Edmund M.K. Lui

De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis

BackgroundAmerican ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway.ResultsIn this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8%) unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450) and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA) inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay.ConclusionsWe demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community that is interested in the molecular genetics and functional genomics of American ginseng.

The Yin and Yang actions of North American ginseng root in modulating the immune function of macrophages

BackgroundImmuno-modulatory effects of ginseng, including both immuno-stimulatory and immuno-suppressive effects, have been widely reported. This study aims to determine whether the paradoxical immuno-modulatory effect is related to unique phytochemical profiles of different North American (NA) ginseng, namely aqueous (AQ) and alcoholic (ALC) extracts.MethodsAQ and ALC extracts were prepared and their immuno-bioactivity were studied in vitro in murine macrophages (Raw 264.7) through measuring the direct stimulatory production of pro-inflammatory mediator and cytokines as well as the suppression of lipopolysaccharide (LPS)-stimulatory response by the two extracts. Gel permeation chromatography was used to fractionate and isolate phytochemicals for characterization of ginseng extracts.ResultsAQ extract up-regulated the production of nitric oxide (NO), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) while ALC extract did not. ALC extract but not AQ extract suppressed LPS-induced macrophage NO and TNF-α production. These immuno-stimulatory and suppressive effects were exhibited at similar extract concentrations. Moreover, the macrophage-stimulating activity of the AQ extract was inhibited in the presence of ALC extract. Fractionation of AQ extract revealed the presence of two major peaks at 230 nm with average molecular weights of 73,000 and 37,000 Da. The first fraction had similar elution volume as the crude polysaccharide (PS) fraction isolated from the AQ extract, and it was the only bioactive species. Parallel fractionation study of ALC extract yielded similar elution profiles; however, both sub-fractions were devoid of PS. Fraction I of the ALC extract suppressed LPS-induced NO production dose-dependently.ConclusionALC extract of NA ginseng, which was devoid of PS, was immuno-inhibitory whereas the AQ extract, which contained PS, was immuno-stimulatory. These extract-related anti-inflammatory and pro-inflammatory effects may be considered as the Yin and Yang actions of ginseng.

Preventive effects of North American ginseng (Panax quinquefolium) on diabetic nephropathy.

PURPOSE Ginseng has been used as an herbal medicine and nutritional supplement in East Asia for thousands of years and gained popularity in the west because of its various pharmacological properties. Panax ginseng (Asian ginseng) and Panax quinquefolium (North American ginseng) both are reported to possess antihyperglycemic properties. The aim of the present study is to evaluate the preventive effects of North American ginseng on diabetic nephropathy (DN) and the underlying mechanisms of such effects. METHODS Models of both type 1 (C57BL/6 mice with STZ-induced diabetes) and type 2 diabetes (db/db mice) and age- and sex-matched controls were examined. Alcoholic ginseng root (200mg/kgbodywt, daily oral gavage) extract was administered to the diabetic mice (type 1 and type 2) for two or four months in order to evaluate its effects on DN. RESULTS Dysmetabolic state in the diabetic mice was significantly improved by ginseng treatment. In the kidneys of diabetic animals, ginseng significantly prevented oxidative stress and reduced the NF-κB (p65) levels. Diabetes-induced up-regulations of ECM proteins and vasoactive factors in the kidneys were significantly diminished by ginseng administration. Furthermore, albuminuria and mesangial expansion in the diabetic mice were prevented by ginseng therapy. CONCLUSION North American ginseng has preventive effects on DN and it works through a combination of mechanisms such as antihyperglycemic and antioxidant activities.

Developmental profiles of antioxidant enzymes and trace metals in chick embryo.

It has been previously well documented that partial pressure of oxygen (PO2) and weight-specific rate of O2 consumption in chick embryo (Gallus gallus domesticus) transiently increase midway through the 21-day in ovo incubation period. The present study found that these oxidative changes were paralleled by the concentrations of glutathione (GSH) and Zn in liver and by the specific activity of superoxide dismutase (SOD) in brain. Levels of antioxidant enzymes and their trace metal cofactors were markedly higher in liver than in brain. Hepatic catalase activity changed in parallel with the concentration of its cofactor, Fe. However, the relative abundance of metal cofactors did not appear to be the determining influence on other antioxidant enzyme activities. Rates of extra-mitochondrial hydrogen peroxide release were also much greater in liver than in brain. Taken together, the results of this initial study of embryonic chick antioxidant systems suggest that certain antioxidants may be regulated by PO2 and rate of oxidative metabolism during fetal development.

Preventive effects of North American Ginseng (Panax quinquefolius) on Diabetic Retinopathy and Cardiomyopathy

Ginseng (Araliaceae) has multiple pharmacological actions because of its diverse phytochemical constituents. The aims of the present study are to evaluate the preventive effects of North American ginseng on diabetic retinopathy and cardiomyopathy and to delineate the underlying mechanisms of such effects. Models of both type 1 (C57BL/6 mice with streptozotocin‐induced diabetes) and type 2 diabetes (db/db mice) and age‐matched and sex‐matched controls were examined. Alcoholic ginseng root (200 mg/kg body weight, daily oral gavage) extract was administered to groups of both type 1 and type 2 diabetic mice for 2 or 4 months. Dysmetabolic state in the diabetic mice was significantly improved by ginseng treatment. In both the heart and retina of diabetic animals, ginseng treatment significantly prevented oxidative stress and diabetes‐induced upregulations of extracellular matrix proteins and vasoactive factors. Ginseng treatment in the diabetic animals resulted in enhancement of stroke volume, ejection fraction, cardiac output, and left ventricle pressure during systole and diastole and diminution of stroke work. In addition, mRNA expressions of atrial natriuretic factor and brain natriuretic factor (molecular markers for cardiac hypertrophy) were significantly diminished in ginseng‐treated diabetic mice. These data indicate that North American ginseng prevents the diabetes‐induced retinal and cardiac biochemical and functional changes probably through inhibition of oxidative stress. Copyright

Biochemical mechanism of metallothionein-carbon tetrachloride interaction in vitro

To elucidate the mechanism underlying the protective effect of metallothionein (MT) against carbon tetrachloride (CCl4) toxicity, in vitro experiments were carried out to study the interaction of metallothionein and CCl4. Results from this study showed that incubation of Cd,Zn-MT with CCl4 in the presence of hepatic microsomes and NADPH resulted in a time-dependent depletion of MT thiols with a concurrent reduction in the metal-binding sites of the protein. Moreover, this reaction also released Zn and Cd from MT. Results from experiments conducted to determine whether or not the CCl4-induced decrease in MT-thiol content was due to the scavenging of CCl4 metabolite(s) showed that the trichloromethyl radical, chloroform and phosgene as well as the products of CCl4-induced microsomal lipid peroxidation were not directly involved. Although covalent binding of 14CCl4 to MT was detected following incubation in the presence of a microsomal bioactivation system, it did not account for the CCl4-induced loss of MT thiol groups for the following reasons: (i) prior oxidation of sulfhydryl groups of MT by hydrogen peroxide did not alter the binding; and (ii) anaerobiosis did not alter the extent of covalent binding but obliterated the inhibitory effect of CCl4 on MT thiol content. Measurement of the thiol content of CCl4-treated MT after treatment with 1,4-dithiothreitol revealed that all the thiol groups that were lost subsequent to CCl4 treatment could be regenerated. These data suggest that CCl4-linked oxidation of MT, rather than the covalent binding of 14CCl4 metabolite(s), may be responsible for the CCl4-induced loss of metal binding sites of MT with the concurrent release of Zn and Cd. However, the precise role of the metal released during the oxidation of MT in CCl4 toxicity remains to be defined.

Antioxidant effect of zinc and zinc-metallothionein in the acute cytotoxicity of hydrogen peroxide in Ehrlich ascites tumour cells

This study was concerned with the role of zinc (Zn) and zinc-metallothionein (Zn-MT) in oxidative stress. Hydrogen peroxide-induced oxidative injury was examined in Ehrlich ascites tumour cells isolated from control host mice, mice pretreated with 10 mg/kg ZnSO4 (i.p.) to increase cellular Zn/Zn-MT levels, and mice exposed to Zn-deficient diet to reduce the cellular Zn/Zn-MT levels. The results of the present study showed that Ehrlich cells with seven-fold differences in Zn-MT concentrations could be obtained by manipulating the Zn status of host mice and that high Zn and Zn-MT levels can make Ehrlich cells more resistant to H2O2-induced oxidative injury (cell viability, lipid peroxidation, [Ca2+]i) while cells with reduced Zn/Zn-MT levels were more susceptible to this treatment. H2O2 treatment resulted in oxidation of MT thiolate groups and loss of its metal binding capacity with translocation of Zn released from oxidized MT to other cellular sites. Preincubation of Ehrlich cells with ZnSO4 in vitro also conferred some degree of resistance to H2O2 toxicity, suggesting the inherent antioxidative property of Zn ions. These data suggested that Zn-MT can be considered as an antioxidant by virtue of its thiolate groups and its Zn ions that are released in the presence of oxidative stress.

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities.

PURPOSE Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs). METHODS Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25mmol/l for 24h. l-Glucose was used at a concentration of 25mmol/l as a control. RESULTS Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB(+)FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment. CONCLUSION These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.

North American ginseng protects the heart from ischemia and reperfusion injury via upregulation of endothelial nitric oxide synthase

Emerging evidence suggests ginseng has therapeutic potential in cardiovascular disease. The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the cardioprotective effects of ginseng during myocardial ischemia and reperfusion (I/R). Treatment with ginseng extract significantly increased Akt phosphorylation and eNOS protein levels in cultured neonatal cardiomyocytes. Upregulation of eNOS was blocked by LY294002, a PI3-kinase inhibitor, suggesting a PI3-kinase/Akt-dependent mechanism. To simulate I/R, cultured neonatal cardiomyocytes from eNOS(-/-) and wild-type (WT) mice were subjected to anoxia and reoxygenation (A/R). Ginseng treatment inhibited A/R-induced apoptosis in WT, but not in either eNOS(-/-) cardiomyocytes or WT cardiomyocytes treated with LY294002. To further study the cardioprotective effects of ginseng in vivo, WT and eNOS(-/-) mice were pretreated with ginseng extract (50mg/kg/day, oral gavage) for 7 days before they were subjected to myocardial I/R. Treatment with ginseng significantly increased Akt phosphorylation and eNOS protein levels in the myocardium. Furthermore, ginseng-induced myocardial eNOS expression was inhibited by LY294002. Strikingly, ginseng treatment significantly decreased infarct size and myocardial apoptosis following I/R in WT mice, but not in either eNOS(-/-) mice or WT mice treated with LY294002. We conclude that ginseng treatment protects the heart from I/R injury via upregulation of eNOS expression. Our study suggests that ginseng may serve as a potential therapeutic agent to limit myocardial I/R injury.

Ginseng Reverses Established Cardiomyocyte Hypertrophy and Postmyocardial Infarction-Induced Hypertrophy and Heart Failure

Background—A major challenge in the treatment of heart failure is the ability to reverse already-established myocardial remodeling and ventricular dysfunction, with few available pharmacological agents prescribed for the management of heart failure having demonstrated successful reversal of the remodeling and hypertrophic processes. North American ginseng (Panax quinquefolius) has previously been shown to effectively prevent cardiomyocyte hypertrophy and heart failure. Here, we determined whether North American ginseng can reverse established cardiomyocyte hypertrophy in cultured myocytes as well as hypertrophy and left ventricular dysfunction in experimental heart failure secondary to coronary artery occlusion. Methods and Results—Ginseng was administered in drinking water (0.9 g/L) ad libitum to rats after 4 weeks of sustained coronary artery ligation when heart failure was established or to angiotensin II- (100 nmol/L), endothelin-1- (10 nmol/L), or phenylephrine- (10 µmol/L) induced hypertrophic cultured neonatal ventricular cardiomyocytes. Echocardiographic and catheter-based measurements of hemodynamic parameters 4 weeks after starting ginseng treatment (8 weeks postinfarction) revealed nearly complete reversibility of systolic and diastolic abnormalities. Similarly, ginseng administration to hypertrophic cardiomyocytes resulted in a complete reversal to a normal phenotype after 24 hours as determined by cell surface area and expression of molecular markers. The effects of ginseng both in vivo and in cultured cardiomyocytes were associated with reversal of calcineurin activation and reduced nuclear translocation of the transcription factor NFAT3 (nuclear factor of activated T cells 3) in cultured myocytes. Moreover, the beneficial effect of ginseng was associated with normalization in the gene expression of profibrotic markers, including collagen (I and III) and fibronectin. Conclusions—This study demonstrates a marked ability of ginseng to reverse cardiac hypertrophy, myocardial remodeling, and heart failure, which was associated with and likely mediated by reversal of calcineurin activation. Ginseng may offer a potentially effective approach to reverse the myocardial remodeling and heart failure processes, particularly in combination with other treatment modalities.