| AimsFufang Qinghuangsan Jiaonang is composed of six kinds of traditional Chinese medicine and is usd to treat myelodysplastic syndromes in the clinic.Clinical application showed a good efficacy,but the pharmacokinetics and the study on the mechanism of K562 cells has not been carried out.Preclinical pharmacy research includs process route design,extraction process operation and optimization,three batches pilotscale and the quality standard;it observed differences in pharmacokinetic parameters of total arsenic in whole blood of ICR mouse after the administration of realgar,Qinghuang San,Fufang Qinghuangsan Jiaonang;it observed the difference of the total arsenic permeation coefficient and cumulative permeation amount across MDCK-MDR1 monolayer membrane after using realgar,Qinghuang San,Fufang Qinghuangsan Jiaonang;it investigated the dissolution rate of realgar,Qinghuang San in artificial stomach and intestinal juice;it hypothesized the structural responses of the main components of realgar and indigo naturalis,alpha-tetrasulphide tetraarsenic(α-tetrasulphate tetraarsenic)and indirubin(C16H10N2O2)of realgar and indigo naturalis,using computational quantum chemistry methods,and it used infrared spectrometer to scan the suspension of realgar and indirubin in the artificial stomach and intestinal fluid;to study the inhibition of protein tyrosine phosphatase SHP2 in vitro by different forms of arsenic,it srudied the apoptosis-inducing effect and related pathways of realgar on chronic myeloid leukemia cell line K5621;it investigated the difference of cytotoxicity on K562 cells with the incubation of realgar and combination of realgar and indigo naturalis;Methods1 With volatile oil extraction as the index,it investigated the steam distillation process of volatile oil from pericarpium citri reticulatae and its β-cyclodextrin inclusion process.Taking Paeoniflorin,Cimicifin,and 5-O-methylstemazolidine as indicators,orthogonal experiments were conducted to examine the water extraction process and the alcohol precipitation process of paeoniae radix alba.Realgar powder and indigo naturalis was mixed by grinding,mixing and convection,and the mixing uniformity of the two drugs was investigated.It investigated dry granulation process and its parameters of decoction powder,inclusion complex,realgar and indigo naturalis.Three batches of pilot tests and long-term toxicity test samples were carried out on the above process parameters.Three batches pilotscales and long-term toxicity test samples were produce production,following with quality standards study.2 Whole blood was digested by microwave digestion instrument.Total arsenic concentration was determined by inductively coupled plasma mass spectrometry(ICP-MS).Realgar,Qinghuang San,Fufang Qinghuangsan Jiaonang were respectively administered to ICR mice.Blood was collected at 0,0.083,0.25,0.5,0.75,1,2,4,8,12,24 h after administration(6 animals per time point),and blood arsenic concentrations at various time points were measured.3 MDCK-MDR1 cells were used as a monolayer cell model,and was incubated on polyester transwell membranes.The integrity of cell monolayers was assessed by measuring the tranepithelial electrical resistance(TEER)with an electric resisrtance meter everyday.Cell monolayers were formed after about 4-5 days.Under the safe drug concentration,Realgar,Qinghuang San,Fufang Qinghuangsan Jiaonang were administrated to MDCK-MDR1 cells.The transmembrane transport mechanism for MDCK-MDR1 monolayer cell model was assayed by adding drugs to an apical insert for the apical to basolateral(AP-BL)transport and to a basolateral well for the basolateal to apical(BL-AP)transport.4 Using artificial gastric juice and artificial intestinal fluid as the dissolution medium,the release of arsenic from realgaer and Qinghuang San was performed by using a slurry dissolution apparatus.The proportion of soluble arsenic in the amount of of total arsenic was investigated within 24 hours for two groups of drugs.The dissolving rate of soluble arsenic in two groups of drugs was investigated at different times and different media.5 The Gaussian software was used to hypothesize the structural responses of α-As4S4 and indirubin which are the major components of realgar and indigo naturalis.Infrared spectrum were used to investigate the mixture of α-As4S4 and indirubin in artificial gastric and intestinal fluids.6 Equivalent molar concentrations of arsenic with different species and valences(arsenous acid iAsⅢ,arsenic acid iAsⅤ,monomethylarsonous acid MMAⅢ,monomethylarsonic acid MMAⅤ,dimethylarsinic acid DMAⅤ,and realgar’s artificial gastric fluid)were prepared.The solution of protein tyrosine phosphatase SHP2 at a certain concentration was prepared.Arsenic with different species and valences were mixed and reacted with SHP2 solution.The absorbance of SHP2 solution was detected by microplate reader.MTT assay and LDH assay were used to detect the cytotoxicity of K562 which was treated by realgar.Flow cytometry was used to detect the number of viable,apoptotic and dead cells of K562.The expression levels of BCR-ABL,SHP2,JAK2,STAT3,STAT5 were detected by Western blot assay.Results and conclusions1 The optimal steam distillation process parameters of volatile oil.from pericarpium citri reticulatae:extracting with 6 folds of water for 8 hours;the optimal volatile oil inclusion process parameters:inclusion over 3 hours at 40℃,and the proportion of volatile oil andβ-CD is 1:8;the optimal water extraction process parameters:extracting with 10 folds of water for 2 times,1.5 hours per time;the filtrate was concentrated to a relative density range of 1.05～1.10(60℃),the concentration temperature was controled below 85℃,ethanol was added to attain the 50%alcohol concentration and stand for 12 hours at 4℃,and the supernatant was concentrated to a relative density of 1.15～1.30(60℃)below 85 ℃.Dry extract was obtained through vacuum drying.After three batches of pilot,dry extract and inclusions were mixed with realgar powder and indigo naturalis powder.The granules were prepared by dry granulation and encapsuled.2 After the treatment with realgar,the total arsenic concentration in blood reached the peak plasma concentration Cmax of 2472.41 μg/L at 45 min,and the area under the blood drug concentration-time curve AUC(0-24)is 18992.00 h μg L-1,AUC(0-∝)is 22039.20 h μg L-1,the half-life t1/2 is 8.58 h,and the average residence time MRT is 12.97 h.After the administration of Qinghuang San,the total arsenic concentration in the blood reached the peak plasma concentration Cmax of 1681.32 μg/L at 45 min,and the area under the blood drug concentration-time curve AUC(0-24)is 24057.60 h μg L-1,AUC(0-∝)is 30343.00 h μg L-1,the half-life t1/2 is 9.91 h,and the average residence time MRT is 15.57 h.After Fufang Qinghuangsan Jiaonang was administered,the total arsenic concentration in the blood reached the peak plasma concentration Cmax 645.79 μg/L at 8 h,and the area under the blood drug concentration-time curve AUC(0-24)was 10296.80 h μg L-1,AUC(0-∝)is 21817.50 h μg L-1,the half-life t1/2 is 23.12 h,and the average retention time MRT is 35.41 h.3 In the AP-BL direction:the apparent permeation coefficient of realgar group was(7.63 ±0.0016)× 10-6 cm/s,and the apparent permeation coefficient of Qinghuang San group was(10.45 ± 0.0009)× 10-6 cm/s,and the apparent permeation coefficient of Fufang Qinghuangsan Jiaonang group was(6.13± 0.0016)×10-6 cm/s.In the BL-AP direction:the apparent permeation coefficient of realgar group was(5.17±0.0045)× 10-6 cm/s,the apparent permeation coefficient of Qinghuang San group was(6.53±0.0056)× 10-6 cm/s,and the apparent permeation coefficient of Fufang Qinghuangsan Jiaonang group was(3.57 ±0.0032)× 10-6 cm/s.The efflux rate of realgar group was 0.68,the efflux rate of combination grouop of realgar and indigo naturali was 0.63,and the efflux rate of Fufang Qinghuangsan Jiaonang group was 0.58.The cumulative permeability of transmembrane transport or the apparent permeability coefficient:Qinghuang San group>realgar group>Fuang Qinghuangsan Jiaonang group.The compound Qinghuang powder capsule group has the lowest value.The Papp of total arsenic in MDCK-MDR1 cells is greater than 2×10-6 and less than 20×10-6 cm/s,determining that arsenic is a medium permeability drug.4 The amount of soluble arsenic in realgar accounted for 1.389%of the total arsenic of realgar in the artificial gastric juice,but accounted for 1.288%in the artificial intestinal guice.After the combination of realgar and indigo naturalis,the amount of soluble arsenic released from realgar accounted for 1.034%of the total arsenic of realgar in the artificial gastric juice,while accounted for 0.895%in the artificial intestinal juice.The curve is plotted,taking the dissolution time as the abscissa and the percentage of the cumulative dissolution of soluble arsenic and the theoretical soluble arsenic content as the ordinate.The results showed that in two dissolution media,the dissolution amount of soluble arsenic in realgar was higher than that of Qinghuang San.5 In the[As4S4…Indirub in]complex,the calculated distance of As-O distance d(lAs-270)is 3.105,2.934 and 2.892(?) at the HF/6-31G*,B3LYP/6-31G*and,wB97XD/6-31+G*levels of theory,respectively,which is larger than the sum of the covalent radii of the As and O atoms(1.84(?)),but shorter than the sum of the van der waals radii of the As and O atoms(3.37(?)),indicating that the α-As4S4 and indirubin(C16H10N2O2)may form a complex of[AS4S4…Indirubin]by the non-covalent interaction.The predicted binding energy with ZPE and BSSE corrections are 0.3,0.3,and-5.8 kcal/mol at the HF/6-31G*,B3LYP/6-31G*,and wB97XD/6-31+G*levels of theory,respectively.Considering the importance of using larger basis sets as well as adequately correlated methods to compute properties of non-covalent interactions,the predicted binding energy of-5.8 kcal/mol at the wB97XD/6-31+G*level of theory is more reasonable than the other two.The infrared spectrum showed that there is difference between indirubin and the combination of indirubin and realgar.6 Monomethylarsonous acid MMAⅢ had no effect on SHP2 protein;the half-life t1/2 of monomethylarsonic acid MMAⅤ on SHP2 was 129.326 min,and half-life t1/2 of arsenic acid iAsv and dimethylarsinic acid DMAⅤ on SHP2 was almost the same,respectively 118.012 min and 117.204 min.The half-life t1/2 of realgar dissolving substance in the artificial gastric juice was 93.297 min;arsenous acid iAsⅢ had the strongest inhibition on SHP2 protein,of which half-life t1/2 was 86.191min.When K562 cells were exposed to different concentrations of realgar working solution(5-80 μm),the proliferation of cells were significantly inhibited with the increase of drug concentration and the prolongation of administration time.The half maximal inhibitory concentration(IC50)of 6 hours,12 hours and 24 hours incubation were 138.994,74.197 and 11.953 μm,respectively.In addition,as the increasing concentration of realgar working solution,the release of LDH in K562 cells increased.After 24 hours of dosing,the percentage of the viable cells decreased from 92.43%±2.14%to 22.13%±6.47%.The proportion of early apoptotic cells increased from 4.40%±0.07%to 46.37%±12.98%,while the proportion of dead cells and late apoptotic cells increased from 3.17%±1.44%to 31.53%±6.58%.These data indicate that realgar can induce apoptosis on K562 cells with a dose-dependent manner.The expression level of BCR-ABL decreased significantly after apoptosis.The expression level of p-SHP2 decreased slightly when the drug concentration reached 80 μm.JAK2 was strongly expressed in K562 cells,and after treated with realgar,the expression level of JAK2 was significantly decreased.The expression level of STAT3 was evident in K562,and the level of STAT3 was significantly decreased after dosing.There was no obvious expression of STAT5 in K562 cells. |
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