A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method was established for simultaneous determination of seven characteristic components from the active fraction of Alpiniae Officinarum Rhizoma in rat plasma, including galangin, kaempferol, kaempferide, pinocembrin, 1,7-diphenyl-4-en-3-heptanone, 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-heptanone(DHPA), and 7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-4-en-3-heptanone(DPHB). The new developed HPLC-MS/MS method was applied to study the pharmacokinetics of the 7 characteristic components in rats with Helicobacter pylori gastritis. A Waters Sunfire C_(18) column(2.1 mm×150 mm, 3.5 μm) was used. The acetonitrile-aqueous solution(containing 0.1% formic acid) was adopted as the mobile phase for gradient elution. Seven components and internal standard(chlorogenic acid) were separated within 12 min. Mass spectrometric detection was performed in multiple reaction monitoring(MRM) mode using electrospray ionization(ESI) source with fast switching between positive and negative ions. The method was verified by specificity, linearity, precision, accuracy, recovery, matrix effect, and stability and met the requirements of pharmacokinetic study on the 7 components in rat plasma. Pharmacokinetic results showed that the average peak time(T_(max)) of the 7 components was 0.31-2.19 h, their elimination half-life(t_(1/2)) was 5.26-16.65 h, and the average residence time(MRT) was 6.29-31.03 h after the oral administration of the active fraction of Alpiniae Officinarum Rhizoma to rats with H. pylori gastritis. The plasma exposure levels of galangin and DHPA were higher than those of the other components. The concentration-time curves of four detected flavonoids showed obvious double peaks. This study elucidated the pharmacokinetic characteristics of 7 characteristic components from the active fraction of Alpiniae Officinarum Rhizoma in rats with H. pylori gastritis, providing a scientific basis for the identification of the pharmacodynamic substances of Alpiniae Officinarum Rhizoma for treatment of H. pylori gastritis and the clinical application of Alpiniae Officinarum Rhizoma in the prevention and treatment of H. pylori gastritis.
Animals ; Rats ; Chromatography, High Pressure Liquid/methods* ; Tandem Mass Spectrometry/methods* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Helicobacter pylori/drug effects* ; Alpinia/chemistry* ; Rats, Sprague-Dawley ; Gastritis/metabolism* ; Helicobacter Infections/metabolism* ; Flavonoids/blood* ; Rhizome/chemistry* ; Liquid Chromatography-Mass Spectrometry

Objective To investigate the effect and mechanism of baicalin on blood lipid metabolism and immune function in rats with gestational diabetes mellitus (GDM). Methods Female rats fed with high-fat and high-sugar diet and male rats fed with ordinary diet were caged together to prepare pregnant rats, and the GDM rat model was established by intraperitoneal injection of streptozotocin (35 mg/kg). GDM rats were randomly divided into a model group, a fasudil (FA) (RhoA/RocK inhibitor) group (10 mg/kg), low-dose (100 mg/kg) and high-dose (200 mg/kg) baicalin groups, and a high-dose baicalin combined with LPA (RhoA/RocK activator) group (200 mg/kg baicalin+1 mg/kg LPA ), with 12 rats in each group. Another 12 pregnant rats fed with high-fat and high-sugar diet were selected as the control group. After 2 weeks of corresponding drug intervention in each group, the level of fasting blood glucose (FBG) was detected by blood glucose meter. The level of fasting insulin (FINS) in serum was detected by ELISA, and the insulin resistance index (HOMA-IR) was calculated. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) in serum, and the levels of immunomodulator tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-10 in peripheral blood were detected by the kit. The histopathological changes of liver were observed by HE staining. The proportion of T lymphocyte subsets in peripheral blood was detected by flow cytometry. The mRNA and protein expressions of Ras homolog gene family member A (RhoA), Rho associated coiled-coil forming protein kinase 1 (ROCK1), and ROCK2 in liver tissue were detected by real-time quantitative PCR and Western blot. Results Compared with the control group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8⁺T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the model group were higher; the level of HDL-C in serum, the percentage of IL-10 levels, CD3⁺T cells, CD4⁺T cell, and CD4⁺T/CD8⁺T ratio in peripheral blood were lower. Compared with the model group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8⁺T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the the FA group and low-dose and high-dose baicalin groups were lower; the level of HDL-C in serum, IL-10 level, the percentage of CD3⁺T cells, CD4⁺T cell, and CD4⁺T/CD8⁺T ratio in peripheral blood were higher. LPA could obviously weaken the improvement effects of baicalin on blood lipid metabolism and immune function in GDM rats. Conclusion Baicalin may improve blood lipid metabolism and immune function in GDM rats by inhibiting the RhoA/ROCK pathway.
Animals ; Female ; Diabetes, Gestational/metabolism* ; Pregnancy ; rho-Associated Kinases/genetics* ; Flavonoids/pharmacology* ; Rats ; rhoA GTP-Binding Protein/genetics* ; Lipid Metabolism/drug effects* ; Male ; Signal Transduction/drug effects* ; Rats, Sprague-Dawley ; Blood Glucose/metabolism* ; Lipids/blood* ; Tumor Necrosis Factor-alpha/blood* ; rho GTP-Binding Proteins

OBJECTIVE: To comprehensively evaluate the effect of icariin in alleviating reproductive function damage (RFD) in male mice via in vitro and in vivo experiments. METHODS: We isolated Leydig cells from 60 KM male mice in vitro, and examined the toxic effect of icariin on the Leydig cells using Cell Counting Kit-8 (CCK-8). We equally randomized the mice into six groups: normal control, RFD model control (made by intraperitoneal injection of busulfan at 10 mg/kg combined with cyclophosphamide (CP) at 120 mg/kg), positive control, and low-, medium- and high-dose icariin. After modeling, we treated the mice in the positive control group with Wuziyanzong Pills and those in the low-, medium- and high-dose icariin groups by intragastrical administration of icariin at 20, 40 and 80 mg/kg-1, respectively, for 30 successive days. Then we obtained the weight and visceral coefficients of the reproductive organs, calculated the sperm count, observed the pathological changes in the testis tissue by HE staining, measured the serum testosterone (T) level by ELISA, determined the indexes of testicular oxidative stress and nitric oxide (NO) signaling pathway by colorimetric assay, and detected the expression levels of the pro-apoptotic genes Fas and Bax by qRT-PCR. RESULTS: CCK-8 assay confirmed that icariin had no toxic effect on the isolated Leydig cells of the mice, and could effectively reduce busulfan- and CP-induced cytotoxicity and promote the secretion of serum T. Icariin at 80 mg/kg significantly increased the visceral coefficient of the testis and promoted spermatogenesis (P<0.05), but had little effect on the visceral coefficient of the epididymis in the RFD model mice. Testicular histomorphometric observation revealed significantly improved testis structure, intact boundary membrane of seminiferous tubules and increased numbers of various types of spermatogenic cells of the model mice after treated with icariin. Compared with the mice in the model control group, those treated with high-dose icariin showed a significantly reduced content of malondialdehyde (MDA) (by 35.3%, P<0.01), elevated total antioxidant capacity (TAOC) and superoxide dismutase (T-SOD) activity (P<0.05), and decreased NO content and nitric oxide synthase (NOS) activity in the testis tissue (P<0.01). In addition, icariin exhibited an evident inhibitory effect on the expressions of the pro-apoptotic genes Bax and Fas. CONCLUSION Icariin can ameliorate oxidative stress-induced damage to the testicular function and protect spermatogenesis of male mice by elevating TAOC, decreasing NOS activity, inhibiting the NO level in the testis, and suppressing busulfan- and CP-induced apoptosis of testicular cells.
Animals ; Male ; Cyclophosphamide/adverse effects* ; Mice ; Busulfan/adverse effects* ; Flavonoids/pharmacology* ; Leydig Cells/drug effects* ; Oxidative Stress/drug effects* ; Testis/drug effects* ; Apoptosis/drug effects* ; Testosterone/blood*

To study the binding capacity of active ingredients of Daidai lipid-lowering flavonoid extract and plasma protein,investigate the ways to improve the traditional formula for calculating protein binding rates based on ultrafiltration,and increase the stability and reliability of the experimental results. UPLC-MS/MS was used to establish a quantitative analysis method for simultaneous determination of active ingredients( neohesperidin and narngin) in ultrafiltrate. The protein binding rates were calculated by the traditional ultrafiltration formula. The correction factors( F) were introduced later,and the binding rates calculated with the correction factors were compared with those without the correction factors. The binding capacity of the extract and plasma protein was evaluated. The quantitative analysis method established by UPLC-MS/MS had a good specificity. The standard curve and linear range,method accuracy,precision and lower limit of quantitation all met the requirements. The method met the requirement for quantitative detection of the active ingredients in ultrafiltrate after the rat plasma was filtrated in the ultrafiltration tube. Under the experimental conditions,the binding rates of both active ingredients( neohesperidin and narngin) were higher than 90%. The active ingredients and rat plasma protein were bound in a concentration-dependent manner,with statistically significant differences( P<0. 01). There was no statistically significant difference between the protein binding abilities of the two active ingredients with rat plasma protein. Therefore,the active ingredients of Daidai lipid-lowering flavonoid extract had a relatively strong binding strength with rat plasma protein,and they were bound in a concentration-dependent manner. Additionally,when calculating protein binding rates by the traditional ultrafiltration formula,the correction factors could be introduced to effectively reflect the errors of multiple ingredient groups in traditional Chinese medicine extracts.This correction method could provide a reference thinking and practical reference for the improvement of the determination method of the traditional Chinese medicine plasma protein binding ability based on ultrafiltration.
Animals ; Blood Proteins ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Hypolipidemic Agents ; pharmacology ; Lipids ; Rats ; Reproducibility of Results ; Tandem Mass Spectrometry

OBJECTIVES: The rate of metabolic syndrome (MetS) in women diagnosed as they age is one of the main concerns of health cares. Recently new strategies used to prevent progressions of MetS toward the diagnosis of diabetes have focused on plant flavonoids. This study was aimed to investigate the beneficial effects of flavonoids fraction of Mespilus germanica leaves (MGL) on MetS in ovariectomized (OVX) rats. METHODS: Twenty-four adult female Wistar rats, weighing 200 to 250 g, were divided into 3 groups: Sham surgery, OVX + Salin, or OVX + Flavonoid. Three weeks after ovariectomy, animals displayed MetS criteria received flavonoid injection (10 mg/kg, intraperitoneally) for 21 days. Then the body weight, body mass index, waist circumference, visceral fat, fasting blood glucose, serum insulin, lipid profiles and tumor necrosis factor-α (TNF-α) were measured. RESULTS: Treatment with flavonoids fraction of MGL significantly decreased serum level of insulin (P = 0.011), glucose (P = 0.024), TNF-α (P = 0.010), also MetS Z score (P = 0.020) and homeostasis model assessment of insulin resistance (P = 0.007). Lipid profiles and visceral fat showed insignificant reduction. CONCLUSIONS: Flavonoids of MGL attenuates some of the MetS components possibly via reduction in TNF-α inflammatory cytokine.
Adult ; Animals ; Blood Glucose ; Body Mass Index ; Body Weight ; Diagnosis ; Fasting ; Female ; Flavonoids ; Glucose ; Homeostasis ; Humans ; Insulin Resistance ; Insulin ; Intra-Abdominal Fat ; Menopause ; Necrosis ; Ovariectomy ; Plants ; Polyphenols ; Rats ; Rats, Wistar ; Waist Circumference

OBJECTIVETo study the effect of total flavonoids of Astmgali Radix (TFA) on liver cirrhosis induced with dimethylnitrosamine (DMN) in rats, and the effect on peroxisome proliferator-activated receptor γ (PPARγ), uncoupling protein 2 (UCP2) and farnesoid X receptor (FXR).

METHODSFifty-three Sprague-Dawley rats were randomly divided into a control group (10 rats) and a DMN group (43 rats). Rats in the DMN group were given DMN for 4 weeks and divided randomly into a model group (14 rats), a low-dosage TFA group (14 rats) and a high-dosage TFA group (15 rats) in the 3rd week. Rats were given TFA for 4 weeks at the dosage of 15 and 30 mg/kg in the low- and high-TFA groups, respectively. At the end of the experiment blood and liver samples were collected. Serum liver function and liver tissue hydroxyproline content were determined. hematoxylin-eosin (HE), Sirus red and immunohistochemical stainings of collagen I, smooth muscle actin (α-SMA) was conducted in paraffinembedded liver tissue slices. Real time polymerase chain reaction (PCR) was adopted to determine PPARγ, UCP2 and FXR mRNA levels. Western blot was adopted to determine protein levels of collagen I, α-SMA, PPARγ, UCP2 and FXR.

RESULTSCompared with the model group, TFA increased the ratio of liver/body weight (low-TFA group P<0.05, high-TFA group P<0.01), improved liver biochemical indices (P<0.01 for ALT, AST, GGT in both groups, P<0.05 for albumin and TBil in the high-TFA group) and reduced liver tissue hydroxproline content (P<0.01 in both groups) in treatment groups significantly. HE staining showed that TFA alleviated liver pathological changes markedly and Sirus red staining showed that TFA reduced collagen deposition, alleviated formation and extent of liver pseudolobule. Collagen I and α-SMA immunohistochemical staining showed that staining area and extent markedly decreased in TFA groups compared with the model group. TFA could increase PPARγ, it regulated target UCP2, and FXR levels significantly compared with the model group (in the low-TFA group all P<0.05, in the high group all P<0.01).

CONCLUSIONTFA could improve liver function, alleviate liver pathological changes, and reduce collagen deposition and formation of liver pseudolobule in rats with liver cirrhosis. The antifibrotic effect of TFA was through regulating PPARγ signal pathway and the interaction with FXR.

Actins ; metabolism ; Animals ; Blotting, Western ; Body Weight ; drug effects ; Collagen Type I ; metabolism ; Dimethylnitrosamine ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Flavonoids ; pharmacology ; therapeutic use ; Hydroxyproline ; metabolism ; Liver ; drug effects ; pathology ; Liver Cirrhosis ; blood ; drug therapy ; genetics ; pathology ; Male ; Organ Size ; drug effects ; PPAR gamma ; genetics ; metabolism ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Uncoupling Protein 2 ; genetics ; metabolism

PURPOSE: This study aimed to investigate the association of total dietary antioxidant capacity (TAC) with oxidative stress and metabolic markers among patients with metabolic syndrome according to gender. METHODS: A total of 346 subjects aged 30~59 years with two or more risk factors of metabolic syndrome were recruited from a general hospital near Seoul in South Korea between 2010 and 2012 based on data from the medical checkup. Biochemical indices for oxidative stress and metabolic markers were measured. Food consumption data from 3-day food records were linked with the antioxidant capacity database for commonly consumed Korean foods to estimate individual's TAC. RESULTS: Average dietary TAC of the study subjects was 132.0 mg VCE/d/1,000 kcal in men and 196.4 mg VCE/d/1,000 kcal in women. Levels of γ-glutamyltransferase (GGT), systolic blood pressure, diastolic blood pressure, and blood triglycerides were reduced significantly according to increasing TAC in men, but there was no significant trend in women. Intakes of total flavonoids and carotenoids were significantly negatively correlated with GGT (p < 0.05) and d-ROMs (p < 0.01) in men, whereas those of α-tocopherol (p < 0.05) and γ-tocopherol (p < 0.05) were positively correlated with biological antioxidant potential (BAP) in women. The odds ratio of high oxidative stress indices and abnormal metabolic markers according to TAC level were not significant in either men or women. CONCLUSION: The results show that dietary TAC was partially associated with oxidative stress and metabolic markers among patients with metabolic syndrome. Further research is required for elucidating the association between dietary TAC and incidence of metabolic syndrome and chronic diseases within a large population in prospective studies.
Blood Pressure ; Carotenoids ; Chronic Disease ; Female ; Flavonoids ; Hospitals, General ; Humans ; Incidence ; Korea ; Male ; Odds Ratio ; Oxidative Stress* ; Prospective Studies ; Risk Factors ; Seoul ; Triglycerides

The present study was designed to develop and validate a rapid, sensitive, and reliable ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of five major active constituents in the traditional Chinese medicinal preparation Xingxiong injection (XXI) in rat plasma, including quercetin 3-O-rutinoside (QCR), kaempferol 3-O-rutinoside (KFR), isorhamnetin 3-O-rutinoside (ISR), bilobalide (BB), and ligustrazine (LGT). The plasma samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was achieved on a Waters Symmetry C analytical column (2.1 mm × 100 mm, 3.5 μm) with a mobile phase of 0.1% aqueous formic acid (A)-acetonitrile (B). Quantitation of the five bioactive constituents was achieved. Naringin was used as the internal standard (IS). All the calibration curves showed good linearity (r > 0.996) over the concentration range, with the lowest limit of quantification (LLOQ) between 2-18 ng·mL. The intra- and inter-day accuracy and precision of the analytes were both within acceptable limits. Moreover, satisfactory extraction recoveries (90.92%-104.03%) were obtained by protein precipitation. The validated method was successfully applied to a pharmacokinetic study of XXI in rats after intravenous administration at three doses. The pharmacokinetic parameters of the five compounds varied in a dose-dependent manner within the tested dosage range. The present study was the first report of pharmacokinetic study for XXI.
Animals ; Bilobalides ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; methods ; Disaccharides ; blood ; pharmacokinetics ; Drugs, Chinese Herbal ; administration & dosage ; analysis ; pharmacokinetics ; Flavonoids ; blood ; pharmacokinetics ; Glucosides ; blood ; pharmacokinetics ; Kaempferols ; blood ; pharmacokinetics ; Pyrazines ; blood ; pharmacokinetics ; Quercetin ; analogs & derivatives ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; methods

To investigate the effect of icariin total flavonoids capsules (ITFC) on bone mineral density (BMD) and bone histomorphometry in growing rats and its anti-osteoporosis mechanism.Thirty female SD rats were randomly divided into 3 groups:normal control group, ITFC-1 group and ITFC-2 group. Rats in ITFC-1 group and ITFC-2 group were fed with 50 mg·kg·dor 100 mg·kg·dITFC, respectively, and those in normal control group were fed with equal volume of distilled water. The whole body BMD was measured after 4, 8 and 12 weeks, and BMDs of the right femur and lumbar vertebrae were measured after 12 weeks. The serum levels of tartaric acid phosphatase 5b (TRACP 5b) and bone alkaline phosphatase (BALP) were measured by ELISA. Bone morphometry was performed on the right tibia.There were no significant differences in the body weight increase between normal control group and two ITFC groups (all>0.05). There were also no significant differences in whole body BMDs after 4 and 8 weeks between normal control group and ITFC groups (all>0.05). After 12 weeks, the whole body BMD, BMD of bone, serum BALP level and trabecular area in ITFC-1 group and ITFC-2 group were significantly higher, trabecular separation was significantly lower than that in normal control group (all<0.05); and the trabecular width and the number in ITFC-2 group were also significantly higher, and serum TRACP 5b level was significantly lower than that in normal control group (all<0.05). The BMD of bone, serum BALP level, trabecular number and area in ITFC-2 group were significantly higher, and serum TRACP 5b level was significantly lower than that in ITFC-1 group (all<0.05).ITFC can prevent osteoporosis by increasing bone density and bone formation, decreasing bone resorption and improving microstructure of bone.
Alkaline Phosphatase ; blood ; drug effects ; Animals ; Bone Density ; drug effects ; Bone Resorption ; drug therapy ; Cancellous Bone ; anatomy & histology ; Dose-Response Relationship, Drug ; Female ; Femur ; anatomy & histology ; Flavonoids ; pharmacology ; Lumbar Vertebrae ; anatomy & histology ; Osteogenesis ; drug effects ; Osteoporosis ; prevention & control ; Rats ; Rats, Sprague-Dawley ; growth & development ; Tartrate-Resistant Acid Phosphatase ; blood ; drug effects ; Tibia ; anatomy & histology

OBJECTIVETo investigate whether Epimedium brevicornu Maxim (EB) and icariin could exert their protective effects on hydrocortisone induced (HCI) rats by regulating the hypothalamus-pituitary-adrenal (HPA) axis and endocrine system and the possible mechanism.

METHODSMale 10-week-old Sprague Dawley (SD) rats were allotted to 6 groups (A-F) with 12 each, group A was injected normal saline (NS) 3 mL/kg day intraperitoneally, group A and B were given NS 6 mL/kg day by gastrogavage, group B-F were injected hydrocortisone 15 mg/kg intraperitoneally, group C and D were given EB 8 or 5 g/(kg day) by gastrogavage, group E and F were given icariin 25 or 50 mg/(kg day) by gastrogavage. Gene expressions of hypothalamus corticotropin releasing hormone (CRH) and pituitary proopiomelanocortin (POMC) were detected by reverse transcription-polymerase chain reaction (RT-PCR), and protein of pituitary POMC by Western-blot.

RESULTSThe serum T4, testosterone, cortisol and POMC mRNA expression were increased after treatment with EB or icariin in HCI rats, the serum CRH and the hypothalamus CRH mRNA expression released from hypothalamus corticotropin decreased compared with group B (P<0.05).The treatment with only icariin increased serum adrenocorticotropic hormone (ACTH) compared with group B (P<0.05).

CONCLUSIONEB and icariin might be therapeutically beneficial in the treatment of HCI rats through attuning the HPA axis and endocrine system which was involved in the release of CRH in hypothalamic, and the production of POMC-derived peptide ACTH in anterior pituitary, the secretion of corticosteroids in adrenal cortex.

Adrenocorticotropic Hormone ; blood ; Animals ; Blotting, Western ; Corticotropin-Releasing Hormone ; blood ; genetics ; Epimedium ; Flavonoids ; administration & dosage ; pharmacology ; therapeutic use ; Gene Expression ; Hydrocortisone ; pharmacology ; Hypothalamo-Hypophyseal System ; drug effects ; Hypothalamus ; chemistry ; Male ; Pituitary-Adrenal System ; drug effects ; Plant Extracts ; pharmacology ; Pro-Opiomelanocortin ; chemistry ; genetics ; Proteins ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction