| This topic is an extension of the task of "Study on Safety and Controllability of 10 Topical Preparations Containing Mineral Drugs" in National public welfare industry research projects of "Study on Commonly-used traditional Chinese medicine processing techniques related to clinical syndromes and decreasing toxicity through the compatibility".Realgar which comes from the minerals sulfide realgar family,tastes acrid and its character is warm.It can enter the channels of the large intestine and liver.But it is poisonous.The clinical application of realgar has a long history,and it was first recorded in the 2000-year-old classic,Shen Nong Ben Cao Jing.Traditionally,it has been used for the treatment of cystic acne,snake bite,abdominal palsy,epilepsy,malaria,etc.,and modern medicine has applied it to the treatment of leukemia and tumors,and has achieved remarkable results.The fact that common Chinese medicines such as Niuhuangjiedu tablets and Angongniuhuang pills also contain realgar indicates realgar has been widely used in modern clinics.Realgar,as a toxic traditional Chinese medicine,has hepatotoxicity and nephrotoxicity,neurotoxicity,hematological toxicity,and genetic toxicity,etc.It is generally processed for internal and external use.However,realgar has many methods of processing.Water-grinding was first published in"Key to the Therapeutics of Children’s Diseases" of the Song Dynasty:"Water-grinding".And it is realgar’s method of processing which was prescribed in the 2015 edition of the "Chinese Pharmacopoeia"(Part 1).Grinding is also a realgar’s method of processing,which is currently used more often,and is included in many processing specifications of provinces and cities.Water washing which developed in modern times is most similar to water-grinding,and it is favored by many companies because of its high efficiency and low consumption.However,the quality,toxicity and efficacy of the different realgar’s processed products manufactured by the above-mentioned commonly used processing methods are still unclear and need to be studied.Our previous research shows that particle size is an important factor affecting the quality of realgar products.Therefore,controlling particle size to eliminate particle size’s interference is very important for studying the influence of different processing methods on realgar products.Therefore,this study will compare the appearance characteristics,chemical composition content,toxicological differences and differences in efficacy of different processed products with uniform particle size processed by the three methods of water-grinding,grinding,and water-washing to clarify the influence of different processing methods on the quality,toxicity and efficacy of the realgarproducts and the significance of water-grinding.Objective:1.Comparing the particle size and color model of the realgar water-grinding products,grinding products,and water-washing products,in order to accurately and objectively describe the differences in the appearance characteristics of the three processed products,and to clarify the effects of water-grinding,grinding,and water-washing on appearance characteristics of the realgar products.2.Comparing the total amount of arsenic and the type and content of elements in water-grinding products,grinding products and water washing-products to clarify the effects of water-grinding,grinding,and water-washing on the quality of the realgar products.Comparing of the morphology of realgar water-grinding products,grinding products and water washing-products under scanning electron microscopy in order to achieve rapid identification of the above three types of realgar processed products,and to provide relevant information for differentiating realgar powders processed by different methods.3.Establish a hydride generation-atomic absorption spectrophotometer method for the determination of soluble arsenic in realgar,compare the soluble arsenic content in realgar water-grinding products,grinding products and water washing-products,and clarify the influence of three processing methods on the efficacy of realgar products and the significance of water-grinding.4.Compare the actual hepatotoxicity and nephrotoxicity of realgar water-grinding products and grinding products,and verify whether the actual toxicity of water-grinding products and grinding products is consistent with the theoretical toxicity.5.Compare the efficacy of realgar water-grinding products and grinding products.Methods:1.The 12 batches of realgar medicinal materials collected were processed according to water-grinding,grinding,and water-washing,and were made into water-grinding products,grinding products and water washing-products.2.The particle size and color model were respectively used as the objective quantitative index of the powder size and color of realgar water-grinding products,grinding products and water washing-products.The particle size and color of the three processed products were respectively measured by a laser particle size distribution analyzer and a colorimeter.Statistical analysis software SAS 8.2 was used for statistical analysis of data,and statistical methods for single-factor analysis of variance were used to statistically analyse the particle size data,L*,a*,b*,C.*,h,and ΔE values of water-grinding products,grinding products and water washing-products;Principal component analysis of L*,a*,b*values of realgar products was performed using the analysis software SIMCA12.0.3.The total amount of arsenic in realgar water-grinding products,grindingproducts and water washing-products shall be determined by the volumetric method which is the determination method of the realgar content in the 2015 edition of the "Chinese Pharmacopoeia"(Part 1).The total amount of arsenic was compared and analyzed.4.The morphological characteristics of the realgar water-grinding products,grinding products and water washing-products were observed under a 10,000-fold microscope by scanning electron microscopy.Elemental analysis of the surface was performed by using an energy spectrometer.And analyze the morphology,element types and contents of the three processed products.5.By investigating the amount of sample,extraction solvent,extraction time,pore size of the microporous membrane,optimize the preparation method for the test sample.A hydride generation-atomic absorption spectrophotometer forthe determination of soluble arsenic in realgar was established.The realgar sample was treated with the best sample preparation method and the soluble arsenic content of the realgar water-grinding products,grinding products and water washing-products was determined by the established soluble arsenic content determination method.The soluble arsenic content of the three processed products was compared and analyzed.6.Pharmacological experiments were conducted on the realgar water-grinding products,grinding products.The rats were divided into groups and administered by gavage.The general conditions of the rats in each group were observed and recorded.The body weights of the rats before and after the experiment were weighed.Weigh the weight of liver and kidney after dissection in each group rats,calculate the organ coefficient of liver and kidney.Detect liver and kidney function indexes in serum of each group rats,detect the oxidation and antioxidation indexes of liver and kidney of each group rats.The pathological changes of liver and kidney in each group were observed by light microscope.Statistical analysis software SAS8.2 was used for statistical analysis of data.Single-factor analysis of variance was used to statistically analyse the weight change and organ coefficient of each group rats.The contents of blood biochemical indicators ALT,AST,BUN,Cr and liver and kidney homogenate GSH,MDA,SOD were statistically analyzed.7.Pharmacological experiments were conducted on the realgar water-grinding products,grinding products.The mice were divided into groups and administered by gavage.The left ear of the mice was swollen with xylene and was punched in the left and right ears with a 6 mm diameter punch.The wafers were weighed and the degree of swelling was calculated.Statistical analysis software SAS8.2 was used for statistical analysis of data.Single-factor analysis of variance was used to statistically analyse the ear swelling of each group mice.Results:1.The statistical results of the particle sizes of realgar water-grinding products,grinding products and water washing-products are respectively 10.25± 1.48μm,10.71 ± 1.99μm and 11.33± 1.90μm.The particle sizes of the three processed products are at the same level.The L*,a*,and b*values of the three realgar products are all positive.The hue angle h value and the color saturation C*value are all sorted as water-grinding products>water washing-products>grinding products,indicating that the color of realgar products is biased toward red and yellow.And the grinding products in the three realgar products should be the most red and brightness.Water-grinding products should be more yellowish and shallow.However,this result is only applicable to the comparison of water-grinding products,grinding products and water washing-products processed by the same ore-derived realgar medicinal materials,and the comparison among the three processed products processed by different’sources of realgar medicinal materials is incompatible.The results of principal component analysis showed that there was no significant difference in color among the realgar water-grinding products,grinding products and water washing-products.The three processed products could not be distinguished by color discrimination.2.The total amount of arsenic in the three processed products is ranked as follows:water-grinding products>water washing-products>grinding products.The appearance of water-grinding products and water washing-products are similar,and the surface of them are clear and smooth,and has a blunt circular shape.The surface is attached with fine powder.The grinding products are mostly polygonal,with sharp-edged corners,and their morphology is different from that of the other two processed products.Some grinding products contain silicon and the other two processed products don’t contain.The ranking of the oxygen content of the three processed products showed the trend of grinding products>water washing-products>water-grinding products.3.A hydride generation-atomic absorption spectrophotometer was established for the determination of soluble arsenic in realgar.The concentration and absorbance of the method are linear in the linear range.The precision,reproducibility,and stability are high.The results are accurate.reliable.The soluble arsenic content of realgar water-grinding products,grinding products and water washing-products showed the trend of water-grinding products>water washing-products>grinding products.4.After one month of high-dose administration of realgar water-grinding product,the rat’s general condition was not abnormal.Only orange stools were frequently discharged.The liver organ coefficient and the content of blood biochemical index ALT were significantly increased.The contents of GSH and SOD were significantly decreased,and the content of MDA was increased.Pathological sections of liver tissue in rats showed hepatocellular edema,disordered hepatic cord arrangement,and hepatic sinusoidal stenosis.The liver is damaged.One week after discontinuation,liver biopsy showed normal liver structure,liver organ coefficient and the content of blood biochemical index ALT,AST were at normal levels.The levels of GSH,SOD,MDA in liver homogenate are also normal levels.After one month of low-dose administration of realgar water-grinding product,high-dose or low-dose administration of realgar grinding product,the liver tissue of the rat showed normal liver structure without obvious pathological changes.And the liver organ coefficient and the contents of blood biochemical index ALT,AST were normal.The contents of antioxidant index SOD and oxidative damage index MDA in the liver homogenate were also normal,and only the content of antioxidant index GSH was decreased.At the high or low-doses of realgar water-grinding product and grinding product in this experiment,one month after administration,the kidney organ organ coefficient,the content of blood biochemical index Cr,and the contents of GSH,SOD,and MDA in the kidney homogenate were measured,all the contents were normal.But the content of blood biochemical index BUN increased significantly.However,pathological section of renal tissue in rats showed normal renal structure without obvious pathological changes.5.When high-dose realgar water-grinding product and realgar grinding product were administered,the swelling degree of the ear of mice was significantly reduced,and the high dose of water-grinding product significantly reduced the degree of ear swelling of mice compared with grinding product;In addition,low-dose administration of water-grinding product and grinding product reduced the ear swelling of mice.Conclusions:1.The water-washing does not have a statistically significant effect on the particle size of the realgar.The particle sizes of realgar water washing-products and grinding products both met the requirements of the processing technology and reached the corresponding particle size of water-grinding products.2.The main factor influencing the color of realgar products is the source of realgar ore,followed by processing methods.3.The use of scanning electron microscopy-energy spectrometer can quickly identify the realgar water-grinding products and grinding products,providing some technical support for different realgar products.Among the three processing methods,the water-grinding has the best effect of removing impurities and eliminating impurities.The quality of water-grinding products is better than that of grinding products and water washing-products,which shows that water-grinding has certain scientific basis for processing realgar.4.The hydride generation-atomic absorption spectrophotometer can be used to determine the soluble arsenic content with precision and reproducibility,high stability,and accurate and reliable results.Among the three processing methods,the content of soluble arsenic in water-grinding products is the highest,the synergistic effect of water-grinding is the strongest,and the quality of water-grinding products is better than that of grinding products and water washing-products.The processing method is an important factor that affects the content of soluble arsenic in realgar.5.Combining the results of oxygen content and soluble arsenic content in realgar products,it can be shown that partially dissolved As2S2 may be the main source of realgar soluble arsenic,and other trace toxic arsenic compounds will also bring some soluble arsenic.Realgar can not only remove some other trace toxic arsenic compounds but also increase the dissolution of arsenic in As2S2 after water-grinding and water-washing.This part of dissolved arsenic may be the medicinal component of realgar and its toxicity is relatively high small.6.The realgar water-grinding products with a soluble arsenic content of 90.92 ng/mL had hepatotoxicity without nephrotoxicity at a dose of 1.92 g/kg administration for one month,had no hepatotoxicity and no nephrotoxicity at a dose of 0.64 g/kg administration for one month.The realgar grinding products with a soluble arsenic content of 80.35ng/mL had no hepatotoxicity and no nephrotoxicity after one month administration regardless of the dose.However,the anti-inflammatory drug efficacy of the realgar water-grinding products was better than that of the realgar grinding products.Soluble arsenic is a component of the toxicity and efficacy of realgar.The toxicity of realgar is related to the daily intake of soluble arsenic and the duration of use.When the daily intake of soluble arsenic exceeds the safe range of soluble arsenic,toxic reactions will occur after a certain period of time.The soluble arsenic content of water-grinding products made from the same realgar medicinal materials is generally larger than that of grinding products and the efficacy is good,but the premise is to ensure that the dose of water-grinding products is within a safe range,while the safe dose of water-grinding products is determined by the safe range of soluble arsenic.Therefore,the research on the safe range of soluble arsenic is very important. |
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