Quzhou Fructus Aurantii Extract suppresses inflammation via regulation of MAPK, NF-κB, and AMPK signaling pathway.

The anti-inflammatory activity of Quzhou Fructus Aurantii Extract (QFAE) has been reported recently. Thus, present study aims to explore the mechanism of anti-inflammation of QFAE in vitro and in vivo to develop a lung phylactic agent. The anti-inflammatory mechanism of QFAE in RAW 264.7 cells and acute lung injury (ALI) mice model was determined by cytokines analysis, histopathological examination, Western blot assay, immunofluorescence, and immunohistochemistry analysis. The results showed that QFAE restrained mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways in LPS-induced RAW 264.7 cells, whereas AMP-activated protein kinase (AMPK) signaling pathways were activated, as revealed by prominent attenuation of phosphorylation of ERK, JNK, p38, p65, IκBα, RSK and MSK, and overt enhancement of phosphorylation of ACC and AMPKα. The levels of pro-inflammatory cytokines TNF, IL-6, and IL-1ß were suppressed, whereas the level of anti-inflammatory cytokine IL-10 increased after pretreatment with QFAE in vivo and in vitro. Moreover, QFAE prevented mice from LPS-provoked ALI, bases on alleviating neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) and mitigatingpulmonary histological alters, as well as hematological change. The MAPK and NF-κB signaling pathways in LPS-stimulated ALI mice were dampened by QFAE pretreatment, whereas AMPK signaling pathways were accelerated, as testify by significant restraint of phosphorylation of ERK, JNK, p38, p65, and IκBα, and distinct elevation of phosphorylation of ACC and AMPKα. The remarkable anti-inflammatory effect of QFAE is associated with the suppression of MAPK and NF-κB signaling pathways and the initiation of AMPK signaling pathway.

Role of Quzhou Fructus Aurantii Extract in Preventing and Treating Acute Lung Injury and Inflammation.

Quzhou Fructus Aurantii (QFA) is an authentic herb of local varieties in Zhejiang, China, which is usually used to treat gastrointestinal illnesses, but its effects on respiratory inflammation have not been reported yet. In our study, the anti-inflammatory activity of QFA extract (QFAE) was evaluated on copper sulfate pentahydrate (CuSO4·5H2O)-induced transgenic neutrophil fluorescent zebrafish model. QFAE showed a significant effect of anti-inflammation in CuSO4·5H2O-induced zebrafish by reducing the neutrophil number in the inflammatory site. We investigated the anti-inflammatory activity of QFAE on lipopolysaccharide (LPS)-induced acute lung injury (ALI) mice models and RAW 264.7 cells. QFAE had an anti-inflammatory effect on reducing total cells, neutrophils, and macrophages in BALF and attenuated alveolus collapse, neutrophils infiltration, lung W/D ratio, myeloperoxidase (MPO) protein expression and other pulmonary histological changes in lung tissues, as well as hematological changes. Levels of pro-inflammatory cytokines, including TNF, IL-6, IFN-γ, MCP-1, and IL-12p70, were decreased, whereas anti-inflammatory cytokine IL-10 was increased after treatment with QFAE both in vivo and in vitro. In summary, our results suggested that QFAE had apparent anti-inflammatory effects on CuSO4·5H2O-induced zebrafish, LPS-induced ALI mice, and RAW 264.7 cells. Furthermore, QFAE may be a therapeutic drug to treat ALI/ARDS and other respiratory inflammations.

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.

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.

Modulatory Effect of Fermented Papaya Extracts on Mammary Gland Hyperplasia Induced by Estrogen and Progestin in Female Rats.

Fermented papaya extracts (FPEs) are obtained by fermentation of papaya by Aspergillus oryzae and yeasts. In this study, we investigated the protective effects of FPEs on mammary gland hyperplasia induced by estrogen and progestogen. Rats were randomly divided into 6 groups, including a control group, an FPE-alone group, a model group, and three FPE treatment groups (each receiving 30, 15, or 5 ml/kg FPEs). Severe mammary gland hyperplasia was induced upon estradiol benzoate and progestin administration. FPEs could improve the pathological features of the animal model and reduce estrogen levels in the serum. Analysis of oxidant indices revealed that FPEs could increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, decrease malondialdehyde (MDA) level in the mammary glands and serum of the animal models, and decrease the proportion of cells positive for the oxidative DNA damage marker 8-oxo-dG in the mammary glands. Additionally, estradiol benzoate and progestin altered the levels of serum biochemical compounds such as aspartate transaminase (AST), total bilirubin (TBIL), and alanine transaminase (ALT), as well as hepatic oxidant indices such as SOD, GSH-Px, MDA, and 8-oxo-2'-deoxyguanosine (8-oxo-dG). These indices reverted to normal levels upon oral administration of a high dose of FPEs. Taken together, our results indicate that FPEs can protect the mammary glands and other visceral organs from oxidative damage.

Oral subchronic toxicity evaluation of a novel antitumor agent 25-methoxydammarane-3, 12, 20-triol from Panax notoginseng in Sprague-Dawley rats.

Panax notoginseng and its main active ingredients ginsenosides have long been used as medicines and food additives in China. Comparing with the extensive uses and active researches of P. notoginseng and its products, the side effect and probable toxicity were rare. 25-Methoxydammarane-3,12,20-triol (25-OCH3-PPD), a novel dammarane-type triterpene sapogenin that was first isolated from the extract of P. notoginseng, was proven to have strong antitumor activities against prostate cancer, breast cancer and lung cancer. The aim of the present study was to investigate the potential subchronic toxicity of 25-OCH3-PPD after it was repeatedly orally administered to Sprague-Dawley rats (5/sex/group/each time-point) at dose levels of 0, 150, 300 or 600 mg/kg/day for 13 weeks and 4-week recovery. No mortality and treatment-related toxicity effects were observed as a result of the administration of 25-OCH3-PPD at any dose level (150, 300 and 600 mg/kg) for 92 consecutive days. Although there were some statistical changes, such as increased weights in female rats and decreased organ weights and coefficients of the liver, spleen, kidney, and adrenal gland compared with the control group at the corresponding time, the autopsy and histopathological examination of the target organs did not show any abnormal responses. As a result, 25-OCH3-PPD was well tolerated by SD rat at doses of up to 600 mg/kg and that it is a potential candidate for therapeutic use.

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).

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.

Protective effect of Lycium barbarum polysaccharides against doxorubicin-induced testicular toxicity in rats.

The present study aimed to investigate whether Lycium barbarum polysaccharides (LBP) would protect against doxorubicin (DOX)-induced testicular toxicity. Male Sprague-Dawley rats were treated with distilled water (4 mL/kg) or LBP (200 mg/kg, p.o.) daily for 10 days and followed by saline (0.9 %, 10 mL/kg) or DOX (10 mg/kg) intravenous injection at day 7. Pretreatment with LBP ameliorated DOX-induced reduction in the testicular weights, sperm concentrations and percentage of motile sperms, as well as the increase in abnormal sperm rate. LBP administration to DOX-treated rats successfully reversed the changes in MDA and GHS-Px levels. Compared with the control, pretreatment with LBP significantly increased the plasma testosterone level in the LBP + DOX group. The histopathology examinations further confirmed that LBP effectively attenuated DOX-induced severe degenerative changes of seminiferous tubules. This study illustrated the capability of LBP in attenuating testicular oxidative stress and protecting testis-specific toxicity in DOX-exposed rats.

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.

Angelica sinensis: a novel adjunct to prevent doxorubicin-induced chronic cardiotoxicity.

Doxorubicin is an anthracycline antibiotic agent used in the treatment of a variety of solid and haematopoietic tumours, but its use is limited by formation of metabolites that induce acute and chronic cardiac toxicities. Angelica sinensis has been widely used to treat cardiovascular and cerebrovascular diseases in China. In the present study, we used an in vivo mouse model to explore whether A. sinensis could protect against doxorubicin-induced chronic cardiotoxicity. Male ICR mice were treated with distilled water or water extraction of A. sinensis (15 g/kg, orally) daily for 4 weeks, followed by saline or doxorubicin (15 mg/kg, intravenously) treatments weekly. Cardiotoxicity was assessed by electrocardiograph, antioxidant activity in cardiac tissues, serum levels of creatine kinase, aspartate aminotransferase (AST) and histopathological change in cardiac tissues. A cumulative dose of doxorubicin (60 mg/kg) caused animal death and myocardial injury characterized by increased QT interval and decreased heart rate in electrocardiograph, decrease of heart antioxidant activity, increase of serum AST, as well as myocardial lesions. Pre-treatment with A. sinensis significantly reduced mortality and improved heart performance of the doxorubicin-treated mice as evidenced from normalization of antioxidative activity and serum AST, preventing loss of myofibrils as well as improving arrhythmias and conduction abnormalities. Furthermore, the in vitro cytotoxic study showed that A. sinensis did not compromise the antitumour activity of doxorubicin. These results suggested that A. sinensis elicited a typical cardioprotective effect on doxorubicin-related oxidative stress, and could be a novel adjunct in the combination with doxorubicin chemotherapy.

Protective effect of Lycium barbarum on doxorubicin-induced cardiotoxicity.

The objective of this work was to explore the hypothesis that Lycium barbarum (LB) may be protective against doxorubicin (DOX)-induced cardiotoxicity through antioxidant-mediated mechanisms. Male SD rats were treated with distilled water or a water extract of LB (25 mg/kg, p.o.) daily and saline or DOX (5 mg/kg, i.v.) weekly for 3 weeks. Mortality, general condition and body weight were observed during the experiment. DOX-induced cardiotoxicity was assessed by electrocardiograph, heart antioxidant activity, serum levels of creatine kinase (CK) and aspartate aminotransferase (AST) and histopathological change. The DOX group showed higher mortality (38%) and worse physical characterization. Moreover, DOX caused myocardial injury manifested by arrhythmias and conduction abnormalities in ECG (increased QT and ST intervals and ST elevation), a decrease of heart antioxidant activity, an increase of serum CK and AST, as well as myocardial lesions. Pretreatment with LB significantly prevented the loss of myofibrils and improved the heart function of the DOX-treated rats as evidenced from lower mortality (13%), normalization of antioxidative activity and serum AST and CK, as well as improving arrhythmias and conduction abnormalities. These results suggested that LB elicited a typical cardioprotective effect on DOX-related oxidative stress. Furthermore, in vitro cytotoxic study showed the antitumor activity of DOX was not compromised by LB. It is possible that LB could be used as a useful adjunct in combination with DOX chemotherapy.