# Acute and Repeated Toxicological Study of Anti-Inflammatory Herbal Formula, Yeosinsan, in Rats
## 4 Discussion  
In this study, Yeosinsan, a traditional herbal formula composed of _P. lactiflora_ and _C. rotundus_, was evaluated for acute and chronic oral toxicity using female and male SD rats. In the acute toxicity experiment, the main findings (single administration of 0, 500, 1000, or 2000 mg/kg of Yeosinsan) indicated no significant alteration in mortality, body weight, general clinical symptoms, or gross pathological signs in either male or female rats. Thus, these results suggest that an approximate acute lethal dose of Yeosinsan is over 2000 mg/kg in rats. In the chronic toxicity experiment, the main findings indicated no significant alteration in mortality, body weight, general clinical symptoms, food uptake, ophthalmological condition, organ weight, or gross pathological signs following daily treatment with Yeosinsan (0, 500, 1000, or 2000 mg\(\cdot\)kg\({}^{-1}\cdot\)d\({}^{-1}\) for 13 weeks). However, the hematological, clinical, pathological, and histopathological observations demonstrated several significant changes in these groups. In the histopathological experiments, a minimal to slight squamous hyperplasia of the forestomach was found in the 2000 mg\(\cdot\)kg\({}^{-1}\cdot\)d\({}^{-1}\) groups. This observation was accompanied by vesiculation or neutrophil infiltration in the limiting ridge of the forestomach. Thus, the change was considered an adverse effect of inflammatory irritation stimulated by the experimental material, according to guidelines and a previous study [23; 24]. Previous studies on the toxicity and safety of _P. lactiflora_ and _C. rotundus_, the component herbs of Yeosinsan, did not report any severe toxicity [14; 22]. A network pharmacology study revealed that _P. lactiflora_ has a protective effect against ulcerative colitis [25]. In addition, the combination of _P. lactiflora_ and _Inula britamica_ L. has a protective effect against gastric ulcers induced via the administration of HCI/EOH solution [26]. An herbal formula composed of _C. rotundus_, _Holarhena antidysenterica_ L., and _Zingiber officinal_ Rose. has been used for abdominal pain [14]. However, no previous study reported the protective or deteriorating effect of _C. rotundus_ on the stomach. The concentration of 2000 mg\(\cdot\)kg\({}^{-1}\cdot\)d\({}^{-1}\) of Yeosinsan is far from the clinical dose of the ingredient herbs, _P. lactiflora_ and _C. rotundus_[14; 22; 27]. Thus, in general clinical use, Yeosinsan could be regarded as a safe herbal formula up to its usual dose. In the histopathological observation of the spleen, mild extramedullary hematopoiesis was found in the female 500 mg\(\cdot\)kg\({}^{-1}\cdot\)d\({}^{-1}\) group and all groups at or over 1000 mg\(\cdot\)kg\({}^{-1}\cdot\)d\({}^{-1}\). The change was characterized as the increase in erythroid lineage cells in the red pulp. These findings were considered to be a regenerative reaction to the decrease in the RBC and increases in RETA, RET%, MCV, and MCH. In addition, very weak increases in pigmentation were observed in the groups at or over 500 mg\(\cdot\)kg\({}^{-1}\cdot\)d\({}^{-1}\). This appears to be related to the clinicopathological findings such as an increase in TBIL in the serum, increases in BILu and UBG in the urine, and an increase in the relative weight of the spleen. These results appear to be related to hemolytic anemia because damaged RBCs are generally removed by macrophages in the spleen [28]. The effects of _P. lactiflora_ or _C. rotundus_ on hemolytic anemia and histopathological changes in the spleen were not previously reported. _P. lactiflora_ has been regarded as a remedy for anemia caused by iron deficiency and hemolysis [29; 30]. Paeoniflorin and albiflorin, the major compounds of _P. lactiflora_, exhibited a hematopoietic effect on a chemotherapy- or radiotherapy-induced murine myelosuppression model [31; 32]. The pathological changes in the spleen inducedby chemical hemolysis were also reduced by treatment with _P. lactisiflora_ or its component glucosides [30; 33]. _C.