Preliminary study of Realgar and arsenic trioxide on gut microbiota of mice / 中国中药杂志

The aim of this paper was to observe the effect of Realgar and arsenic trioxide on gut microbiota. The mice were divided into low-dose Realgar group(RL), medium-dose Realgar group(RM), high-dose Realgar group(RH), and arsenic trioxide group(ATO), in which ATO and RL groups had the same trivalent arsenic content. Realgar and arsenic trioxide toxicity models were established after intragastric administration for 1 week, and mice feces were collected 1 h after intragastric administration on day 8. The effects of Realgar on gut microbiota of mice were observed through bacterial 16 S rRNA gene sequences. The results showed that Lactobacillus was decreased in all groups, while Ruminococcus and Adlercreutzia were increased. The RL group and ATO group were consistent in the genera of Prevotella, Ruminococcus, and Adlercreutzia but different in the genera of Lactobacillus and Bacteroides. Therefore, the effects of Realgar and arsenic trioxide with the same amount of trivalent arsenic on gut microbiota were similar, but differences were still present. Protective bacteria such as Lactobacillus were reduced after Realgar administration, causing inflammation. At low doses, the number of anti-inflammatory bacteria, such as Ruminococcus, Adlercreutzia and Parabacteroides increased, which can offset the slight inflammation caused by the imbalance of bacterial flora. At high doses, the flora was disturbed and the number of Proteobacteria was increased, with aggravated intestinal inflammation, causing edema and other inflammatory reactions. Based on this, authors believe that the gastrointestinal reactions after clinical use of Realgar may be related to flora disorder. Realgar should be used at a small dose in combination with other drugs to reduce intestinal inflammation.

Synergistic effect and different toxicities of adjuvant components of Realgar–Indigo Naturalis formula / 中草药·英文版

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, the LD50 was 2756.73 mg/kg (equivalent to 23.6 mg/kg As2O3). However, the LD50 dropped to 936.90 mg/kg when Realgarwas used with I. naturalis. By contrast, the LD50 increased to 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 theRealgar/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.

Quality of realgar and its influencing factors based on toxicity / 中国中药杂志

The results of a toxicity analysis showed differences from those of the existing experimental data. Therefore, HPLC-ICP-MS was used to analyze the soluble arsenic content at different valences in realgar prepared with water grind processing, which were collected from 3 companies. The results showed that the free arsenic of the 3 companies did not exceed the limit of Chinese Pharmacopoeia. However, if the free arsenic was calculated based on the total value of As(Ⅲ) + As(Ⅴ), free arsenic of 1 company exceeded the limit of Chinese Pharmacopoeia. The method of determining free arsenic in Chinese Pharmacopoeia. was ancient Cai's arsenic detection method, which had a certain limitation and failed to effectively avoid the toxicity of remaining arsenics except for trivalent arsenic. Then, we examined the effects of water and temperature on the content and form of soluble arsenic in realgar. The results showed that the content of soluble arsenic increased with the rise of water content, and the form of soluble arsenic did not change, there were only As (Ⅲ) and As (Ⅴ); With the simple temperature factor, there was an increasing trend in the content of soluble arsenic in the samples, the maximum increment was As (Ⅲ) 2.489 mg•g⁻¹ and As (Ⅴ) 0.546 mg•g⁻¹; When water and temperature played an synergistic effect, the increase of soluble arsenic in the samples significantly changed, the maximum increment was As (Ⅲ) 23.690 mg•g⁻¹, As (Ⅴ) 0.468 mg•g⁻¹, respectively. Through comprehensive analysis, we believed that the quality of realgar was susceptible to water content and temperature. Both of the single effect of water content and the synergistic effect of water and temperature can significantly change the content of soluble arsenic in realgar, and the water content was a high-risk factor. In the current Chinese Pharmacopoeia 2015 version, the free arsenic detection method had limitations, hence new techniques shall be introduced; At the same time, realgar does not have a water content inspection item in the current pharmacopoeia, which shall be added. However, due to the limit of water content, more in-depth studies are required.

Effect of clinical doses of Realgar-Indigo Naturalis formula and large-dose of realgar on CYP450s of rat liver / 中国中药杂志

To investigate the effect of clinical dose of Realgar-Indigo Naturais formula (RIF) and large-dose of Realgar on main drug-metabolizing enzymes CYP450s of rat liver, as well as its regulatory effect on mRNA expression. Wistar rats were administrated orally with tested drugs for 14 days. A Cocktail method combined with HPLC-MS/MS was used in the determination of 4 cytochrome P450 isozymes (CYP1A2, CYP2B, CYP3A and CYP2C) in liver of the rats, and the mRNA expression levels of the above subtypes were detected by real-time fluorescent quantitative PCR. The results showed that RIF can significantly induce CYP1A2 and CYP2B enzyme activity, and inhibit CYP3A enzyme activity. This result was consistent with the mRNA expression. However, its single compound showed weaker or even contrary phenomenon. Different doses of Realgar also showed significant inconsistencies on CYP450 enzymes activity and mRNA expression. These phenomena may be relevant with RIF compatibility synergies or toxicity reduction. The results can also prompt drug interactions when RIF is combined with other medicines in application.