Evidences for the mechanism of Shenmai injection antagonizing doxorubicin-induced cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a widely used antitumor drug. However, its clinical application is limited for its serious cardiotoxicity. The mechanism of DOX-induced cardiotoxicity is attributed to the increasing of cell stress in cardiomyocytes, then following autophagic and apoptotic responses. Our previous studies have demonstrated the protective effect of Shenmai injection (SMI) on DOX-induced cardiotoxicity via regulation of inflammatory mediators for releasing cell stress. PURPOSE: To further investigate whether SMI attenuates the DOX-induced cell stress in cardiomyocytes, we explored the mechanism underlying cell stress as related to Jun N-terminal kinase (JNK) activity and the regulation of autophagic flux to determine the mechanism by which SMI antagonizes DOX-induced cardiotoxicity. STUDY DESIGN: The DOX-induced cardiotoxicity model of autophagic cell death was established in vitro to disclose the protected effects of SMI on oxidative stress, autophagic flux and JNK signaling pathway. Then the autophagic mechanism of SMI antagonizing DOX cardiotoxicity was validated in vivo. RESULTS: SMI was able to reduce the DOX-induced cardiomyocyte apoptosis associated with inhibition of activation of the JNK pathway and the accumulation of reactive oxygen species (ROS). Besides, SMI antagonized DOX cardiotoxicity, regulated cardiomyocytes homeostasis by restoring DOX-induced cardiomyocytes autophagy. Under specific circumstances, SMI depressed autophagic process by reducing the Beclin 1-Bcl-2 complex dissociation which was activated by DOX via stimulating the JNK signaling pathway. At the same time, SMI regulated lysosomal pH to restore the autophagic flux which was blocked by DOX in cardiomyocytes. CONCLUSION: SMI regulates cardiomyocytes apoptosis and autophagy by controlling JNK signaling pathway, blocking DOX-induced apoptotic pathway and autophagy formation. SMI was also found to play a key role in restoring autophagic flux for counteracting DOX-damaged cardiomyocyte homeostasis.

Protective effect of Shenmai injection on doxorubicin-induced cardiotoxicity via regulation of inflammatory mediators.

BACKGROUND: Doxorubicin (DOX) is a chemotherapy drug for malignant tumors. The clinical application of DOX is limited due to its dosage relative cardiotoxicity. Oxidative damage and cardiac inflammation appear to be involved in DOX-related cardiotoxicity. Shenmai injection (SMI), which mainly consists of Panax ginsengC.A.Mey.and Ophiopogon japonicus (Thunb.) Ker Gawl, is widely used for the treatment of atherosclerotic coronary heart disease and viral myocarditis in China. In this study, we investigated the protective effect of Shenmai injection on doxorubicin-induced acute cardiac injury via the regulation of inflammatory mediators. METHODS: Male ICR mice were randomly divided into seven groups: control, DOX (10 mg/kg), SMI (5 g/kg), DOX with pretreatment with SMI (0.5 g/kg, 1.5 g/kg or 5 g/kg) and DOX with post-treatment with SMI (5 g/kg). Forty-eight hours after the last DOX administration, all mice were anesthetized for ultrasound echocardiography. Then, serum was collected for biochemical and inflammatory cytokine detection, and heart tissue was collected for histological and Western blot detection. RESULTS: A cumulative dose of DOX (10 mg/kg) induced acute cardiotoxicity in mice manifested by altered echocardiographic outcome, and increased tumor necrosis factor, interleukin 6 (IL-6), monocyte chemotactic protein 1, interferon-γ, and serum AST and LDH levels, as well as cardiac cytoplasmic vacuolation and myofibrillar disarrangement. DOX also caused the increase in the expression of IKK-α and iNOS and produced a large amount of NO, resulting in the accumulation of nitrotyrosine in the heart tissue. Pretreatment with SMI elicited a dose-dependent cardioprotective effect in DOX-dosed mice as evidenced by the normalization of serum inflammatory mediators, as well as improve dcardiac function and myofibril disarrangement. CONCLUSIONS: SMI could recover inflammatory cytokine levels and suppress the expression of IKK-α and iNOS in vivo, which was increased by DOX. Overall, there was evidence that SMI could ameliorate DOX-induced cardiotoxicity by inhibiting inflammation and recovering heart dysfunction.

Simultaneous determination and pharmacokinetics of eight ginsenosides by LC-MS/MS after intravenously infusion of 'SHENMAI' injection in dogs.

SHENMAI injection, a prescription comprised of Panax ginseng and Ophiopogon japonicas, is being extensively applied in the field of cardio-protection and immune-modulation in China. Ginsenosides are the main active components in SHENMAI injection. In order to capture and analyze the pharmacokinetic profile of major ginsenosides of SHENMAI injection in Beagle dogs, liquid chromatography equipped with electro-spray ionization and tandem mass spectrometry method was applied in simultaneous determination for protopanaxatriol type ginsenoside (Re, Rf, Rg1), protopanaxadiol type ginsenoside (Rb2, Rb1, Rd, Rc) and oleanolic acid type ginsenoside (Ro). A C18 column (150 × 2.1mm, 5µm) and a linear gradient program were used to achieve chromatographic separation, with 0.02% acetic acid solution and acetonitrile. I.S. and ginsenosides were detected by LC-MS/MS in selective reaction mode. Good linearity spanning 5- 1500ng/mL was achieved with the R2 values higher than 0.99 for all analytes. Limit of quantification of all analytes were 3ng/mL. Intra- and inter-day precisions ranges from 0.47 to15.68 % and accuracies were within the range of 85.27-117.57%. Validated analyzing method was then used in the pharmacokinetic experiment for SMI in dogs. The results showed that the pharmacokinetic profile of protopanaxadiol, protopanaxatriol and oleanolic acid type ginsenoside were significant difference in dogs. Protopanaxadiol type ginsenosides exhibited an extremely higher level of exposure and a much slower elimination process. Whereas protopanaxatriol type ginsenosides were quickly eliminated. We concluded that 20 (S) - protopanaxadiol type ginseno sides could be a potential pharmacokinetic marker of SHENMAI injection.

Investigation of the protective effect of Paeonia lactiflora on Semen Strychni-induced neurotoxicity based on monitoring nine potential neurotoxicity biomarkers in rat serum and brain tissue.

Semen Strychni has been widely used as a traditional Chinese herb medicine, but its clinical use was limited for its potential neurotoxicity and nephrotoxicity. This study aimed to investigate S. Strychni-induced neurotoxicity and the neuro-protective effect of Paeonia lactiflora based on monitoring nine potential neurotoxicity biomarkers in rat serum and brain tissue. A sensitive liquid chromatography-tandem mass spectrometry method was developed and validated to monitor serotonin, tryptophan, dopamine, tyrosine and glutamate in serum and five brain regions (prefrontal cortex, hippocampus, striatum, cerebellum and hypothalamus). Analytes were separated on a CAPCELL CORE PC column (150 mm × 2 mm, 2.7 µm) with a gradient program of acetonitrile-water (0.2 % formic acid) and a total runtime of 7.5 min. In addition, enzyme-linked immunosorbent assay was conducted to determine four kinds of protein (tryptophan hydroxylase, tyrosine hydroxylase, endogenous brain-derived neurotrophic factor and nerve growth factor). Results demonstrated that the administration of S. Strychni could cause certain endogenous substances disorder. These analytes were found significantly changed (p < 0.05) in serum (except glutamate) and in certain tested brain regions in S. Strychni extract group. Pretreatment of P. lactiflora could significantly reverse the S. Strychni-induced neurotoxicity and normalize the levels of such endogenous substances. The study could be further used in predicting and monitoring neurotoxicity caused by other reasons, and it was expected to be useful for improving clinical use of S. Strychni through pretreatment with P. lactiflora.

Potential accumulation of protopanaxadiol-type ginsenosides in six-months toxicokinetic study of SHENMAI injection in dogs.

SHENMAI injection (SMI), derived from famous Shen Mai San, is a herbal injection widely used in China. Ginsenosides are the major components of SMI. To monitor the exposure level of SMI during long-term treatment, a 6-month toxicokinetic experiment was performed. Twenty-four beagle dogs were dived into four groups (n = 6 in each group): a control group (0.9% NaCl solution) and three SMI groups (2, 6 or 3 mg/kg). The dogs were i.v. infused with vehicle or SMI daily for 180 d. Blood samples for analysis were collected at specific time points as follows: pre-dose (0 h); at 10, 30, and 60 min during infusion; and at 10, 30, 60, 90, 120, 240, and 300 min post-administration. Concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 in the plasma were determined simultaneously by liquid chromatography-tandem mass spectrometry. Non-compartmental parameters were further calculated and analyzed. Significant differences were found between the kinetic behavior of 20(S)-protopanaxadiol-type (PPD-type) and 20(S)-protopanaxatriol-type (PPT-type) ginsenosides. Increasing in the exposure level of PPD-type ginsenosides was observed in dogs during the experiment. Therefore, PPD-type ginsenosides are closely related to the immunity modulation effect of SMI. Increased PPD-type ginsenoside exposure level may present potential toxicity and induce drug-drug interaction risks during SMI administration. As such, PPD-type ginsenoside accumulation must be carefully monitored in future SMI research.

Promotion of classic neutral bile acids synthesis pathway is responsible for cholesterol-lowing effect of Si-miao-yong-an decoction: Application of LC-MS/MS method to determine 6 major bile acids in rat liver and plasma.

Si-miao-yong-an decoction (SMYAD), a traditional Chinese medicine formula, significantly reduced plasma TC, LDL-c levels and increased HDL-c level in hyperlipidemia rats. Liver function test and tissue section examination indicated that SMYAD improved liver function and reduced fat accumulation in hyperlipidemia rat liver. A LC-MS/MS method was established and well validated to evaluate major bile acids derived from cholesterol metabolism through the classic neutral pathway and the alternative acidic pathway (cholic acid, chenodeoxycholic acid and their taurine and glycine conjugates) in liver and plasma. Increased total 6 bile acids concentrations in both liver and plasma were observed after oral administration of 12g/kg/d, 24g/kg/d and 36g/kg/d of SMYAD in a dose dependent manner which contributed to eliminate of cholesterol. Cholic acid, taurocholic acid and glycocholic acid act as the main products of bile acid classic neutral synthesis pathway and show sharp increase (p<0.01) after treatment of SMYAD at dosage of 24-36g/kg/d. For liver samples, taurocholic acid level act as the largest growth section, while in plasma samples, cholic acid act as the largest growth section after SMYAD treatment, compared with Model group. By contrast, the main products of alternative acidic pathway (chenodeoxycholic acid and its glycine and taurine conjugates) show no significant increase after treatment of SMYAD. In conclusion, the cholesterol lowing effect of SMYAD may be related with the accelerated transformation of cholesterol into bile acids through the classic neutral pathway.

An analytical strategy to investigate Semen Strychni nephrotoxicity based on simultaneous HILIC-ESI-MS/MS detection of Semen Strychni alkaloids, tyrosine and tyramine in HEK 293t cell lysates.

A Previous metabolomics study has demonstrated that tyrosine metabolism might be disrupted by treating with Semen Strychni on the cell nephrotoxicity model. To investigate the relationship between Semen Strychni alkaloids (SAs) and endogenous tyrosine, tyramine under the nephrotoxicity condition, an HILIC-ESI-MS/MS based analytical strategy was applied in this study. Based on the established Semen Strychni nephrotoxicity cell model, strychnine and brucine were identified and screened as the main SAs by an HPLC-Q Exactive hybrid quadrupole Orbitrap mass system. Then, a sensitive HILIC-ESI-MS/MS method was developed to simultaneously monitor strychnine, brucine, tyrosine and tyramine in cell lysate. The analytes were separated by a Shiseido CAPCELL CORE PC (150mm×2.1mm, 2.7µm) HILIC column in an acetonitrile/0.1% formic acid gradient system. All the calibration curves were linear with regression coefficients above 0.9924. The absolute recoveries were more than 80.5% and the matrix effects were between 91.6%-107.0%. With the developed method, analytes were successfully determined in cell lysates. Decreased levels of tyrosine and tyramine were observed only in combination with increased levels of SAs, indicating that the disturbance of tyrosine metabolism might be induced by the accumulation of SAs in kidney cell after exposure of Semen Strychni. The HILIC-ESI-MS/MS based analytical strategy is a useful tool to reveal the relationships between the toxic herb components and the endogenous metabolite profiling in the toxicity investigation of herb medicines.

Development of quercetin-phospholipid complex to improve the bioavailability and protection effects against carbon tetrachloride-induced hepatotoxicity in SD rats.

Quercetin (QT) is a natural flavonoid with various biological activities and pharmacological actions. However, the bioavailability of QT is relatively low due to its low solubility which severely limits its use. In this study, we intended to improve the bioavailability of QT by preparing quercetin-phospholipid complex (QT-PC) and investigate the protective effect of QT-PC against carbon tetrachloride (CCl4) induced acute liver damage in Sprague-Dawley (SD) rats. The physicochemical properties of QT-PC were characterized in terms of infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (XRPD) and water/n-octanol solubility. FTIR, DSC and XRPD data confirmed the formation of QT-PC. The water solubility of QT was improved significantly in the prepared complex, indicating its increased hydrophilicity. Oral bioavailability of QT and QT-PC was evaluated in SD rats, and the plasma QT was estimated by HPLC-MS. QT-PC exhibited higher Cmax (1.58±0.11 vs. 0.67±0.08µg/mL), increased AUC0-∞ (8.60±1.25 vs. 2.41±0.51mg/Lh) and t1/2z (7.76±1.09 vs. 4.81±0.87h) when compared to free QT. The greater absorption of QT-PC group suggested the improved bioavailability. Moreover, biochemical changes and histopathological observations revealed that QT-PC provided better protection to rat liver than free QT at the same dose. Thus, phospholipid complexation might be one of the suitable approaches to improve the oral bioavailability of QT and obtain better protective effects against CCl4 induced acute liver damage in SD rats than free QT at the same dose level.

Effects of phospholipid complexes of total flavonoids from Persimmon (Diospyros kaki L.) leaves on experimental atherosclerosis rats.

The total flavonoids from Persimmon leaves (PLF), extracted from the leaves of Diospyros kaki L. Dispryosl and Ebenaceae, is reported to possess many beneficial health effects. However, the oral bioavailability of PLF is relatively low due to its poor solubility. In the present study, the phospholipid complexes of total flavonoids from Persimmon leaves (PLF-PC) was prepared to enhance the oral bioavailability of PLF and to evaluate its antiatherosclerotic properties in atherosclerosis rats in comparison to PLF. A HPLC-MS method was developed and validated for the determination of quercetin and kaempferol in rats plasma to assess the oral bioavailability of PLF-PC. The effect of PLF (50mg/kg/d) and PLF-PC (equivalent to PLF 50mg/kg/d) on atherosclerosis rats induced by excessive administration of vitamin D (600,000IU/kg) and cholesterol (0.5g/kg/d) was assessed after orally administered for 4 weeks. The relative bioavailabilities of quercetin and kaempferol in PLF-PC relative to PLF were 242% and 337%, respectively. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) in serum were measured by an automatic biochemistry analyzer. The morphological changes of aorta were observed with optical microscopy. According to the levels of biochemical parameters in serum and the morphological changes of aorta, PLF-PC showed better therapeutic efficacy compared to PLF. Thus, PLF-PC holds a promising potential for increasing the oral bioavailability of PLF. Moreover, PLF-PC exerts better therapeutic potential in the treatment of atherosclerotic disease than PLF.

Simultaneous Quantification of 13 Compounds in Guanxin Shutong Capsule by HPLC Method.

Guanxin Shutong (GXST) capsule, which is frequently used in clinical therapy, has a certain and positive therapeutic effect against coronary heart disease. However, the existing quality standard of GXST capsule is inadequate to control the quality of GXST capsule. In this paper, a new high-performance liquid chromatographic (HPLC) method for simultaneous determination of 13 compounds (gallic acid, danshensu, protocatechuic acid, procatechuic aldehyde, ellagic acid, rosmarinic acid, salvianolic acid A and salvianolic acid B, eugenol, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA) in GXST capsule was developed and validated. The analytes were successfully separated and quantified with an Agilent TC-C18 column (250 × 4.6 mm, 5 µm) by gradient elution using 0.05% phosphoric acid and acetonitrile as mobile phase. The flow rate was 1 mL/min and the detection wavelength was set at 280 nm. All the compounds showed good linearity (r > 0.9991) in a relatively wide concentration range. The intra-and the inter-day variability were in the range of 0.85-2.68 and 1.47-2.86%, respectively. The recoveries of the selected compounds were in the range of 95.24-104.75%. This method was successfully applied to quantify the 13 components in GXST capsule and was conducive to controlling the quality of GXST capsule.

The comparative pharmacokinetics of four bioactive ingredients after administration of Ramulus Cinnamomi-Radix Glycyrrhizae herb pair extract, Ramulus Cinnamomi extract and Radix Glycyrrhizae extract.

Ramulus Cinnamomi (RC)-Radix Glycyrrhizae (RG) is a classic herb pair, which is commonly used as a fixed form to treat cardiovascular disease in the clinic. Our work aimed to compare the pharmacokinetic difference of cinnamic acid, liquiritin, isoliquiritigenin and glycyrrhetinic acid in rats after oral administration of the RC-RG herb pair extracts [Guizhigancao Decoction (GGD) and Lingguizhugan Decoction (LGZGD)] and the single RC or RG extract. A HPLC-MS method was developed and validated to study comparative pharmacokinetics. The pharmacokinetic parameters (Cmax , AUC, MRT) of four compounds between the RC-RG herb pair group and the single herb (RC or RG) group showed significant differences (p < 0.05). Compared with the single herb (RC or RG) group, higher peak concentration, slower elimination and larger exposure could be observed after giving the RC-RG herb-pair extracts. The pharmacokinetic differences might indicate the relativity of remedy in the RC-RG herb pair and provide scientific information for rational administration of the drug in the clinic. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of depression biomarkers in rat plasma by liquid chromatography-mass spectrometry for the study of the antidepressant effect of Zhi-Zi-Hou-Po decoction on rat model of chronic unpredictable mild stress.

Zhi-Zi-Hou-Po decoction (ZZHPD), a traditional Chinese medicine (TCM) formula, has been used for treatment of depressive-like symptoms for centuries. However, studies of its antidepressant effect and mechanism are challenging, owing to the complex pathophysiology of depression and complexity of ZZHPD with multiple constituents acting on different metabolic pathways. The present study was designed to develop a liquid chromatography-mass spectrometry (LC-MS) method for simultaneous determination of depression biomarkers: tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), indole-3-acetic acid (IAA), hippuric acid (HA), phenaceturic acid (PA), creatinine (Cr), glutamic acid (Glu), succinic acid (SA) and γ-aminobutyric acid (GABA), as well as to study the antidepressant effect and potential mechanism of ZZHPD based on holistic view of an organism. The analysis was performed on a CAPCELL PAK C18 column with methanol and 0.01% formic acid in water using gradient elution. The method showed a good linearity (r(2)>0.99) with the other validation parameters were within acceptance range. The results demonstrated Trp, Phe, Tyr, IAA, HA, Cr, Glu and SA levels were significant lower in model group, while PA and GABA were significant higher than those in control group. The rats with ZZHPD treatment showed a tendency of bringing the levels of all these biomarkers to normal except Cr and Glu. It could be a powerful manner to provide mechanistic insights into the therapeutic effects of complex prescriptions and further understand pathophysiology of depression to assist in clinical diagnosis.

Cell-based screening identifies the active ingredients from Traditional Chinese Medicine formula Shixiao San as the inhibitors of atherosclerotic endothelial dysfunction.

In this study, we performed a phenotypic screening in human endothelial cells exposed to oxidized low density lipoprotein (an in vitro model of atherosclerotic endothelial dysfunction) to identify the effective compounds in Shixiao San. After investigating the suitability and reliability of the cell-based screening method using atorvastatin as the positive control drug, this method was applied in screening Shixiao San and its extracts. The treatment of n-butanol fraction on endothelial cells exhibited stronger healing effects against oxidized low density lipoprotein-induced insult when compared with other fractions. Cell viability, the level of nitric oxide, endothelial nitric oxide synthase and endothelin-1 were measured, respectively. The assays revealed n-butanol fraction significantly elevated the survival ratio of impaired cells in culture. In parallel, n-butanol fraction exhibited the highest inhibition of inflammation. The generation of prostaglandin-2 and adhesion molecule (soluble intercellular adhesion molecule-1) was obviously declined. Furthermore, n-butanol fraction suppressed the production of reactive oxygen species and malondialdehyde, and restored the activity of superoxide dismutase. Compounds identification of the n-butanol fraction was carried out by ultra high liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. The active ingredients including quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside revealed the ability of anti-atherosclerosis after exposing on endothelial cells. The current work illustrated the pharmacology effect of Shixiao San and clearly indicated the major active components in Shixiao San. More importantly, the proposed cell-based screening method might be particularly suitable for fast evaluating the anti-atherosclerosis efficacy of Traditional Chinese Medicines and screening out the interesting ingredients of Traditional Chinese Medicines.

Evaluation of the indicative roles of seven potential biomarkers on hepato-nephrotoxicity induced by Genkwa Flos.

ETHNOPHARMACOLOGICAL RELEVANCE: Genkwa Flos, a classical traditional Chinese medicine, is used for the definite antitumor activity and tends to be taken overdose or long term in these years. While the excessive application can result in damage to liver and kidney. In this study, the indicative roles of seven potential biomarkers were evaluated to investigate hepato-nephrotoxicity in the early stages after oral administration of Genkwa Flos for 14 days. MATERIALS AND METHODS: Histopathology, serum biochemistry and seven potential biomarkers in serum or urine from male Sprague-Dawley rats were monitored. Hepatic and renal tissues were histopathologically examined to identify specific changes occurring. Routine serum biochemical parameters were tested by using standard clinical laboratory methods. Seven biomarkers including cholic acid, taurine, 5-oxoproline, hippuric acid, uric acid, 3-indoxyl sulfate and kynurenic acid were detected by a developed LC-MS method. RESULTS: The histopathological alterations and the increased levels of serum biochemistry were detected on the 8th day after Genkwa Flos treated. The seven analytes were also found significantly changed in Genkwa Flos treated group, especially cholic acid, taurine, 5-oxoproline and hippuric acid which were changed on the 2nd or 4th day. CONCLUSIONS: Although serum biochemistry and histopathology suggested that Genkwa Flos was responsible for the hepato-nephrotoxicity that occurred following the ingestion of this medicinal herb, evaluation of these biomarkers might be more beneficial for the early detection of liver and kidney injuries. This study could be further used in hepatic and renal failures caused by other reasons in the following research works.

One-month toxicokinetic study of SHENMAI injection in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: 'SHENMAI' injection (SMI) has been widely used in cardioprotection and modulation of the immune system because of its great efficacy. SMI primarily comprises the saponins from Panax ginseng and Ophiopogon japonicas. The profiles of saponins in SMI during long-term toxicokinetics remain unclear. MiR-146a possesses excellent sensitivity as a bio-marker in the innate immunity modification effect of SMI. AIM OF THE STUDY: Is to monitor the exposure level of SMI during a one-month toxicokinetic experiment, an analytical method involving ESI-LC-MS/MS technology was developed to determine 20 (S)-protopanaxadiol-type ginsenoside (Rb1, Rb2, Rc, Rd), 20 (S)-protopanaxatriol-type ginsenoside (Rg1, Re, Rf), oleanolic acid-type ginsenoside (Ro), and ophiopogonin D in rats. The levels of AST, CK, ALT, SOD, GSH-pX, MDA, miR-146a, and ECG were measured to explore the effects of SMI in cardiologic function and immune activity. RESULTS: Results show that the levels of AST, CK, and MDA decreased upon the administration of SMI. The level of miR-146a increased upon the administration of SMI dosage. During the administration of SMI, increasing exposure levels of 20 (S)-protopanaxadiol-type ginsenosides were also observed. CONCLUSION: The 20 (S)-protopanaxadiol-type ginsenosides were considered potential PK/TK markers because of their high exposure levels that continuously increased. Oxidative stress was slightly alleviated during the toxicokinetic study. Based on the level of miR-146a, negatively regulated innate immunity was observed. The regulation became more serious with increasing exposure levels of 20 (S)-protopanaxadiol-type ginsenosides. Negatively regulated innate immunity could be induced by long-term administration of SMI (>0.4g/kg).

A study of Semen Strychni-induced renal injury and herb-herb interaction of Radix Glycyrrhizae extract and/or Rhizoma Ligustici extract on the comparative toxicokinetics of strychnine and brucine in rats.

Recently, the renal injury caused by Semen strychni and its major toxic constituents, strychnine and brucine, was reported in many clinical cases. Hence, this study was conducted to investigate the renal injury induced by Semen Strychni and the protective effects of Radix Glycyrrhizae and Rhizoma Ligustici. The protective mechanisms were related to the comparative toxicokinetics of strychnine and brucine. Serum and urine uric acid and creatinine were used as renal function markers to evaluate the condition of kidney, and renal injury was directly reflected by histopathological changes. Compared with rats in blank group and protective herb groups, rats in Semen Strychni high-dose group showed significant differences in the results of renal function markers, and various glomerular and tubular degenerations were found in the histopathological study. The decreased AUC (only strychnine) and Cmax, the increased Tmax by Radix Glycyrrhizae and the decreased T1/2 by Radix Glycyrrhizae and Rhizoma Ligustici were found in model groups. Results indicated that high dose of Semen Strychni might induce renal injury. Radix Glycyrrhizae and Rhizoma Ligustici might work together and have effects on the elimination of strychnine and brucine. The protective effects of Radix Glycyrrhizae might also be explained by the slow absorption of the alkaloids.

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni.

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure, 12 typical biomarkers are selected and a simple LC-MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3-indoxyl sulfate, indole-3-acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C18 column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3-indoxyl sulfate, and indole-3-acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.

Simultaneous determination of five free and total flavonoids in rat plasma by ultra HPLC-MS/MS and its application to a comparative pharmacokinetic study in normal and hyperlipidemic rats.

A simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) method has been developed for the simultaneous determination of five free flavonoids (amentoflavone, isorhamnetin, naringenin, kaempferol and quercetin) and their total (free and conjugated) forms, and to compare the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats. The free and total forms of these flavonoids were extracted by liquid-liquid extraction with ethyl acetate. The conjugated flavonoids were deconjugated by the enzyme ß-Glucuronidase and Sulfatase. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution. Detection was performed on a 4000Q uHPLC-MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were 2.0-5.0ng/mL for all the analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from -9.3% to 11.0%, and the mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 81.7%. The validated method was successfully applied to a comparative pharmacokinetic study of five free and total analytes in rat plasma. The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration-time curves.

Protective effect of Salvia miltiorrhizae injection on N(G)-nitro-D-arginine induced nitric oxide deficient and oxidative damage in rat kidney.

N(G)-nitro-D-arginine (d-NNA) could convert into N(G)-nitro-L-arginine (l-NNA) in vivo, and kidney is the major target organ. In the chiral inversion process, a number of reactive oxygen species (ROS) were generated and NOS activity was inhibited, which may cause renal damage. Salvia miltiorrhiza (SM), a traditional Chinese drug, was used in the treatment of cardiovascular diseases and chronic renal failure. The aim of the present study was to investigate the kidney damage caused by D-NNA administration for 12 weeks and to evaluate the effects of treatment with SM on D-NNA-induced kidney damage. The rats, induced with D-NNA for period of 12 weeks, showed significant elevation of Blood Urea Nitrogen (BUN), Creatinine (Crea) and MDA levels, and significant decrease of SOD and GSH-Px activities, as compared with control group. In addition, the kidney of rats induced with D-NNA only showed remarkable histopathology, including severe mononuclear cell infiltration, mild tubular dilatation and congestion, and moderate interstitial desmoplasia. After 4 weeks SM treatment, the activity of SOD, GSH-Px and iNOS and the production of NO were significantly higher (P<0.05), and the levels of BUN, Crea and MDA were significantly lower than that of D-NNA only group (P<0.05). In addition, treatment with SM showed histopathological protection in tubular dilatation, congestion, mononuclear cell infiltration and interstitial desmoplasia. The present results indicate that the toxicity of D-NNA relates to its ability to generate oxidative stress and upregulate NOS activity in rat kidney. SM probably ameliorates D-NNA-induced nephrotoxicity in rats according to scavenging free radical and upregulating NOS activity.

Electrocardiographic and biochemical evidence for the cardioprotective effect of antioxidants in acute doxorubicin-induced cardiotoxicity in the beagle dogs.

Doxorubicin (DOX) is a potent antitumor agent, but the cardiotoxicity mediated by the formation of reactive oxygen species limit its clinical use. The present study aims to explore electrocardiographic and biochemical evidence for the cardioprotective effect of two antioxidants, Lycium barbarum polysaccharides (LBP, the main antioxidant in Lycium barbarum) and edaravone (a potent free radical scavenger, EDA) against DOX-induced acute cardiotoxicity in beagle dogs. In this study, male beagle dogs received daily treatment of either LBP (20 mg/kg, per os (p.o.)) or EDA (2 mg/kg, intravenously (i.v.)) for 7 d and then followed by an intravenous injection of DOX (1.5 mg/kg). DOX (15 mg/kg) significantly induced acute cardiotoxicity in dogs characterized by conduction abnormalities (including decreased heart rate, ST segment elevation, QT intervals prolongation, inverted T wave, arrhythmia, and myocardial ischemia) and increased serum creatine kinase (CK) and aspartate aminotransferase (AST). Pretreatment with LBP or EDA effectively alleviated both DOX-associated conduction abnormalities and increased serum CK and AST. Moreover, physiological and serum biochemical evidences demonstrated that EDA is more effective than LBP in alleviating these abnormalities produced by DOX in heart. All these results confirm and extend previous observations in rats concerning the effectiveness of LBP or EDA against DOX-induced cardiomyopathy.