugs. Because of concern for the complications of hypertension, a right nephrectomy was performed on the sixth hospital day. At laparotomy, there was no evidence of mechanical compression of the renal artery by the tumor. The tumor, about 8 cm in diameter, was confined within the renal capsule without involvement of the renal blood vessels at the hilum. Histopathologically, it was characteristic Wilms' tumor of favorable histology. On electron microscopy, the tumor cells contained many electron dense secreting granules in the cytoplasm, suggesting that the tumor itself was the source of the renin and cause the clinical manifestations. Shortly after nephrectomy, signs and symptoms were relieved dramatically, and plasma renin activity and aldosterone concentration were also decreased to normal.