Study on Optimization of Ethanol Reflux Extraction of Phenolic Acids from Salvia miltiorrhiza ()
1. Introduction
1.1. Salvia miltiorrhiza and Its Efficacy
Salvia miltiorrhiza is the name of traditional Chinese medicine. It is the dry root and rhizome of Salvia miltiorrhiza Bge. It is distributed in most regions of the country [1]. The main effects of salvia miltiorrhiza are to promote blood circulation and remove blood stasis, smooth menstruation and relieve pain, clear the heart and remove annoyance, cool blood and eliminate carbuncle [2].
1.2. Main Chemical Components of Salvia miltiorrhiza
The main chemical components of Salvia miltiorrhiza can be divided into fat-soluble components and water-soluble components. The fat-soluble components are mainly tanshinones, and the water-soluble components are mainly phenolic acids [3]. Fat-soluble components belong to quinone and ketone structure, including tanshinone, cryptotanshinone, isotanshinone, isocryptotanshinone, hydroxytanshinone, methyl tanshinate, methylenetanshinone, dihydrotanshinone, etc. The water-soluble phenolic acid compounds are: salvia acid A, B, C; Salvianolic acid A, B, C, D, E, G; Rosmarinic acid, methyl rosemary acid, methyl shikonate, dimethyl shikonate, ethyl shikonate, shikonate B, protocatechuic aldehyde, etc. [4].
1.3. Main Extraction Methods of Salvia miltiorrhiza
The extraction methods of the main components of Salvia miltiorrhiza include water decoction, ethanol reflux, gradient percolation, ultrasound, CO2 supercritical extraction, etc. [5]. Among them, the gradient percolation method has the advantages of a large concentration gradient, good leaching effect and less solvent consumption [6]. The ethanol reflux method is a classical extraction method for extracting the main components of Salvia miltiorrhiza, which is widely used in industrial extraction. In this experiment, the ethanol reflux method was used to extract the main components of Salvia miltiorrhiza, and the ethanol concentration, reflux time, and material-to-liquid ratio were optimized, and the optimal extraction process was studied [7].
2. Experimental Contents and Methods
2.1. Experimental Design
2.1.1. Experimental Drugs
Salvia miltiorrhiza slices, absolute ethanol, deionized water, sodium nitrite, aluminum nitrate, sodium hydroxide, etc.
2.1.2. Equipment Required for Experiment
Electronic balance, centrifuge, thermostatic water bath, spectrophotometer, thermostatic drying instrument, pulverizer, etc.
2.1.3. Instruments Required for the Experiment
Conical flask, condensate return pipe, beaker, round bottom flask, pipette, filter paper, absorbent paper, volumetric flask, gauze, iron stand, centrifuge tube, cuvette, etc.
2.1.4. Test Steps
1) Standard curve is made with salvianolic acid B standard. 2) The temperature of thermostatic water bath is set at about 80˚C. 3) Weigh Danshen powder. 4) The ethanol solution for reflux extraction was prepared according to the experimental design (60%, 80%, 100%). 5) According to the experimental design, the ratio of material-to-liquid is 1:10, 1:15, 1:20 respectively, and Salvia miltiorrhiza powder and ethanol are mixed evenly. 6) The extraction time was different according to the experimental design (1 h, 1.5 h, 2 h). 7) After the reflux is completed, the mixed solution is preliminarily filtered with 4 layers of gauze. 8) Take the filtrate for centrifugation (centrifuge parameters: speed 6000 r/min, time 10 min). 9) Use a pipette to absorb 0.4 mL of the centrifuged extract and put it into a clean test tube. Repeat the operation twice and label it. Then use a pipette to absorb 0.4 mL of deionized water for blank adjustment. 10) Add the reagent components of the color developing agent in order, and conduct dark treatment. a) Add 2.5 mL of 1% sodium nitrite solution, shake well, and conduct dark treatment for 5 min. b) Add 0.25 mL of 20% aluminum nitrate solution, shake well, and conduct dark treatment for 5 min. c) Add 4 mL of 1 mol of sodium hydroxide solution, shake well, add sterile distilled water to 10 mL of liquid in the test tube, and conduct dark treatment for 10 min. 11) Measure the OD value and record the original data.
2.2. Experimental Process
2.2.1. Treatment and Preparation of Experimental Materials
1) Salvia miltiorrhiza slices are crushed into powder, dried in a thermostatic dryer, sealed and stored away from light. 2) Prepare color developing agent: 1% sodium nitrite solution, 20% aluminum nitrate solution, 1 mol/L sodium hydroxide solution.
2.2.2. Selection of Measurement Wavelength
Take salvianolic acid B standard sample and add color developing agent for treatment. Use a spectrophotometer to detect it at the wavelength of 300 - 500 nm. It is found that the absorption value at 493 nm is the most obvious. It is decided to use 493 nm as the wavelength for determination.
2.2.3. Draw Standard Curve of Salvianolic Acid B
1) Preparation of standard solution: Accurately weigh 2 g of reference substance of salvianolic acid B, fix the volume with a 10 mL volumetric flask, and obtain 0.2 g/mL of standard solution of salvianolic acid B. 2) Drawing of standard curve: Take 0, 0.4, 0.8, 1.2, 1.6, 2.0 mL of standard solution, and dilute to 4 mL. Carry out spectrophotometer measurement and record the OD value [7]. With the sampling amount of salvianolic acid B as the abscissa and the OD value as the ordinate, the standard curve is drawn, and the regression equation is Y = 0.4958X − 0.0261, r = 0.9973. The results were recorded in Table 1.
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Table 1. OD value of salvianolic acid B standard.
2.2.4. Single Factor Experiment
1) Effect of reflux time on the extraction rate of effective components of Salvia miltiorrhiza
Take the ethanol solution with the concentration of 60%, mix the Salvia miltiorrhiza powder and ethanol solution according to the ratio of material-to-liquid 1:20, and set the reflux time as 1 h, 1.5 h and 2 h respectively. Other operations were performed according to the experimental procedures. Measure the corresponding OD values, and record the data in Table 2 [8].
2) Effect of material liquid ratio on the extraction rate of effective components of Salvia miltiorrhiza
Accurately weigh three parts of dried Salvia miltiorrhiza powder, 6 g, 8 g and 12 g respectively, and add 120 ml of ethanol solution with a concentration of 60%. The ratio of material-to-liquid is 1:20; 1:15; 1:10. Other operations were performed according to the experimental procedures. Measure the corresponding OD values, and record the data in Table 3.
3) Effect of ethanol concentration on the extraction rate of effective components of Salvia miltiorrhiza
Use deionized water to prepare ethanol solutions with ethanol concentration of 60%, 80% and 100%, take 120 mL each, and mix them with 6 g Danshen powder evenly. Other operations were performed according to the experimental procedures. Measure the corresponding OD values, and record the data in Table 4.
2.2.5. Orthogonal Test
On the basis of single factor experiment, the technological conditions of ethanol reflux extraction of Salvia miltiorrhiza were further optimized. Parameter conditions: reflux time, material liquid ratio, ethanol concentration. The orthogonal test and results were recorded in Table 5 and Table 6.
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Table 2. OD values of salvianolic acids extracted by ethanol reflux at different reflux times.
Note: Control variables: material liquid ratio 1:20, ethanol concentration 60%.
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Table 3. OD Values of Salvia miltiorrhiza phenolic acid compounds extracted by ethanol reflux at different material-to-liquid ratios.
Note: Control variable: reflux time 1 h, ethanol concentration 60%.
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Table 4. OD values of salvianolic acid compounds extracted by ethanol reflux at different ethanol concentrations.
Note: Control variable: reflux time 1 h, material liquid ratio 1:20.
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Table 5. Orthogonal test factor level table.
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Table 6. Orthogonal experiment results.
2.3. Original Record of Experimental Data
The OD values of Salvia miltiorrhiza extract at different reflux time, ratio of solid to liquid and ethanol concentration were recorded in Tables 2-4 respectively.
2.4. Experimental Data Processing and Analysis
According to the average value of OD data obtained in Tables 2-4, histograms were made in Figures 1-3. The effects of reflux time, ratio of solid to liquid and concentration of ethanol on the absorbance of Salvia miltiorrhiza extract were shown respectively. On the basis of single factor experiment, the orthogonal design was carried out for the extraction of phenolic acids from Salvia miltiorrhiza. The results of the orthogonal design were recorded in Table 6.
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Figure 1. Broken line diagram of influence of reflux time on extraction rate of salvianolic acids.
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Figure 2. Broken line diagram of the influence of material liquid ratio on the extraction rate of phenolic acid compounds in Salvia miltiorrhiza.
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Figure 3. Broken line diagram of the influence of ethanol concentration on the extraction rate of phenolic acids in Salvia miltiorrhiza.
3. Result and Discussion
3.1. Results
1) From the analysis of experimental data in Experiment 1, it is concluded that when other experimental conditions are the same, the longer the refluxing time is, the better. Under the extraction condition of 1.5 h refluxing time, the content of salvianolic acid compounds in the extraction solution is the highest. 2) From the analysis of experimental data in Experiment 2, it can be concluded that when other experimental conditions are the same, the higher the multiple of the material-to-liquid ratio is, the better. Under the extraction condition of the material-to-liquid ratio of 1:10, the content of salvianolic acid compounds in the extraction solution is the highest. 3) From the analysis of experimental data in Experiment 3, when other experimental conditions are the same, the higher the ethanol concentration is the better. Under the extraction condition of 60% ethanol concentration, the content of salvianolic acid compounds in the extraction solution is the highest. This should be related to the fact that the salvianolic acids in Salvia miltiorrhiza are water-soluble substances. When the water content is relatively high, the contact probability between the phenolic acids in Salvia miltiorrhiza and water molecules is increased. 4) According to the orthogonal test analysis, the main factor affecting the extraction rate of salvianolic acid compounds is the ratio of material-to-liquid, and the other two factors have weak effects on the extraction rate of salvianolic acid compounds.
3.2. Analysis and Discussion
In this experiment, the ethanol reflux method was selected to extract phenolic acid compounds, the main components of Salvia miltiorrhiza, and the process was optimized. The main reason was that the ethanol reflux method was widely used in the extraction of traditional Chinese medicine, with simple operation, low cost and higher research significance. The conclusions obtained in this experiment can provide a certain reference for the ethanol reflux extraction process of salvianolic acids in industry, and can also provide data reference for other researchers.
4. Conclusion
In summary, salvianolic acids have poor stability in water, but good stability in ethanol and other organic solutions. Therefore, with a longer extraction time, salvianolic acid will not decompose. The optimal scheme of ethanol reflux extraction process optimization of phenolic acid compounds in Salvia miltiorrhiza in this experiment is as follows: reflux time is 1.5 h, material liquid ratio is 1:10, and ethanol concentration is 60%.