Normal Rat Liver Research Articles

Extraction of Phenolic Compounds from Tabernaemontana catharinensis Leaves and Their Effect on Oxidative Stress Markers in Diabetic Rats.

The aim of this study was to evaluate the most effective extraction condition (temperature, solvent type and time) for recovery of high-value phytochemicals present in the Tabernaemontana catharinensis leaves (TC) and to assess their effect on biochemical parameters in streptozotocin-induced diabetic rats. The extraction of phenolic compounds from TC using a factorial design (FD) 2³, high performance liquid chromatography (HPLC), response surface methodology (RSM) and principal component analysis (PCA) were studied. It was found that the optimal conditions for extraction of phenolics were higher temperature (65 °C) and time (60 min) using ethanol as extractor solvent. In this condition of extraction (A8), total phenolic compounds (TPC) and antioxidant activity (AA) were determined. Additionally, this extract was used to evaluate their effect on antioxidant enzyme activities (superoxide dismutase (SOD) and catalase (CAT)) as well as lipid peroxidation (LP) and protein thiols level (PSH) in the liver and kidneys of normal and diabetic rats. As result, T. catharinensis extract presented TPC content of 23.34 mg EAG/g (equivalent gallic acid) and AA of 34.26 μmol Trolox/g. Phenolic acids (ferulic acid and coumaric acid) and flavonoids (quercetin, rutin and pinocembrin) could be recovered and identified by HPLC. This study indicated an important role of the T. catharinensis extract on free radical inactivation and on the antioxidant defense system in diabetic rats. In fact, the use of T. catharinensis extract restored the normal activity of SOD (p < 0.05) and suppressed malondialdehyde levels in liver and kidney tissues. Thus, the T. catharinensis extract, rich in phenolic compounds, can be responsible for the recover the enzymatic changes in the liver and kidney tissues provoked by diabetes in rats. In addition, the lipid peroxidation rate decreased in the diabetic rats treated with T. catharinensis.

Toxic interactions of Romiplostim and Rituximab in normal and thrombocytopenic Rats

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by immunologic destruction of normal platelets and insufficient production of platelets, as mediated by autoantibodies. Because of the progression of the disease and its multiple causes a combination of treatments used for management of ITP would be a point of interest. Co-administration of therapies may be useful in patients who are refractory to monotherapies and may result in a booster response because they target multiple mechanisms. Romiplostim is a potent drug used for the management of acute ITP and used in combination with other treatment options for ITP. The combination of Rituximab and Romiplostim may inhibit platelet destruction and at the same time increase platelet production. This combination can lead to a strong and a massive increase in platelet counts. In Preclinical studies of Romiplostim safety margins could not be reliably estimated. Although some clinical studies provided some information about the safety profile of this drug. The goal of this study is to investigate the safety of co-treatment of Romiplostim with Rituximab as compared to each of these drugs in normal and thrombocytopenic rats. The measured safety parameter was evaluated for the liver and kidney function in normal and diseased rats. The safety of this regimen should be taken in consideration, so that a balance between the harmful effects and beneficial response could be attained. Future studies are necessary to investigate the safety and efficacy balance of Romiplostim and Rituximab alone or in combination with each other for management of thrombocytopenia.

The Performance of Composite Nanoparticles Based on Fe3O4@SiO2/PLGA/PFOB in Magnetic Resonance Imaging and Photoacoustic Imaging

Polylactic acid glycolic acid (PLGA), a polymer material, is used as a shell and perfluorooctane (PFOB) as the core. Fe3O4@SiO2 nanoparticles are distributed in the shell to prepare a magnetic phase change multifunctional nano emulsion contrast agent (Fe3O4@SiO2/PLGA/PFOB). The ultrasound, magnetic resonance imaging and CT imaging effects of the contrast agent on normal rat liver parenchyma are investigated. Methods: Single emulsification method is used to prepare Fe3O4@SiO2-loaded polymer Fe3O4@SiO2/PLGA/PFOB nanoparticles encapsulated with PLGA. In addition, polymer Fe3O4@SiO2/PLGA nanoparticles loaded with Fe3O4@SiO2 nanoparticles, PLGA@PFOB nanoparticles coated with PLGA and blank PLGA nanoparticles are prepared as experimental control group. Its surface morphology, internal structure, particle size, potential and other general characteristics are tested. RAW264.7 mouse mononuclear macrophages are cultured in vitro. Prussian blue staining is used to observe macrophage phagocytosis after Fe3O4@SiO2/PLGA/PFOB contrast agent is added to incubate for 3 hours. MTT method is used to detect the effect of contrast agent on cell activity. The macrophage suspension engulfed with Fe3O4-PFOB is examined by ultrasound, magnetic resonance and CT. The results showed that Fe3O4@SiO2/PLGA/PFOB is brown emulsion after being dissolved in double distilled water. Under the light microscope and scanning electron microscope, Fe3O4@SiO2/PLGA/PFOB is spherical, with regular shape, uniform size, good dispersity and smooth surface. Macrophages are dependent on the phagocytosis of the contrast medium without affecting the activity of macrophages. Moreover, in vitro imaging experiments, compared with the control group, macrophages phagocytized by Fe3O4@SiO2/PLGA/PFOB have obvious enhancement effects in ultrasound, magnetic resonance and CT imaging. In conclusion, Fe3O4@SiO2/PLGA/PFOB has good stability in vivo, can be engulfed by liver macrophages, and can enhance ultrasound, magnetic resonance and CT imaging of liver parenchyma. It is a safe and effective multifunctional ultrasound contrast agent.

Genomic Study, Phytochemical Characterization,and Antiproliferative Activity of Two Different Genotypes of Jatropha curcas L. Obtained by a Breeding Program

Jatropha curcas (Euphorbiaceaefamily) is a multipurpose plant with considerable potential in biodiesel production, and in cosmetic and medicinal uses. The part of J. curcas usually used is the oil obtained from its seeds, whereas the leaves generally represent the waste material. The aim of this study was to characterize the composition and to preliminary investigate the biological activity ofJ. curcas leaves obtained from plants by a breeding program. To describe genomic structure, molecular markers were applied. For biological study, two genotypes (JA and JB) that are genetically divergent were selected by cluster analysis. A phytochemical analysis was carried out to characterize the chemical composition of the extracts, which resulted rich in biologically active compounds, whereas toxic phorbol esters were absent. Biological assays showed an antiproliferative effect on the Buffalo normal rat liver cell line (BRL-3A), with genotype B being more potent than that of the counterpart (JA).The purified compounds isolated did not show antiproliferative activity, suggesting that the effect observed was due to the phytocomplex and should involve several secondary metabolites. This study highlights that a plant of the same genus and same species that has been cultivated in the same soil and climatic conditions can be characterized by a high variability. This is what makes research in pharmacognosya complex process.

Phenotypic Polymorphism of Normal Rat Liver Kupffer Cells

The aim of study was to evaluate the immunophenotype of resident macrophages of the liver, Kupffer cells, in rats in the norm.Material and methods. The study included male Wistar rats’ samples (n=6) that presented fragments of the middle lobe of the liver taken under ether anesthesia. The obtained samples were fixed in liquid nitrogen, after that cryosections 5–7 μm thick were prepared. Histological slides were used to detect the expression of a number of macrophage markers with an antibody kit: CD68, CD206, CD 163, CD86. After the first antibodies, sections were stained with antibodies conjugated to FITC, cell nuclei were detected using DAPI, the obtained preparations were studied using a fluorescence microscope.Results. When analyzing the expression of CD68 in the rat liver, it was found that normally about 20% of the cells in the field of vision appeared to be CD68+ cells, which was consistent with the earlier study results of the authors. The number of CD163+ and CD206+ cells coincided with the number of CD68+ macrophages, while CD86+ macrophages were significantly less.Conclusions. Under normal conditions, the population of resident macrophages of the rat liver is represented by cells with pronounced expression of CD68, CD163 and CD206. A large number of CD163+ and CD206+ macrophages allows concluding that Kupffer cells are close to the M2 pro-regenerative phenotype. However, the detection of CD86+ resident macrophages indicates the presence of M1 macrophages, or the presence of normal macrophages with an intermediate M1 and M2 phenotype, in the rat liver. The revealed high content of macrophages expressing CD163 and CD206 in the liver evidences not only pro-regenerative properties of Kupffer cells, but also the close connection of macrophages with liver functions, since these receptors are involved in the utilization of hemoglobin and a number of hormones.

Evaluation of the Effects of Fullerene C60 Nanoparticles on Oxidative Stress Parameters at Liver and Brain of Normal Rats

Evaluation of the Effects of Fullerene C60 Nanoparticles on Oxidative Stress Parameters at Liver and Brain of Normal Rats

DNA methylation and RASSF4 expression are involved in T-2 toxin-induced hepatotoxicity

T-2 toxin is a secondary metabolite produced by Fusarium species and commonly contaminates food and animal feed. T-2 toxin can induce hepatotoxicity through apoptosis and oxidative stress; however, the underlying mechanism is not clear. Recent studies indicated that RASSF4, a member of the RASSF family, participates in cell apoptosis and some cancers due to its inactivation via DNA hypermethylation. However, its role in T-2 toxin-induced liver toxicity is poorly understood. Therefore, in this study, female Wistar rats were given a single dose of T-2 toxin at 2 mg/kg b.w. and were sacrificed at 1, 3 and 7 days post-exposure. A normal rat liver cell line (BRL) was exposed to different concentrations of T-2 toxin (10, 20, 40 nM) for 4, 8, 12 h, respectively. Histopathological analysis revealed with apoptosis in some liver cells and clear proliferation under T-2 toxin exposure. Expression analysis by immunohistochemical assays, quantitative real-time PCR (qPCR) and western blot demonstrated that T-2 toxin activated PI3K-Akt/Caspase/NF-κB signaling pathways. Additionally, DNA methylation assays revealed that the expression of RASSF4 was silenced by promoter hypermethylation after exposure to T-2 toxin for 1 and 3 days as compared to the control group. Moreover, joint treatment of 5-Aza-2′-deoxycytidine (DAC) (5 μM) and T-2 toxin (40 nM) increased expression of RASSF4 and PI3K-Akt/caspase/NF-κB signaling pathways-related genes, inducing cell apoptosis. These findings for the first time demonstrated that DNA methylation regulated the RASSF4 expression under T-2 toxin, along with the activation of its downstream pathways, resulting in apoptosis.

Evidence for Toxic Advanced Glycation End-Products Generated in the Normal Rat Liver.

Glucose/fructose in beverages/foods containing high-fructose corn syrup (HFCS) are metabolized to glyceraldehyde (GA) in the liver. We previously reported that GA-derived advanced glycation end-products (toxic AGEs, TAGE) are generated and may induce the onset/progression of non-alcoholic fatty liver disease (NAFLD). We revealed that the generation of TAGE in the liver and serum TAGE levels were higher in NAFLD patients than in healthy humans. Although we propose the intracellular generation of TAGE in the normal liver, there is currently no evidence to support this, and the levels of TAGE produced have not yet been measured. In the present study, male Wister/ST rats that drank normal water or 10% HFCS 55 (HFCS beverage) were maintained for 13 weeks, and serum TAGE levels and intracellular TAGE levels in the liver were analyzed. Rats in the HFCS group drank 127.4 mL of the HFCS beverage each day. Serum TAGE levels and intracellular TAGE levels in the liver both increased in the HFCS group. A positive correlation was observed between intracellular TAGE levels in the liver and serum TAGE levels. On the other hand, in male Wister/ST rats that drank Lactobacillus beverage for 12 weeks—a commercial drink that contains glucose, fructose, and sucrose— no increases were observed in intracellular TAGE or serum TAGE levels. Intracellular TAGE were generated in the normal rat liver, and their production was promoted by HFCS, which may increase the risk of NAFLD.

DNA methylation is involved in pro-inflammatory cytokines expression in T-2 toxin-induced liver injury

Currently, T-2 toxin has been reported to cause liver toxicity with the effects of oxidative stress and inflammation; however, the underlying mechanism of T-2 toxin-induced liver injury is not fully understood. Increasing lines of evidence show that DNA methylation affects the expression of inflammatory cytokine, and plays a crucial role in autoimmune diseases. Nevertheless, the potential role of DNA methylation in the hepatotoxicity of T-2 toxin has not been explored. In this study, female Wistar rats were given a single dose of T-2 toxin at 2 mg/kg b.w. and were sacrificed at 1, 3 and 7 days post-exposure. In vitro, a normal rat liver cell line (BRL) was exposed to different concentrations of T-2 toxin. Histopathological analysis was used to investigate damage to the liver, which was detected at the molecular level by RT-PCR, Western blot and immunohistochemical assays, methylation-specific PCR (MSP), bisulfite sequencing (BSP), and flow cytometry. The results showed that T-2 toxin significantly increased the levels of DNA methyltransferases (DNMT1, DNMT3A), which were mainly concentrated at the site of liver injury. The 5-methylcytosine (5-mC) level of genomic DNA was also raised in T-2 toxin-treated rat livers. The expression of inflammatory cytokines (IL-6, IL-1β, IL-11, IL-1α, and TNF-α) increased both in vivo and in vitro under T-2 toxin treatment. Notably, DNA demethylation directly increased the expression of cytokines IL-11, IL-6, IL-α, and TNF-α under T-2 toxin exposure. DNA methylation inhibitors combined with T-2 toxin directly or indirectly induced the production of inflammatory cytokines and aggravate cell apoptosis. Our study uncovered for the first time that DNA methylation is related to the expression of inflammatory cytokines in T-2 toxin-induced liver injury. These findings suggested that DNA methylation is a potential mechanism of T-2 toxin-induced hepatotoxicity.

Daming capsule, a hypolipidaemic drug, lowers blood lipids by activating the AMPK signalling pathway

Background/aimsHyperlipidaemia is a major risk factor for cardiovascular and cerebrovascular diseases. Daming capsule (DMC), a medicine for lowering blood lipids, is marketed in China; however, its mechanism is unclear. The present study aimed to investigate the mechanism by which DMC reduces blood lipids. Methods and resultsA rat model of hyperlipidaemia was established by feeding rats a high-fat diet (HFD), and the serum lipid levels were detected with an automatic biochemical analyser. DMC (162 mg/kg) and atorvastatin calcium (10 mg/kg) were orally administered to the hyperlipidaemic rats for 4 weeks. HFD feeding markedly induced increases in the levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-c); however, DMC treatment significantly decreased the levels of TC, TG, and LDL-c in rats serum. Meanwhile, the hepatic TC and TG levels, liver weight/body weight ratio, and body weight were significantly lower in the DMC-treated rats than in the HFD rats. Moreover, DMC significantly alleviated hepatomegaly, hepatic lipid deposition, and hepatic steatosis. The protein expression level of phospho-adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172) in HFD rat livers was lower than that in normal rat livers, whereas it increased in the liver of the DMC-treated rats; however, the protein expression level of total-AMPK in the liver was not different among the groups. The AMPK-activating effect of DMC was blocked by Compound C (a specific AMPK inhibitor) in HepG2 cells. Additionally, DMC considerably increased peroxisome proliferator-activated receptor-alpha (PPARα) protein expression and lipoprotein lipase (LPL) transcription and concentration in the liver. This effect of DMC was also inhibited by Compound C in HepG2 cells. DMC also promoted LDL receptor (LDLR) protein expression by activating AMPK. We further found that DMC reduced the levels of TC and TG in oleic acid-treated HepG2 cells, and it restored the expression levels of p-AMPK, PPARα, LPL, and LDLR compared to the decreased levels observed in oleic acid-treated HepG2 cells. ConclusionDMC lowered lipids in serum and the liver by activating AMPK. On the one hand, the activation of AMPK enhanced PPARα expression and LPL transcription to lead to the hydrolysis of TG; on the other hand, it increased LDLR protein expression to promote lipid metabolism.

Abstract 4296: Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells

Abstract Background: Although interfering with the actions of microtubule-associated proteins (MAP’s) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP’s in OVCA cells is lacking. We sought to identify cancer-specific MAP’s in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing. Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5’ terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification. Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment. Citation Format: Ahmed Fadiel, Viswam S. Nair, Peter Aceto, Frederick Naftolin. Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4296.

Measurement of temperature dependence of sound velocity in biological tissues

Transcatheter arterial chemo-embolization using Lipiodol® is one of the treatments for hepatocellular carcinoma. During this procedure, it is important to monitor the Lipiodol® deposited inside tumors. This study proposes a method for measurement of the temperature dependence of the velocity of sound in biological tissues. An ultrasonic pulse-echo method was used to measure the velocity of sound. The temperature coefficients of the velocity of sound for Lipiodol® was −2.82 m s−1 °C−1. Values of 1.07 and 1.59 m s−1 °C−1 were obtained for normal rat liver and cancerous liver tissue, respectively. Thus, this procedure may potentially allow for the evaluation of the infiltration of Lipiodol® inside tumors.

Bile Acid Transporters Are Expressed and Heterogeneously Distributed in Rat Bile Ducts.

Background/AimsCholangiocytes are capable of reabsorbing bile salts from bile, but the pathophysiological significance of this process is unclear. To this end, we detected the expression and distribution of bile acid transport proteins in cholangiocytes from normal rat liver and analyzed the possible pathophysiological significance.MethodsBile duct tissues of Sprague-Dawley rats were isolated by enzymatic digestion and mechanical isolation, and then divided into large and small bile duct tissues. Immunohistochemistry, real-time polymerase chain reaction and Western blotting were used to determine the expression of the apical sodium-dependent bile acid transporter (ASBT), ileal bile acid binding protein (IBABP), and basolateral organic solute transporter α (Ostα) in the biliary tract system of rats. Differences in the expression and distribution of these proteins were analyzed.ResultsIn cholangiocytes, ASBT and IBABP were mainly expressed in cholangiocytes of the large bile ducts, in which the expression of both was significantly higher than that in the small ducts (p<0.05). Ostα was simultaneously expressed in cholangiocytes of both the large and small bile ducts, showing no significant difference in expression between the two groups of bile ducts (p>0.05).ConclusionsBile acid transporters are expressed and heterogeneously distributed in rat bile ducts, indicating that bile acid reabsorption by cholangiocytes might mainly occur in the large bile ducts. These findings may help explore the physiology of bile ducts and the pathogenesis of various cholangiopathies.

Hypolipidemic Effect of Chrysophyllum albidum Peel Extract and Its Underlying Antioxidant Mechanisms in Normal and Triton-X-100-Induced Hyperlipidemic Rats

Chrysophyllum albidum peel ethanol extract (CAPEE) was investigated for its antioxidant and hypolipidemic effects in rats. Dried peel was extracted in 70% ethanol and tested for phenolic content and in vitro antioxidant properties. Acute toxicity effect of CAPEE was tested at dose level of 2,000 mg/kg. The antioxidant and hypolipidemic effects in serum and hepatic tissues were evaluated in normal and triton-X-100-injected rats following 14 days of repeated treatment with CAPEE (50 and 200 mg/kg). Total phenolic content (TPC) and total flavonoid content (TFC) of CAPEE were 49.49 ± 2.48 mg GAE/g of sample and 14.71 ± 0.90 mg RE/g sample, respectively. The extract scavenged DPPH radical in a concentration-dependent manner with an IC50 of 63.1 ± 0.5 µg/mL. CAPEE (2,000 mg/kg) showed no sign of toxicity in mice. CAPEE (50 and 200 mg/kg) did not alter lipid profile in normal rats but ameliorated triton-X-100-induced increases in triglycerides and cholesterol and decrease in high-density lipoprotein (HDL). CAPEE showed hepatoprotective as well as antioxidant activity in serum and liver of normal and hyperlipidemic rats. The results revealed that Chrysophyllum albidum fruit peel ethanol extract possesses antioxidants and hypolipidemic properties.

Exploration of the hepatoprotective chemical base of an orally administered herbal formulation (YCHT) in normal and CCl4-intoxicated liver injury rats. Part 1: Metabolic profiles from the liver-centric perspective

Ethnopharmacological relevanceYin-Chen-Hao Tang (YCHT), derived from “Treatise on Febrile Diseases” in ancient China, has been a very popular hepatoprotective three-herb formula in China and Japan, although its chemical base remains unclear. Aim of this studyAs the first step in revealing the hepatoprotective chemical base of YCHT, we aimed to clarify the absorbed ingredients and associated metabolic pathways for orally dosed YCHT in both normal and liver injury rats from a liver-centric perspective. Materials and methodsWith the aid of 10 reference compounds, the absorbed ingredients and generated metabolites were systematically characterized by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF) in the portal vein plasma (the plasma before hepatic disposition) – liver – systemic plasma (the plasma after hepatic disposition), following oral administration of YCHT in normal and CCl4-induced liver injury rats. ResultsA total of 38 compounds with six chemical structures, consisting of 10 prototypes and 28 metabolites generated through 9 biotransformations, were absolutely or tentatively identified, and 25 compounds were first reported on YCHT treatments. Among them, 8 compounds were absolutely confirmed by comparing with standard substances, and some had published hepatoprotective activities. Compared with the 35, 15, and 29 compounds identified in the portal vein plasma, liver, and systemic plasma of normal rats, respectively, the corresponding numbers of characterized compounds were 37, 13 and 29 in the liver injury rats. ConclusionsSulfation and glucuronidation were the predominant biotransformations, and intestinal metabolism, prior to hepatic metabolism, occurred for most compounds. CCl4-induced liver injury caused only slight changes in the metabolic profiles of rats orally administered YCHT. These results provided the precondition for further quantitative analysis and pharmacodynamic screening of compounds in YCHT.

Enhanced Anti-diabetic Effect of Berberine Combined With Timosaponin B2 in Goto-Kakizaki Rats, Associated With Increased Variety and Exposure of Effective Substances Through Intestinal Absorption.

Objective: Inspired by the traditionally clinical application of herb pair Zhimu-Huangbo to treat diabetes, a combination of plant ingredients, timosaponin B2 (TB-2) and berberine (BBR), was evaluated for their anti-diabetic efficacy and cooperative mechanisms.Methods: The efficacy and pharmacokinetics of orally administered TB-2 (33.3 mg/kg/day), BBR (66.7 mg/kg/day), and TB-2+BBR (100 mg/kg/day) were evaluated in spontaneously non-obese diabetic Goto-Kakizaki (GK) rats, and metformin (200 mg/kg/day) was used as a positive control. The comparative exposure of the parent drugs, timosaponin A3 (TB-2 metabolite), and M1–M5 (BBR metabolites) was quantified in the portal vein plasma (before hepatic disposition), liver, and systemic plasma (after hepatic disposition) of normal rats on single and combination treatments. Cooperative mechanism of TB-2 and BBR on intestinal absorption and hepatic metabolism was investigated in Caco-2 cells and primary hepatocytes, respectively.Results: After a 6-week experiment, non-fasting and fasting blood glucose levels and oral glucose tolerance test results showed that TB-2+BBR treatments (100 mg/kg/day) displayed significantly anti-diabetic efficacy in GK rats, comparable to that on metformin treatments. However, no significant improvement was observed on TB-2 or BBR treatments alone. Compared to single treatments, combination treatments led to the increased circulating levels of BBR by 107% in GK rats. In normal rats, the hepatic exposure of BBR, timosaponin A3, and M1–M5 was several hundred folds higher than their circulating levels. Co-administration also improved the levels in the plasma and liver by 41–114% for BBR, 141–230% for TB-2, and 12–282% for M1–M5. In vitro, the interaction between TB-2 and BBR was mediated by intestinal absorption, rather than hepatic metabolism.Conclusion: Combining TB-2 and BBR enhanced the anti-diabetic efficacy by increasing the in vivo variety of effective substances, including the parent compounds and active metabolites, and improving the levels of those substances through intestinal absorption. This study is a new attempt to assess the effects of combined plant ingredients on diabetes by scientifically utilizing clinical experience of an herb pair.

Off-line two-dimensional liquid chromatography coupled with diode array detection and quadrupole-time of flight mass spectrometry for the biotransformation kinetics of Ginkgo biloba leaves extract by diabetic rat liver microsomes

Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (Km) value of genistein was obvious increased while its maximal velocity (Vmax) and intrinsic clearance (CLint) values were significantly decreased by diabetic RLMs, and the Vmax and CLint values of kaempferol and isorhamnetin were notably enhanced while their Km values were markedly reduced. For the half-time (t1/2) values of four target compounds and the Km, Vmax and CLint values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE.

Toxicity of some broad-spectrum antibacterials in normal rat liver: the role of mitochondrial membrane permeability transition pore

Ciprofloxacin (CIP) and Amoxycillin/Clavulanate (AC) are broad-spectrum antibiotics that are commonly administered for treatment of various bacterial infections. Studies have reported the antiproliferative and apoptotic activities of CIP in several cancer cell lines while AC has been implicated in drug–induced liver injury. We investigated the influence of CIP and AC on mitochondrial Permeability Transition (mPT) pore, ATPase activity, and cytochrome C release of normal Rat Liver Mitochondria (RLM) spectrophotometrically. In vitro, CIP and AC induced the opening of the mPT pore in a concentration-dependent manner with evidence of cytochrome C release maximally at 70 µg/ml by 13 and 10 folds, respectively. In vivo, CIP (100, 200 mg/kgbw) significantly induced mPT pore opening with induction folds of 2.4 and 2.6, respectively. However, low dose of AC (10 mg/kgbw) had no effect whatsoever on the mPT pore while higher dose (30 mg/kgbw) significantly induced pore opening by 3.4 folds. Similarly, CIP(100 mg/kgbw) and AC (30 mg/kgbw), significantly enhanced RLM ATPase activity, induced cytochrome C release and increased levels of RLM malondialdehyde generation and triggered the activation of caspases-9 and 3 in liver post-mitochondrial fraction. There were also significant (p<0.05) elevation in levels of serum aminotransferases and white blood cell count. Our results show that prolonged use of Ciprofloxacin and Amoxicillin Clavulanate could result in mitochondrial membrane breakdown via induction of opening of mPT pore leading to expulsion of cytochrome C, lipid peroxidation and decrease in energy content in healthy liver cells. These drugs should therefore be used with caution.

Effect of Milk Protein Hydrolysate on Some Hematological Parameters of Type II Diabetic Rats

Milk protein-derived bioactive peptides could be used in human nutrition in both newborns and adults, since they are claimed to be health-improving components that can be used to reduce the risk of disease or to enhance certain physiological functions. This study investigated the effect of oral intake of milk protein concentrate (MPC) and milk protein concentrate hydrolysate (MPCH) on body weight, organ relative weight, and hematological parameters in alloxan-induced diabetes rats. The animals were divided into six major groups after stabilization of diabetes for one week. Diabetic and non-diabetic rats groups were received intra-gastric dose of MPC or MPCH (150mg/kg B.W) for six weeks. Feed treatment by MPC and MPCH significantly (<i>P</i><0.05) increased the final body weight of normal rats and improved the body weight of diabetic rats and significantly protective (<i>P</i><0.05) the liver, kidney, heart and lung of normal rats and reduced the fallers of these organs in diabetic rats. The diabetes groups treated by MPCH showed improvement in RBC, Hb, Ht and PLT values. Meanwhile WBC not affected. The oral intake of MPCH protected the normal range of Lymphocytes and S. Nutrophils in healthy and diabetic rats. The oral intake of MPCH caused significant (<i>P</i><0.05) decrease of Monocytes in diabetic and non-diabetic rats, while the oral intake of MPC had no affected on Monocytes of diabetic and non-diabetic rats. MPC and MPCH reduced the harmful effect of T2D on body weight, organs weights of rats, and improvement most hematological parameters of normal and diabetes rats.

Functional Teas from the Stems of Penthorum chinense Pursh.: Phenolic Constituents, Antioxidant and Hepatoprotective Activity.

Penthorum chinense Pursh (PCP), a medicinal and edible plant, is traditionally used for liver protection and treatment of liver diseases. In this study, we compared the differences of composition and activity of flowers, stems and leaves of PCP to select a bioactive part. The stems of PCP with stronger antioxidant activity (6.25-100μg/mL) and lower cytotoxicity (25-200μg/mL) than the flowers and leaves were a better bioactive part. Then the chemical composition and hepatoprotective effects of an aqueous extract and an 70% ethanolic extract made with stems of PCP were investigated. We found that the 70% ethanolic extract enriched more polyphenols and flavonoids and possessed significantly stronger hepatoprotective activity than the aqueous extract in the dose range of 25-200μg/mL, which indicated that 70% ethanol is the better solvent of PCP in extraction technology. Moreover, ethyl acetate extract of stems of PCP (PSE) was used to evaluate the hepatoprotective ability of PCP against oxidative damage using an in vitro model of a normal rat's liver cell (BRL-3A). Besides, 12 phenolic compounds were identified from PSE by ultra-performance liquid chromatography followed by electrospray ionization mass spectrometry (UPLC-ESI-MS). Obtained results strongly support the traditional use of PCP and prove stems of PCP to be an important source of bioactive compounds associated with hepatoprotective activity.