Table2_Toxicological safety evaluation of Qin-Zhi-Zhu-Dan formula in rats during the treatment and recovery periods.XLSX

Background: Qinzhi Zhudan Formula (QZZD), optimized from Angong Niuhuang Wan, consists of Radix Scutellariae, Fructus Gardeniae and Pulvis Fellis Suis. We had investigated the neuroprotective effects of QZZD and its active components, and demonstrated that it could treat cerebral ischemia and dementia through multiple pathways and mechanisms. Nevertheless, toxicological data on this formula still remains limited. In the study, we sought to examine the toxicological effects of QZZD during the treatment and recovery periods.
Methods: We investigated potential toxicities of QZZD in Sprague-Dawley (SD) rats via 28-day gavage administration. SD rats were randomly divided into control group and treatment groups of A (0.5 g/kg/d QZZD), B (1.5 g/kg/d QZZD), and C (5.0 g/kg/d QZZD). The 56-day course includes treatment period (administration with water or QZZD once a day for 28 consecutive days) and recovery period (28 days). The rats received daily monitoring of general signs of toxicity and mortality, as well as weekly determination of body weight and food consumption. Moreover, the complete blood cell count, biochemistry, coagulation, and urine indicators, organ weights, and histopathological report were analyzed respectively at the end of the treatment and recovery periods.
Results: There was no death related to the active pharmaceutical ingredients of QZZD during the treatment period. The maximum no observed adverse effect level (NOAEL) was 0.5 g/kg/d, which is approximately 16.7 times of the equivalent dose of clinical dose in rats. In group TB (1.5 g/kg/d QZZD) and TC (5.0 g/kg/d QZZD), there were adverse effects of blue coloring of tail skin, weight loss, a significant increase of total bilirubin (TBIL), blackening of liver and kidney in gross examination, hyperplasia of bile duct and karyomegaly of hepatocytes in histopathological examination. Besides, in females rats, the food consumption was reduced, while in male rats, there was decrease in triglycerides (TG) and slight increase in white blood cell (WBC) count and neutrophils. In group TC (5.0 g/kg/d QZZD), the indicators of red blood cell (RBC) count, hemoglobin (HGB) and hematocrit (HCT) were decreased slightly, while the platelet count (PLT) was increased. However, these changes were not considered to be toxicologically significant because they resolved during the recovery period.
Conclusion: Overall, QZZD exhibited a good safety profile. The maximum no observed adverse effect level was 0.5 g/kg/d, and no target organs toxicity were identified. The present findings might confirm the safety of QZZD in clinical practices.

背景：芩芝竹丹方（Qinzhi Zhudan Formula, QZZD）是由安宫牛黄丸优化得到的方剂，其组成成分为黄芩（Radix Scutellariae）、栀子（Fructus Gardeniae）与猪胆粉（Pulvis Fellis Suis）。本团队此前已对QZZD及其活性成分的神经保护作用进行了研究，证实其可通过多通路、多机制发挥脑缺血与痴呆治疗作用。然而，目前针对该方剂的毒理学数据仍较为有限。本研究旨在探究QZZD在给药与恢复期的毒理学效应。方法：本研究通过28天灌胃给药方式，考察芩芝竹丹方对斯普拉格-道利（Sprague-Dawley, SD）大鼠的潜在毒性。将SD大鼠随机分为对照组与给药组A（0.5 g/kg/d QZZD）、B（1.5 g/kg/d QZZD）、C（5.0 g/kg/d QZZD）。实验周期共56天，分为给药期（每日一次灌胃给予生理盐水或QZZD，连续28天）与恢复期（28天）。每日监测大鼠的一般毒性体征与死亡率，每周测定体重与摄食量。此外，分别于给药期末与恢复期结束时，采集全血细胞计数、生化指标、凝血功能、尿液指标、脏器重量及组织病理学检查结果进行分析。结果：给药期内无与QZZD活性成分相关的死亡事件。本品最大未观察到有害作用剂量（no observed adverse effect level, NOAEL）为0.5 g/kg/d，约为大鼠等效临床剂量的16.7倍。与对照组相比，B组（1.5 g/kg/d QZZD）与C组（5.0 g/kg/d QZZD）大鼠出现尾部皮肤蓝染、体重下降、总胆红素（total bilirubin, TBIL）显著升高；大体解剖可见肝肾发黑，组织病理学检查可见胆管增生与肝细胞核肥大。此外，雌性大鼠摄食量降低，雄性大鼠甘油三酯（triglycerides, TG）水平下降，白细胞计数（white blood cell, WBC）与中性粒细胞数轻度升高。在C组（5.0 g/kg/d QZZD）中，红细胞计数（red blood cell, RBC）、血红蛋白（hemoglobin, HGB）与红细胞比容（hematocrit, HCT）轻度降低，血小板计数（platelet count, PLT）轻度升高，但上述变化在恢复期均恢复正常，因此被认为无统计学毒理学意义。结论：总体而言，芩芝竹丹方具有良好的安全性，其最大未观察到有害作用剂量为0.5 g/kg/d，未发现靶器官毒性。本研究结果可为QZZD的临床应用安全性提供实验依据。