Penthorum chinense Pursh is rich in gallic acid, which has antioxidant, anti-inflammatory, anti-fungal and antitumor activities. In order to optimize their extraction conditions, various extraction parameters were chosen to identify their effects on gallic acid extraction. With extraction amount of gallic acid as index, based on single factor analysis, influence of solid/liquid ratio, ethanol concentration, fetch time and extraction temperature on extraction technology were investigated by orthogonal test. The optimization conditions for gallic acid extraction were determined as follows: ethanol concentration 60%, extraction time 2.5 h, temperature 90 °C and solid/liquid ratio 1:30. The corresponding gallic acid content was 4.85%. This optimized extraction process was stable and feasible.
Penthorum chinense Pursh is the species belonging to Saxifragaceae Penthorum perennial herb, and the whole plant is popularly used as medicine in china. It is mainly distributed in Hunan, Sichuan and Guizhou and other provinces. It is a rooted vascular plant, which has the functions of detoxification, diuresis, and promoting blood circulation. In addition, abundant studies have shown that they have anti-aging, anti-viral, anti-mutation activities [
Dried Penthorum chinense Pursh was purchased from Gulin Hongan Pharmaceutical Company (Sichuan Province, China). gallic acid (reference standard) was purchased from National Instisutes for Food and Drug Control in China; all the other chemicals used were of analytical grade.
An electronic balance was measured on quality (Jingtian electronic instrument company, Shanghai, China, JT202N); An UV-vis spectrophotometer was used for quantification of gallic acid (Analysis of general instrument company, Beijing, China, T6)
Gallic acid standard substance (25.4 mg) was accurately weighed, dissolved and filled into a 100 mL volumetric flask. Then 60% ethanol was added into the volumetric flask. 0.0 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL from 100 mL volumetric flask was filled into 6 volumetric flasks, respectively. Then 60% ethanol was added into these volumetric flasks and stood for 10 minutes in dark place. At last, absorbance values were recorded by a T6 spectrophotometer at the wavelength of 279 nm. At the same time, the 60% ethanol was measured as blank control in an identical way. The regression equation of the gallic acid standard curve was obtained as A = 50.098C + 0.1587 (R2 = 0.9946), exhibiting a good linear relationship within the rang of 0.005 - 0.025 mg∙mL−1 (see
Penthorum chinense Pursh (6.0 g) was treated by water bath for extraction of gallic acid under the designed conditions. Extraction conditions were adjusted to different ethanol concentrations (30% - 80%), ratios of solid/liquid (g∙mL−1) (1:10 - 1:35), extraction times (1 - 3.5 h), and water bath temperatures (50˚C - 100˚C). When one of the conditions was changed, the other conditions were adjusted accordingly. Penthorum chinense Pursh was extracted twice. After the extracts were combined and filtered, the obtained concentrated solution was dissolved in ethanol (60%) to 200 mL, then, 2.0 mL was taken out to a defined volume of 50 mL for analysis.
3.0 mL from the standard solution was filled into 50 mL volumetric falsk, measured 6 times according to the method of 2.1, RSD = 1.02%. The precision of this method was good.
3.0 mL from the gallic acid sample solution was filled into 50 mL volumetric falsk every 20 minutes, and measured 6 times according to the method of 2.1, RSD = 1.02%. It showed that the samples were in good stability within 120 minutes.
Six pieces of P. chinense (2.0 g) was treated by water bath for extraction of gallic acid under the designed conditions. The experiment was repeated six times, and measured 6 times according to the method of 2.1, RSD = 1.34%. It showed that the repeatability of the method was good.
Six pieces of P. chinense (6.0 g) were weighed and added a certain amount of gallic acid standard, then measured absorbance values of gallic acid according to the method of 2.1. The experiment was repeated six times, and calculated the recovery of 98.0%, RSD = 1.42%. It showed that the recovery of the method was good. (see
In this work, the effect of solid/liquid ratio on extraction yield of gallic acid from P. chinense was investigated, and the results were listed in
The effect of ethanol concentration on extraction yield of gallic acid from P. chinense was shown in
The effect of extraction time on extraction yield of gallic acid from P. chinense was shown in
No | Original/mg | Added/mg | Detected/mg | Recovery/% | X/% | RSD/% |
---|---|---|---|---|---|---|
1 | 2.7878 | 0.7260 | 3.500 | 98.2 | ||
2 | 2.7864 | 0.7075 | 3.478 | 97.9 | ||
3 | 2.7841 | 0.7278 | 3.513 | 100.2 | ||
4 | 2.7856 | 0.7131 | 3.490 | 98.8 | 98.0 | 1.42 |
5 | 2.7834 | 0.7023 | 3.463 | 96.8 | ||
6 | 2.7822 | 0.7045 | 3.460 | 96.2 |
In this work, the effect of different extraction temperature on extraction yield of gallic acid from P. chinense was investigated, and the results were listed in
The first step in the extraction parameters of gallic acid from P. chinense was to optimize the operating conditions to obtain an efficient extraction of the target compounds. Since various parameters potentially affected the extraction process, the optimization of the experimental conditions was a critical step in the development of a solvent extraction method. In fact, the extraction temperature, extraction time, solid/liquid ratio and ethanol concentration were generally considered to be the most important factors. Optimization of the suitable extraction conditions in the gallic acid extraction could be carried out by using an experimental design. In the present study, all selected factors were examined using an orthogonal L9 (34) test design. The total evaluation index was used to analysis by statistical method. Factors and levels are presented in
The results of experiments presented in
Under the condition of the optimization process, the experiment was repeated six times, and measured the average yield of gallic acid of 4.85%, RSD = 1.02%. It showed that the process was stable.
Chen [
Levels | Ethanol concentration (%) | Solid/liquid ratio | Extraction time (h) | Extraction temperature (˚C) |
---|---|---|---|---|
A | B | C | D | |
1 | 60% | 1:20 | 2.5 | 70 |
2 | 70% | 1:25 | 3.0 | 80 |
3 | 80% | 1:30 | 3.5 | 90 |
Levels | A | B | C | D | Extraction efficiency (%) |
---|---|---|---|---|---|
1 | 1 | 1 | 1 | 1 | 5.16 |
2 | 1 | 2 | 2 | 2 | 5.01 |
3 | 1 | 3 | 3 | 3 | 5.21 |
4 | 2 | 1 | 2 | 3 | 5.15 |
5 | 2 | 2 | 3 | 1 | 4.76 |
6 | 2 | 3 | 1 | 2 | 5.20 |
7 | 3 | 1 | 3 | 2 | 4.40 |
8 | 3 | 2 | 1 | 3 | 4.47 |
9 | 3 | 3 | 2 | 1 | 4.67 |
I | 15.38 | 14.63 | 14.83 | 14.59 | - |
II | 15.11 | 14.71 | 14.83 | 14.61 | - |
III | 13.54 | 15.08 | 14.37 | 14.83 | - |
R | 1.84 | 0.45 | 0.46 | 0.24 | - |
Factors | Sum of squares | F | F value | F critical value | Significant |
---|---|---|---|---|---|
A | 0.674 | 2 | 44.636 | 19 | ** |
B | 0.059 | 2 | 3.9072 | 19 | |
C | 0.083 | 2 | 5.5333 | 19 | |
D | 0.011 | 2 | 0.7285 | 19 |
Note: F(0.1) = 9, F(0.05) = 19.
1:30 and 60%, respectively. Four methods had their advantages and disadvantages, but the percolation method consumed time longer, was not suitable for the manufacturing. So gallic acid from Penthorum chinense pursh extracted with aqueous ethanol was studied. With extraction amount of gallic acid as index, based on single factor analysis, influence of solid/liquid ratio, ethanol concentration, fetch time and extraction temperature on extraction technology were investigated by orthogonal test. The yield of the gallic acid of 4.85% was obtained when extraction temperature, extraction time, solid/liquid ratio and ethanol concentration were 90˚C, 2.5 h, 1:30 and 60%, respectively. In this experiment, the aqueous ethanol extraction could shorten extracting time, was more suitable for the industrial production. In the experiment, gallic acid content was determined by simple and fast UV. The method was suitable for quality control in actual manufacturing, and precision, stability, repeatability was good.
This work was supported by the Fund of Doctor’s Program in Chongqing Normal University (12XLB006) and the Open Program of Engineering Research Center of Biotechnology of Active Materials (Ministry of Education) in Chongqing Normal University.
In this experiment, our work got these teachers’ help (He Daiping, Xiao Feng), thanks!