Microbial transformation of curcumin to its derivatives with a novel Pichia kudriavzevii ZJPH0802 strain.

Curcumin, a polyphenolic compound, has shown a wide range of pharmacological activities and has been widely used as a food additive. However, the clinical use of curcumin is limited to some extent because of its poor water solubility and low bioavailability. To overcome these problems, many approaches have been attempted and structural modification of curcumin by microbial transformation has been proven to be an alternative. In this study, we isolated a novel yeast strain Pichia kudriavzevii ZJPH0802 from a soil sample, which is capable of converting curcumin to its derivatives. The transformed products by this strain were evaluated by HPLC, (+) electrospray ionization (ESI)-MS(n), and (1)H nuclear magnetic resonance methods. Compared with controls, two new peaks of the transformed broth appeared at retention times of 26 min (I) and 62 min (II) by HPLC analysis. The two transformed products were then further identified by (+) ESI-MS(n). The spectrum showed that compound I had an accurate [M+H+NH3](+) ion at m/z 392, [M+H](+) ion at m/z 375, [M+H-H2O](+) ion at m/z 357, and (+) ESI-MS(3) spectrum showed that ion at m/z 357 could further form fragment ions at m/z 339, 177, and 163; compound II had an accurate [M+H](+) ion at m/z 373, [M+H-H2O](+) ion at m/z 355, and (+) ESI-MS(3) spectrum showed that ion at m/z 355 could further form fragment ions at m/z 219, 179, 177, 163, and 137. These two transformed products thereby were confirmed as hexahydrocurcumin (I) and tetrahydrocurcumin (II).

[Effect of cold and cool herbs on liver mitochondria proteome of rats with heat symptom].

In the 1960s, modern science began involving the essence of heat syndrome, but there have still no in-depth systematic studies on pathological mechanisms of heat syndrome and action mechanisms of cold and cool herbs. In this study, the animal model with heat syndrome was set up by feeding herbs with hot property, and then cold and cool herbs was applied in the experimental therapy. The two-dimensional electrophoresis and mass spectrometry technologies were adopted to compare the liver mitochondria proteome of the rats of the heat syndrome model and the ones treated with cold and cool herbs, so as to discover specificity-related proteins after heat syndrome and treatment with cold and cool herbs.

Dihydroartemisinin-induced inhibition of proliferation in BEL-7402 cells: an analysis of the mitochondrial proteome.

Artemisinin, the active ingredient of the Chinese medicinal herb Artemisia annua L., and its derivatives (ARTs) are currently widely used as anti-malarial drugs around the world. In this study, we found that dihydroartemisinin (DHA), one of the main active metabolites of ARTs, inhibited the proliferation of human hepatocarcinoma BEL-7402 cells in a concentration-dependent manner. To interpret the mechanisms involved, an analysis of the mitochondrial proteome was performed employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Seven mitochondrial proteins including fumarate hydratase, 60 kDa heat shock protein, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, two subunits of ATP synthase and NADPH:adrenodoxin oxidoreductase were identified to be differentially expressed between the control and DHA-treated groups. Our results indicate that the imbalance of energy metabolism induced by DHA may contribute, at least in part, to its anti-cancer potential in BEL-7402 cells.

[Proteomic research on anti-atherosclerosis mechanism of curcumin in RAW264.7].

OBJECTIVE: Two-dimensional difference gel electrophoresis and mass spectrum were used to study the anti-atherosclerosis mechanism of curcumin. METHOD: The proteins from RAW264.7 cell and RAW264.7 cell treated with 25 micromol x L(-1) curcumin were labeled with Cy3 or Cy5 randomly. Each Cy3-labeled sample and Cy5-labeled sample was mixed on the same 2-D gel along with a Cy2-labeled mixture of all samples as an internal standard and run on the same gel. The gels were scanned under different wave-length light after electrophoresis. All images were analyzed by DeCyder 6.5 software, and the different proteins were identified by mass spectrum. RESULT: The expression of ATP synthesis H+ transporting, MHC class II, non-muscle myosin alkali light chain and cytochrome b5 increased in the RAW264.7 cell treated with 25 micromol x L(-1) curcumin, while the expression of phosphodiesterase 4D, elF-3, Hnrpf protein, vimentin, nucleophosminl and Ranbp 1 decreased. CONCLUSION: The anti-atherosclerosis mechanism of curcumin is the result of enhancement of the cell inflammation, antioxidant activity and inhibition of cholesterol transport, reduce of the accumulation of intracellular cholesterol and other factors. In addition, curcumin also had the effect of anti-tumor through regulated tumor cell differentiation and apoptosis.

[Effects of warm and tonify kidney-yang herbs on liver mitochondria proteome of kidney-yang deficiency rats].

OBJECTIVE: To study the effects of warm and tonify kidney-yang herbs on the liver mitochondria proteome of the thyroidectomized kidney-yang deficiency rats. METHOD: Twelve rats were divided into normal group, model group and treated group; each group had four rats. The rats of model group and treated group were excised the two side thyroid gland, and the rats of normal group were done the homologous operation, but didn't excise the thyroid gland. After seven days, the rats of model group and treated group appeared the symptoms of kidney-yang deficiency. From the eighth day, the rats of treated group were fed warm and tonify kidney-yang herbs 6.7 g x kg(-1) once daily, and the rats of other two groups were fed the equal normal. All rats of three groups were killed by decollation after six days of treatment, and liver mitochondria proteins were separated. Each liver mitochondria protein sample was labeled with Cy3 or Cy5 randomly, and one Cy3-labeled sample and one Cy5-labeled sample were mixed on the same 2-D gel along with a Cy2-labeled mixture of all samples as an internal standard and run on the same gel. The gels were scanned under different wavelength light after electrophoresis. All images were analyzed by DeCyder 6. 5 software, and the different proteins were identified by mass spectrum. RESULT: The expression of HSP60, catalase, glutathione peroxidase, carbamoylphosphate synthetase I, ATP synthase, lactotransferrin, H(+)-transporting two-sector ATPase, alpha-ETF and calpain 12 were increased in the thyroidectomized kidney-yang deficiency rats, while the expression of oxoisovalerate dehydrogenase, Acyl-CoA dehydrogenase, ornithine aminotransferase, and GTP-binding regulatory protein were decreased. After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs, the expression of HSP60, catalase, glutathione peroxidase, oxoisovalerate dehydrogenase, Acyl-CoA dehydrogenase, ATP synthase, lacto-transferrin, H(+)-transporting two-sector ATPase, alpha-ETF and GTP-binding regulatory protein were increased, and the expression of carbamoyl-phosphate synthetase I and calpain 12 were decreased. CONCLUSION: The warm and tonify kidney-yang herbs perform its therapeutical effect by regulating the metabolism, protecting the stability of mitochondrial membrance and maintaining the signal conduction in the cells.

[Study of the property of lipids reducing of curcumin on hyperlipidemia mice after fermented by Monascus purureus].

OBJECTIVE: To study the lipids reducing property of curcumin on Hyperlipidemia mice after fermented by Monascus purureus. METHODS: The stain Monascus purureus was used for microbial transformation, and both substrate control and strain control were set. The mice were reared with high lipid and cholesterol feed for 15d to establish the Hyperlipidemia models. The models were treated with fermented curcumin in 500 mg/kg, 200 mg/kg, 100 mg/kg, substrate control and strain control in 500 mg/kg. Positive and Normal group were treated with natural saline. The general situation was observed and the changes of TG, TC, HDL-C levels in serum and liver were tested after 10 d. RESULTS: Fermented curcumin could significantly reduce the serum TC, TG of Hyperlipidemia mice all in high, middle and low doses. Serum TC was reduced by 38.7%, 34.5%, 32.7% and TG was reduced by 38.3%, 28.6%, 30.1%, respectively while substrate control and strain control had no effect. Fermented curcumin also could reduce the TC, TG in liver but no effect of curcumin substrate at the same dose. CONCLUSION: The property of lipids reducing of curcumin is significantly enhanced after fermentation.

Curcumin up-regulates LDL receptor expression via the sterol regulatory element pathway in HepG2 cells.

Plasma low-density lipoprotein-cholesterol (LDL-C) is mainly taken up and cleared by the hepatocellular LDL receptor (LDL-R). LDL-R gene expression is regulated by the sterol regulatory element binding proteins (SREBPs). Previous studies have shown that curcumin reduces plasma LDL-C and has hypolipidemic and anti-atherosclerotic effects. Herein, we investigated the effect of curcumin on LDL-R expression and its molecular mechanism in HepG2 cells. Curcumin increased LDL-R expression (mRNA and protein) and the resultant uptake of DiI-LDL in a dose- and time-dependent manner. Using a GFP reporter system in a transfected HepG2/SRE-GFP cell line, we found that curcumin activated the sterol regulatory element of the LDL-R promoter. In HepG2/Insig2 cells, curcumin reversed the inhibition of LDL-R expression induced by Insig2 overexpression. These data demonstrate that curcumin increases LDL-R protein expression and uptake activity via the SREBPs pathway. These findings contribute to our further understanding of the cholesterol-lowering and anti-atherosclerotic effects of curcumin.

Progress of research in treatment of hyperlipidemia by monomer or compound recipe of Chinese herbal medicine.

Hyperlipidemia (HLP) is the No.1 risk factor for patients with atherosclerosis (AS) and is directly related to the occurrence of coronary artery disease (CAD) and cerebrovascular disease. Therefore, prevention and treatment of AS is of great importance and of practical significance in controlling the incidence and mortality of CAD. With its peculiar syndrome-dependent therapy, traditional Chinese medicine (TCM) has accumulated abundant practical experiences in this field and good clinical effects have been achieved. Chinese herbal medicine, with its particularly unique advantages and high potentials yet to be tapped, displays its huge strength in HLP prevention and treatment. The progress of studies concerning prevention and treatment of HLP by Chinese herbal medicines, in the form of monomers or compound recipes, is reviewed in this paper.

Regulation of LDL receptor expression by the effect of curcumin on sterol regulatory element pathway.

To investigate the molecular mechanisms of the effect of curcumin on up-regulation of LDL receptor expression, a sterol regulatory report system was established in Xenopus laevis oocytes by microinjection of a plasmid pLXRN-4SRE-fPA, in which green fluorescence protein (GFP) gene was constructed downstream from the sterol regulatory element-1 (SRE-1), in the nucleus of the oocytes. The oocytes were treated with different kinds of natural medicines: curcumin, tea polyphenols, tanshinone, ligustrazine, ginkgolide, astragalosides. The expression of GFP was induced only by curcumin. In addition, it was also proved that the curcumin can increase the expression of functional LDL receptors in human liver hepatoma cell line HepG2. In conclusion, curcumin may up-regulate the expression of LDL receptor by its effect on SRE pathway.

Regulation of lipoprotein lipase expression by effect of hawthorn flavonoids on peroxisome proliferator response element pathway.

To investigate the possibility that natural medicines affect lipid metabolism by regulating lipoprotein lipase (LPL) expression, a green fluorescent protein (GFP) gene was constructed downstream of the peroxisome proliferator response element (PPRE) and the constructed plasmid was microinjected into Xenopus oocytes to establish a PPRE regulatory reporter system. Using this system, hawthorn flavonoids were quickly selected from a panel of natural medicines and found to up-regulate GFP expression by an effect on PPRE. To confirm the effect of hawthorn flavonoids, we treated mice orally with water (control), hawthorn flavonoids, and pioglitazone and measured the LPL levels in serum, adipose tissue, and muscle by an enzyme-linked immunosorbent assay. The serum LPL levels were no different from the controls after treatment with either hawthorn flavonoids or pioglitazone, but LPL increased significantly in muscular tissues and decreased in adipose tissues. These results demonstrate that hawthorn flavonoids meditate LPL expression in mice with tissue-specific differences. A novel PPRE regulatory report system was established for rapid and effective selection and evaluation of LPL-mediating drugs.

Effect of curcumin on the expression of LDL receptor in mouse macrophages.

To investigate the molecular mechanisms of lipid-lowering drug, Rhizoma Curcumae Longae, we treated the mouse macrophages with curcumin, which was purified from the ethanol extraction of Rhizoma Curcumae Longae. The LDL receptors expressed in the macrophages were determined by ELISA, FLISA and assay of LDL uptake. Here for the first time, we found that curcumin obviously up-regulated the expression of LDL receptor in mouse macrophages, and the dose-effect relationship was demonstrated.

[Effect of curcumin on the gene expression of low density lipoprotein receptors].

OBJECTIVE: To investigate the molecular mechanisms and effective target points of lipid-lowering drug, Rhizoma Curcumae Longae, and study the effect of curcumin on the expression of low density lipoprotein (LDL) receptors in macrophages in mice. METHODS: Macrophages in mice were treated with curcumin, which was purified from the ethanolly extraction of Rhizoma Curcumae Longae for 24 h. The LDL receptors expressed in the macrophages were determined by enzyme-linked immunosorbent assay (ELISA) and assay of DiI labeled LDL uptake by flow cytometer. RESULTS: It was found for the first time that 10 micromol/L-50 micromol/L curcumin could obviously up-regulate the expression of LDL receptor in macrophages in mice, and a dose-effect relationship was demonstrated. CONCLUSION: One of the lipid-lowering mechanisms of traditional Chinese medicine, Rhizoma Curcumae Longae, was completed by the effect of curcumin through the up-regulation of the expression of LDL receptor.

A peroxisome proliferator response elements regulatory system in xenopus oocytes and its application.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element (PPRE), a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-gamma ligands on the PPRE-mediated pathway regulatory system. METHODS: Two plasmids were constructed: pXOE-PPARgamma, in which the human PPARgamma gene was in the downstream of TFIIIA gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein (EGFP) gene was subcloned into PPRE. The xenopus oocytes were injected with these two plasmids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE-PPARgamma plasmid into the oocytes, which then treated with prostaglandin E1 and Hawthorn flavonoids. The mass of expressed lipoprotein lipase (LPL) in the cells was determined by enzyme labeling linked immunosorbent assay (ELISA). RESULTS: The expression of EGFP was only induced by prostagalandin E1, pioglitazone, Hawthorn flavonoids. A concentration-response relationship was seen between expressed EGFP and Hawthorn flavonoids. The levels of LPL in both Hawthorn flavonoids groups and PPARgamma ligand prostagalandin E1 group injected with pXOE-PPARgamma plasmid increased significantly (< 0.001) compared with controls, and a concentration-response relationship was observed between LPL mass and Hawthorn flavonoids. CONCLUSIONS: It is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARgamma ligand. Hawthorn flavonoids can increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.

[Effect of curcumin on expression of human low density lipoprotein receptors in Xenopus Laevis oocytes].

OBJECTIVE: To investigate the molecular mechanism of curcumin in reducing blood lipids by establishing gene expression system of human low density lipoprotein receptors (LDL-R) in Xenopus Laevis oocytes (XLO). METHODS: The expression of LDL-R on cytomembrane was determined using immuno-fluorescent, ligand-fluorescent and immune colloidal gold techniques after human LDL-R containing p3.7 LDL plasmid was led into nucleus. And the expression of LDL-R gene in XLO was quantitatively determined by ELISA after being interfered with different concentrations of curcumin. RESULTS: The human LDL-R gene could be expressed on XLO, which could be significantly enhanced by curcumin in a dose-dependent manner. Conclusion One of the paths of curcumin in reducing blood lipids and anti-atherosclerosis was improving LDL-R gene expression and increasing the LDL-cholesterol absorption of cells.

Galactose-specific asialoglycoprotein receptor is involved in lipoprotein (a) catabolism.

Lp(a) [lipoprotein (a)] is a highly atherogenic plasma lipoprotein assembled from low-density lipoprotein and the glycoprotein apolipoprotein (a). The rate of Lp(a) biosynthesis correlates significantly with plasma Lp(a) concentrations, whereas the fractional catabolic rate does not have much influence. So far, little is known about Lp(a) catabolism. To study the site and mode of Lp(a) catabolism, native or sialidase-treated Lp(a) was injected into hedgehogs or ASGPR (asialoglycoprotein receptor)-knockout (ASGPR-) mice or wild-type (ASGPR+) mice, and the decay of the plasma Lp(a) concentration was followed. COS-7 cells were transfected with high- (HL-1) and low-molecular-mass ASGPR subunits (HL-2), and binding and degradation of intact or desialylated Lp(a) were measured. In hedgehogs, one of the few species that synthesize Lp(a), most of the Lp(a) was taken up by the liver, followed by kidney and spleen. Lp(a) and asialo-Lp(a) were catabolized with apparent half-lives of 13.8 and 0.55 h respectively. Asialo-orosomucoide increased both half-lives significantly. In mice, the apparent half-life of Lp(a) was 4-6 h. Catabolism of native Lp(a) by wild-type mice was significantly faster compared with ASGPR- mice and there was a significantly greater accumulation of Lp(a) in the liver of ASGPR+ mice compared with ASGPR- mice. The catabolism of asialo-Lp(a) in ASGPR- mice was 8-fold faster when compared with native Lp(a) in wild-type mice. Transfected COS-7 cells expressing functional ASGPR showed approx. 5-fold greater binding and 2-fold faster degradation of native Lp(a) compared with control cells. Our results for the first time demonstrate a physiological function of ASGPR in the catabolism of Lp(a).

[Effects of xuefuzhuyu decoction on collagen synthesis and proliferation of cardiac fibroblasts].

OBJECTIVE: To study the effects of XueFuZhuYu Decoction (XFZYD) on collagen synthesis and proliferation of cardiac fibroblasts and provide academic bases of remedying kinds of heart disease aroused by proliferation of cardiac fibroblasts. METHODS: By adding medicine directly and serum pharmacological method. RESULTS: Compared with the control group, 1 g/ml XFZYD(dilution 1:80) and 10 percent serum of rat contained XFZYD could significantly inhibit the collagen synthesis and the proliferation of cardiac fibroblasts. Moreover, 1 g/ml XFZYD(dilution 1:80) could significantly affect collagen secrtion of cardiac fibroblasts (P < 0.05), up to 17.9%. CONCLUSION: XFZYD could inhibit both proliferation and collagen secretion of cardiac fibroblasts. It could resist cardiac muscle fibrosis.

Study on the Relationship between Lipoprotein(a) Receptor and LDL Receptor.

Lp(a) receptor and LDL receptor on rhesus monkey liver cellular membrane were studied by Western blotting, to investigate whether Lp(a) and LDL metabolize through the same route. The experiment demonstrated Lp(a) receptor ( 300kD) and LDL receptor ( 185kD) to be different kinds of receptors. The result reveals that Lp(a) has its own metabolic pathway.