Preliminary researches on conditions to extract alchol soluble proteins of leaves of Wedelia trilobata (L.) Hitchc.

. The conditions to extract alchol soluble proteins of leaves of Wedelia trilobata (L.) Hitchc. were discussed. When the extractant was alcohol, the weight of leave(g) -to- volume of 75% alcohol solution (mL) ratio was 1:10 , temperature for extraction was 50℃and the extraction period was 2hrs, the concentration of alcohol solution needed for the best extraction effect was 95% (V/V). When the extractant was 75% alcohol solution, the temperature for extraction was 50 ℃and the extraction period was 2hrs, the weight of leave(g) -to- volume of 75% alcohol solution (mL) ratio needed for the best extraction effect was 1:20. When the extractant was 75% alcohol solution, the weight of leave(g) -to- volume of 75% alcohol solution (mL) ratio was 1:10 and the extraction period was 2 hrs, the temperature needed for the best extraction effect was 55℃. When the extractant was 75% alcohol solution, the weight of leave(g) -to-volume of 75% alcohol solution (mL) ratio was 1:10 and the temperature for extraction was 50℃, the extraction period needed for the best extraction effect was 2hrs.


The method to extract alchol soluable proteins from leaves of Wedelia trilobata (L.) Hitchc
2.0 g leaves of Wedelia trilobata (L.) Hitchc were mixed with 20 mL extractant and a littlt of quartz sand, and were ground. And after being dealt with at 50℃ for 2hrs, the mixture above was centrifugated at 10000 rpm for 10 minutes. The supernatant was collected for determination.

The influences of alcohol on the effects to extract alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc at different concentrations
The experiments were done basicaly according to the method 3.1, but the alcohol concentration was used as 60%, 75% and 95% respectively , according to the volume of alcohol -to-volume of alcohol mixed with DDW ratio.

The influences of 75% alcohol solution on the effects to extract alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc with different leaf-to-solution ratios
The experiments were done basicaly according to the method 3.1, but the leaf-to-solution ratio used was changed as 1:5,1:10,1:15 and 1:20 respectively, according to the weight of leave(g) -to-volume of 75% alcohol solution (mL) ratio.

The influences of 75% alcohol solution on the effects to extract alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc at different temperatures
The experiments were done basicaly according to the method 3.1, but only the 75% alcohol solution was used at 35℃, 42℃, 45℃, 50 ℃ and 55℃ respectively.

The influences of 75% alcohol solution on the effects to extract alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc for different extraction periods
The experiments were done basicaly according to the method 3.1, but only the 75% alcohol solution was used for 30mins, 60 mins, 90 mins and 120 mins respectively.

Measurement of protein content
The experiments were done basicaly according to the Bradford`s method [22]. When measuring the protein content of the extracts, the extractants used were used as contrasts in stead of DDW.

The influences of alcohol on the effects to extract alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc at different concentrations
According to Table 1, when extracting alcohol soluble proteins using different alcohol solution from leaves of Wedelia trilobata (L.) Hitchc ,the extraction effect by 95% alcohol solution was the best, and the one by 75% alcohol was the second. The one by 60% alcohol was the lowest.

The influences of 75% alcohol solution on the effects to extract alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc with different leaf-to-solution ratios
According to Table 2, when extracting alcohol soluble proteins using 75% alcohol solution from leaves of Wedelia trilobata (L.) Hitchc , the different ratio between the weight of leave(g) and the volume of 75% alcohol solution (mL) could influence the extraction effects. When the ratio was 1(g):20 (mL), the extraction effect was the best. And when the ratio became 1(g):5 (mL) ,1(g):10 (mL) and 1(g):15 (mL) respectively, the extraction effects declined respectively.

The influences of 75% alcohol solution on the effects to extract alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc for different extraction periods
According to Table 4, extraction periods could influence the extraction effects of alcohol soluble proteins from leaves of Wedelia trilobata (L.) Hitchc using 75% alcohol solution.When extraction periods changed from 30mins, 60mins, 90mins to 120mins, the extraction effects gradually rised, and the best effect could be get when leaves of Wedelia trilobata (L.) Hitchc were treated by 75% alcohol solution for 120mins.

Discussion
Liu et al [23 ] got new process of preparing zein with the optimum extraction conditions including ratio of liquid to solid 14:1(ml/g), temperature 50℃, pH 9.0 and time 2hr. Ao et al [10] got the the optimal conditions to extract alcohol soluble proteins from Gnaoderma lucidum, with concentration of ethanol 60%, pH 2.5, material-liquid ratio 1:40 and extration temperature 60 ℃. Ma et al [24] studied the the optimal conditions to extract alcohol soluble proteins from corn gluten meal, showing that liquid-solid ratio and time of microwave-treating could have a greater impact on extraction of the zein than microwave power and particle size. And the optimal conditions including that concentration of ethanol was 80%, one-time extract, liquid-solid ratio was 14: 1, particle size was 20 , microwave power was 420W, extraction times was 8 and the total time was 360s .Wang et al [25] showed that the best conditions to extract alcohol soluble proteins from gluten included 70% of ethanol concentration ， solid-liquid ratio of 1:4， extraction temperature of 50 ℃， pH of 5.0 and soaking time of 6 hours. Xu et al [26] showed that the best conditions to extract alcohol soluble proteins from goose millet included ethanol concentration 65%, solid/liquid (goose millet/ ethanol solution) ratio 1:6, temperature 75℃ and time 1h. Feng et al [14] got the the best conditions to extract alcohol soluble proteins from (Phaseolus vulgaris L.) , showing that the highest extraction effect was obtained when ethanol concentration, solid-liquid ratio, extraction temperature and extraction time were 70%, 1:10, 30℃ and 3.0 h, respectively. Jiang et al [15] showed that the best conditions to extract alcohol soluble proteins from beer grains included 81% of ethanol concentration， solid-liquid ratio of 1:21 (g/mL),extraction temperature of 48 ℃ and oscillation time of 50 min. Otherwise, Li et al [5] found that the complex reagent consisting of 2-mercaptoethanol (1% ) and urea (12% ) was the best for extracting prolamins from P. tabulaeformis seeds for acidic polyacrylamide gel electrophoresis (A-PAGE).Wang et al [6] found that 60 %τ-butanol plus 4 % DTT at a sample-weight to extraction-solution ratio of 1:8 (g: mL) was optimum for kafirin extraction for SDS-PAGE. Deng et al [27] found that 70% propylalcohol supplemented with 0.2% DTT was the best extraction solution and the ratio of sample-weight to extraction-solution 1:8 (g / m L) could give optimal results to extract alcohol soluble proteins from seeds of Sorghum sudanense for SDS-PAGE.
It is obvious that different extraction conditions are fit for different materials for different researches. For its low prices and innocuity, maybe ethanol will be an important extractant to extract alcohol soluble proteins in the future. For the special applications of alcohol soluble proteins, the conditions to extract them will be studied further.