Fruits pulps of S. mukorossi were collected from Tiantai of Zhejiang Province, China, in November 2008. Prior to all extractions, fruits pulp was dried at 60˚C for 48 h and was ground in a Wiley mill to pass a 0.5 mm poresize screen. Chromatograph solvents used during the study were of HPLC grade and the other solvents and reagents used during the study were of AR grade.

1 kg ground fruits pulps of S. mukorossi was mixed with 6 times the amount by weight of 85% EtOH, homogenized and transferred to an extraction flask. Reflux extraction was carried out at 85˚C water bath temperature for 2 hours. After cooling the extract solution is filtered through a folded filter. The filtrate is concentrated to dryness in a rotary evaporator at 75˚C water bath. The sample of saponins were washed sequentially with chloroform, ethyl acetate and then dried. The solid matter of sample was re-dissolved in 85% EtOH and mixture was centrifuged at 6000 g for 10 min at room temperature. The supernatant was dried by lyophilization. The samples were prepared for the following test (Du et al., 2014) .

SPF Wistar rats, weight 175 - 213 g, provided by Zhejiang Provincial Research Center for Experimental Animals. Quarantined and observed for one week before testing. Caged feeding, five rats in one cage (20˚C ± 2˚C, 40% ± 5%). Grain whole-nourishment feed for Experimental animals. Environment is held constant. We ensure the welfare of Experimental Animals.

Rats were randomly assigned to 5 dosage groups. See Table 1. Density of the sample is 1.05 g/ml, Oral dosing capacity is 10 ml/kg BW in the 2150, 4640, 10,000 mg/kg BW dosage groups, in which the sample is diluted to 215, 464, 1000 mg/ml by distilled water. In 21,500 mg/kg BW group the oral dasing capacity is 20.48 ml/kg BW without dilution of the sample. Feed is withheld 14 hour before dosing with supply of water. The rats are weighed before administration. The sample is administration through oral intubations. Feed is given two hours after dosing. In the first hour after oral administration continuous observation is conducted, followed by two times of observation per day till 14^th day. The character of poisoning, recovery and death were recorded in detail. All dead rats were anatomized and examined.

Rats were weighed into one dosage group. See Table 2. Fur on the back of rats is removed by electric clippers 24 hours before dosing. The sample is smeared on a 5 × 6 cm² area of the naked skin at a dose of 0.95 ml/200 gBW (Using the sample directly) for 24 hours, with plastic covered and rubberized fabric fixed. Day after exposure terminated and the skin is cleaned with water. In the first hour after oral administration continuous observation is conducted, followed by two times of observation per day till 14^th day. The character of poisoning, recovery and death were recorded in detail. All dead rats were anatomized and examined.

Fur on the back of rabbits is removed by electric clippers 24 hours before dosing. The sample is smeared on a 5 × 6 cm² area of the naked skin at a dose of 0.95 ml/200 g BW (Using the sample directly) for 24 hours, with plastic covered and rubberized fabric fixed. Day after exposure terminated and the skin is cleaned with water. In the first hour after administration continuous observation is conducted, followed by two times of observation per day till 14^th day. The character of poisoning, recovery and death were recorded in detail. All dead rats were anatomized and examined.

The results are using Horn’s table to find LD₅₀.

30 minutes after administration, poisoning sign of sagged spirit were observed. Death of rats happen from 4 hours to 4 days after administration, swollen stomach and intestine were found by anatomy. Most of the survived rats were recovered from poisoning symptom 7 days after administration. No other significant pathogenic alteration was observed by naked eye. The result of acute oral toxicity test showed that LD₅₀ of saponins from Sapindus mukorossi is 9260 mg/kg (95% confidence interval is 6360 - 13,500 mg/kg) and 7940 mg/kg (95% confidence interval is 4890 - 12,900 mg/kg) in female and male Wistar rats respectively (Table 3).

No obvious intoxication sign was observed and death happened. The result of acute dermal toxicity test showed that LD₅₀ of saponins from Sapindus mukorossi is more than 5000 mg/kg in both female and male Wistar rats (Table 4).

The result of dermal irritation test in rabbits showed that the average score of dermal irritation per day of each rabbit is zero after 14 days of continuous dermal irritation by the saponins from Sapindus mukorossi (Table 5).