Effects of Asparagus officinalis Extracts on Liver Cell Toxicity and Ethanol Metabolism

ABSTRACT:  Asparagus officinalis  is a vegetable that is widely consumed worldwide and has also long been used as a herbal medicine for the treatment of several diseases. Although  A. officinalis  is generally regarded as a supplement for the alleviation of alcohol hangover, little is known about its effects on cell metabolism. Therefore, this study was conducted to analyze the constituents of the young shoots and the leaves of asparagus and to compare their biochemical properties. The amino acid and inorganic mineral contents were found to be much higher in the leaves than the shoots. In addition, treatment of HepG2 human hepatoma cells with the leaf extract suppressed more than 70% of the intensity of hydrogen peroxide (1 mM)-stimulated DCF fluorescence, a marker of reactive oxygen species (ROS). Cellular toxicities induced by treatment with hydrogen peroxide, ethanol, or tetrachloride carbon (CCl₄) were also significantly alleviated in response to treatment with the extracts of  A. officinalis  leaves and shoots. Additionally, the activities of 2 key enzymes that metabolize ethanol, alcohol dehydrogenase and aldehyde dehydrogenase, were upregulated by more than 2-fold in response to treatment with the leaf- and shoot extracts. Taken together, these results provide biochemical evidence of the method by which  A. officinalis  exerts its biological functions, including the alleviation of alcohol hangover and the protection of liver cells against toxic insults. Moreover, the results of this study indicate that portions of asparagus that are typically discarded, such as the leaves, have therapeutic use.     

河蚬营养成分的提取及其生理功能研究进展

河蚬(Corbicula fluminea)是一种双壳类软体动物,富含蛋白质、多糖、不饱和脂肪酸、矿物质及维生素等营养物质,具有清热解毒、醒酒护肝、利尿明目等多种生理功能。近年来,研究发现河蚬提取物能够抑制肝癌细胞HepG-2的生长,清除O2^-·、·OH、DPPH·自由基,降低血清胆固醇,甘油三酯的含量,促进胆固醇代谢,减少动脉粥样硬化,调控乙醇代谢过程中的关键酶细胞色素P4502E1(CYP2E1)的表达,降低对人体有害自由基的生成,抑制脂质过氧化反应,减少脂质过氧化物的生成,可以有效缓解酒精对肝脏造成的损伤。本文主要阐述了河蚬的营养成分、提取方式、生理功能及其开发研究现状,为实现河蚬精深加工以及工业化生产提供理论依据。     

魔芋葡甘聚糖抗醉解酒作用机理研究

以不同剂量的魔芋葡甘露聚糖(Konjac Glucomannan,KGM)灌胃昆明种小鼠,采用56%vol红星二锅头灌胃方法进行造模,研究KGM对小鼠的抗醉解酒作用及机理。结果表明:中(240mg/kg)、高(400 mg/kg)剂量组可有效延长醉酒潜伏期,显著缩短醉酒小鼠的睡眠时间和醒酒时间,且高、中剂量组的血清乙醇浓度显著低于模型组小鼠,高剂量组小鼠肝组织匀浆中乙醇脱氢酶(Alcohol dehydrogenase,ADH)、乙醛脱氢酶(Acetaldehyde dehydrogenase,ALDH)和细胞色素P450(Cytochrome P450,P450)含量均显著升高;中、高剂量组小鼠胃黏膜组织和血清中丙二醛(Methane dicarboxylic aldehyde,MDA)含量显著降低,过氧化物歧化酶(Superoxide Dismutase,SOD)活力、一氧化氮(Nitric Monoxide,NO)含量、前列腺素E2(Prostaglandin E,PGE2)含量显著升高。其解酒机理:(1)可能是其抑制酒精的吸收,降低血清中乙醇浓度;(2)小鼠胃黏膜和血清中MDA含量的降低,SOD活力、NO和PGE的升高减轻了酒精对胃黏膜的损伤,并提高了肝脏中ADH、ALDH、P450含量,通过乙醇脱氢酶和乙醇氧化酶系统加速酒精代谢,发挥其防醉解酒作用。     

白酒醇酸酯比率对小鼠酒后乙醇代谢及行为能力的影响

控制白酒中醇、酸、酯的含量是白酒生产企业的关键技术。该研究模拟白酒中主要醇类、酸类和酯类的含量及比例配制成实验酒样,测定小鼠酒后在不同时间点的血液乙醇含量,计算小鼠的乙醇代谢能力;同时通过Morris水迷宫实验测定小鼠相应的行为能力,并测定小鼠酒后2 h时体内乙醇代谢相关酶活性以及急性酒精性肝损伤情况。结果表明,杂醇对小鼠的乙醇代谢及行为记忆能力有较大影响;过高的杂醇（尤其是异戊醇）含量,会抑制乙醇在体内代谢,主要原因是抑制了乙醇脱氢酶和乙醛脱氢酶的活性;同时降低小鼠记忆能力,显著延长潜伏逃脱时间（P＜0.05）。乙酸能较好的促进乙醇代谢,减缓其他组分对乙醇脱氢酶和乙醛脱氢酶活性的抑制作用,且会刺激小鼠记忆能力,显著降低潜伏逃脱时间（P＜0.05）。     

益生菌联合白藜芦醇对小鼠慢性酒精性肝损伤的改善作用及机制研究

利用C57BL/6J小鼠构建慢性酒精性肝损伤模型,探究益生菌联合白藜芦醇对慢性酒精性肝损伤的保护作用及可能机制。小鼠被随机分为空白对照组（Con组)和酒精模型组(Mod组）,三组益生菌分别联合白藜芦醇（Resveratrol,Res）干预组（Lactobacillus paracasei J5+Res（J5+Res）、Lactobacillus casei YRL577+Res（YRL577+Res）、Bifidobacterium animalis F1-7+Res（F1-7+Res））和阳性药物硫普罗宁组（LP组）。实验结束后,通过分析小鼠肝脏脂质含量、酒精代谢酶活性、氧化应激水平等指标,对益生菌联合白藜芦醇的作用效果进行评价。为了进一步探究联合作用机制,对肝脏中氧化应激相关基因CYP2E1、核因子 E2 相关因子 2（Nuclear factor erythroid-2-related factor 2, Nrf2）、血红素加氧酶1（Heme oxygenase-1,HO-1）的mRNA表达进行分析。结果表明,相较于Mod组,益生菌联合白藜芦醇能够显著降低小鼠肝脏中甘油三脂、总胆固醇含量、血清谷草转氨酶和谷丙转氨酶活力（P<0.05）,提高肝脏乙醇脱氢酶、乙醛脱氢酶活性并抑制肝脏CYP2E1活性及其mRNA表达,显著提高肝脏还原型谷胱甘肽含量和超氧化物歧化酶、过氧化氢酶活性（P<0.05）,并能有效激活Nrf2/HO-1通路,其中,Nrf2 mRNA表达量在J5+Res、YRL577+Res、F1-7+Res三组益生菌联合干预组中分别被上调了2.6、3.7和2.7倍,HO-1 mRNA表达量被上调了2.0、6.2和4.0倍。因此,益生菌联合白藜芦醇可能通过激活Nrf2/HO-1途径预防慢性酒精性肝损伤。     

菊苣醇提物对对乙酰氨基酚诱导小鼠肝损伤的保护作用

目的研究菊苣醇提物对对乙酰氨基酚(acetaminophen,APAP)诱导的小鼠肝损伤的保护作用。方法将56只昆明小鼠随机分为7组:分别是阴性对照组,APAP模型组,水飞蓟宾阳性对照组,菊苣醇提物单独给药组(200 mg/kg体重),菊苣醇提物低、中和高剂量(50、100和200 mg/kg体重)与APAP联用组。各剂量组小鼠灌胃给予对应药物连续7 d后,一次性腹腔注射APAP(300 mg/kg体重)构建肝损伤模型,24 h后采集肝组织样品和血清。分别测定肝组织和血清的多种氧化指标以及细胞色素P450 2E1酶(CYP2E1)的蛋白表达水平。结果菊苣醇提物能够显著降低血清中谷丙转氨酶(alanine aminotransferase,ALT)和谷草转氨酶(aspartate aminotransferase,AST)水平。降低肝组织中过氧化氢(hydrogen peroxide,H2O2)和谷胱甘肽(glutathione,GSH)的含量以及提高抗氧化酶系中过氧化氢酶(catalase,CAT)和总超氧化物歧化酶(total superoxide dismutase,T-SOD)活性;同时,体外抗氧化实验亦表明其具有良好的清除自由基的能力。另外,菊苣醇提物还能够显著抑制CYP2E1的蛋白表达。结论菊苣醇提物有助于维持小鼠体内酶防御系统功能,提高机体清除自由基的能力,并通过减少CYP2E1的蛋白表达对APAP所致小鼠肝损伤具有明显保护作用。     

Alcohol metabolizing and antioxidant activities of complex herbal extracts from medicinal plants

The objective of this study was to evaluate the alcohol-metabolizing and antioxidative activities of complex herbal extract (CHE). The alcohol-metabolizing activity of CHE was evaluated by assessing alcohol dehydrogenase (ADH) activity, acetaldehyde dehydrogenase (ALDH) activity, and protective effect against alcohol induced damage in in vitro and in vivo models. In this study, CHE treatment significantly increased ADH and ALDH activities and reduced cell death in alcohol-induced liver cell damage. Moreover, it also significantly reduced the serum alcohol and acetaldehyde concentrations in alcoholfed rats. To define the effect of CHE on alcohol metabolism, its antioxidative activities were estimated by measuring radical scavenging activity, ferric-reducing antioxidant potential ability, and thiobarbituric acid reactive substance, and the results demonstrated a higher antioxidative capacity of CHE than the vitamin C control at a high dose (10 mg/mL). Therefore, this study suggests that CHE can be a potential natural resource for the management of ethanol-induced liver toxicity.     

Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions,using alcohol dehydrogenase as sensor reaction

With the current quantitative metabolomics techniques, only whole‐cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde‐3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH‐catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose‐limited chemostats under steady‐state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose‐limited steady‐state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.     

Acetaldehyde: an intermediate in the formation of ethanol from glucose by lactic acid bacteria.

Group N streptococci formed acetaldehyde and ethanol from glucose. As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate. A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present. Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and alpha-carboxylase were absent. Phosphoketolase activity was induced in Streptococcus lactis subsp. diacetylactis after growth on D-xylose. Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.     

The role of free radicals and antioxidants: How do we know that they are working?

This review briefly discusses how free radicals are formed and the possible participation of free radicals in disease. The review describes the basic radical reactions and the types of products that are formed from the free‐radical reactions of cellular constituents. In many cases, in vivo free‐radical oxidation can be detected by measuring products that were derived from radical reactions. Since aerobic organisms generate oxygen‐containing free radicals during oxygen metabolism, they carry chemicals and enzymes that reduce the threat posed by these radicals. The more common sources of in vivo free radicals are described in the article as well as the methods used by cells to protect themselves from free‐radical damage. Generation of free radicals in vivo also may be the result of exposure to certain chemical agents present in the environment Many of these agents cause pathologic changes to the exposed tissues and organs by initiating free‐radical reactions.     

浓香型白酒风味成分对乙醇代谢及关键酶活性影响的体内实验研究

选用不同产地的四种浓香型白酒样品（编号为SC1、SC2、SC3、JS）,建立小鼠灌胃模型,分别灌胃白酒、高醇白酒、高酯白酒和相同浓度的酒精溶液,随后测定小鼠的行为指标,血液中乙醇和乙醛含量以及肝脏中乙醇脱氢酶（ADH）和乙醛脱氢酶（ALDH）活性。结果表明,灌胃给药后,白酒中高浓度异丁醇、正戊醇和异戊醇显著降低了小鼠的协调能力（P＜0.05）;血液中乙醇含量（3 692～23 237 mg/L）和乙醛含量（18～84 mg/L）均升高;增加白酒中大多数醇类和酯类含量均能抑制ADH（30.93～45.73 U/L）和ALDH（87.98～104.61 U/L）活性,且对ADH抑制作用更显著（P＜0.05）;增加SC2和SC3酒样中丁酸乙酯含量可以同时促进ADH（34.73 U/L、35.11 U/L）和ALDH（104.61 U/L、103.52 U/L）的活性。体内实验结果表明,不同产地的白酒及其差异化风味成分（增量变化）对乙醇代谢和关键代谢酶有不同程度的影响。     

Antioxidant activity of blueberry anthocyanin extracts and their protective effects against acrylamide‐induced toxicity in HepG2 cells

In this study, the antioxidant activities of blueberry anthocyanin extracts from ten blueberry varieties were evaluated based on the methods of scavenging activities for DPPH radicals, ABTS radicals, hydroxyl radicals and ferric reducing antioxidant power (FRAP) assay. Among the ten blueberry varieties, Polaris had the highest antioxidant abilities and the largest amounts of anthocyanins identified by HPLC‐MS. The protective effects of anthocyanin extracts from Polaris (AEP) against acrylamide (AA)‐induced toxicity in HepG2 cell models were also evaluated due to the neurotoxic, genotoxic and potentially carcinogenic effects of AA. The protective effects of AEP on the damage of HepG2 cells were explored from the aspects of cell viability, T‐SOD and CAT activity and MDA level. The AEP (5, 10, 20 μg mL^?1) could significantly increase cell viability (P < 0.01) and inhibit AA‐induced cytotoxicity. Polaris also markedly promoted the activity of SOD, CAT and inhibited MDA level. The results showed that AEP had strong antioxidant activities, presenting high protective effects against AA‐induced cell damage in HepG2 cell models.     

酒精对人原代培养肝细胞的氧化损伤与CYP 2E1关系的研究

目的 研究急性酒精暴露下对人原代培养肝细胞中CYP 2E1依赖的毒性作用和氧化损伤。方法 分离培养人原代肝细胞，以25-100mmol/L乙醇作用于人原代肝细胞9h及100mmol／L乙醇作用于人原代肝细胞0～24h后，检测人原代肝细胞中CYP 2E1的含量，并研100mmol/L乙醇作用于人原代肝细胞0～24h后，天冬氨酸转胺酶(aspartate transaminase，AST)的释放量及肝细胞中谷胱甘肽(Glutathione，GSH)、丙二醛(Malonclialdehyde，MDA)的含量。结果 急性酒精暴露导致人原代肝细胞中CYP 2E1的释放增加，并呈明显的剂量效应和时间效应关系；在100mmol/L乙醇作用下，AST和MDA明显升高，在0～24h内呈明显的时间效应关系，而GSH含量在6h后明显降低。结论 100mmol／L乙醇急性暴露可导致人原代培养肝细胞明显的氧化损伤，这种损伤与CYP 2E1活性的变化直接相关。     

Anti‐obesity effect of Liupao tea extract by modulating lipid metabolism and oxidative stress in high‐fat‐diet‐induced obese mice

Liupao tea (LPT) is traditional dark Chinese tea. The effect of LPT extract on high‐fat‐diet‐induced obese mice was investigated systematically. The results showed that LPT extract could reduce body weight and significantly alleviate liver damage and fat accumulation. LPT could also decrease the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (AKP), total cholesterol (TC), triglycerides (TG), and low‐density lipoprotein cholesterol (LDL‐C) and increase the level of high‐density lipoprotein cholesterol (HDL‐C) in the liver. It also decreased the serum levels of inflammatory cytokines, including tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), interleukin (IL)‐1β, and IL‐6 and increased the serum levels of anti‐inflammatory cytokines, including IL‐10 and IL‐4. Moreover, LPT improved the levels of total superoxide dismutase (T‐SOD), glutathione peroxidase (GSH‐Px), and catalase (CAT) and reduced the level of malondialdehyde (MDA) in the liver. Moreover, LPT could upregulate the mRNA and protein expressions of peroxisome proliferator‐activated receptor alpha (PPAR‐α), lipoprotein lipase (LPL), carnitine palmitoyltransferase 1(CPT1), and cholesterol 7 alpha‐hydroxylase (CYP7A1) and downregulate those of PPAR‐γ and CCAAT/enhancer‐binding protein alpha (C/EBP‐α) in the liver. It also increased the mRNA expression of copper/zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), CAT, gamma‐glutamylcysteine synthetase 1 (GSH1), and GSH‐Px. The components of LPT extract include catechin, rutin, taxifolin, and astragalin, which possibly have a wide range of biological activities. In conclusion, our work verified that LPT extract possessed an anti‐obesity effect and alleviated obesity‐related symptoms, including lipid metabolism disorder, chronic low‐grade inflammation, and liver damage, by modulating lipid metabolism and oxidative stress.     

Protective effects of protein hydrolysate from marine microalgae Navicula incerta on ethanol-induced toxicity in HepG2/CYP2E1 cells

Ethanol is independently known to cause tissue damage through various mechanisms. This study was designed to evaluate the protective effect of marine microalgae, Navicula incerta protein enzymatic hydrolysates (NEHs) against ethanol-induced hepatotoxicity in HepG2 cells transfected with human CYP2E1. Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. When the alcohol-induced cells were treated with NEHs at various concentrations, there was a dose-dependent decrease of gamma-glutamyl transpeptidase (GGT) activity in the culture media and loss of cell viability. Among the NEHs constituents the hydrolysates which were obtained by papain (P-NEH), pronase-E (PR-NEH) and α-chymotrypsin (A-NEH) activity attenuated the ethanol cytotoxicity effectively, respectively. The activity appeared to be a GGT inhibitor. Therefore, the cytoprotective effects at alcohol-induced HepG2/CYP2E1 cells could be attributed to the inhibition of GGT activity by NEHs. This study suggests that NEHs have enough potential to be considered as highly active compounds against ethanol toxicity which leads NEHs to be significant nutraceuticals.     

Glucose metabolism,enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum

The physiology of Hanseniaspora uvarum K₅ was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h^?1, glucose was completely metabolized in biomass and CO₂. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO₂ were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.     

METABOLISM OF ACETALDEHYDE—A METABOLIC BRANCHING POINT II. EFFECT OF ETHANOL

Acetaldehyde is an intermediate in alcoholic fermentation. The major part of it is reduced to ethanol but a minor fraction is normally oxidized to acetic acid. Owing to the kinetic properties of alcohol dehydrogenase and aldehyde dehydrogenase, the oxidized fraction decreased as the intracellular concentration was increased. Since alcohol dehydrogenase catalyses a reversible reaction, the distribution between oxidation and reduction of acetaldehyde was sensitive to the concentration of ethanol; an increased concentration of ethanol reduced the formation of acetic acid.     

Effects of Graptopetalumparaguayense extract on tert‐ butylhydroperoxide‐induced oxidative stress

BACKGROUND: Graptopetalum paraguayense E. Walther is used in folk medicine to lower blood pressure, protect liver function and relieve pain and infection. The effect and mechanism of its 50% ethanolic extract (GE50) were investigated in tert‐butylhydroperoxide (t‐BHP)‐induced Wistar rats. The serum was analysed for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities while the tissue cytosols were analysed for lipid peroxidation, antioxidant level and enzyme activities. RESULTS: The liver was the primary target organ while the heart appeared to be the least responsive organ for t‐BHP treatment among the three tissues investigated. t‐BHP treatment increased serum AST and ALT activities, which are indicators of liver toxicity, while GE50 pretreatment reduced t‐BHP‐induced liver damage. t‐BHP treatment induced lipid peroxidation in the liver and brain but not in the heart. GE50 pretreatment prevented t‐BHP‐induced lipid peroxidation by maintaining superoxide dismutase (SOD) activity as well as glutathione (GSH) and vitamin C levels in the liver and vitamin E level in the brain. Although t‐BHP treatment did not induce lipid peroxidation in the heart, it caused a decrease in antioxidant level. GE50 pretreatment prevented the t‐BHP‐induced decrease in vitamin C level in the heart. CONCLUSION: GE50 pretreatment in rats protects the liver and brain from possible damage by t‐BHP‐induced lipid peroxidation. Copyright © 2007 Society of Chemical Industry     

First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation

Dekkera bruxellensis is continuously changing its status in fermentation processes, ranging from a contaminant or spoiling yeast to a microorganism with potential to produce metabolites of biotechnological interest. In spite of that, several major aspects of its physiology are still poorly understood. As an acetogenic yeast, minimal oxygen concentrations are able to drive glucose assimilation to oxidative metabolism, in order to produce biomass and acetate, with consequent low yield in ethanol. In the present study, we used disulfiram to inhibit acetaldehyde dehydrogenase activity to evaluate the influence of cytosolic acetate on cell metabolism. D. bruxellensis was more tolerant to disulfiram than Saccharomyces cerevisiae and the use of different carbon sources revealed that the former yeast might be able to export acetate (or acetyl‐CoA) from mitochondria to cytoplasm. Fermentation assays showed that acetaldehyde dehydrogenase inhibition re‐oriented yeast central metabolism to increase ethanol production and decrease biomass formation. However, glucose uptake was reduced, which ultimately represents economical loss to the fermentation process. This might be the major challenge for future metabolic engineering enterprises on this yeast.