Berberine protects liver from ethanol-induced oxidative stress and steatosis in mice

Alcohol consumption is customary in many cultures and it is a common human behavior worldwide. Binge ethanol and chronic alcohol consumption, two usual drinking patterns of human beings, produce a state of oxidative stress in liver and disturb the liver function. However, a safe and effective therapy for alcoholic liver disease in humans is still elusive. This study identified the natural product berberine as a potential agent for treating or preventing ethanol-induced liver injury. We demonstrated that berberine attenuated oxidative stress resulted from binge drinking in liver by reducing hepatic lipid peroxidation, glutathione exhaust and mitochondrial oxidative damage. Furthermore, berberine also prevented the oxidative stress and macrosteatosis in response to chronic ethanol exposure in mice. Either the total cytochrome P450 2E1 or the mitochondria-located cytochrome P450 2E1, which is implicated in ethanol-mediated oxidative stress, was suppressed by berberine. On the other hand, berberine significantly blunted the lipid accumulation in liver due to chronic alcohol consumption, at least partially, through restoring peroxisome proliferator-activated receptor α/peroxisome proliferator-activated receptor-gamma Co-activator-1α and hepatocyte nuclear factor 4α/microsomal triglyceride transfer protein pathways. These findings suggested that berberine could serve as a potential agent for preventing or treating human alcoholic liver disease.     

Development and validation of an HPLC method for quality control of Pueraria lobata flower

Pueraria lobata, also known as Kudzu (Japan) or Ge (China), is a medicinal plant widely used in Oriental traditional medicine. In this study the development, optimization and validation of an HPLC ethod for quality control of Pueraria flower plant material is presented. By means of this analytical method the three major compounds, i. e., the isoflavones tectorigenin 7- O-[beta- D-xylopyranosyl-(1 - 6)-beta- D-glucopyranoside], tectorigenin 7- O-beta- D-glucopyranoside and tectorigenin, were quantified, using the isoflavones genistin and genistein as external standards. The extraction procedure, the extraction solvent, the extraction yields and the HPLC conditions were evaluated and optimized. The samples were analyzed on an RP C18 column, and eluted with a binary system consisting of water and methanol using a linear gradient; detection was at 262 nm. Tectorigenin used in the recovery experiments was isolated and purified in the laboratory. The final method was fully validated according to the ICH guidelines in terms of linearity, precision and accuracy. The validation data showed that the precision, (RSD% (betweendays) of 3.1, 2.84 and 1.77 for the three major compounds, respectively), and the accuracy (recovery of 104.2 %) were acceptable. These validation results demonstrate the suitability of the method for the quality control of this crude drug.     

HPLC法同时测定野葛花中4种异黄酮成分的含量

目的建立野葛花中葛花苷、尼泊尔鸢尾异黄酮、鸢尾黄素-7-O-木糖葡萄糖苷、鹰嘴豆芽素A4种异黄酮成分含量测定方法。方法采用HPLC法。色谱柱为Kromasil C18柱(200 mm×4.6 mm,5μm),以体积分数为1%的乙酸水溶液-甲醇-乙腈为流动相,线性梯度洗脱,流速为0.8 mL.min-1,检测波长为254 nm,柱温35℃。结果葛花苷、尼泊尔鸢尾异黄酮、鸢尾黄素-7-O-木糖葡萄糖苷、鹰嘴豆芽素A质量浓度分别在42.8~856 mg.L-1、9.12~182.4 mg.L-1、13.2~264 mg.L-1、4.20~840 mg.L-1内呈良好线性关系,相关系数分别为0.999 9、0.999 9、0.999 1、0.999 3,平均回收率分别为100.7%(RSD=2.2%,n=9)、99.1%(RSD=2.4%,n=9)、98.6%(RSD=1.6%,n=9)和98.3%(RSD=1.5%,n=9)。结论该方法简便、准确、重现性好,可用于野葛花的质量控制。不同花期和不同药用部位的野葛花中4种指标成分的含量差异较大。     

Isoflavonoid glycosides from the flowers of Pueraria lobata

Pueraria lobata flower is a medicinal herb for treating intoxication, hepatic, and gastrointestinal tract lesions induced by alcohol. This study aims to investigate the isoflavonoid glycosides in P. lobata flowers. Two new isoflavone compounds were isolated from the extract of P. lobata flowers. Their structures were determined to be 5,6,7,4′-tetrahydroxyisoflavone-6,7-di-O-β-d-glucopyranoside and 5,6,7-trihydroxy-4′-methoxyisoflavone-6,7-di-O-β-d-glucopyranoside on the basis of spectroscopic means including HR-ESI-MS, UV, IR, ¹H, and ¹³C NMR.     

Isoflavonoid glycosides from the flowers of Pueraria lobata

Pueraria lobata flower is a medicinal herb for treating intoxication, hepatic, and gastrointestinal tract lesions induced by alcohol. This study aims to investigate the isoflavonoid glycosides in P. lobata flowers. Two new isoflavone compounds were isolated from the extract of P. lobata flowers. Their structures were determined to be 5,6,7,4'-tetrahydroxyisoflavone-6,7-di-O-β-D-glucopyranoside and 5,6,7-trihydroxy-4'-methoxyisoflavone-6,7-di-O-β-D-glucopyranoside on the basis of spectroscopic means including HR-ESI-MS, UV, IR, 1H, and 13C NMR.     

Identification of isoflavones in the roots of Pueraria lobata

The isoflavones of the roots of Pueraria lobata (Willd.) Ohwi (Puerariae Radix) were investigated by high-performance liquid chromatography (HPLC) coupled to photodiode array (PDA) and to mass spectroscopy (MS) using atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) in combination with collision-activated decomposition (CAD) (HPLC-APCI-CAD-MS or ESI-CAD-MS) for identification of glycosides and HPLC-APCI-CAD-MS for identification of aglycones. The major glycosides are derived from daidzein ( 9) and most are 8- C-glycosides. 3'-Hydroxypuerarin-4'- O-deoxyhexoside ( 2B) and 3'-methoxy-6'- O- D-xylosylpuerarin ( 6) were identified as new constituents. MS data were obtained for puerarin-4'- O- D-glucoside ( 1), 3'-hydroxypuerarin ( 2A), puerarin ( 3), 3'-methoxypuerarin ( 4), 6'- O- D-xylosylpuerarin ( 5), daidzin ( 7) and 3'-methoxydaidzin ( 8), which were previously characterized by NMR analysis. Isoflavones identified in Puerariae Radix comprise 3'-methoxydaidzein ( 10), genistein ( 12), daidzein-7- O-methyl ether ( 13A), 3'-methoxydaidzein-7- O-methyl ether or 3'-methoxyformononetin ( 13B) and biochanin A ( 15), while previous characterization of daidzein ( 9) and formononetin ( 14) was substantiated by MS data. The structure of compound 11 could not be established by MS techniques. The estrogenic activity was mainly located in the aglycone fraction.     

Major isoflavonoid contents of the phytoestrogen rich-herb Pueraria mirifica in comparison with Pueraria lobata

Pueraria mirifica tubers collected from 28 out of 76 provinces of Thailand and Pueraria lobata tubers collected from Guangzhou province, China were submitted to HPLC analysis with the established gradient system comprising 1.5% acetic acid and acetonitrile. Five major isoflavonoids, including puerarin, daidzin, genistin, daidzein and genistein, were adopted as authentic standards. P. mirifica tubers showed intra- as well as inter-provincial differences in isoflavonoid and total isoflavonoid contents. The difference in both cases should be mostly influenced by genetic and environmental factors. In comparison with P. lobata, P. mirifica population exhibited differences only with a lower amount of daidzein.     

旋转组合设计优选葛根中葛根素提取工艺

目的:优选葛根中葛根素提取工艺。方法:以乙醇用量、提取时间、乙醇浓度为考察因素,以葛根素提取率为指标,采用旋转组合设计结合三维图优选最佳提取工艺。结果:最佳提取工艺为乙醇用量14倍,提取时间118min,乙醇浓度为56%。结论:旋转组合设计法可用于葛根有效成分的提取。     

Quercetin, a flavonoid antioxidant, prevents and protects against ethanol-induced oxidative stress in mouse liver

This study evaluates whether quercetin (25, 50 and 75 mg/kg body weight) treatment has a protective effect on the pro-oxidant-antioxidant state following chronic ethanol treatment in mice. Pretreatment (quercetin 25, 50 and 75 mg/kg body weight for 15 d+co-treatment of ethanol 18%+quercetin for 15 d and ethanol 18% for the 15 d) increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione (GSH) in comparison to the ethanol group. No significant differences from the ethanol group were observed in the group after post-treatment (ethanol 18% for 30 d+quercetin 25, 50 and 75 mg/kg body weight for 15 d) with quercetin. A significant increase in lipid peroxidation (malondialdehyde, MDA) products was observed in liver tissue after administration of ethanol, which was attenuated by pre- and post-treatment with a high dose of quercetin. GSH levels increased and oxidized glutathione (GSSG) levels decreased in groups of ethanol-exposed mice that received quercetin for 15 d prior to ethanol exposure. In conclusion, pre-treatment of quercetin may protect against ethanol-induced oxidative stress by directly quenching lipid peroxides and indirectly by enhancing the production of the endogenous antioxidant GSH. There was no protective effect on post-treatment with quercetin.     

均匀设计法优选葛根总黄酮提取工艺

目的:筛选超声法提取葛根总黄酮的最佳工艺条件。方法:采用均匀设计法考察乙醇浓度、乙醇用量、提取时间、提取温度、超声提取次数等5个因素对葛根总黄酮提取量的影响;利用紫外-可见分光光度法测定葛根总黄酮的含量;以提取率和提取物中葛根总黄酮含量为指标优选工艺。结果:最佳提取工艺为乙醇浓度75%,乙醇用量20mL.g-1,提取时间40min,提取温度68℃,超声提取5次。结论:该提取工艺合理、可行,可为工业化生产提供理论依据。     

Preparation of total flavonoids from loquat flower and its protective effect on acute alcohol-induced liver injury in mice

This study aimed to research the preparation techniques of total flavones from loquat flower (TFLF), its anti-oxidation capacity, and its protective effect on hepatic injury. The best extraction parameters by orthogonal experimentation were water at 100°C, extraction time 2.5 hours, solid/liquid ratio 1:20, and three decoctions. The chromogenic reaction to the flavones showed that loquat flowers mainly contained flavone, flavonol, and flavanone compounds combining orthophenolic hydroxyl group structure in the 10–30% ethanol fraction. The anti-oxidant capacity of O2- was 26.09% and of OH⁻· was 83.01% by salicylic acid and pyrogallol auto-oxidation. Compared with the model group, TFLF lowered the levels of alanine aminotransferase, aspartate aminotransferase, triglyceride, and malondialdehyde and liver index significantly, and upregulated the expression of adipose triglyceride lipase and Heine oxygenase-1 mRNA. The present findings suggest that TFLF has protective effect on acute alcoholinduced liver injury in mice and may be related to its antioxidant and free-radical scavenging activity.     

Isolation and properties of Sarothamnoside, a novel isoflavone glycoside from various Sarothamnus species

Sarothamnoside (genistein 7,4'-di-O-[4-O-beta-D-glucopyranosyl-beta-D-apiofuranoside]), a novel isoflavone glycoside, has been isolated from Sarothamnus scoparius and S. patens seeds. The structure, in agreement with acid and enzymatic hydrolysis experiments, has been unambiguously determined, using spectral methods: SM, (1)H NMR and (13)C NMR. (1)H NMR spectra showed the beta configuration of the two apioses, whereas (13)C NMR provided evidence of the sugar linkages.     

PI3K/Akt pathway activation was involved in acute ethanol-induced fatty liver in mice

Accumulating evidences support the important roles of sterol regulatory element-binding protein-1 (SREBP-1) activation in ethanol-induced fatty liver, but the underlying mechanisms for its activation are not fully understood. Recent studies have demonstrated that phosphatidylinositol 3 kinase (PI3K)/Akt pathway activation could enhance SREBP-1 activity. The current study was designed to investigate the potential roles of PI3K/Akt pathway in acute ethanol-induced fatty liver in mice. In the first experiment, mice were treated with ethanol (2.5 or 5 g/kg bw) or isocaloric/isovolumetric maltose-dextrin solution, and sacrificed at several time points after ethanol exposure. As expected, ethanol dose-dependently increased the hepatic triglyceride (TG) levels and the protein levels of the mature form of SREBP-1 (n-SREBP-1). The phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β) was significantly increased in mice treated with ethanol (5 g/kg bw), while the protein levels of PI3K-p85 were significantly reduced. To confirm the roles of PI3K/Akt pathway, mice were then pretreated with wortmannin (0.7 or 1.4 mg/kg bw), a specific PI3K/Akt pathway inhibitor, before exposure to ethanol. Interestingly, a dual effect of wortmannin was observed. Low dose of wortmannin significantly reduced the hepatic TG levels, while high dose of wortmannin aggravated ethanol-induced fatty liver. The ratio of LC3II/LC3I of wortmannin (1.4 mg/kg bw) group mice was significantly increased, while the p62 protein level was significantly decreased compared to those of ethanol group, which indicated that wortmannin (1.4 mg/kg bw) might suppress the lipid degradation by autophagy. These results supported the hypothesis that PI3K/Akt activation might be involved in acute ethanol-induced fatty liver, and PI3K/Akt inhibitors might have therapeutic potential for the treatment of ethanol-induced fatty liver.     

Anti-diabetic effects of puerarin,isolated from Pueraria lobata (Willd.), on streptozotocin-diabetogenic mice through promoting insulin expression and ameliorating metabolic function

Diabetes is a chronic disease characterized by the metabolic disorder in specific tissues. Our present study was designed to assess the potential benefits of puerarin (PR) on hypoglycemic and hypolipemic effects in diabetic mice induced by streptozotocin (STZ). The results achieved from these experiments showed that glycemia in STZ-diabetogenic mice were significantly reduced following the PR administration, while serum insulin concentration was increased. In addition, PR contributed to improving the dyslipidemia conditions. Histopathological examination indicated that the STZ-lesioned pancreas tissue in PR-administrated mice was effectively alleviated. Meanwhile, intrapancreatic protein levels of insulin receptor substrate-1 (IRS-1) and insulin-like growth factor-1 (IGF-1) were up-regulated, respectively. On the other hand, endogenous mRNA levels of skeletal muscle insulin receptor (InsR) and peroxisome proliferators-activated receptor α (PPARα) were increased after administration of PR. Taken together, these findings reveal that puerarin effectively exerts the hypoglycemic and hypolipemic roles, which its potential anti-diabetic activity is associated with elevating insulin expression and maintaining metabolic homoeostasis in STZ-diabetogenic mice.     

复方枳椇子制剂对乙醇所致小鼠肝损伤的保护作用

目的研究复方枳椇子制剂(CPH)对乙醇所致小鼠肝损伤的保护作用。方法采用56%乙醇灌胃的方法来诱导小鼠急性肝损伤模型。将小鼠随机分为空白组,模型组,CPH(1,2,4 g.kg-1)组和阳性(XJGGK)组。测定小鼠血清中谷丙转氨酶(ALT),谷草转氨酶(AST),甲胎蛋白(AFP),以及肝组织中丙二醛(MDA),超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px)的含量或活性,并进行小鼠肝病理组织学检查。结果 CPH可显著降低饮酒小鼠血清中ALT、AST含量的升高,降低MDA含量,增加SOD活性和GSH-Px含量,并能改善乙醇损伤小鼠肝病理组织结构,但CPH提取物对饮酒小鼠AFP水平没有明显影响。结论 CPH对乙醇所致小鼠肝损伤具有保护作用,其机制可能与清除氧自由基和抗脂质过氧化有关,为将CPH开发成安全、有效的功能食品提供依据。     

Effects of triterpenoids from Pueraria lobata on immunohemolysis: beta-D-glucuronic acid plays an active role in anticomplementary activity in vitro

The anticomplementary properties of kaikasaponin III (4) and soyasaponin I (8) from Pueraria lobata and their hydrolytic analogs were investigated in vitro. Diglycosidic saponins [kaikasaponin I (3), soyasaponin III (7)] showed most potent anticomplementary activities, followed by monoglycosidic saponins [soyasapogenol B monoglucuronide (6), sophoradiol monoglucuronide (2)] and triglycosidic saponins [soyasaponin I (8), kaikasaponin III (4)], whereas sophoradiol (1) and soyasapogenol B (5) showed enhancement of hemolysis under the presence of serum on the classical pathway of complement system. But all of them showed very weak or no anticomplementary activities on the alternative pathway of complement system. The anticomplementary activity of the saponins was influenced by the nature of glucuronic acid, where the free acid forms (-COOH) showed much more potent activity than the sodium salt forms (-COO-Na+) or methyl ester forms (-COOCH3), and the reduced forms (-CH2OH) decreased the activity significantly.     

Isolation and properties of sarothamnoside, a novel isoflavone glycoside from various sarothamnus species.]

Sarothamnoside (genistein 7,4'-di-O-[4-O-beta-D-glucopyranosyl-beta-D-apiofuranoside]), a novel isoflavone glycoside, has been isolated from SAROTHAMNUS SCOPARIUS and S. PATENS seeds. The structure, in agreement with acid and enzymatic hydrolysis experiments, has been unambiguously determined, using spectral methods: SM, (1)H NMR and (13)C NMR. (1)H NMR spectra showed the beta configuration of the two apioses, whereas (13)C NMR provided evidence of the sugar linkages.     

葛根异黄酮抑制去卵巢大鼠胫骨骨矿和微量元素丢失

目的 :研究葛根总异黄酮 (TIP)对去卵巢引起的骨质疏松症大鼠骨骼骨矿和微量元素含量的影响。方法 :5 2只雌性SD大鼠分为假手术、模型、尼尔雌醇 (CEE3 )、TIP 10 0和TIP 2 0共 5组。除假手术组外均切除双侧卵巢 ,7d后分别灌胃给予 0 .5 %CMC Na(假手术和模型组 )、CEE3 1.0mg·kg 1·wk 1、TIP 10 0和 2 0mg·kg 1·d 1,连续 7个月。化学法测定胫骨钙和磷含量 ,原子吸收分光光度法测定铁、锌、镁、锰、铜、钴、铬和钼等微量元素的含量。结果 :大鼠在去卵巢后胫骨的表观密度及灰密度分别下降了 5 .8%和 11.4 % ,钙、磷、铁、锌和镁等元素的含量也显著减少。经TIP 10 0和 2 0mg·kg 1·d 1治疗 7个月后 ,胫骨表观密度增加 7.5 %和 7.1% ,灰密度增加 9.6 %和 4 .6 % ;钙密度增加 9.8%和 6 .1% ,磷密度增加 10 .1%和 6 .2 % ,对钙磷比值无明显影响。铁、锌和镁含量恢复到接近正常水平 ,锰和钼含量略高于正常组 ,而铜比正常组高 39%和 75 % ,铬比正常组高313%和 2 6 5 % ,钴含量则无明显变化。结论 :TIP对雌激素缺乏引起的骨矿和微量元素丢失具有明显的防治作用