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

Abstract

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.

Keywords: biochemical; gardenia extract; organ coefficient; pulvis fellis suis; reversibility study; scutellaria baicalensis extract; sub-chronic toxicity.

FIGURE 1

Representative experimental scheme. ‘AP’ indicates 14 days of suitability rearing before the experiment; ‘FDR’ was defined as trial day 1 (D1), body weight and ophthalmic examinations were performed. The whole experiment lasted for 56 days, it was divided into treatment period (TP) and recovery period (RP). During the trial, cage observation daily; detailed clinical signs observation, body weight and food consumption were performed weekly. Ophthalmic examination and urine examination are performed before the end of the treatment and the recovery periods (D26 and D54). Hematologic, biochemical and coagulation examinations were tested at the end of the treatment period and the recovery period (D28 and D56), and necropsy, organ weight and histopathological examination were performed after the animals were euthanized.

FIGURE 2

Effects of different doses of QZZD on the body weight in female (A) or male (B) rats and food consumption in female (C) or male (D) rats. In the treatment period, n = 18 in each group, and two female rats (4110 and 4111) death caused by faulty gavage operation, the total number was 16 (n = 16) in TC female group. In the recovery period, n = 6 in each group for female or male.

FIGURE 3

Effects of three different doses of QZZD on absolute organ weights, organ coefficients and organ/brain coefficients in male or female rats at the end of the treatment period or recovery period. In the treatment period, n = 12 in female group (n = 10 in TC) (A–C), and n = 12 in male group (D–F). In the recovery period, n = 6 in each group, including six females (G–I) and six males (J–L). Compare with the control group *p < 0.05.

FIGURE 4

The representative histopathological sections of rat liver at the end of the treatment period. (A) The CN and TA rats liver showing normal hepatocytes and hepatic vasculature; The TB and TC rats liverr showing bile duct proliferation and karyomegaly of hepatocytes (×100 magnification). (B) The CN and TA rats liver showing normal hepatocytes and hepatic vasculature; The TB and TC rats liver showing bile duct proliferation and karyomegaly of hepatocytes (×400 magnification).