A proper amount of macro-porous resin (1.2g) was taken into a beaker; 50ml of 95% ethanol was added for 12hrs, and then washed. Washing was repeated 3 times. After washing 30 ml of a 3% HCl solution was used; the solution was soaked for 5 hrs, washed to neutral pH, and then soaked in a 0.5 mol/L NaOH solution for 5 hours, and washed with distilled water to neutral pH (Liu et al., 2013).

1.2 g of eight solid materials were taken into a conical flask, 15 mL of camellia oil were added to a gas bath at 25 ^ºC, at 200 r/min, and 6 mL of the supernatant oil samples were shocked in a round flask for 5 hrs. After adding 30 mL of ethanol solution (1 mol/L NaOH) in a boiling-water bath, it was returned to liquid after 1 hr, and then the ethanol was removed by vacuum distillation at about 100 mL at 40 ºC. The resulting liquid obtained from the 5mL was diluted 20 times. The absorbance of 0.5 mL diluted solution was measured and the value was reported. The content of tea saponin in camellia oil was calculated according to the following formula:

Where:

Q: the adsorption amount in mg/mL; A: 550 nm under the conditions of the absorbance value of the liquid soap; R: the adsorption Rate % (Liu et al., 2013).

A quantity of 1.2 grams resin were pre-treated (wet weight, surface water was dried with filter paper) in a conical flask, then added to 15 mL of camellia oil under control conditions: shock speed 200 r/min, shock adsorption 4hrs, temperatures of 20 ^°C, 30 ^°C, 40 ^°C, 50 ^°C, 60 ^°C, respectively. Then spectrum-photometry was used to determine the absorption value and the tea saponin adsorption rate was calculated (Liu et al., 2016a).

The camellia oil and resin after pre-treatment, in liquid-solid ratios (mL:g) of 9:1, 10:1, 11:1, 12:1, 13:1 were subjected to shock conditions: speed 200 r/min, temperature 25 ^ºC, time 5 hours. Under these conditions and after the shock absorption, a spectrophotometer was used to measure the absorbance value, and then tea saponin adsorption rate was calculated (Liu et al., 2013).

In optimal conditions of adsorption, the adsorption resin stays in the upper oil phase. As tea saponin is insoluble in acetone, camellia oil was dissolved in acetone. The acetone was washed with the attached oil resin, then filtered. After the resin was packed, 20%, 50%, 70% and 90% ethanol solutions were used. The flow rate of 1 mL/min was controlled and the liquid-to-solid ratio 25:1 was eluted. The spectrophotometric method was used to determine the eluent absorption value; and the elution rate of tea saponin was calculated. The elution rate of tea saponin was calculated according to the following formula:

W₁: tea saponin content, eluted from the resin of mg; m₁: elution resin dosage (g). W₂: Tea saponin content, resin adsorption mg; m₂: adsorption resin (g) (Liu et al., 2013).

A 90% ethanol solution was used to incorporate the resin at a controlled flow rate of 1 mL/min, 1.5 mL/min, 2 mL/min and 3 mL/min, at a liquid-to-solid ratio elution of 20:1. After applying the spectrophotometric method for the determination of the eluent absorption value of A, the elution rate of tea saponin was calculated (Liu et al., 2013).

Design-Expert software and response surface methodology were used to explain the interaction between the factors of response surface analysis and response surface curve. The correlation between independent and dependent factors was graphically supported by 3D reaction surface and 2D form plots produced by a prototype. The software generated the optimum saponin adsorption rate to reveal the conditions which gave the most extreme results (Shen and Wu, 2017).

60 healthy male Kunming mice, five weeks old, 25 to 30 g weight, SPF (specific pathogen free), purchased from the Center for Disease Control (CDC), Hubei province, China were used. The animal number was No. 42000600002931. Mice feed was provided by the experimental animal research center of Hubei province and the qualified number was 42000500000068. The mice were fed with tea oil containing tea saponin for 90 days; they were housed in a room with temperature (18 to 24 ^°C), and relative humidity (40% to 60%). All the experiments were performed at the College of Food Science and Technology; Huazhong University; Hubei Provence; Wuhan; China. The mice were separated into various cages, each with 10 mice. They were stamped, weighed, and fed with essential feed, with versatile feeding and monitored for seven days. The mice were then arbitrarily partitioned into six groups of 10 mice each, all male sex. A normal group (control A), rapeseed oil group (control B) were taken as control groups. Tea saponin/water (C), low (D), medium (E) and high (F)-dose groups were also assigned. All systems were led as per the standards and methodology illustrated in the National Institutes of Health Guide on the Care and Use of Laboratory Animals (NIH, 1997). All the experiments were approved and reviewed by the Institutional Animal Welfare and Research, Ethics Committee guidelines of Huazhong Agricultural University, Wuhan, China (Approval number: 31273519).

All mice had free food and water intake; they were given an intra-gastric administration every morning from 8:00–9:00 once a day. The normal group (control A) was given standard saline; and control B was given rapeseed oil (0.01 mL). The other groups were given the comparing test substance (tea oil containing tea saponin) at 28, 255, 382.5, and 510 mg/kg. bw. The mice were weighed every week and the activities of the mice, feeding, presence of poisoning and death, were observed every day. Drug dose was balanced by body weight.

The method of Abliz et al., (2014) was followed with some modifications. The mice were treated for 90 days, and fasted one day before the end of the trial. On the last day, they were weighed. They were fixed, and blood was gathered from their eyes. All the blood was left at room temperature for 0.5 hr, and was then centrifuged at 3000 rpm for 10 min. Serum was pipetted for the estimation of serum lipid concentration and the serum blood was determined using the automatic blood cell analyzer. After blood gathering, the heart, liver, spleen, and kidney were immediately taken and washed with super-cold saline; blotted on filter paper and weighed. The organ indexes were calculated by the following formula:

The organs were washed and dried, and three examples were taken from each one. They were immediately put into glass vials loaded with 4% formaldehyde, and cooled to 4 °C the for histopathological examination (Abliz et al., 2014). Serum was used in the biochemical assays. Triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL), and low density lipoprotein (LDL) were determined using the TG kit, TC kit, HDL kit and LDL kit (Beijing wantai DRD CO., LTD., Beijing, PR China) (Shen, and Wu, 2017).

The effects of tea saponins of camellia oil on blood lipids in mice are shown in Table 2. In comparison with control group A, TC and TG were the important indexes which reflected the body lipid metabolism, and LDL was increased significantly in the model control group. HDL was increased in the model groups except for group F (high ratio of tea saponin/oil). All the treatment groups had significantly higher TC, TG and LDL. All the measured parameters were dose-dependent and did not have a significant impact, which could explain the effect of the camellia oil with tea saponin on mice lipid metabolism.

Table 3A shows the effect of tea saponins from camellia oil on alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total protein (TP) in the serum of mice for 90 days to assay toxicity. The doses of 28, 255, 382.5 and 510 mg/kg/bw increased the ALT, AST, and TP levels in serum compared with the two control groups. The overall results suggested that AST, ALT and TP activities were increased, but as a general observation, the tea saponin did not show toxicity in mice serum.

The body weights of the mice were recorded each day after tea oil saponin treatment. No changes in eating, drinking or exploratory behavior were seen in the mice treated with tea oil saponin. The body weight plot demonstrated that the weights of the mice expanded typically and no distinction was seen among the control and tea oil saponin-treated groups (data not shown). This suggested that the tea oil saponin did not have a harmful impact to the body weight of the mice. Following 90 days of administrations, the mice were sacrificed and the significant organs were gathered. The organ indexes (organ weight/body weight) of the heart, liver, spleen, and kidney were ascertained and compared (Table 3B). The outcome demonstrated that the tea oil saponin had no undesirable impact on the organ indexes. Non significant differences were detected between the control and treated groups in kidney, spleen and heart, but significant differences were detected between the two control groups and groups C and F with regards to the liver. The livers were bigger in all groups compared to other organs.

The anatomy of mice organs (liver, kidney, spleen and heart) was observed by the naked-eye; and no differences were found between the control groups (A and B) and dose groups (C, D, E and F) concerning edema, hyperplasia, atrophy lesions, with bright-color appearance, as the normal organs and without any hitopathological changes.

Figure 6a shows the heart histopathological tissue in Kunming mice. The heart tissue showed no changes in the control and dose groups and no abnormal accumulation or precipitation and no cavitations. Figure 6b shows kidney histopathological tissue in Kunming mice. The results for kidney tissues in the dose groups were roughly the same as the control groups with no significant differences, where there was clear renal tubular structure, complete epithelium, no crystals within the lumen, and no inflammatory cells. Figure 6c shows liver pathological tissue in Kunming mice. The figure shows ordinary liver tissue, with no changes in the appearance of the controls or the dose groups. There were big nucleus, neat veins closely surrounding cell tissues, and regular lobular, beside no cavitations, no abnormal accumulation or precipitation. Generally, the dose groups showed a normal liver tissue, fatty degeneration was not found in the liver either (Ghule et al., 2009), with no toxic effects. Figure 6d shows spleen histopathological tissue in Kunming mice. The figure shows tissue morphology; and all the groups were characterized by a clear organizational structure, some large volume and irregular polygonal cells, scattered distribution in the edge area of the spleen cells, red pulp spleen sonne and a lymph follicle germinal center can be clearly seen. All the treatment groups compared to the control groups were presented as normal, and there were no significant differences. Therefore, as the general results, camellia oil containing tea saponin did not produce any adverse effects on mice heart, kidney, liver or spleen physiologies.