Effects of isopsoralen on tibial fracture and vascular healing in mice / 中国骨伤

OBJECTIVE@#To explore effects of isopsoralen (ISO) with different doses on fracture and vascular healing in mice.@*METHODS@#Sixty 2-month-old male C57BL/6 mices with body mass of (20±2) g were selected and divided into 4 groups by random number table method:model group (model), low dose group (isopsoralen-low dose, ISO-L), medium dose group (isopsoralen-medium dose, ISO-M) and high dose group (isopsoralen-high dose, ISO-H), with 15 animals in each group. The right tibial fracture model was established. After operation, ISO-L group, ISO-M group and ISO-H group were given ISO concentration of 10 mg·kg-1, 20 mg·kg-1 and 40 mg·kg-1, respectively. Model group was given same volume of normal saline once a day for 28 days. Weighed once a week. X-ray was performed on 7, 14, 21 and 28 days, respectively, and modified I.R. Garrett scoring method was used to evaluate callus growth. After 28 days, the main organs were stripped and weighed, and organ coefficients were calculated. Hematoxylin eosin staining (HE staining) was performed on the organs to observe whether there were pathological structural changes. Micro-computed tomography (Micro-CT) was used to scan fracture area and conduct three-dimensional reconstruction to obtain the effect map, and quantify bone volume fraction (bone volume/total volume, BV/TV). After decalcification, the tibia was embedded in paraffin wax and sectioned. The healing and shape of fracture end were observed by HE staining and ferruxin solid green staining. The right tibia was removed and decalcified after intravascular infusion of Microfil contrast agent. Micro-CT was used to scan the callus microvessels in the fracture area, and the vascular volume fraction and vessel diameter were quantified.@*RESULTS@#After 28 days of administration, there was no significant difference in body mass and organ coefficient among all groups (P>0.05), and no significant pathological changes were found in HE staining of organs. The results of X-ray and improved I.R. Garrett score showed that ISO-M group was higher than that of Model group at 28 days (P<0.05). Scores of ISO-H group at 14, 21 and 28 days were higher than those of the other 3 groups (P<0.05). Micro-CT results showed intracavitary callus in ISO-M group was significantly reduced, which was lower than that in Model group (P<0.05), most of the callus in ISO-H group were subsided, and BV/TV in ISO-H group was lower than that in the other 3 groups (P<0.05). The results of HE staining and ferrubens solid green staining showed fracture area of ISO-H group was closed, continuous laminar bone had appeared, and the fracture healing process was higher than that of other groups. Angiographic results showed vascular volume fraction in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05), and the vascular diameter in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05).@*CONCLUSION@#In the concentration range of 10-40 mg·kg-1, ISO has no obvious toxic and side effects, and could improve bone microstructure, promote formation of callus microvessels, and accelerate healing of fracture ends in a concentration-dependent manner.

Formulation optimization, in situ intestinal absorption and permeability of psoralen and isopsoralen / 中草药·英文版

Objective: To optimize a self-emulsifying drug delivery system (SEDDS) formulation for psoralen and isopsoralen (PSO and IPSO) isolated from Psoraleae Fructus. Methods: A D-optimal design was used to investigate the influence of oil percentage, surfactant percentage and cosurfactant percentage on several properties of SEDDS including particle size, polydispersity, equilibrium solubility, in situ intestine absorption rate and intestinal permeability. Furthermore, the desirability function approach was applied to obtain the optimal formulation for the system. Results: The oil percentage, surfactant percentage and cosurfactant percentage were optimized to be 53.6%, 35.7% and 10.7%, respectively, which means the model is available. Conclusions: The D-optimal design is valuable to optimize the SEDDS formulation and understand formulation compositions’ functions on SEDDS properties.

Analysis of standard decoction of salt-processed Psoraleae Fructus / 中国中药杂志

The traditional Chinese medicine standard decoction is prepared on the basis of the theory of traditional Chinese medicine and clinical application. With reference to the modern extraction method,the single decoction of traditional Chinese medicine is prepared by the standardized process,and the establishment of its quality standards is conducive to standardizing clinical medication. This research is to set an evaluation standard for the quality of salt-processed Psoraleae Fructus standard decoction. Twelve batches of salt-processed Psoraleae Fructus standard decoctions were prepared. The contents of psoralen and isopsoralen were determined,the transfer and extract rates were calculated,and the pH value was measured; HPLC fingerprint method was established for analysis. The results of the 12 batches of samples revealed that the transfer rates of psoralen and isopsoralen were 17. 10%-26. 40%,14. 70%-22. 70%,respectively; the extract rate was between 14. 7%-27. 0%,and the pH value was between 5. 4-6. 9. Moreover,7 common chromatographic peaks were determined based on fingerprint by using similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine( 2012 A).The similarities of the 12 batches of samples were analyzed and compared,and the results showed that the similarities were all higher than0. 9. In this study,the preparation method for salt-processed Psoraleae Fructus decoction was standard,with high similarities in fingerprint. This study build a convenient and reliable method of comprehensive quality evaluation,with a high precision,stability and repeatability,which can provide a reference for the quality control of salt-processed Psoraleae Fructus dispensing granules.

Psoralen and isopsoralen improve lipid metabolism disorder via inhibition of NF-κB activation in LO2 cells / 中国中药杂志

The aim of this paper was to investigate the mechanism and effect of psoralen and isopsoralen in the treatment of lipid accumulation in LO2 cells. Human LO2 cells nonalcoholic fatty liver models were established by using palmitic acid( PA). Then psoralen and isopsoralen were administered for intervention. Intracellular triglyceride( TG) and total cholesterol( TC) content,the cell supernatant alanine aminotransferase( ALT) and aspartate aminotransferase( AST) levels were determined by enzyme method. Cell supernatant proinflammatory cytokines( IL-6,TNF-α) and chemokines( IL-8,MCP-1) were determined by ELISA method. Western blot method was conducted to detect the protein expression of intracellular nuclear factor( NF-κB) p65 phosphorylation( p-p65),nonphosphorylated protein( p65),and transforming factor TGF-β1. Result showed that as compared with the model group,intracellular TG and TC levels,the cell supernatant ALT and AST levels,proinflammatory cytokines and chemokines were decreased( P < 0. 01,P <0. 05); the p-p65/p65 ratio and TGF-β1 protein expression were also significantly decreased( P< 0. 01,P< 0. 05) in psoralen intervention group. As compared with the model cells,intracellular TG content had no significant changes,but all the other indexes were reduced( P<0. 01,P<0. 05) in the cells of isopsoralen intervention group. Psoralen exhibited better effect than isopsoralen( P< 0. 01,P<0. 05). It is concluded that psoralen could improve the adipogenesis of LO2 cells induced by PA; both psoralen and isopsoralen are effective in ameliorating LO2 cells injury induced by PA,reducing inflammation via inhibiting the activation of NF-κB and down-regulating the expression of TGF-β1.

Effect of isopsoralen on hepatic function and bile acid transporters in SD rats with different administration periods / 中国药理学通报

Aim: To investigate the effect of isopsoralen, the active ingredient of Psoralea corylifolia, on rat liver and bile acid transporters after oral administration for different time. Methods: Rats were randomly divided into four groups; the control group and the groups treated with 60 mg · kg-1 isopsoralen for 1, 3 or 7 days. After the experiment, the body weight and liver weight were measured. The levels of ALT, AST, ALP, TBA, TC, TG and TBIL in rat serum were examined by different kits. The mRNA levels of BSEP, NTCP, MRP2, MRP4, MDR1, ABCG5, ABCG8 and OSTa in rat liver were detected by real-time PCR. Results: Compared with control group, isopsoralen could induce the increase of liver weight and liver/body weight ratio. The levels of ALT, AST, TBA, TG and TBIL significantly increased after administration of isopsoralen. The content of ALP did not change significantly and the content of TC decreased remarkably. Furthermore, the level of AST significantly increased after one day of isopsoralen administration. After treatment with isopsoralen, the mRNA levels of BSEP, NT-CP, MRP2, MDR1, ABCG5, ABCG8 and OSTa were distinctly reduced. The mRNA levels of MRP4 showed no significant difference. Conclusions: The administration of isopsoralen for 1 to 3 days may cause obvious liver injury. The mechanism underlying isopsoralen-induced injury may be associated with the interference of bile acid transporters.

Simultaneous determination of 16 chemical components in Psoraleae Fructus based on quantitative analysis of multi-component with single-marker / 中草药

Objective: The purpose of this study was to establish a QAMS analytical method for 16 compounds in Psoraleae Fructus, and attempt to evaluate the quality difference among different batches of Psoralen Fructus by chemometrics. Methods: All experiments were performed on three different HPLC instruments. Isopsoralen was used as the internal reference substance to determine the relative correction factors of the other 15 compounds. The robustness and durability of the measured relative correction factors of the 15 compounds were evaluated on different chromatograph instruments and columns; And the measurement result deviation was compared between QAMS method and the external standard method. Results: Under the established chromatographic conditions, the relative correction factors of 15 compounds in Psoraleae Fructus had high accuracy, good durability, and good reproducibility under different experimental conditions. The results obtained from two analysis methods showed no significant deviation. The results obtained by the new established QAMS analytical method showed that the consistency of compound types among different batches of Psoraleae Fructus were better, but the content of different componounds was relatively different. Conclusion: The newly established QAMS analytical method for simultaneous determination of 16 compounds in Psoraleae Fructus provides a more efficient method to evaluate the comprehensive quality of Psoraleae Fructus from different sources.

Quality evaluation system for standard decoction of salt-processed Psoraleae Fructus / 中草药

Objective To establish a quality evaluation system for standard decoction of salt-processed Psoraleae Fructus (PF). Methods Fifteen batches of crude drugs were collected and processed into decoction pieces with salt complied with the 2015 Edition of the Chinese Pharmacopoeia, and then the standard decoctions of salt-processed PF were prepared as freeze-dried powders. Based on the established HPLC fingerprint, seven common peaks were recognized and the main chemical constituents were identified in combination with time-of-flight mass spectrometry. Results Benzofuran glycosides and furanocoumarins turned out to be the main composition of standard decoction of salt-processed PF. The dry extract yielding rate varied from 16.31% to 20.59%, while the total contents of psoralen and isopsoralen were 1.17%—1.50% with a transfer rate ranging from 13.55% to 23.57%, and the fingerprint similarity of 15 batches of samples were all higher than 0.9. Conclusion This stable and reliable method of comprehensive quality evaluation for standard decoction of salt-processed PF may provide reference for quality control of salt-processed PF dispensing granules and related preparations.

In vitro dissolution and anti-vitiligo activity of isopsoralen nanostructured lipid carriers / 中草药

Objective To evaluate the in vitro dissolution characteristic of IPRN-NLC and to study its effects on B16F10 cells proliferation, melanin synthesis, and tyrosinase activity. Methods The dynamic dialysis was employed to compare the in vitro dissolution of IPRN and IPRN-NLC; MTT assay was used to detect the proliferation of B16F10; The tyrosinase activity was determined by L-DOPA-oxidation; The melain content was determined by GENMED Cell Melanin Quantitative Assay Kit. Results The accumulation dissolution of IPRN-NLC was 67.31% within 72 h, which showed sustained release; While the dissolution of IPRN-suspension, IPRN-physical mixture, and IPRN-DMSO were 53.34%, 90.30%, and 98.67%, respectively. The IPRN-NLC could significantly promote the proliferation, tyrosinase activity and melanin content compared with IPRN DMSO groups (P < 0.05) at the same concentration. Conclusion IPRN-NLC could increase the solubility of the drug with sustained release, and showed good cell biology intermiscibility, which could significantly increase the effects on B16F10 cells.

Simultaneous determination of nine bioactive components in Sishen Pills by HPLC-ESI-MS/MS / 中草药

Objective To develop and validate an high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method for simultaneously qualitative and quantitative determination of nine major bioactive components (deoxyschizandrin, γ-schizandrin, schizandrin, schizandrol B, schisantherin A, psoralen, isopsoralen, evodiamine, and rutaecarpine) in Sishen Pills. Methods The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C18 column (100 mm × 4.6mm, 3.5 μm) with a gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.4 mL/min, and the injection volume was 20 μL. The nine major bioactive components were detected using an electrospray ionization source in positive ionization mode (ESI+) and quantified by multiple reaction monitor (MRM) scanning at the same time. Results The linear ranges of deoxyschizandrin, γ-schizandrin, schizandrin, schizandrol B, schisantherin A, psoralen, isopsoralen, evodiamine, and rutaecarpine were 8.50-850.00 ng/mL (r = 0.999 7), 1.32-132.00 ng/mL (r = 0.997 4), 9.60-960.00 ng/mL (r = 0.999 8), 12.00-1 200.00 ng/mL (r = 0.999 3), 11.50-1 150.00 ng/mL (r = 0.997 9), 21.70-2 170.00 ng/mL (r = 0.999 7), 23.80-2 380.00 ng/mL (r = 0.999 6), 10.70-1 070.00 ng/mL (r = 0.999 5), 8.54-854.00 ng/mL (r = 0.998 0), and the average recoveries were 98.3% (RSD = 2.21%), 100.3% (RSD = 1.78%), 99.2% (RSD = 2.19%), 100.4% (RSD = 2.23%), 99.1% (RSD = 2.18%), 97.7% (RSD = 3.03%), 99.0% (RSD = 2.51%), 98.9% (RSD = 2.72%), and 100.3% (RSD = 2.10%), respectively. The contents of eight batches of the nine major bioactive components were 67.6-425.6, 0-131.5, 2.1-258.0, 0-71.2, 23.2-678.8, 806.4-1310.8, 718.5-1293.7, 11.5-123.2, and 10.9-62.4 μg/g, respectively. Conclusion The developed method is simple, specific, and sensitive, and it can be applied for the determination of nine major bioactive components and the quality control of Sishen Pills collected from different production batches.

Simultaneous Determination of 4 Constituents in Yaotong Capsules by HPLC / 中国药房

OBJECTIVE:To establish a method for the simultaneous determination of paeoniflorin,cinnamaldehyde,psoralen and isopsoralen in Yaotong capsules.METHODS:HPLC method was adopted.The determination was performed on Agilent-C18 column with mobile phase consisted of acetonitrile-water (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelengths were set at 240 nm (paeoniflorin,psoralen,isopsoralen) and 290 nm (cinnamaldehyde).The column temperature was 35 ℃,and sample size was 10 μL.RESULTS:The linear ranges of paeoniflorin,cinnamaldehyde,psoralen and isopsoralen were 0.004 02-0.100 6 mg/mL(r=0.999 8),0.024 50-0.612 7 mg/mL(r=0.999 8),0.005 24-0.131 0 mg/mL(r=0.999 9),0.005 11-0.127 8 mg/mL(r=0.999 9),respectively.LOQ were 37.17,1.47,21.76,25.57 ng,respectively;LOD were 11.15,0.44,6.53,7.67 ng,respectively.RSDs of precision,stability and reproducibility tests were all lower than 2.0％.The recoveries were 99.02％-100.83％ (RSD=0.68％,n=6),98.58％-100.99％ (RSD=0.83％,n=6),99.78％-101.69％ (RSD=0.89％,n=6),100.06％-102.46％ (RSD=0.92％,n=6),respectively.CONCLUSIONS:The method is simple,accurate and suitable for simultaneous determination of paeoniflorin,cinnamaldehyde,psoralen and isopsoralen in Yaotong capsules.

Simultaneous Determination of 7 Kinds of Components in Qing'e Pills by HPLC / 中国药房

OBJECTIVE:To develop a method for simultaneous determination of psoralen,isopsoralen,bergapten,imperato-rin,trimethylpsorale,neobavaisoflavone and bavachin in Qing'e pills. METHODS:HPLC method was adopted. The separation was performed on Kinetex-C18 column with mobile phase consisted of methanol-0.1% glacial acetic (gradient elution) at the flow rate of 1.0 mL/min. The detection wavelength was set at 248 nm. The column temperature was 30 ℃. The sample size was 20 μL. RESULTS:The linear ranges were 1.012-101.2 μg/mL for psoralen(r=0.9999),1.007-100.7 μg/mL for isopsoralen(r=0.9997), 1.010-101.0μg/mL for bergapten(r=0.9999),1.021-102.1μg/mL for imperatorin(r=0.9999),1.002-100.2μg/mL for trimethyl-psorale(r=0.9996),1.008-100.8 μg/mL for neobavaisoflavone(r=0.9999),1.025-102.5 μg/mL for bavachin(r=0.9998),re-spectively. The limits of quantitation were 0.15,0.15,0.30,0.30,0.15,0.30,0.30 μg/mL,and the limits of detection were 0.05, 0.05,0.10,0.10,0.05,0.10,0.10 μg/mL,respectively. RSDs of precision,stability and reproducibility tests were lower than 2.0%. The recoveries were 95.3%-99.6%(RSD=1.9%,n=6),96.1%-99.3%(RSD=1.2%,n=6),95.2%-98.4%(RSD=1.4%,n=6),95.4%-99.2%(RSD=1.5%,n=6),96.1%-99.3%(RSD=1.5%,n=6),95.6%-98.9%(RSD=1.4%,n=6), 95.2%-99.6%(RSD=1.6%,n=6),respectively. CONCLUSIONS:The method is simple and accurate,can be used for simultane-ous determination of 7 kinds of components in Qing'e pills.

Comparative Study on the Processing Technology of Psoralea corylifolia by Salt Frying and Salt Steaming / 中国药房

OBJECTIVE:To compare the contents of 2 index components in salt frying and salt steaming samples of Psoralea corylifolia,and select better processing technology. METHODS:Under the conditions that ratio of salt and medicinal materials was 1:50,4-5 times salt of adding water,brine run time was 2 h,the amounts of psoralen and isopsoralen in salt frying samples after 10,12,14 min of frying under 150 ℃,170 ℃,190 ℃ and in salt steaming samples after 1,1.5,2,2.5 h of steaming were re-spectively investigated,and the effects of 2 processing technology were compared. RESULTS:Under fixed condition,the contents of index components was the highest in salt frying samples by fried for 12 min under 150 ℃ and in salt steaming samples by steamed for 1 h;the content in salt steaming samples (1.49%) was higher than that in salt frying samples (1.29%)(P=0.011). CONCLUSIONS:According to the comparison of index components'contents in processing samples,salt steaming is superior to salt frying of P. corylifolia.

Study on quality standard improving of Shenmai Dihuang Pills / 中草药

Objective: To develop the quality standard for Shenmai Dihuang Pills (SDP). Methods: The TLC methods applied to identify MoutanCortex, CorniFructus, Alismatis Rhizoma, and GlehniaeRadix in the formulation were carried out according to the methods recorded in general rules of Chinese Pharmacopeia (2015 edition, volume 4). The contents of psoralen, iso psoralen, and paeonol were analyzed by high performance liquid chromatography on a Diamonsil C18 column (250 mm ×4.6 mm, 5μm) with the mobile phase of acetonitrile (A)-0.1% formic acid (B) by gradient elution at a flow rate of 1.0 mL/min. The detection wavelength was set at 254 nm and the column temperature was controlled at 30℃. Results: There were good specificities of TLC for MoutanCortex, CorniFructu, Alismatis Rhizoma, and Glehniae Radix. The methodology validation for the assay of psoralen, iso psoralen, and paeonol presented that they were in good linear correlation in the ranges of 6.531-58.78, 6.115-55.04 and 20.40-183.6 μg/mL, with the regression equations of Y =1.523×104X-1.209×104 (r = 0.999 9), Y =1.809×104 X-1.523×104 (r = 0.999 9), and Y =2.760 ×104 X-1.683 ×104 (r = 0.999 9), respectively. The average recoveries were 100.4% (RSD 1.1%), 101.5% (1.3%), and 101.5% (0.79%).The content ranges of psoralen, isopsoralen, and paeonol from five different batches of SDP were 322.1-331.0, 298.0-350.1, and 929.5-982.7 μg/g, respectively. Conclusion: The established quality of TLC and HPLC methods are specific, reliable, accurate, and suitable, which can be successfully applied to the quality control for the preparation.

Preparation of isopsoralen loaded nanostructured carrier and its in vitro transdermal permeation characteristics / 中国中药杂志

To increase the permeation and retention of isopsoralen in skin, and improve its bioavailability.Isopsoralen loaded nanostructure liquid carrier (IPRN-NLC) was prepared by high pressure homogenization andoptimized by orthogonal experiment with the encapsulation efficiency, drug loading and average particle size as the evaluation indexes. The in vitro transdermal permeation of IPRN-NLC was evaluated by Franze diffusion cells.The results showed that solid-liquid lipid ratio of optimum IPRN-NLC formulation was 7∶3,drug-lipid ratio of 1∶30, 1% surfactant. Under these conditions, IPRN-NLC had an average encapsulation of （90.25±0.73）%,drug loading of （1.56±0.27）% and an average particle size of (305±1.57) nm.The in vitro transdermal permeation results showed that IPRN-NLC could increase the amount of IPRN permeated though skin, with 3 times of the epidermal retention as compared with IPRN solution. From the results we can know that the IPRN-NLC prepared by high pressure homogenization can improve the permeation andaccumulation of IPRN in the skin, with wide application prospects in the field of transdermal administration.

Analysis on pharmacokinetics-pharmacodynamics correlation of effective ingredients of Qing'e wan / 中国中药杂志

To study the pharmacokinetics of three active ingredients in Qing'e wan, namely geniposidic acid, psoralen and isopsoralen, in rats, in order to investigate their correlation in the anti-osteoporotic effect. The rats were taken blood from their eye sockets at different time points after being orally administered with raw and salt-processed Qing'e wan. Geniposidic acid, psoralen and isopsoralen in rats plasma were determined by means of UHPLC-MS/MS to draw the concentration-time curve. The proliferation rate of osteoblasts was taken as the pharmacodynamic index, and determined by MTT method to draw effect-time curve. In comparison between the effect-time curve and the concentration-time curve, the blood concentrations of geniposidic acid and psoralen were close to the peak when the cell proliferation rate reached its peak, indicating a good correlation between them. The peak blood concentration of isopsoralen was slightly lagging behind the peak of efficacy. According to the correlation analysis after fitting the effect-time curve and the concentration-time curve, salt-processed Qing'e wan had a better correlation than the raw one. The above experimental results showed that the effect-time curve and the concentration-time curve of geniposidic acid and psoralen had a good correlation, and the correlation of salt-processed Qing'e wan was better than the raw one.

Effect of XuanHuSuoSan and its disassembled prescriptions on dissolution of psoralen and isopsoralen / 国际药学研究杂志

Objective To investigate the effect of XuanHuSuoSan and its disassembled prescriptions on dissolution of psoralen and isopsoralen. Methods The dissolution of psoralen and isopsoralen in XuanHuSuoSan and its disassembled prescriptions were determined by HPLC, using ES Industries Epic Polar C18 column (250 mm×4.6 mm, 5 μm) with methanol and water (55: 45) as mobile phase at 25 °C with a flow rate of 1.0 ml/min and detection wavelength of 246 nm. Results The dissolution of psoralen and isopsoralen had great changes among XuanHuSuoSan and its disassembled prescriptions, and the analysis of variance and SNK showed that there were significant differences among XuanHuSuoSan and its disassembled prescriptions (P < 0.05). Conclusion Radix Angelicae Sinensis and Radix Achyranthis Bidentatae are helpful to the dissolution of psoralen and isopsoralen from Fructus Psoraleae, while Rhizoma Corydalis can inhibit the dissolution of psoralen and isopsoralen.

Simultaneous Determination of 4 Components in Danzhi Qing’e Tablet by HPLC / 中国药房

OBJECTIVE:To establish a method for the contents determination of psoralen,isopsoralen,psoralenoside and isop-soralenoside in Danzhi qing’e tablet. METHODS:HPLC performed on the column of Eclipse XDB-C18 with mobile phase of metha-nol-water(51∶49,V/V)(isocratic elution,for psoralen and isopsoralen)and acetonitrile-0.1% formic acid(12∶88,V/V)(isocratic elution,for psoralenoside and isopsoralenoside)at a flow rate of 1.0 ml/min,the detection wavelength was 246 nm,column tem-perature was 30℃,and injection volume was 10 μl. RESULTS:The linear range was 3.138-200.8 μg/ml for psoralen(r=0.999 9), 3.175-203.2μg/ml for isopsoralen(r=0.999 9),3.181-101.8μg/ml for psoralenoside(r=0.999 9)and 3.169-101.4μg/ml for isopso-ralenoside (r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 2%;limits of quantitation were 0.627 5 ng,0.635 0 ng,3.181 0 ng and 3.169 0 ng,the limits of detection were 0.251 0 ng,0.254 0 ng,1.273 0 ng and 1.268 0 ng;recoveries were 95.68%-102.80%(RSD=2.4%,n=6),95.91%-102.10%(RSD=2.3%,n=6),98.64%-99.13%(RSD=0.23%,n=6) and 100.20%-101.70%(RSD=0.69%,n=6),respectively. CONCLUSIONS:The method is simple and accurate, and can be used for the simultaneous determination of psoralen,isopsoralen,psoralenoside and isopsoralenoside in Danzhi qing’e tablet.

Simultaneous Determination of 5 Components in Chanlong Dingchuan Mixture by HPLC / 中国药房

OBJECTIVE:To establish a method for the simultaneous determination of psoralen,isopsoralen,epimedin B,epi-medin C and icariin in Chanlong dingchuan mixture. METHODS:HPLC was performed on the column of Welch Materials C18 with mobile phase of acetonitrile- methanol(1∶1,V/V)-water(gradient elution)at a flow rate of 0.9 ml/min,the detection wavelength was 246 nm(psoralen,isopsoralen)and 270 nm(epimedin B,epimedin C and icariin),column temperature was 30 ℃,injection volume was 20 μl. RESULTS:The linear range was 8.24-164.80 μg/ml for psoralen(r=0.999 7),5.15-103.00 μg/ml for isopso-ralen(r=0.999 3),4.06-81.20 μg/ml for epimedin B(r=0.999 6),5.88-117.60 μg/ml for epimedin C(r=0.999 5)and 4.90-98.00μg/ml for icariin(r=0.999 8);the limits of quantitation were 0.385 μg/ml,0.179 μg/ml ,0.124 μg/ml,0.218 μg/ml and 0.348 μg/ml, limits of detection were 0.127μg/ml,0.059μg/ml ,0.041μg/ml,0.072μg/ml and 0.115μg/ml;RSDs of precision,stability and re-producibility tests were lower than 2.0%;recoveries were 97.93%-100.06%(RSD=0.80%,n=6),96.91%-100.16%(RSD=1.37%,n=6),96.95%-99.63%(RSD=0.98%,n=6),96.69%-99.33%(RSD=1.03%,n=6) and 96.76%-98.53%(RSD=0.70%,n=6),respectively. CONCLUSIONS:The method is simple and reliable,and suitable for the simultaneous determination of psoralen,isopsoralen,epimedin B,epimedin C and icariin in Chanlong dingchuan mixture.

Psoralen induced bile acid accumulation and cytotoxicity by inhibiting MRP2 and MRP3 in HepG2 cells / 中国药理学通报

Aim To investigate the toxicity of isopsor-alen in HepG2 cells and its effects on bile acid, bile acid synthesis and transport. Methods Cell viability was evaluated by MTT assay and bile acid was deter-mined inside HepG2 cells, with exposure to various isopsoralen for 24h. The mRNA transcription of BSEP, MRP2, MRP3, NTCP, OATP2, OSTα, CYP7A1, CYP27 A1 , FXR and PXR were assessed by real-time PCR. Results The cell viability was decreased dose-dependently with isopsoralen in HepG2 cells, and IC50 was 118. 1μmol·L-1 exposure to isopsoralen for 24h. Bile acid inside cells significantly increased with 100 and 400 μmol · L-1 isopsoralen. Isopsoralen caused the down-regulation of MRP2 , MRP3 , CYP7 A1 mRNA at 25 μmol · L-1 . Beside these, the up-regulation of OATP2,OSTα,CYP27A1,FXR,PXR with 100 μmol· L-1 isopsoralen, but there was no significant change of BSEP and NTCP. Conclusion The results show that isopsoralen induces bile acid accumulation and cytotox-icity which may be associated with the down-regulation of MRP2, MRP3 in HepG2 cells.

Enzyme kinetics of psoralen and isopsoralen in rat and human liver microsomes / 中国药理学与毒理学杂志

OBJECTIVE To investigate and compare the enzyme kinetic characters of psoralen (PRN)and isopsoralen(IPRN)in rat and human liver microsomes. METHODS PRN and IPRN in liver microsomes incubates were determined using LC-MS/MS. The enzyme kinetic and metabolic stability of PRN and IPRN were investigated by employing the optimized rat and human liver microsomes incubations. The Vmax and Km values were calculated using the nonlinear regression method. RESULTS The quanti?tative method showed good linearity within the range of 0.1-50.0 μmol · L-1 and was suitable for the assay in biological samples. The in vitro elimination was linear with the substrate concentrations lower than 1 μmol,the protein concentration within 0.5 g · L-1,and the incubation time within 40 min. The t1/2 values of PRN and IPRN in rat and human liver microsomes were 74.5,95.0,74.5 and 173.3 min, respectively. The Vmax values of PRN in rat and human liver microsomes were(1.140±0.080)μmol·min-1·g-1 protein,(0.620±0.060)μmol·min-1·g-1 protein,while Km values of PRN in rat and human liver microsomes were (12.9 ± 0.3)μmol · L- 1,(7.4 ± 1.3)μmol · L- 1,respectively. The Vmax values of IPRN in rat and human liver microsomes were(0.251±0.012)and(0.103±0.014)μmol·min-1·g-1 protein,while Km values of IPRN in rat and human liver microsomes were (3.0 ± 0.4)μmol · L-1,(3.4 ± 0.7)μmol · L-1,respectively. CONCLUSION The enzyme kinetic characters and metabolic stability of PRN and IPRN show species and chemical structures related differences. Interestingly,the metabolic eliminations of PRN and IPRN are similar in rats. However,the metabolic elimination of IPRN in humans involved in CYP enzymes may be much slower than that of PRN.