As arsenic widely exists in nature and has been used in the pharmaceutical preparations, the traditional Chinese medicine(TCM) with arsenic include realgar(As_2S_2 or As_4S_4), orpiment(As_2S_3), and white arsenic(As_2O_3). Among the above representative medicine, the TCM compound formulas with realgar are utilized extensively. Just in Chinese Pharmacopoeia(2020 edition), there are 37 Chinese patent medicines including realgar. The traditional element analysis focuses on the detection of the total amount of elements, which neglects the study on the speciation and valence of elements. The activity, toxicity, bioavailability, and metabolic pathways of arsenic in vivo are closely related to the existence of its form, and different forms of arsenic have different effects on organisms. Therefore, the study on the speciation and valence of arsenic is of great importance for arsenic-containing TCMs and their compound formulas. This paper reviewed four aspects of the speciation and valence of arsenic, including property, absorption and metabolism, toxicity, and analytical assay.
Arsenic/analysis* ; Arsenicals/analysis* ; Sulfides ; Arsenic Trioxide ; Medicine, Chinese Traditional ; Drugs, Chinese Herbal/analysis* ; Biological Products

Sepiae Endoconcha is a common marine animal medicine,which generally contains high concentration of arsenic( As).The Chinese Pharmacopoeia( 2010 edition,part I) stipulated that the total As content of Sepiae Endoconcha should not exceed 2 mg·kg~(-1),while this limit was revised to 10 mg·kg~(-1) in the 2015 edition. So far,there is no research on the speciation of As in Sepiae Endoconcha,which made it hard to accurately evaluate its security risk. In this study,32 batches of Sepiae Endoconcha from different sources were collected. The safety risk assessment was carried out by determining the total As content and As speciation,inorganic As[As( Ⅲ),As( Ⅴ) ]and organic As( MMA,DMA,As C,As B) by HPLC-ICP-MS,and then the limit standard was discussed. The results showed that As B was the main form of As in Sepiae Endoconcha,followed by DMA and As( Ⅴ) ． Of the 32 batches of Sepiae Endoconcha,9 batches( accounting for 28%) were detected possessing i As. The maximum concentration of As( Ⅲ) was 103. 3 μg·kg~(-1),and the maximum concentration of As( Ⅴ) was 222. 4 μg·kg~(-1). According to the limit of i As in food,18. 75% of the samples exceeded the standard. The results indicate that there is no simple positive correlation between total As and As morphology in Sepiae Endoconcha. Besides,there is a risk in the total As limit,especially after the relaxation of the total As limit. The problem of high i As content caused by pollution and other factors is difficult to regulate. Since the toxicity of inorganic As is much higher than that of organic As,it is of great practical significance to establish inorganic As form limits in Sepiae Endoconcha.
Animals ; Arsenic/analysis* ; Arsenicals/analysis* ; Chromatography, High Pressure Liquid ; Drug Contamination ; Environmental Pollution ; Mass Spectrometry ; Medicine, Chinese Traditional ; Sepia/chemistry*

This study aims to establish a method for the determination of As B,As C,DMA,As( Ⅲ),MMA and As( Ⅴ) by using HPLC-ICP-MS. A Dioncx Ion PacTMAS7( 4 mm×250 mm) column was used for the HPLC-ICP-MS method. The mobile phase was 100 mmol·L-1 ammonium carbonate-1. 5 mmol·L-1 ammonium dibasic phosphate( gradient elution) at a flow rate of 1 m L·min-1. The injection volume was 10 μL. The linear relationships of As B,As C,DMA,As( Ⅲ),MMA,As( Ⅴ) were good with the concentration of10-500 μg·L-1. The average recovery rates( n = 6) were 105. 7%,100. 5%,102. 9%,105. 7%,100. 2%,92. 69%. The RSD were0. 50%,2. 4%,0. 93%,1. 3%,0. 89%,1. 5%. The precision and repeatability of this method were good. In this study,six forms of arsenic were separated effectively by this method. With methodological validation and sample determination,this method can be used to determine the morphological valence of arsenic in content determination.
Arsenic/analysis* ; Arsenicals/analysis* ; Chromatography, High Pressure Liquid ; Mass Spectrometry

OBJECTIVETo explore apoptosis-inducing effects of realgar nanoparticle (nano-realgar) on drug-sensitive leukemia cells.

METHODPreparation of nano-realgar was mechanical milled using a high-energy planetary ball mill. Using drug-sensitive leukemia cells (K562) as target cells, MTT assay was used to detect the proliferating activity of K562 cells, and the cellular apoptosis was investigated with double staining of FITC-Annexin V and propidium iodide (PI) by flow cytometry. Flow cytometry (FCM) was employed to detect expression of intracellular Bax, Bcl-2, P-53 protein and the activity of Caspase-3.

RESULTThe raw realgar was made to ultra-fine powder by ball milling, and the average diameter of the nanoparticle was (72.72 +/- 22.18) nm measured with electron microscopes. Nano-realgar significantly inhibited the proliferation of K562 cells, Treated for 24, 48 and 72 hours, the 50% inhibitory concentration (IC50) was 43.48, 20.52, 16.07 mg x L(-1). After exposure to 20 mg x L(-1) and 50 mg x L(-1) nano-realgar for 48 hours, the apoptosis of K562 cells detected by Annexin V/PI staining was increased, the apoptotic rate of K562 cells was 10. 52% and 73.25%. After the target cells were treated with 20 mg x L(-1) and 50 mg x L(-1) nano-realgar for 48 h, the expression of P-53, Bax, Bcl-2 markedly increased in a time and dose-dependent manner. After administration of 20 mg x L(-1) and 50 mg x L(-1) nano-realgar for 48 h, the percentage of BCRP+, P-gp+ and co-expressing P-gp and BCRP cell population in K562 cells incrased dramatically.

CONCLUSIONNano-Realgar significantly induced apoptosis of drug-sensitive leukemia cells.

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Proliferation ; drug effects ; Humans ; K562 Cells ; Leukemia ; drug therapy ; pathology ; Nanotechnology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Sulfides ; pharmacology ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVETo establish the comparative proteomic profiles of retinoid acid (RA) resistant human acute promyelocytic leukemia (APL) NB4-R1 cells before and after apoptosis induced by realgar (tetra-arsenic tetra-sulfide, As4S4).

METHODSFirst a serial of assays were performed using MTT, transmission electron microscopy, Annexin V FITC/PI double-stain, flow cytometry and confocal laser scanning microscopy to qualitatively and quantitatively observe the in vitro apoptosis inducing effect of realgar on RA-resistant cells. Then the comparative proteomic profile before and after NB4-R1 apoptosis was established using high-resolution two-dimensional electrophoresis system.

RESULTSThe inhibition effect of realgar on NB4-R1 cell growth was dose and time dependent. The 24-h 50% inhibiting concentration (IC50) was 24.06 +/- 0.19 micromol/L, and the 48-h IC50 9.50 +/- 0.13 micromol/L, and 72-h IC50 6.55 +/- 0.03 micromol/L, respectively. 24 h and 48 h were the early and late phase of major NB4-R1 apoptotic cell populations induced by 25 micromol/L realgar respectively. Differential proteomic profiles before and after realgar induced NB4-R1 apoptosis were successfully established. Averagely 1069, 975 and 893 spots could be detected of the untreated group (R0), the 24-h treatment group (R24), and the 48-h treatment group (R48), respectively by ImageMaster 2D Platinum Software. The matching rate between R24 and R0 was 79.94% and that between R48 and R0 69.33%, and that between R24 and R48 71.91%.

CONCLUSIONDifferential proteomic profiles of realgar induced NB4-R1 apoptosis were successfully established for the first time, which provided a basis for comprehensively understanding the signal transduction of realgar induced apoptosis in RA-resistant APL cells, also for screening new bio-markers and drug targets of hematopoietic malignant tumor.

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; drug effects ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Proteome ; analysis ; Sulfides ; pharmacology ; Tretinoin ; pharmacology

OBJECTIVETo explore arsenic accumulation and toxicity mechanism following long-term use of realgar and provide scientific basis for safety use of realgar in clinic.

METHODThe realgar which was used in the study contains 90% insoluble asenic sulfide (As2S2) and 1.696 mg x kg(-1) soluble arsenic. Two separate experiments were performed: 1) Twenty-eight fasting SD rats were orally given a single dose of realgar at the dose of 0.8 g x kg(-1) and the other four rats were given ultra-filtrated water served as control group. Blood, hearts, livers, kidneys, lungs and brains of four rats were taken out at 0.5, 1, 2, 4, 8, 16, 36 h respectively after treatment. Asenic quantity of each organ or blood sample was measured. 2) Forty SD rats were randomly divided into four groups: control group and realgar 0.02, 0.08, 0.16 g x kg(-1) groups, each group containing 5 females and 5 males. The rats were intra-gastrically treated with realgar once a day for successively 90 days, while the control group was given ultra-filtrated water. Asenic amount in blood, liver, kidney and brain of each rat was measured in fasting rats at 16 h after last dosing.

RESULTAsenic amount of blood, liver, kidney, heart, lung and brain increased after single dosing of realgar at dose of 0.16 g x kg(-1), with the order from high to low blood > kidney > lung > liver > heart > brain. Asenic amount was much higher in blood than that in other organs. The feature of asenic distribution in blood following realgar administration may be the basis for its use for leukemia Ninety-day oral treatment of realgar led to significant accumulation of asenic in blood, kidney, liver and brain. The highest asenic accumulation times was found in kidney followed by liver, which was assumed to be associated with nephrotoxicity and hepatotoxicity of realgar. The highest amount of asenic was observed in blood after 90 day's administration of realgar, and the amount of asenic in organs was in the order of blood > kidney > liver > brain.

CONCLUSIONAsenic can be absorbed and extensively distributed in various organs or tissesses after realgar administration in rats. Long-term use of realgar caused high asenic accumulation in various tissueses, including blood, kidney, liver, and brain. The nephrotoxicity and hepatotoxicity of realgar could be associated with the asenic accumulation in relative organs. Blood is the target of the most highest distribution and accamulation of asenic after realgar treatment, that could be associated with the efficacy of realgar on the treatment of leakemia.

Animals ; Arsenic ; analysis ; chemistry ; pharmacokinetics ; toxicity ; Arsenicals ; administration & dosage ; adverse effects ; chemistry ; Female ; Male ; Rats ; Rats, Sprague-Dawley ; Solubility ; Sulfides ; administration & dosage ; adverse effects ; chemistry ; Time Factors

OBJECTIVETo determine the effects of Angong Niuhuang pill (AGNHW) on lipopolysaccharide (LPS)-induced neuroinflammation and further to investigate the role of realgar and cinnabar on AGNHW-mediated neuroprotection.

METHODPrimary rat midbrain neuron-glia cultures were used as an in vitro model to examine the effects of AGNHW on LPS-induced dopamine (DA) neuronal damage. Cultures were divided randomly into five groups: control, LPS, LPS plus AGNHW, LPS plus realgar and LPS plus cinnabar. Dopaminergic neurotoxicity was measured by [3H] DA uptake assay. The production of intracellular reactive oxygen species (ROS) was quantified via the DCFH-DA probe. Real-time RT-PCR was applied to detect the mRNA expression of pro-inflammatory factors. Then the protein levels of these factors were determined by ELISA and western blot assay.

RESULTCompared with the control group, LPS apparently decreased DA uptake capacity (P < 0.05); induced the production of intracellular ROS (P < 0.05); enhanced the mRNA expression of TNF-alpha, iNOS, IL-13 and COX-2 (P < 0.05) and the release of TNF-alpha, IL-1beta and PGE2 in the supernatant of cultures (P < 0.05); and also increased the level of iNOS protein (P < 0.05). Compared with the LPS group, AGNHW and realgar significantly inhibited LPS-induced reduction of DA uptake (P < 0.05); attenuated the production of intracellular ROS (P < 0.05) and the mRNA expression of TNF-alpha, iNOS, IL-1beta and COX-2 (P < 0.05) and the release of TNF-alpha, IL-1beta and PGE2 (P < 0.05) and the level of iNOS protein (P < 0.05). However, there was no significant difference between LPS group and LPS plus cinnabar group.

CONCLUSIONAGNHW is effective in protecting against LPS-induced neuroinflammation, and realgar is one of active components for AGNHW to produce anti-inflammatory effects.

Animals ; Arsenicals ; analysis ; pharmacology ; Cytokines ; genetics ; metabolism ; Female ; Gene Expression Regulation ; drug effects ; Inflammation ; prevention & control ; Intracellular Space ; drug effects ; metabolism ; Lipopolysaccharides ; pharmacology ; Neuroglia ; drug effects ; metabolism ; pathology ; Neuroprotective Agents ; analysis ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfides ; analysis ; pharmacology ; Superoxides ; metabolism

OBJECTIVETo investigate the effect of post-remission therapy mainly with Compound Huangdai Tablet (CHDT) on long-term survival of patients with acute promyelocytic leukemia (APL).

METHODSOne hundred and twelve APL patients were treated after remission mainly with CHDT administered alternately with chemotherapeutic projects such as HACP, HAOP, HAEP and HAMP. The relapse rate and relapse-free survival (RFS) rate in them were estimated by bone marrow examination.

RESULTSThe total relapse rate was 14.29% (16/112), and the median time of relapse was 12.5 (4-67) months. Patients were followed up for 1-72 months, the median follow-up time being 59 months. The actual RFS rate of 1-, 2-, 3-, 4-, 5- and 6-year was 92.86%, 89.29%, 88.39%, 87.50%, 86.61% and 85.71%, respectively, while the estimated RFS rate (%) of corresponding year was 92.45 +/- 2.57, 88.25 +/- 3.20, 87.09 +/- 3.36, 85.89 +/- 3.52, 84.44 +/- 3.75 and 82.78 +/- 4.03 respectively; the relapse rate in patients who received treatment after complete response for <10 courses group was 34.29%, while in those treated for > or = 10 courses was 5.19%; and the RFS rate in them was 65.71% and 94.81% respectively, the difference between groups was statistically significant (P<0.01).

CONCLUSIONThe post-remission therapy mainly with CHDT is an effective and feasible program for the treatment of APL.

Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Arsenicals ; administration & dosage ; Child ; Child, Preschool ; Disease-Free Survival ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; Male ; Middle Aged ; Phytotherapy ; Remission Induction ; Survival Analysis ; Young Adult

OBJECTIVETo study the fibrinolytic activity in patients with acute promyelocytic leukemia (APL) and its alteration in all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) treatment.

METHODSPlasma fibrinogen concentration was determined with the conventional method, and the levels of fibrin degradation products (FDP) and D-dimer were quantified with ELISA. Plasminogen was measured by chromogenic assay. Cell surface expression of Annexin II and u-PAR and their mRNA levels were measured by flow cytometry and real time-PCR, respectively.

RESULTSThe levels of FDP and D-dimer in APL were remarkably higher in APL patients than that in normal controls, while fibrinogen and plasminogen were lower. Both Annexin II and u-PAR were highly expressed on APL cells, which declined after treatment with ATRA and/or ATO, but remained higher than those on normal bone marrow mononuclear cells.

CONCLUSIONAbnormally high levels of Annexin II and u-PAR expression on APL cells may contribute to the increased production of plasmin, leading to primary hyperfibrinolysis in APL. ATRA and ATO therapy induces down-regulation of Annexin II and u-PAR expression, which may be contribute, at least in part, to the relief of the hemorrhagic complications in APL.

Adolescent ; Adult ; Aged ; Annexin A2 ; analysis ; Arsenicals ; therapeutic use ; Female ; Fibrinolysis ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; physiopathology ; Male ; Middle Aged ; Oxides ; therapeutic use ; RNA, Messenger ; genetics ; Tretinoin ; therapeutic use ; Urokinase-Type Plasminogen Activator ; analysis ; Young Adult

OBJECTIVEConstruct the gene library of apoptosis related genes in acute promyelocytic leukemia (APL) cell line NB4 cells treated by arsenic trioxide to clarify the apoptotic mechanism of NB4 cells.

METHODAPL cell line NB4 cells treated with or without arsenic trioxide for 24 hours. Total RNA was extracted and suppress subtractive hybridization (SSH) was conducted according to the manual. With the cDNA of the apoptosis cells as the tester and that of control cells as the driver, forward and reverse hybridization was performed. Differentially expressed genes were linked with pGEM-Teasy cloning vector and transformed into E. coli DH5alpha. The positive clones were screened by blue and white spot. PCR were used to amplify these genes.

RESULTThe subtractive cDNA libraries related with apoptosis of NB4 cells were successfully constructed.

CONCLUSIONThe constructed subtractive libraries are suitable for further study on the functional genes associated with apoptosis ofNB4 cells induced by arsenic trioxide.

Apoptosis ; drug effects ; genetics ; Arsenicals ; pharmacology ; Cell Line, Tumor ; DNA, Complementary ; analysis ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Library ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; Oxides ; pharmacology ; Sequence Analysis, DNA