Acid Water-ground Nano-realgar Is Superior to Crude Realgar in Promoting Apoptosis of MCF-7 Breast Cancer Cells

Objective:Realgar is a traditional mineral Chinese medicine with antitumor effects,but it has high toxicity and low efficacy in its crude form.The purpose of this study was to optimize realgar to increase its efficacy and therapeutic potential.Methods:Crude realgar(CR)was mechanically ground to obtain nano-realgar(NR),and then nano-realgar processed products(NRPPs)were obtained using three different traditional Chinese medicine processing methods:grinding in water,acid water,and alkali water,respectively.Results:By analyzing the size distribution of nanoparticles and the content of arsenic trioxide(As_(2)O_(3);ATO),we found that acid water-ground NRPPs had the characteristics of high purity and low toxicity.The effects of CR,NR,and NRPPs on proliferation,cell cycle,and apoptosis of MCF-7 cells were detected,and the ability of NRPPs to induce apoptosis in MCF-7 cells was analyzed.The results showed that the average particle size of acid water-ground NRPPs was 137.7 nm,and the content of ATO was 2.83 mg/g.Acid water-ground NRPPs showed better effects on inhibiting proliferation,cell cycle,and apoptosis of MCF-7 cells than CR and NR.Western blot assays further confirmed that acid water-ground NRPPs upregulated the protein expression of TP53,Bax,cytochrome c,caspase-9,and caspase-3 in MCF-7 cells(P<0.05)and inhibited the expression of Bcl-2(P<0.05).Conclusion:These results suggest that acid water-ground NRPPs can induce apoptosis of MCF-7 cells through regulating mitochondrial-mediated apoptosis,providing evidence for the clinical application of realgar.

Induction of SiHa Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism

The effects of nanometer realgar uterine cervix cancer cell line SiHa cells and suspension on proliferation and apoptosis of human oncogenic genes HPV16E6/E7 were investigated. A ＂micro-jet effiux＂ strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations （6.25, 12.5, 25 and 50 mg/L） for different durations （12, 24, 48 and 72 h）. The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry （FCM）. The expression of HPV 16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25--50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group （P〈0.01）, and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

The Research of Nanocrystallized Realgar for the Treatment of Skin Cancer

Objective: To observe the effects and mechanisms of nanocrystallized realgar for the treatment of skin cancer. Methods: The clinical part was observing the therapeutic effect for the skin ulceration by externally using the nanocrystallized realgar. The experimental part was culturing the human squamous cell carcinoma A431 cells (A431) in vitro environment, estimating the effects of proliferation and apoptosis of A431 by MTT method and flow cytometry, and observing the effects on the expression of Survivin, Caspase-3 by RT-PCR method. Results: External treatment with nanocrystallized realgar had a therapeutic effect for the skin ulceration;it can promote the ulcers of skin cancer and skin metastasis healing. The experiments confirmed that nanocrystallized realgar can inhibit the A431 proliferation, induce the cells apoptosis, promote the expression of Caspase-3 and reduce the expression of Survivin. And the experiments also found that the effects were in a concentration-dependent manner, and they have a synergistic effect with cis-Dichlorodiamineplatinum (DDP). Conclusions: External treatment with nanocrystallized realgar for the patients of skin cancer or skin metastasis achieved satisfactory therapeutic effects by inhibiting A431 proliferation and inducing these cells apoptosis. The mechanism might be associated with promoting the expression of Caspase-3 and reducing the expression of Survivin.

Secondary Minerals of Weathered Orpiment-Realgar-Bearing Tailings in Shimen Carbonate-Type Realgar Mine,Changde, Central China

Formation and dissolution of secondary arsenic minerals often play significant roles in controlling arsenic mobility in contaminated environments, especially in sulfide mines. Weathering of the orpiment and realgar-bearing tailings from the Shimen realgar deposit, the largest realgar deposit in Asia, were studied. An integrated mineralogical analysis by using X-ray powder diffraction (XRD), Raman spectrum, scanning electron microscope (SEM) and transmission electron microscope (TEM) reveals four kinds of As-bearing secondary minerals including arsenic oxides, arsenates, As-gypsum, and As-Fe minerals. The precipitation of arsenates is due to interaction of As-bearing run-off waters and the underlying carbonate rocks, or the transformation of gypsum into arsenates or As-bearing gypsum through SO42-/HAsO42- substitution. Ca-arsenates are mainly weilite and pharmacolite with Ca/As atomic ratio of 1. Scanning transmission X-ray microscope (STXM) and X-ray absorption fine structure (XAFS) reveal that the valence of arsenic is mainly +3 and +5.

High-pressure investigations on the isostructural phase transition and metallization in realgar with diamond anvil cells

The high-pressure structural,vibrational and electrical properties for realgar were investigated by in-situ Raman scattering and electrical conductivity experiments combined with first-principle calculations up to~30.8 GPa.It was verified that realgar underwent an isostructural phase transition at~6.3 GPa and a metallization at a higher pressure of~23.5 GPa.The isostructural phase transition was well evidenced by the obvious variations of Raman peaks,electrical conductivity,crystal parameters and the As–S bond length.The phase transition of metallization was in closely associated with the closure of bandgap rather than caused by the structural phase transition.And furthermore,the metallic realgar exhibited a relatively low compressibility with the unit cell volume V_(0)=718.1.4Å^(3)and bulk modulus B_(0)=36.1 GPa.

Effect of realgar on telomerase activity and hTERT-mRNA expression in NB4 cells

Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.

Sixty-one Cases of Angina Pectoris due to Coronary Heart Disease Treated by External Use of the Paste of Nitrum and Realgar Powder on Zhiyang(GV 9)

In order to inquire into the therapeutic effects of Xiao Shi Xiong Huang San (硝石雄黄散the Nitrum and Realgar Powder), one of the Dunhuang Prescription, on angina pectoris due to coronary heart disease (APCHD), the authors have treated 61 cases of APCHD by externally applying paste of the powder on Zhiyang (GV 9), with another 30 cases of APCHD treated with the nitroglycerin paste on Zhiyang as the controls. The results showed that the total effective rate was 82% and markedly effective rate 31.2% in the treatment group (the Paste of Nitrum and Realgar Powder), and the total effective rate was 46.6% and markedly effective rate 23.2% in the control group (the nitroglycerin paste). The difference in therapeutic effects between the two groups was very significant (P<0.01), indicating that the therapeutic effect of the former was significantly superior to that of the latter.

Effects of realgar on stress proteins, inflammatory mediators, and complement in brain tissue and serum of rats with inflammatory brain injury

BACKGROUND：The Chinese herbal compound realgar exerts detoxification effects as an adjuvant. It is suggested that realgar exerts detoxification via the following pathways： in the pathological state, realgar corrects the oxidative stress state by increasing stress levels, activating some endogenous protective factors and antagonizing the excessive release of inflammatory factors, as well as inhibiting complement activation. OBJECTIVE： To observe the changes in stress proteins, inflammatory mediators, and complement in the brain tissue and serum of rats with inflammatory brain injury, which have been treated with the Chinese herbal compound Angong Niuhuang, and to compare the efficacy of Angong Niuhuang with that of realgar, to verify the mechanism of action of realgar. DESIGN, TIME AND SETTING： Randomized, controlled, cytological experiment, performed in the Institute of Clinical Pharmacology, Guangzhou University of Traditional Chinese Medicine in March 2006. MATERIALS： Thirty-six healthy, male, Sprague Dawley rats received 250 U/kg Bordetella pertussis via the common carotid artery within 15 seconds to induce inflammatory brain injury. Reagents and kits were as follows： Realgar and Angong Niuhuang powder （Foshan Second Pharmaceutical Factory, China）, Bordetella pertussis diagnostic antigen （National Institute for the Control of Pharmaceutical and Biological Products, China）, heat shock protein 70 （HSP70） enzyme-labeled immunosorbent assay （ELISA） kit （Stressgen, USA）, tumor necrosis factor-α （TNF-α） ELISA kit （Biosource, USA）, nitric oxide synthase （NOS） kit, Coomassie brilliant blue protein kit （Nanjing Jiancheng Bioengineering Co.,Ltd., China）, and complements C3 and C4 （Shanghai Kehua Dongling Diagnositic Products Co.,Ltd., China）. METHODS： Thirty-six rats were randomly and evenly divided into the following six groups： normal control, model, high-, middle-, and low-dose realgar-treated, and Angong Niuhuang-treated groups. At one hour prior to establishing the model, rats in the high-, middle-, and low-dose realgar-treated groups were administered 300, 150, and 75 mg/kg realgar, respectively; while rats in the Angong Niuhuang group received 1.5 g/kg Angong Niuhuang powder; In the model group, rats were administered Bordetalla pertussis only. MAIN OUTCOME MEASURES： Two hours after the administration of Bordetalla pertussis, the HSP70 level in the brain tissue and serum levels of interleukin-1β （IL-1β）, interleukin-6 （IL-6）, and TNF-α were determined by ELISA tests, hemooxygenase-1 （HO-1） activity in the brain tissue and serum was determined by dual-wavelength spectrophotometry, NOS and inducible nitric oxide synthase （iNOS） activities in the brain tissue were measured by the Bradford assay method, and serum levels of complement C3 and C4 were determined by immunoturbidimetry. RESULTS： In the high-dose realgar and Angong Niuhuang groups, the HSP70 level in the brain tissue of rats with inflammatory brain injury was increased significantly （P 〈 0.01）. In the realgar-treated groups, HO-1 activity in the brain tissue and serum was dose-dependently enhanced with increasing realgar doses. However, no significant difference existed between the realgar groups and the model group （P 〉 0.05）. In the Angong Niuhuang group, HO-1 activity in the brain tissue and serum was increased （P 〈 0.05）. In the realgar and Angong Niuhuang groups, serum levels of IL-1β, IL-6, and TNF-α were significantly decreased; the serum IL-1β level recovered to the normal level and serum levels of IL-6 and TNF-α dose-dependently decreased in the realgar groups. NOS activity in the brain tissue was lower in the high-dose realgar group than in the model group （P 〈 0.05）. iNOS activity was significantly lower in the middle- and high-dose realgar groups than in the model group （P 〈 0.01）. NOS and iNOS activities were significantly lower in the Angong Niuhuang group than in the model group （P 〈 0.01）. The serum C3 level was significantly decreased in the middle-dose realgar and Angong Niuhuang groups （P 〈 0.01）. CONCLUSION： At certain doses, realgar is able to correct the oxidative stress state, by inducing and activating endogenous protective factors, such as HSP70, and inhibiting the excessive release of inflammatory mediators, such as IL-1β, IL-6, and TNF-α. However, it remains unclear whether realgarinhibits the activation of C3 and C4.

INVESTIGATION OF REALGAR IN THE TREATMENT FOR MULTIPLE MYELOMA

Objective To observe the clinical effect of realgar on multiple myeloma and to investigate its mechanism. Methods MTT and double antibody cramped ELISA assay were used to detect the activity of interleukin-6(IL-6) and level of soluble interleukin-6 receptor(sIL-6R) of the bone marrow supernant in 15 multiple myeloma patients treated by realgar or not. Results The activity of interleukin-6 and level of soluble interleukin-6 receptor of multiple myeloma patients were significantly higher than that of control group(P<0.01). They were not apparently decreased after realgar was used for 50 days(P>0.05).The interleukin-6 activity of stage Ⅲ patients were much higher than that of stage Ⅰ or Ⅱ(P<0.05). There was no difference of the level of soluble interleukin-6 receptor between two groups(P>0.05). For the 15 patients who were treated by using realgar, 3 got complete remission(CR), 5 got partial remission(PR), 7 came to not remission(NR). Conclusion Realgar could not decrease the activity of interleukin-6 and level of soluble interleukin-6 receptor so as to inhibit the proliferation of tumor cells.

Realgar transforming solution is involved of JNK, P38 MAPK pathway in down-regulating Ras/MAPK pathway in Caenorhabditis elegans

OBJECTIVE To investigate the effect and the mechanisms of realgar transforming solution(RTS)on down-regulating over activated ras.METHODS we used the optimizing technical processes to obtain the RTS,and eval⁃uate the mechanisms of RTS on down-regulating overactivated ras in Caenorhabditis elegans.RESULTS We found that the mRNA level of let60 and lin45 significantly decreased following exposure to RTS,but mRNA levels of mpk1 were not statistically significant in let60/ras(gf)mutant.RTS together with sorafenib(RAF inhibitors)significantly enhanced the activity of RTS on down-regulating overactivated ras more than RTS only,but 50μmol·L^-1 PD98059,a specific ERK inhibitor did not.Lin45 gene RNAi decreased the ability of RTS on down-regulating overactivated ras significantly,but mpk1 gene RNAi did not,indicating that the activity of RTS on down-regulating overactivated ras mainly through targeting to lin45/raf.In addition,RTS significantly increased mRNA level of pmk1/p38 and jnk1/jnk in let-60/ras(gf)mutant,50μmol·L^-1 SB203580(p38 inhibitor)and SP600125(JNK inhibitor)significantly attenuated the effects of RTS on down-regulating overactivated ras in some degree,and pmk1,jnk1 gene RNAi displayed the similar results,suggesting that P38 and JNK/MAPK pathways in some degree were involved in the effects of RTS on down-regulating overactivated ras in C.ele⁃gans.CONCLUSION Realgar transforming solution down-regulate the Ras/MAPK pathway through JNK and P38 MAPK pathways.

Classification,Minerogenic Models and Prospecting of Realgar/Orpiment Deposits in China

China's realgar/orpiment deposits may be classified into three types, the stratabound, hot-water sedimentary and hydrothermal, according to their mineralizing processes, geological occurrences, tectonic and geochemical features. The three types may be further distinguished into seven subtypes, namely, the Xiaguan, Shuiluo, Jiepaiyu, Songpan, Shixia, Wangzhuang and Ninghshan ones. On this basis three minerogenic models are established, and based on studies of their geochemistry and minerogenic mechanisms the prerequisites for prospecting for these types of deposits are given in the paper.

Realgar induces differentiation through ROS-dependent mitochondrial pathway in HL-60 cells

Realgar （As 4 S 4 ）, as a mineral drug in traditional Chinese medicine, is currently used as the remedy for acute promyelocytic leukemia and has been proven to have relatively milder side effects as compared to the arsenolite （As 2 O 3 ）-based drugs. We have previously demonstrated that realgar induces differentiation in HL-60 cells, and the differentiation is associated with serine/threonine protein phosphatases, MAPK signaling pathways, and mitochondrial transmembrane potential decrease. In this study, we further explore the roles of mitochondrial permeability transition pore and reactive oxygen species （ROS） in realgar-induced differentiation in HL-60 cells. The differentiation was preceded by marked changes in the cellular level of ROS, and could be enhanced by SB202190, a p38 MAPK inhibitor. In addition, the efficacy of realgar was suppressed by closing the MPTP with an inhibitor. Taken together, these findings indicate that the opening of MPTP and the alteration of ROS generation were involved in realgar-induced differentiation.

雄黄治疫古今探微

自新冠疫情暴发以来,由于西医缺乏有效的治疗手段且疫苗研发相对滞后,有着几千年抗疫历史经验的中医药焕发了新的光彩,为维护世界人民的生命健康安全做出了重大贡献。雄黄是古代治疫的常用药物之一,药用历史悠久。通过梳理雄黄治疫的历史源流,使用方法,配伍规律并结合现代药理研究对雄黄的应用前景进行展望,以期推动雄黄未来的开发与应用进程,丰富疫病防治手段。

不同辐照度对雄黄退化的影响

光照对材料的影响是不可避免的,也是不可逆的,而且光照的损害具有积累作用。国际博物馆学会根据材料的光化学稳定性,将材料划分为:不敏感、低敏感、较敏感和极敏感。不同光敏感性材料对光照的承受能力是不同的,因此研究光照对颜料的影响是非常有必要的,这对文物的保护和修复有指导意义。雄黄是众所周知的光敏材料,在可见光的照射下,雄黄退化生成乌宗石(As4S5)和砒石(As_(2)O_(3)),其中乌宗石作为中间产物脱硫后生成副雄黄。光诱导雄黄退化是常见现象,但辐照条件不同,雄黄退化结果和所需时间也不同。拉曼光谱技术是一种无损检测技术,可进行定性、半定量的物质组分分析。在光诱导雄黄退化过程中,根据物质拉曼峰的位置和强度判断物质结构和成分。使用WLED灯照射雄黄,测得不同辐照度下雄黄拉曼信号完全消失的时间,得出辐照度y与雄黄退化时间x的关系为y=e^(9.057)/x^(0.861),表明辐照度越大,雄黄完全退化所需时间越短。且研究还发现雄黄经WLED辐照后的最终产物均是副雄黄和乌宗石共存,这是因为雄黄光照的退化产物副雄黄,在白色光源中含有的蓝光的辐照下,又退化成中间产物乌宗石。在古建筑内部照明中,应尽可能降低光敏颜料表面接收到的辐照度。照明光源不同,对雄黄的影响也有所不同,但该实验采用的照明光源符合目前照明情况,可以根据上述关系式,结合古建筑内部照明的辐照度对古建彩画进行定期修复。

Realgar-Induced Apoptosis of Cervical Cancer Cell Line Siha via Cytochrome C Release and Caspase-3 and Caspase-9 Activation

Objective： To explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells. Methods： The cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry. The activities of caspase-3, -8, and -9 were detected by fluorescence resonance energy transfer technology and colorimetric assay, while the levels of Bcl-2, cytochrome c, and Bax were detected by Western blot method. Results： Induction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner. The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8. Further, the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor, and a caspase-9 inhibitor but not by a caspase-8 inhibitor. Bcl-2 and Bax protein expressions were not changed by realgar. Conclusion： The induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3.

1例口服雄黄制剂致白血病患儿急性砷中毒死亡原因分析

目的 探讨雄黄制剂致急性砷中毒的原因。方法 回顾性分析1例口服雄黄制剂致白血病患儿急性砷中毒的诊治过程,总结雄黄制剂治疗儿童白血病的砷中毒特点及治疗注意事项。结果 患儿雄黄日用量为1 g,远高于单味日用量50~100 mg;临床症状为多器官功能衰竭,尿砷含量为33.56μmol/L,远超过人体尿砷正常值1.8μmol/L,诊断为急性砷中毒。结论 该患儿雄黄日用量属超剂量用药。雄黄制剂的剂量和疗程应符合药品说明书要求,不可长期和超量服用,一旦出现心律失常和心电图QT间期延长等砷中毒症状时应立即停药,保证用药安全。

Liver metabonomics study on the protective effect of glycyrrhetinic acid against realgar-induced liver injury

Glycyrrhetinic acid(GA)is the bioactive ingredient in Glycyrrhizae Radix et Rhizoma.Our previous study has reported that GA has protective effect on realgar-induced hepatotoxicity.However,the details of the hepatoprotective mechanisms of GA on realgar-induced liver injury remain to be elucidated.In the study,mice were divided into control,GA-control,realgar,and co-treated groups.Their liver tissues were used for metabonomics study by ultra-performance liquid chromatography-mass spectrometry(UPLCMS)method.The results illustrate that GA significantly ameliorate the liver injury and metabolic perturbations caused by realgar.Some metabolites,such as phenylalanine,pyroglutamic acid(PGA),proline,carnitine,nicotinamide,choline,lysophosphatidylcholine(LPC)16:0 and LPC 18:2 were found responsible for the hepatoprotective effect of GA.These metabolites are associated with the methylation metabolism of arsenic,cell membrane structure,energy metabolism and oxidative stress.From the results of this study,we infer that the potential hepatoprotective mechanism of GA on realgar-induced liver injury may be associated with reducing arsenic accumulation and its methylation metabolism in the liver,promoting the conjugation of arsenic and GSH to play detoxification effect,and ameliorating the liver metabolic perturbations caused by realgar.

雄黄对宫颈癌细胞增殖、凋亡及对HPVE6/IL-6/STAT3通路的影响

目的探讨雄黄对宫颈癌细胞增殖、凋亡的影响,并研究其与E6和IL-6/STAT3通路的关系。方法采用CCK8法检测不同浓度(0、25μg/mL、50μg/mL、75μg/mL、100μg/mL)的雄黄对宫颈癌HeLa、Caski细胞增殖的影响,流式细胞术检测HeLa、Caski细胞凋亡情况。Western blot检测不同浓度的雄黄对两种细胞中STAT3和p-STAT3蛋白表达的影响,qPCR法检测E6、IL-6、PARP、Bcl-xL基因表达水平。结果雄黄对上述2种宫颈癌细胞的增殖有显著的抑制作用,并促进凋亡,且呈现一定的浓度依赖性。Western blot和qPCR结果显示STAT3、p-STAT3、E6、IL-6、PARP、Bcl-xL表达均下调(P<0.05),并呈一定的浓度依赖性。结论雄黄可以抑制HeLa和Caski细胞的增殖,促进凋亡,且可能与通过E6介导的IL-6/STAT3通路有关。

Advances in the Safety Evaluation of Mineral Medicines-Cinnabar and Realgar

Mineral medicines have a long history in the treatment of diseases. Among these, cinnabar and realgar are the most commonly used. This review discusses the differences between cinnabar and realgar and other mercurials and arsenicals. In addition, it explores the toxicity studies carried out on cinnabar and realgar, which include acute and chronic toxicity, neurotoxicological effects, special toxicity, disposition and accumulation, and the relative mechanism of toxicity. Cinnabar and realgar are seldom used alone but rather in combination with herbs or animal medicines. Thus, in this article, we have also included a study of the effect of toxicity reduction and efficacy enhancement after cinnabar and realgar were combined with other Chinese medicinal materials. This review provides the theoretical evidence for their clinical application, suggesting that the dosage and treatment period using cinnabar and realgar should be rigidly controlled and emphasis should be placed on the drug safety of special populations.

Synergistic effect and different toxicities of adjuvant components of Realgar–Indigo Naturalis formula

Objective：Realgar–Indigo Naturalis formula（RIF）is a well-known arsenic-containing preparation that is used to treat acute promyelocytic leukemia（APL）in China.In recent multicenter clinical trials,complete remission rates in APL patients have ranged from 96.08%to 100%.RIF has a satisfactory therapeutic effect,but its safety is a widespread concern,since the preparation contains arsenic,a wide-ranging and naturally occurring toxicant.In this study,in order to determine the toxic potential of RIF,acute toxicity and sub chronic toxicity assays were performed to evaluate the toxic potential of Realgar and the adjuvant components of RIF in addition to Realgar’s synergy with these adjuvant components.Methods：KM mice and Wistar rats were selected for these experiments.To evaluate acute toxicity,the toxic effects of a single dose of a gradient of concentrations of Realgar were firstly determined.Then,the toxic effects of combinations of gradient doses of Realgar and fixed doses of Indigo naturalis and Salvia miltiorrhiza were evaluated.Results：The results showed that when Realgar was used alone,the2756.73 mg/kg（equivalent to 23.6 mg/kg）.However,theto 936.90 mg/kg when Realgar was used with I.naturalis.By contrast,theto 7538.86 mg/kg when Realgar was used with S.miltiorrhiza.Hence,I.naturalis strengthened the toxicity of Realgar,whereas S.miltiorrhiza displayed the opposite effect.The sub chronic toxicity assessment results revealed a trend that was consistent with acute toxicity.Changes in the levels of different valence states of arsenic were also taken into account.The test results of the effects of in vitro combinations of Realgar and adjuvant components on soluble arsenic dissolution showed that I.naturalis increased the level of soluble arsenic in Realgar extracts and I.naturalis suspensions when the Realgar/I.naturalis ratio was 2,1.5,and 1.0.However,S.miltiorrhiza did not affect it.Conclusion：Based on the collective experimental results presented here,it can be concluded that the toxicity of RIF is the result of the soluble arsenic in Realgar and that the I.naturalis and S.miltiorrhiza in the RIF exert completely opposite effects on the toxicity of Realgar.This maybe an intelligent explanation for the compatibility of this formula,and this RIF study may therefore be viewed as a classic case of traditional Chinese medicine research on compatibility.