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To explore the effect of geniposidic acid (GPA) on the signal pathway of small heterodimer dimer receptor (SHP) and liver receptor homologue 1 (LRH-1) in cholestasis rats induced by alpha-naphthalene isothiocyanate (ANIT). Methods: Fifty SD rats were randomly divided into five groups: a blank group, an ANIT group, an ANIT+GPA (100 mg/kg) group, an ANIT+GPA (50 mg/kg) group, and an ANIT+GPA (25 mg/kg) group (n=10 in each group). The GPA were intragastrically given to rats for 10 days, and the control group and the ANIT group were given normal saline. At the eighth day of administration, all rats except the blank group were given 65 mg/kg ANIT once until the tenth day. After the last administration, serum total cholesterol (TC), triglyceride (TG) and total bile acids (TBA) were measured. The primary hepatocytes (RPH) were isolated from normal rats and cultured. The cells were divided into a blank group, an ANIT (40 μmol/L) group, an ANIT (40 μmol/L)+GPA (4.00 mmol/L) group (A4.00G group), an ANIT (40 μmol/L)+GPA (1.00 mmol/L) group (A1.00G group), and an ANIT (40 μmol/L)+GPA (0.25 mmol/L) group (A0.25G group). The mRNA transcription levels of SHP and cholesterol 7 alpha hydroxylase (CYP7A1) in RPH were detected by real-time-PCR, and the protein levels of SHP and CYP7a1 were detected by Western blotting. In the LRH-1 silence experiment, the RPH were divided into a blank group, a negative transfection group, a siRNA-LRH group (ZR group), a siRNA-LRH+GPA (4.00 mmol/L) group (ZR4.00G group), a siRNA-LRH+GPA (1.00 mmol/L) group (ZR1.00G group) and a siRNA-LRH+GPA (0.25 mmol/L) group (ZR0.25G group). The protein and mRNA levels of SHP, CYP7a1, LRH-1 were detected. In the over-expression experiment, the RPH were also divided into a blank group, a negative transfection group, a LRH-1 over-expression plasmid group (OE group), a LRH-1 over-expression plasmid+GPA (4.00 mmol/L) group (OE4.00G group), a LRH-1 over-expression plasmid+GPA (1.00 mmol/L) group (OE1.00G group), and a LRH-1 over-expression plasmid+GPA (0.25 mmol/L) group (OE0.25G group). The protein and mRNA levels of SHP, CYP7a1 and LRH-1 were detected. Results: Compared with the blank control group, TC and TBA were significantly increased (both P<0.01) in the ANIT group, but there was no difference in TG; compared with the ANIT group, the contents of TC and TBA in the AG100 and AG50 groups were significantly reduced (all P<0.01). Compared with the blank control group, the proteins and mRNA levels of SHP were significantly decreased (P<0.01), while CYP7a1 were dramatically increased (P<0.01) in the ANIT group; compared with the ANIT group, the proteins and mRNA levels of SHP in the A4.00G group and the A1.00G group were significantly increased (both P<0.01), while the levels of CYP7a1 proteins and mRNA levels were evidently decreased in the A4.00G and A1.00G groups (both P<0.01). Compared with the negative transfection group, the proteins and mRNA levels of CYP7a1 and LRH-1 were dramatically restrained (all P<0.01), while there was no change in SHP in the ZR group; compared with the ZR group, the proteins and mRNA levels of SHP were significantly increased (all P<0.01), while LRH-1 and CYP7a1 were not changed in the ZR4.00G, ZR1.00G and ZR0.25G groups. Compared with the negative transfection group, the protein and mRNA levels of CYP7a1 and LRH-1 were significantly suppressed in the OE group (all P<0.01). Compared with the OE group, the protein and mRNA levels of SHP were evidently increased in the OE4G and OE1G groups (all P<0.01), while LRH-1 and CYP7a1 were not changed in the OE4G, OE1G and OE0.25G groups. Conclusion: The over-expression of LRH-1 in RPH can up-regulate the mRNA and protein levels of CYP7a1. GPA can improve the biochemical and liver pathology of ANIT-induced cholestasis rats, which may be related to the decrease of CYP7a1 by activating SHP through LRH-1 in RPH.
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Animals , Rats , Cholestasis , Iridoid Glucosides , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear , Signal TransductionABSTRACT
Objective To observe the damage effect of borneol on rabbit corneal epithelial cells. Methods After the treatment with borneol at 100, 200, 400 μg·mL-1 respectively, the viability of rabbit corneal epithelial cells was determined by methyl thiazolyl tetrazolium ( MTT) assay, cell apoptosis was determined by flow cytometry with Annexin V- fluorescein isothiocyanate/propidium iodide ( FITC/PI) staining, and Caspase-3 mRNA expression was detected with real-time reverse transcription polymerase chain reaction ( RT-PCR) . Results Borneol at the concentrations of 100, 200, 400 μg·mL-1 inhibited the activity of rabbit corneal epithelial cells. Compared with the normal control group, borneol increased the rate of apoptosis, and enhanced the Caspase-3 mRNA expression in rabbit corneal epithelial cells ( P<0.05 or P<0.01) . Conclusion Borneol can inhibit the proliferation of rabbit corneal epithelial cells, induce cell apoptosis through enhancing the expression of apoptosis-related gene Caspase-3 mRNA.
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Objective To establish a HPLC fingerprint analysis method of salt-prepared Cortex Phellodendri. Methods C18 column was used, with gradient elution by the mobile phase consisted of acetonitrile-0. 1% phosphoric acid (contained 0. 2 % triethylamine). The detection wavelength was at 230 nm, and the flow rate was at 0. 8 mL/min. Results Seventeen char-acteristic peaks were selected and the fingerprints on HPLC was set up. Conclusion HPLC fingerprint method is repro-ducible, accurate and stable, and can be used for the quality control of salt-prepared Cortex PheUodendri.
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Objective To explore the optimal method of extraction and determination of rhynchophylline and isorhynchophylline in Ramulus Uncariae cum Uncis. Methods A RP-HPLC was performed on a Phenomenex C18 column (4. 6 mm× 150 mm, 5m) at 30℃. The mobile phase consisted of methanol-0. 01 mmol/L triethylamine solution (70: 30, pH 7.0 adjusted by glacial acetic acid) at the flow rate of 0. 5 mL/min. The automatic sample injector was set at 5℃ and the ultraviolet detector was operated at 245 nm. And then, the effect of different extraction condition on the contents of rhynchophylline and isorhyn-ehophylline in Ramulus Uncariae cum Uncis was investigated. ResultsA good linearity of rhynchophylline was in the range of 2. 5μg/mL to 80. 0μg/mL (Y=76. 7170X-0. 0727,r=0. 999 8),the average recoveries were from 99. 84 ％ to 116. 91％, and the RSD (n=6) were less than 8.8 ％. A good linearity of isorhynchophylline was in the range of 2. 0 μg/mL to 80. 0 μg/mL (Y=87. 4729X-0. 3666, r=0.999 7), the average recoveries were from 87.08 ％ to 104. 97 ％, and the RSD (n=6) were less than 7.3 ％. The contents of rhynchophylline and isorhynchophylline in the methanol-extracted solu- tion were approximately ten times as much as those in water-decocted solution. The main factors which had effect on the ex-traction of rhynchophyUine and isorhynchophylline in methanol solution were supersonic time,methanol amount and cold-dousing time, in a decreasing sequence. The best condition selected by orthogonal experiment wasb as follows: extracting the medicinal material with 20-fold volumes of methanol, cold maceration for 24h and supersonic extraction for 1 h. Conclusion The extraction percentage of rhynchophylline and isorhynchophylline in the extraction condition of cold maceration with methanol and supersonic extraction is superior to that in the water-decocting condition. The method is simple, fast and accurate, and it can be used for the quality control of Ramulus Uncariae cum Uncis.
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BACKGROUND: Borneol can open blood-brain barrier (BBB) but the mechanisms are not very clear. Histamine and 5-hydroxtryptamine can take part in regulation of permeability of BBB. There is not report on the rela tion between the effect of opening BBB of borneol and the regulation of permeability of BBB of histamine (HA)/5-hydroxytryptamine (5-HT). OBJECTIVE: To study the relation between the effect of opening BBB of bornool and the regulation of permeability of BBB of histamine (HA)/5-hydroxytryptamine (5-HT).DESIGN: A completely randomized and controlled study.SETTING: Clinical Pharmacological Institute of University of Traditional Chinese Medicine of Guangzhou.MATERIALS: The experiment was carried out at the Clinical Pharmacological Institute of University of Traditional Chinese Medicine of Guangzhou from September 2003 to February 2004. Totally 104 healthy male SD rats, weighting 230-27.0 g, supplied by Guangdong Medical Experimental Animal Center, were selected.METHODS: The rats were randomly divided into 13 groups: pre-medicine group and post-medicine group (high, moderate and low dosage group and 5, 20, 45 and 60 minutes group in each dosage group) with 8 in each group. Borneol was mixed as 10% millet oil suspension. Rats were fasted before experiment for whole night, and medicine was perfused on the next morning with the high, moderate and low dosage of 0.15, 0.12 and 0.09 g/kg respectively. Hypothalami of rats was selected at various time points to make biological samples. Contents of HA and 5-HT were assayed with HPLC system electrochemical detector.MAIN OUTCOME MEASURES: Changes of level of histamin (HA) and 5-HT in hypothalamus of rats after administration of borneol.RESULTS: Data of totally 104 rats was entered the final analysis. ① Contents of HA and 5-HT in hypothalami were 2.07±0.54 μg/g and 1.45±0.14 μg/g respectively. ② The level of HA in hypothalamus of rats after different doses of borneol were higher than that of before administration. Comparing the level of HA in 20 minutes after moderate dose with before administration, the level of HA in 20 minutes was increased significantly [(3.36±0.21) μg/g, P < 0.01], the others of moderate dose, the 45 minutes after high dose, the 20 and 45 minutes after low dose were also increased significantly than before administration (P < 0.05). ③ After administration of different doses of borneol, the level of 5-HT after high dose were higher than that of before administration [5, 20, 45 and 60 minutes after medicine: (1.90±0.32), (3.28 ±0.25), (2.66±0.46), (2.80±0.34) μg/g, respectively; (P < 0.05 or P < 0.01)]; the level of 5-HT after 5, 20 and 45 minutes of moderate and low dose were increased significantly too (P < 0.05 or P < 0.01).CONCLUSION: Borneol could open BBB by increasing levels of HA and 5-HT in hypothalamus of rats.Borneol mediates opening of BBB by increasing levels of HA and5-HT in rats.
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OBJECTIVE: To observe the therapeutic effect of Wuling Powder extract on rats with renal hypertension and to evaluate the influence of it on the volume of urine and the concentrations of Na(+), K(+), Cl(-). METHODS: Reformed Gold-blatt hypertension rat model (G-2K1C) was established. The rats were divided into 6 groups as follows: sham-operation group; model group, Wuling Powder high dosage group (80 g/kg), Wuling Powder middle dosage group (40 g/kg), Wuling Powder low dosage group (20 g/kg), and hydrochlorothiazide (HCT) group (25 mg/kg). Urine volume of the rats was measured during the experiment. Tail arterial pressure and [Na(+)], [K(+)], [Cl(-)] in serum of the rats were detected after 30 days of treatment. RESULTS: The blood pressure of the G-2K1C rats was decreased in the three Wuling Powder groups (P0.05). Diuretic effect of the three dosages of Wuling Powder was weaker than that of the HCT (P0.05), but [K(+)] of the HCT group was significantly decreased compared with that of the false-operation group and the three Wuling Powder groups (P<0.01). CONCLUSION: Wuling Powder extract had satisfying therapeutic effects in increasing the discharge of urine, decreasing the blood pressure and keeping the balance of the serum electrolyte contents in rats with renal hypertension.
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Objective To observe the protective effects of the extracts of Rhizoma Zingiberis and Pericarpium Citri Reticulatae on myocardial ischemia reduced by isoproterenol(ISO) in rats,and to explore their optimal ratio.Methods Acute myocardial ischemia rat model was established by continuously subcutaneous injection of large-dose.Then the content of the serum creatine kinase(CK) within 24 hours in rats were detected,and the optiaml ratio of Rhizoma Zingiberis and Pericarpium Citri Reticulatae was optimized by uniform design.Results Compared with the control group,the content of CK in the model group was significantly increased(P
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Objective To investigate the effect of borneol administered by gastric gavage or external application on ICAM-1 expression in rat retina with ischemia-reperfusion injury.Methods Rat ischemia-reperfusion model was established.Laser microscope and biological analysis microsoft were used to observe the change of ICAM-1 expression in retina or rats after gastric gavage or external application of borneol.Results Borneol administered by gastric gavage or external application obviously decreased the expression of ICAM-1 after the modeling for 24 hours,the difference being significant as compared with that in the model group(P
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Objective To determine the intestinal absorption characters of zingerone in rats.Methods The concentration of zingerone in the intestine perfusate was determined by HPLC.The absorption kinetics were obtained by the in-situ perfusion method in rats.Results The absorption rate constants(Ka) of zingerone were 0.0137,0.0135 and 0.0139 min-1 at the concentration of 25.0,50.0 and 100.0 ?g?mL-1,respectively;Ka of zingerone in the duodenum,jejunum,ileum and colon were 0.0070,0.0031,0.0032 and 0.0026 min-1,respectively.Conclusion Concentration of zingerone has no significant effect on its absorption kinetics.The best absorption segment of the intestine was duodenum,ileum,jejunum and colon by turns.The intestinal absorption of zingerone has the first-order kinetic characters with passive diffusion mechanism.
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Objective To observe the analgesic action and anti-inflammation effect of borneol. Methods The analgesic action and anti-inflammation effect of borneol were observed by mouse hot-plate test,mouse acetic-acid-induced twisting test,mouse abdominal cavity capillary permeability increase test induced by acetic acid,and rat pedal swelling test induced by carrageenin. Results Borneol reduced the pedal swelling of rats,ED50=0.3242 g?kg-1,increased pain threshold in mice ED50=0.3870 g?kg-1,inhibited twisting response of mice obviously,ED50=0.5813 g?kg-1. In the same dose,the analgesic action of borneol was stronger than the anti-inflammatory effect. Conclusion Borneol has certain analgesic action and anti-inflammation effect.
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Objective To investigate the effects of Rhizoma Zingiberis Extract (RZE) on the cardiac functions of rabbits with heart failure. Methods Forty pure-bred New-Zealand rabbits were randomly allocated to 4 groups: control group, low-dosage RZE group, moderate-dosage RZE group and high-dosage RZE group. The rabbit model of heart failure was established by intravenous dripping of 20 g/L pentobarbital sodium. Oral tube perfusion of RZE was given after model establishment.The hemodynamic changes were observed before and after the modeling and 5,10,15,20,30,45,60,90,120,150 minutes after treatment by a RM-6000 4-graph physiological monitor. Results After treatment, LVSP, lv?dp/dtmax,lv +dp/dtmax,lv-dp/dtmax showed an ascending tend in all the groups, particularly in RZE groups; the differences between the control group and RZE groups were significant 30 minutes after treatment (P
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Objective: To study the effect of Fructus Psoraleae on antipyrine elimination in rats. Methods:After the medication of Fructus Psoraleae decoction once daily for 6 days in a dose of 5. 3 g/kg, antipyrine was fed to rats in a dose of 400mg/kg. Concentration of antipyrine in various time was measured by HPLC, and the pharmacokinetics parameters of antipyrine elimination was caculated. Results: The elimination rate constant (Kel) of antipyrine in rats fed with Fructus Psoraleae decoction was 1.55 times more than that in the control group, and the elimination half - life (t1/2) was shortened by 60. 7 % . Conclusion: Fructus Psoraleae decoction accelerate the elimination of antipyrine in rats by inducing the activities of hepatic oxygenases.
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Objective To develop a HPLC-UV method for the determination of [6]-gingerol in the rat plasma.Methods [6]-Gingerol was extracted from the rat plasma by using liquid-liquid extraction,then was separated on a Hypersil C18 column(250 mm?4.6 mm,5 ?m).The mobile phase was acetonitrile and water(45:55,v/v) with a flow-rate of 1.0 mL/min.The eluate from the HPLC column was monitored by the spectrophotometric detector at 280 nm.The injection volume was 20 ?L.[6]-Gingerol was identified based on its retention time compared with the reference standard.Results The linear calibration curve was obtained in the concentration range of 0.25～5.0 ?g/mL.The low quantitative limit was 0.1 ?g /mL.The RSD of inter-day was
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Objective To establish a HPLC fingerprint analysis method of salt-prepared Cortex Phellodendri. Methods C18 column was used, with gradient elution by the mobile phase consisted of acetonitrile-0.1 % phosphoric acid (contained 0.2 % triethylamine). The detection wavelength was at 230 nm, and the flow rate was at 0.8 mL/min. Results Seventeen characteristic peaks were selected and the fingerprints on HPLC was set up. Conclusion HPLC fingerprint method is reproducible, accurate and stable, and can be used for the quality control of salt-prepared Cortex Phellodendri.
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Objective To develop a high performance liquid chromatography-ultraviolet detection(HPLC-UV) method for the determination of midazolam in rat plasma,and to study the pharmacokinetics of midazolam in single dose intravascular administration in rats.These results can provide a methodological reference for evaluating cytochrome P450 3A(CYP3A) activity.Methods Firstly,common carotid artery(CCA) intubate was set in rats,Midazolam was injected into rat vena caudalis and plasma samples were collected in different time.Then,the sample was extracted using dichlormethane and evaporated to dry thoroughly with N2 soft stream at 37 ℃.Finally,the residues were reconstituted with 150 ?L 30 %methanol and further analyzed by HPLC.A Phenomenex C18 column(4.6?150 mm i.d,5 ?m)was employed at 20 ℃.The mobile phase consisted of(A) methanol-acetonitrile-0.03 %phosphate solution(pH 2.85) in the proportion of 5:10:85(V/V/V) and(B) methanol-acetonitrile-0.03 %phosphate solution(pH 2.85) in the proportion of 5:10:85(V/V/V),using a linear gradient elution of 0 %B～100 %B at 0～18 minutes,then retaining for 5 minutes and returning to A.The flow rate was set at 0.5 mL/min and the ultraviolet detector was operated at 240 nm.Results Midazolam and internal standard were isolated on baseline in plasma apart from all other material.The the linear range was from 0.025～2.0 ?g/mL,and the detection limit was 2.5 ng/mL.The standard addition recoveries were from 99.98 %to 105.71 %and the extraction rates were from 91.29 %to 92.58 %.All of the intraday and interday variations were less then 14 %.The primary pharmacokinetics parameters of rat single dose intravascular administration were as follows:t1/2?was 0.582 h,Vd was 0.214 L,Cl was 0.584 L/h and AUC0→t was 0.419 ?g?h-1?mL-1.Conclusion Midazolam single dose intravascular administration has the characteristics of rapid distribution and elimination in blood and quick transport from blood to tissue.The method is sensitive,simple and suitable for the research of pharmacokinetie parameters of midazolam and description of possible pharmacological interactions of rat CYP3A1/2 or human CYP3A4/5 enzymes.
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Objective To study the interaction of tetramethylpyrazine and human serum albumin(HSA).Methods The fluorescent quenching of HSA with te tramethylpyrazine has been detecte d by fluorimetry.Results It was found that fluo-rescence of HSA were quenched by tetr amethylpyrazine.The fluorescent q uenching data were analyzed accordi ng to Stern -volmer equation and the bindi ng constant was obtained.Conclusion The mechanism of fluorescent quench ing is considered to be the formation of HSA-tetramethylpyrazine complex.
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Objective To investigate the preventive and therapeutic effects of Ginger extraction on heart failure in rabbits.Methods Twenty-seven New Zealand pure-breed rabbits were randomized into 3 groups:group A,group B and group C.Group A was orally administrated with 2 %Ginger extraction 3 days before and after modeling;group B was orally administrated with purified water 3 days before modeling and with 2 %Ginger extraction after modeling;group C was orally administrated with purified water 3 days before and after the establishment.The differences of modeling time and dosage of pentobarbital sodium between groups were compared;hemodynamics parameters before and after administration were detected.Results (1)The modeling time and dosage of Pentobarbital Sodium were remarkably increased in group A,differences being significant compared other 2 groups(P
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Objective SD rat models of stomach-heat syndrome were established to further investigate the physiopathological mechanism of stomach-heat syndrome.Methods Under certain laboratory condition,criteria of stomach-heat syndrome for the animal models were set up firstly.Then decoction of Rhizoma Zingiberis was given to the rats for modeling.Two weeks after modeling,the symptoms of the rats were observed and pathological and biochemical examination was carried out.Weireqing capsules were used to confirm the success of the establishment of stomach-heat syndrome in rats.Results Two weeks after modeling,the model rats had the symptoms of thirst with preference for drinking and reddened tongue.Congestion in gastric mucosa occurred and the levels of 6-keto-PGF1?and TXB2 in the model group were higher than those in the control group.However,in model rats pretreated with Weireqing,the symptoms and signs of stomach-heat syndrome and histological changes were relieved.Conclusion Under the controlled laboratory condition,feeding rats with decoction of Radix Zingiberis can be successfully used to establish animal model of human stomach-heat syndrome .
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Objective To establish the method of measuring quercetin content in semen cuscutae(SC) and to explore its in- vivo pharmacokinetics features.Methods The rapid and sensitive HPLC- UV method was adopted with carbamazepine acting as internal standard. After the extract of SC was given, serum samples of rats were collected and measured on C18 reverse- phase chromatographic column after liquid- liquid extract. Results The linear range of calibration curve were 0.05～ 9? g/mL (r=0.9998). The method showed good precision and recoveries for serum with 86.65～ 99.07% , and extracting recoveries 79.91～ 84.56% .The limit of detection(LOD) as 0.05? g/mL. Conclusion The pharmacokinetic process of quercetin in vivo manifestated an opening two- compartment model with t1/2(Ab)=0.13h, t1/2(? )=0.19h, t1/2(? ) =1.22h, tmax=0.333h. The results may provide evidence of safety and effectiveness for clinical use of SC.
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Objective To evaluate the therapeutic effect of Wuling Powder (WLP) on adriamycin-induced nephrotic syndrome in rats. Methods Rat models of nephrotic syndrome were established by injection of adriamycin into tail veins of rats. The rats were divided into 7 groups: group A(normal control),group B (high-dosage WLP),group C (moderate-dosage WLP),group D (low-dosage WLP),group E (hydrocortisone),group F (hydrocortisone and moderate-dosage WLP extractive) and group G (model control). The influence of 4-week treatment of WLP on 24-hour urinary protein volume,serum levels of albumin,cholesterol and triglycerides as well as pathological changes of kidney tissue were assayed. Results 24-hour urinary protein volume of group B,C,E and F were significanuly lower than that of group G;Serum albumin level of group B,C,E and F differed from that of group G; Serum albumin level of group E and F were higher than that of group B and C;Serum cholesterol level of WLP groups (group B,C,E,F) was much lower than that of group G and the difference was insignificant between group B,F and group A;As for serum triglycerides level,three WLP groups and group F differed from group E while the group G not. Conclusion WLP not only can eliminate tissue edema and decrease urinary protein volume and blood lipid content,but also can increase serum albumin levels,thus it can protect the kidney from injury. There also have been showen that a synergistic action of WLP and hydrocortisone has been shown in the treatment of nephrotic syndrome.