Toxicolological Test of Saponins from Sapindus mukorossi Gaerth

The study has been carried out to investigate acute oral toxicity, acute dermal toxicity in SPF rats and dermal irritation in rabbits. The result shows: 1) acute oral toxicity test shows 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); 2) acute dermal toxicity test shows that LD50 of saponins from Sapindus mukorossi is more than 5000 mg/kg in both female and male Wistar rats; 3) dermal irritation test in rabbits shows that the average score of dermal irritation per day of each rabbit is zero after 14 days of continuous dermal irritation. According to the classification standard of toxicity in “Hygienic Standard for Cosmetics” (2002 version), the sample is classified as “practical nontoxic” and “non dermal irritation”. Thus, we can conclude that the saponin extraction from S. mukorossi Gaerth is safe for cosmetics.


Introduction
The species of the genus of Sapindus belonging to the Sapindaceae has about five to twelve species of shrubs and small trees (FRPS, 1998).Members of the genus are commonly known as soapberries or soapnuts because the fruit pulp is used to make soap (FRPS, 1998).There are about 10% saponins in fruit pulp (FRPS, 1998), which makes it an ideal resource for the extraction of saponins.Saponin, a natural non-ionic surfactant, not only has a good emulsifying, separating and dispersing capability but also is a good foaming and foam stabilizer with a great cleaning capacity (Zhang et al., 1993).Thus, it can be used as foam stabilizer for building concrete (Lin, 1977) pesticide synergist (Hong & Tokunaga, 2000) and antiviral, and reducing blood pressure (Huang et al., 2007;Ibrahim et al., 2006Ibrahim et al., , 2008;;Kuo et al., 2005;Yukiyoshi, 2001;Yata et al., 1986).So it is widely used in daily chemical industries, building materials, food industries and agriculture.In this study we evaluated saponins from Sapindus mukorossi to study acute oral toxicity, acute dermal toxicity in SPF rats and dermal irritation in rabbits.

Plant and Chemical Material
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.

Extraction Process
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).

Experimental Animals
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.

Acute Oral Toxicity Test
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.

Acute Dermal Toxicity Test
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 cm 2 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.

Dermal Irritation Test in Rabbits
Fur on the back of rabbits is removed by electric clippers 24 hours before dosing.The sample is smeared on a 5 × 6 cm 2 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.

Statistical Analysis
The results are using Horn's table to find LD 50 .

Oral Acute Toxicity in Wistar Rats by Saponins
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 LD 50 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).

Acute Dermal Toxicity in Wistar Rats by Saponins
No obvious intoxication sign was observed and death happened.The result of acute dermal toxicity test showed that LD 50 of saponins from Sapindus mukorossi is more than 5000 mg/kg in both female and male Wistar rats (Table 4).

Grading Results of Dermal Irritation Test
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).

Conclusion
The result shows: 1) acute oral toxicity test shows that LD 50 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); 2) acute dermal toxicity test shows that LD 50 of saponins from Sapindus mukorossi is more than 5000 mg/kg in both female and male Wistar rats; 3) dermal irritation test in rabbits shows that the average score of dermal irritation per day of each rabbit is zero after 14 days of continuous dermal irritation.According to the classification standard of toxicity in "Hygienic Standard for Cosmetics" (Ministry of Public Health of China, 2002), the sample is classified as "practical nontoxic" and "non dermal irritation".Thus, we can conclude that the saponin extraction from S. mukorossi Gaerth is safe for cosmetics.

Table 1 .
Dosage and weight of rats in five different dosage groups.

Table 2 .
Dosage and weight of rats.

Table 3 .
Oral acute toxicity in Wistar rats by saponins.

Table 4 .
Acute dermal toxicity in Wistar rats by saponins.

Table 5 .
Grading results of dermal irritation test in rabbits by saponins.