Acute Toxicity Test of Abrus cantoniensis Hance Extract

,2*

1. Youjiang Medical University for Nationalities, Baise 533000, China; 2.Key Laboratory of Guangxi’s Colleges for the Study of Characteristic Ethnic Medicine in Youjiang River Basin, Baise 533000, China

Abstract [Objectives] To investigate the acute toxicity of petroleum ether extract, n-butanol extract, ethyl acetate extract and water extract to mice. [Methods] The medium lethal dose (LD50) in mice could not be determined, so the maximum dose method was used for experiment. Mice were not fed but allowed to drink water for 16 h before the experiment. There were 74 SPF Kunming Mice, half male and half female, and it was divided into extract group and blank control group. The toxicity and death were recorded after intragastric administration and regular close observation. If any mice died, the dead mice were dissected. The changes of heart, liver, spleen, lung, kidney, brain, stomach, small intestine and other organs were observed with the naked eye. At the end of the 14 d observation period, the surviving mice in each group were killed and dissected. The gross pathological changes of the main organs were observed with the naked eye according to the same method mentioned above. [Results] There were no obvious toxic symptoms and no death in the mice. [Conclusions] The maximum doses of petroleum ether extract, n-butanol extract, ethyl acetate extract and water extract in mice were 1.61, 1.71, 1.65 and 1.79 g/L, respectively. Thus it was found that the quarterly tests of petroleum ether extract, n-butanol extract, ethyl acetate extract and water extract suggested that the medicine was safe.

Key words Abrus cantoniensis Hance, Acute toxicity, Maximum dosage

1 Introduction

AbruscantoniensisHance, as a plant in the genus Abrus Adans., is one of traditional Chinese medicines, which contains many ingredients and pharmacological activity. It has the effect of clearing heat and detoxifying, soothing the liver and relieving pain. It can be used to make soup for diet therapy. It is widely used in the treatment of jaundice, hypochondriac discomfort, epigastric distension, acute and chronic hepatitis and cirrhotic ascites[1-2]. It has liver-protecting, antibacterial, anti-virus, anti-tumor, anti-oxidation, anti-inflammation, immune-regulating, and lipid-lowering effects. It originated in Guangxi, Guangdong and other southern provinces and regions, with the largest cultivation area in Guangxi. It is the authentic medicinal material of Guangdong and Guangxi.A.cantoniensisHance is rich in flavonoids, alkaloids, polysaccharides, amides, saponins, organic acids, anthraquinones, chromogenic ketones, lignans, amino acids and other chemical constituents[4]. The anti-tumor active components found in traditional Chinese medicine include polysaccharides, flavonoids, saponins, alkaloids, triterpenoids, phenolic acids and so on[5-8]. It can be seen thatA.cantoniensisHance has a variety of chemical constituents in resisting tumor. The growth of tumor cells depends on a certain environment, and there are usually rich blood vessels around them to maintain their nutritional needs. At this time, medicine can also be transported through blood vessels, to play an anti-inflammatory, detoxification role, and have the inhibitory effect on the growth of tumor cells[3]. The related toxicity ofA.cantoniensisHance has not been reported, but it seriously limits the deep development and utilization ofA.cantoniensisHance. Therefore, the toxicity of petroleum ether extract, n-butanol extract, ethyl acetate extract and water extract fromA.cantoniensisHance was studied. The acute toxicity in SPF Kunming mice was observed by intragastric administration in order to provide a reference for the further development and utilization ofA.cantoniensisHance.

2 Materials and methods

2.1 InstrumentsRE-3000A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), FA1104 electronic analysis balance (Shanghai Balance Instrument Factory), DHG-9070A electric thermostat oven (Shanghai Precision Instrument Co., Ltd.), HHS-21-4 electric thermostat water bath (Jiangsu Jintan Hongkai Instrument Factory), SHB- III circulating water vacuum pump (Shanghai Jiapeng Technology Co., Ltd.).

2.2 MedicineThe dried powder ofA.cantoniensisHance was identified by Associate Professor Qin Daoguang of the Department of Ethnic Medicine of Youjiang Medical University for Nationalities as the whole grass of Abrus Adans. plant. The petroleum ether extract, n-butanol extract, ethyl acetate extract and water extract fromA.cantoniensisHance were prepared by our research group into extract, dimethylsulphoxide (DMSO). The rest of the reagents were analytically pure.

The petroleum ether extract, n-butanol extract, ethyl acetate extract and water extract were prepared as follows. The freshA.cantoniensisHance was rinsed with clean water, dried, and crushed into the powder for preservation. 100 g ofA.cantoniensisHance powder was weighed, and 95% ethanol was added to it at the solid-liquid ratio of 1∶2, and the following method was used to extract. The extraction was carried out 3 times by heating and refluxing with constant temperature water bath, 2 h for the first two and 1.5 h for the third. The filtrate was extracted, merged, and concentrated under decompression with a rotary evaporator. Then the concentrated solution was extracted with petroleum ether, ethyl acetate and n-butanol in turn. Finally, the concentrated solution was dried, and the extract was prepared. When used, it was prepared to the desired concentration.

2.3 AnimalsSPF Kunming mice, weighing 18-22 g, half male and half female. They were provided by the Experimental Animal Center of Youjiang Medical University for Nationalities, Animal Production License No.SCXK Gui 2017-0003.

2.4 Methods

2.4.1Pre-grouping. 36 mice, half male and half female, were weighed separately. According to the principle of similar body weight, 18 male mice were divided into Group 1, 2 and 3 with 6 mice in each group. The mice were marked with picric acid and the corresponding body weight was recorded. The pre-grouping method of 18 female mice was the same as above.

2.4.2Grouping. One mouse was randomly selected from the 18 male mice in Group 1, 2 and 3, respectively, to form a random group. It was repeated 6 times, and 6 experimental groups of mice with various weights were randomly formed, with 3 male mice in each group. They were petroleum ether extract group, ethyl acetate extract group, n-butanol extract group, water extract group, 10% DMSO group, blank control group. In this method, the average body weight and weight distribution of male mice in 6 experimental groups was made consistent as much as possible. The formal grouping method of 18 female mice was the same as above. The experimental groups A, B, C, D, E and F were composed of 6 mice in each group, 3 males and 3 females.

2.4.3Pre-test method. The mice in each group were not fed but allowed to drink water for 16 h and were given intragastric administration with the bearable volume of 0.4 mL/20 g (body weight). The maximum mass concentrations of petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract were 80.43, 82.32, 85.71 and 89.58 g/L, respectively, 2 times per day, with an interval of 4 h each time. The mice were fed routinely and observed continuously for 7 d. During this period, the life and health conditions of the mice were good and there was no death. The medium lethal dose (LD50) in mice could not be determined, so the maximum dose method of the four extracts was carried out[9].

2.4.4Maximum dose. According to the method of pre-grouping, 60 mice, half male and half female, were divided into 6 groups: petroleum ether extract group, ethyl acetate extract group, n-butanol extract group, 10% DMSO group and blank control group with 10 mice in each group, half male and half female. Before the experiment, the mice in each group were not fed but allowed to drink water for 16 h. The petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract with the maximum mass concentrations of 80.43, 82.32, 85.71 and 89.58 g/L, respectively, were administered intragastrically to mice with the bearable volume of 0.4 mL/20 g (body weight), 2 times per day, with an interval of 4 h each time. The blank control group was given normal saline in the same way. After administration, the mice were observed continuously for 14 d.

2.4.5Observation indexes. After administration, the general conditions of mice (fur smoothness, feeding, defecation, eye, nasal secretion, mucosa, respiration and so on) were observed. The activity and autonomic nerve response of mice were observed, such as vocal production, tremor, convulsion, and foraging[10]. The above indexes were observed at 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 12, 16, 20, 24 h, respectively, after intragastric administration. From the second day after administration, it was observed at a fixed time every day and the body weight was measured. After the 14th d, the mice were killed and the main organs of liver, spleen, lung and kidney were taken and observed with naked eyes. If the mice died, the dead mice were dissected and the changes of heart, liver, spleen, lung, kidney, brain, stomach, small intestine and other organs were observed with the naked eye.

3 Results and analysis

3.1 General situationThe appearance and physical signs of the four groups of mice were not abnormal, they moved about freely, and their eating, drinking and feces excretion were in normal conditions. The fur was smooth, white and clean, and the mouth, nose, eyes and anus were clean and free of secretions.

3.2 Dissection and naked eye observation of main organsAll 50 mice survived 14 d after the experiment. There was no obvious toxic reaction in 10% DMSO group, blank group and acute toxicity experimental groups of four extracts. It also did not affect the corresponding tissues, organs and systems. On the 14th d, no abnormal fluid was found in the thoracic cavity and abdominal cavity by dissection. There was no flatulence in the intestines, no color and morphology abnormalities in the heart, liver, spleen, lung, kidney and other main organs, no bleeding spots or other pathological changes. The color of gastric mucosa was normal, there was no bleeding spots, and there was no abnormality in autopsy.

3.3 Death of mice14 d after administration, all the 60 mice in the blank control group, 10% DMSO group and extract group survived, and none of the mice in each group died.

3.4 Maximum doseThe maximum dosage of ethyl acetate extract, water extract, n-butanol extract and petroleum ether extract in mice was 1.65, 1.79, 1.71 and 1.61 g/L, respectively.

3.5 Changes of body weight in miceThe body weight of the mice was measured on the 1st, 3rd, 7th, 10th and 14th d after intragastric administration, and the body weight of the mice in the two groups increased normally. The weight changes are shown in Table 1.

Table 1 Effects of different extracts fromAbruscantoniensisHance on the change of body weight in SPF Kunming mice after administration

GroupDosage∥g/LGenderDaily body weight of mice∥g 1 d3 d7 d10 d14 dEthyl acetate extract1.65♂23.6±3.7327.5±3.1427.8±2.8028.6±2.9729.3±2.30♀23.5±2.7026.3±1.9627.3±2.2727.3±2.3628.4±2.40Water extract1.79♂23.4±9.8126.6±2.4427.4±2.5428.3±2.5628.9±2.56♀26.0±8.1427.7±1.7827.3±2.0427.6±2.7127.6±3.25N-butanol extract1.71♂22.2±2.3825.6±3.4326.8±3.6427.0±3.9127.3±3.79♀24.0±0.4626.4±2.9727.1±2.9927.4±2.7827.9±2.92Petroleum ether extract1.61♂23.6±0.9925.7±2.5626.5±2.1226.8±2.4227.4±2.30♀25.2±1.1028.3±5.6828.6±5.3228.6±5.5828.8±5.43Distilled water-♂23.5±0.7129.9±4.7227.3±3.7728.0±3.5527.5±3.63♀22.5±0.2526.1±1.9826.5±2.1527.3±2.3627.5±2.3610%DMSO-♂24.4±0.5128.5±2.3028.4±2.4229.3±3.0929.8±3.41♀24.1±3.0826.9±2.0126.5±1.6425.7±1.8926.9±1.80

4 Discussions

LD50was not detected by intragastric administration ofA.cantoniensisHance extract to mice. Therefore, mice were given intragastric administration of four kinds of extracts twice a day at the maximum concentration and volume. The mice had normal activity, feeding, good mental state, normal weight gain and no death. After the mice were killed on the 14th d, no abnormality was found in the organs. The maximum dosage of ethyl acetate extract, water extract, n-butanol extract and petroleum ether extract was 1.65, 1.79, 1.71 and 1.61 g/L, respectively. The quarterly tests of petroleum ether extract, n-butanol extract, ethyl acetate extract and water extract suggested that the medicine was safe[11]. Therefore, the clinical use of the medicine was safe.