The main materials and instrument used in this study: tea seed oil, tea saponin, distilled water, concentrated sulfuric acid, 77% sulfuric acid, 8% vanillin, sodium hydroxide (NaOH), ethanol and methanol; UV-2008 (UV- visible spectrophotometer scanning) and HPLC as equipment instruments. The materials and the instruments used in this experiment were from the laboratory of the Academy of Cereals, Oil and Vegetable Protein Engineering, College of Food Science and Technology in Huazhong Agricultural University, Wuhan, China.

1) Saponification solution

6 ml of tea oil was weighed in 100 ml flask; 30 ml 1 mol/L NaOH solution was added and heated for 1 h at 95˚C. Remove the ethanol by used decompression of distilled at normal temperature, collect the solution and completed the volume to 100 ml distilled water [15].

2) Standard curve for tea saponin:

a) Preparation of standard tea saponin solution and method of measurement

The standard tea saponin solution (0.95 mg/ml) was prepared in the following steps: 0.1 g standard tea saponin powder (containing 95% tea saponin) was weighted precisely put into a 100 ml volumetric flask. The saponin powder was dissolved in 50% ethanol solution, and then additional ethanol was added to volume, spinal solution 0.95 mg/ml. The standard solution (0.5 ml) was measured and into 10 ml test tubes with a stopper, then put in an ice-water bath, and then 0.5 ml of 8% vanillin solution and 5 ml of 77% sulfuric acid was added. After shaking, the test tube was transferred to an intermittent warm water bath (60C) for 20 min, then for color development cooled in an ice-water bath for 10 min. The sample was allowed to reach room temperature before detecting the absorbance of the solution was used 1cm cuvette at the wavelength of maximum absorption 550 nm in the UV-visible (V-2008 UV-visible spectrophotometer scanning). The blank solution 1:8 (volume ratio) mixtures of vanillin solution and sulfuric acid were prepared as above [16].

3) Standard curve determination:

Series of the standard solution (0 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml and 0.5 ml) were taken and the mixed solutions were prepared by the method described as above for measuring absorbance values. The resulting standard curve was assessed with Microsoft excel [5].

Standard tea saponin (0.4350 g) was dissolved in 80% ethanol in a 50 ml volumetric flask. For a final concentration of 8.7 g/L. Different weights (0.02, 0.1, 0.2, 0.8, 1 g) was taken placed in 50 ml volumetric flasks, ethanol (80%) was added to the line, shaken, filtered through a 0.45 μm micro porous membrane filter, then placed in cooled storage [17]. Conditions of HPLC equipment are as below: C18 analytical column of TC-C18, 4.6 × 250 mm; column temperature at room temperature; injected sample volume 10 μL; mobile phases 10% - 100% methanol; flow-rate 0.8 ml/min; detection wavelength 280 nm [18].

1) Sample preparation

Saponification solution (1 g) was evaluated and diluted to the mark in 50 ml volumetric flasks with 80% ethanol under sanction 5 min, then filtered using micro membrane and stored under refrigerator at 6˚C - 8˚C, before analysis by HPLC [17].

According to the absorbance of the standard tea saponin solution and the standard curve (see Figure 1 & Table 1)

with vanillin sulfuric acid colorimetric method [20], which is obtained at the maximum absorption wavelength of 550 nm at different concentration of tea saponin, the regression equation is as follows:

Then combined with the standard curve, tea saponin content is calculated with equation:

where:

A: absorbance of oil sample, V: volume of tube solution, N: dilution, W: weight of sample (mg)

Figure 2 & Table 2 explained the chromognic single factor (vanillin and sulfuric acid) in difference volumes 3.5 - 5.5 ml, but the best results at 5 ml for each one.

Figure 3 & Table 3 shown the absorption of tea saponin increased when the temperature rises from 40˚C to 80 ˚C. However, the good absorption began to decline when the temperature higher than 40˚C. Optimum color temperature is 40˚C - 60˚C.

In order to find out the optimum time, several different time tests were conducted. The results (Figure 4 & Table 4) showed that the tea saponin absorption rate increased over time, suggesting balance point of the absorption could reach at that time, which could also be the optimum time is15 - 25 min.

Orthogonal analysis (Table 5) showed that the main determinants order is A > B > C > D. The higher temperature showed greatest complete reaction of the tea saponin. From the results of orthogonal experiment, the best

condition for tea saponin is A3B3C2D3, at temperature is 60˚C, ratio of chromogenic reagent 0.75:3.75, 4.5, and time is 20 minutes.

Mobile phases (water, methanol) were checked at various compositions in an attempt to resolve with standard tea saponin and saponification sample by HPLC. Figure 5 & Table 6 illustrates the universal curve of stock tea saponin by HPLC. Then combined with the standard curve, tea saponin content is calculated with equation:

where:

Y: an area of standard tea saponin, x: different concentration mg/ml

Figure 6 as showed the standard tea saponin, measured at a wavelength 280 nm, a retention time was 45 min; it can be observed that: chromatography peak shape is high; few impurity peaks before the tall peak, higher peak at 21 min.

Figure 7 showed, separation and analysis time is brief, measured at 280 nm wavelengths and in retention time 45 minimums. They are few impurity peaks before the sample peak, the taller peak at 19.637 min; within the time of higher peak of tea saponin. No impurity peaks out after a sample peak. Standard peak time it’s long (21 min), Compared with previous studies; time peaks at (4. 295 min, 14.8 min and 16.3 min) [18] [19] in difference

conditions. However, these compounds have a complex phenolic nature and correspond to a hydroxymethyl derivative [20].