Extraction process of total salvianolic acid and anti-oxidation application of total salvianolic acid

Abstract

The invention discloses an extraction process of total salvianolic acid and antioxidant application of the total salvianolic acid. The extraction process comprises the following steps: (1) precisely weighing salvia miltiorrhiza powder, and sieving with a No.2 sieve for later use; and (2) adding a 20% ethanol solution according to a solid-liquid ratio of 1: 350g / mL, carrying out ultrasonic extraction for 40 minutes, filtering, and collecting the filtrate to obtain the total salvianolic acid extracting solution. According to the present invention, the antioxidant experiment results show that the total salvianolic acid has certain ABTS free radical and. OH free radical scavenging ability and strong reducing power, and can be used for preparing antioxidant drugs or health-care foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracting active ingredients from Chinese medicinal materials, and particularly relates to an extraction process for total phenolic acids from Salvia miltiorrhiza and its antioxidant application. Background Art

[0002] Salvia miltiorrhiza Bunge is a perennial herb of the genus Salvia in the Lamiaceae family. Its roots are the main medicinal parts, and it has the alias of "red root". Salvia miltiorrhiza prefers a climate with mild temperature, sufficient sunlight, humid air, and fertile soil. It grows mostly on slopes, in grass under forests, or beside stream valleys, at an altitude of 120 - 1300 meters. The climate and geographical conditions in many regions of China are suitable for the cultivation of Salvia miltiorrhiza, and its main production areas include the southwest, northwest, and north China regions of China. The medicinal properties of Salvia miltiorrhiza are bitter and slightly cold. Bitter can disperse, and cold can clear heat. It enters the heart and liver meridians. [1] A large number of pharmacological studies have shown that Salvia miltiorrhiza has effects such as improving microcirculation, inhibiting platelet aggregation, and antioxidant activity. [2] In the field of treating cardiovascular and cerebrovascular diseases, the treatment of Salvia miltiorrhiza and its related preparations has a high correlation with the antioxidant activity of its extracts. The principle of its action is that the phenolic acid active components of Salvia miltiorrhiza will inhibit the generation of free radicals, thereby protecting the heart from damage. [3] .

[0003] Previous studies have mostly focused on the extraction and pharmacological activity research of single active components in Salvia miltiorrhiza, resulting in waste of Salvia miltiorrhiza resources. [4] , and only a few scholars have studied the process of simultaneously extracting two active components through orthogonal experiments. [5-6] . However, in previous studies, the span of ethanol concentration was relatively large. Especially for ethanol concentrations greater than 60%, the influence trend on salvianolic acid components was not very clear, and there was no research on the biological activity of the extracted samples, which had certain deficiencies. [7] .

[0004] Different from the traditional method of extracting Salvia miltiorrhiza by boiling with water, the ultrasonic-assisted extraction method adopted in the present invention can extract total phenolic acids from Salvia miltiorrhiza more efficiently and simply. The extraction method is optimized through single-factor experiments and orthogonal experiments to obtain the best alcohol extraction process. At the same time, the free radical scavenging and antioxidant properties of the total phenolic acid samples of Salvia miltiorrhiza are measured. Summary of the Invention

[0005] The purpose of the present invention is to provide an extraction process for total phenolic acids from Salvia miltiorrhiza.

[0006] Another purpose of the present invention is to provide an antioxidant application for total phenolic acids from Salvia miltiorrhiza.

[0007] The present invention is realized through the following technical solutions:

[0008] An extraction process of total phenolic acids from Salvia miltiorrhiza Bunge described in the present invention comprises the following steps:

[0009] (1) Weigh accurately Salvia miltiorrhiza powder, pass through No. 2 sieve, and reserve for use;

[0010] (2) Add 10 - 30% ethanol solution according to a solid - liquid ratio of 1:350 - 750 g / mL, perform ultrasonic extraction for 30 - 70 min, filter, and collect the filtrate to obtain the extraction solution of total phenolic acids from Salvia miltiorrhiza.

[0011] Preferably, the solid - liquid ratio described in step (2) of the present invention is 1:350 - 450 g / mL.

[0012] More preferably, the solid - liquid ratio described in step (2) of the present invention is 1:350 g / mL.

[0013] Preferably, the ethanol concentration described in step (2) of the present invention is 10 - 20%.

[0014] More preferably, the ethanol concentration described in step (2) of the present invention is 20%.

[0015] Preferably, the ultrasonic extraction time described in step (2) of the present invention is 30 - 50 min.

[0016] More preferably, the ultrasonic extraction time described in step (2) of the present invention is 40 min.

[0017] Application of the total phenolic acids from Salvia miltiorrhiza obtained by the extraction process of the total phenolic acids from Salvia miltiorrhiza described in the present invention in the preparation of antioxidant drugs or health foods.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The extraction rate of the target active ingredient, total phenolic acids from Salvia miltiorrhiza, obtained by the extraction process of the present invention is high. Through single - factor experiments, the present invention investigated the effects of different extraction methods, solvent concentrations, solid - liquid ratios, and extraction times on the extraction rate of total phenolic acids from Salvia miltiorrhiza. On the basis of the results of single - factor experiments, an orthogonal optimization experiment was designed. The results showed that the optimal extraction process conditions for total phenolic acids from Salvia miltiorrhiza are a solid - liquid ratio of 1:350 g / mL, an extraction time of 40 min, and an ethanol concentration of 20%. Under these conditions, each gram of Salvia miltiorrhiza contains 35.12 mg of total phenolic acids.

[0020] 2. The active ingredient salvianolic acids obtained by the extraction process of the present invention has certain abilities to scavenge ABTS free radicals and ·OH free radicals and relatively strong reducing power. The results of the antioxidant experiments show that: within the range of solid-liquid ratio (1:300 to 1:1100), the ability of the salvianolic acids extraction solution to scavenge ABTS free radicals increases with the increase of concentration (14% - 73%), and the ability to scavenge ·OH free radicals also increases with the increase of concentration (14% - 78%); with the increase of the concentration ratio, the reducing power of the salvianolic acids extraction solution also increases, and the increase amplitude is relatively large.

[0021] 3. The present invention examines the extraction method, and the results show that compared with reflux extraction, the extraction rate of salvianolic acids obtained by ultrasonic extraction is higher, so ultrasonic extraction is selected. Description of the Drawings

[0022] Figure 1 Effect of different dosages of 10% sodium carbonate on the content of salvianolic acids;

[0023] Figure 2 Effect of different dosages of Folin-Ciocalteu reagent on the content of salvianolic acids;

[0024] Figure 3 Effect of different ethanol concentrations on the content of salvianolic acids;

[0025] Figure 4 Effect of different ethanol dosages on the content of salvianolic acids;

[0026] Figure 5 Effect of different extraction times on the content of salvianolic acids;

[0027] Figure 6 Standard curve of salvianolic acids;

[0028] Figure 7 Relationship between the processing methods of air-drying, sun-drying, freeze-drying, and oven-drying and the content of salvianolic acids;

[0029] Figure 8 Relationship between the processing method of sweating for 1 day at 1 / 2 - sweating for 2 days at 1 / 4 and the content of salvianolic acids;

[0030] Figure 9 Relationship between the processing method of sweating for 3 days at 1 / 2 - sweating for 4 days at 1 / 4 and the content of salvianolic acids;

[0031] Figure 10 Relationship between the processing method of sweating for 5 days at 1 / 2 - sweating for 7 days at 1 / 2 and the content of salvianolic acids;

[0032] Figure 11 Effect of different solid-liquid ratios under different processing methods on the scavenging rate of ABTS free radicals;

[0033] Figure 12 Effect of different liquid-to-material ratios on the scavenging rate of ·OH free radicals under different processing methods;

[0034] Figure 13 Determination results of the total reducing power of the salvianolic acid extract under different processing methods. Specific implementation manners

[0035] The technical solution of the present invention will be further specifically described below through specific embodiments.

[0036] Example 1

[0037] (1) Weigh the salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use;

[0038] (2) Add 20% ethanol solution according to a liquid-to-material ratio of 1:350 g / mL, perform ultrasonic extraction for 40 min, filter, and collect the filtrate to obtain the salvianolic acid extract.

[0039] Example 2

[0040] (1) Weigh the salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use;

[0041] (2) Add 30% ethanol solution according to a liquid-to-material ratio of 1:750 g / mL, perform ultrasonic extraction for 70 min, filter, and collect the filtrate to obtain the salvianolic acid extract.

[0042] Example 3

[0043] (1) Weigh the salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use;

[0044] (2) Add 10% ethanol solution according to a liquid-to-material ratio of 1:350 g / mL, perform ultrasonic extraction for 30 min, filter, and collect the filtrate to obtain the salvianolic acid extract.

[0045] Example 4

[0046] (1) Weigh the salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use;

[0047] (2) Add 20% ethanol solution according to a liquid-to-material ratio of 1:650 g / mL, perform ultrasonic extraction for 60 min, filter, and collect the filtrate to obtain the salvianolic acid extract.

[0048] Example 5

[0049] (1) Weigh the salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use;

[0050] (2) Add 20% ethanol solution according to a liquid-to-material ratio of 1:450 g / mL, perform ultrasonic extraction for 50 min, filter, and collect the filtrate to obtain the salvianolic acid extract.

[0051] Example 6

[0052] (1) Weigh the Salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use.

[0053] (2) Add 20% ethanol solution according to a material-liquid ratio of 1:550 g / mL, extract it by ultrasonic wave for 50 min, filter it, and collect the filtrate to obtain the total phenolic acids extract of Salvia miltiorrhiza.

[0054] Example 7

[0055] (1) Weigh the Salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use.

[0056] (2) Add 10% ethanol solution according to a material-liquid ratio of 1:450 g / mL, extract it by ultrasonic wave for 60 min, filter it, and collect the filtrate to obtain the total phenolic acids extract of Salvia miltiorrhiza.

[0057] Example 8

[0058] (1) Weigh the Salvia miltiorrhiza powder precisely, sieve it through a No. 2 sieve, and reserve it for use.

[0059] (2) Add 30% ethanol solution according to a material-liquid ratio of 1:650 g / mL, extract it by ultrasonic wave for 40 min, filter it, and collect the filtrate to obtain the total phenolic acids extract of Salvia miltiorrhiza.

[0060] To verify the effectiveness of the present invention, the invention team conducted a series of tests as follows:

[0061] I. Optimization of the extraction process of total phenolic acids from Salvia miltiorrhiza by orthogonal test method

[0062] 1 Instruments and materials

[0063] 1.1 Instruments

[0064] The required instruments are listed in Table 1.

[0065] Table 1 Experimental instruments

[0066]

[0067] 1.2 Reference substances and reagents

[0068] The required reference substances and reagents are listed in Table 2.

[0069] Table 2 Reference substances and reagents

[0070]

[0071] 1.3 Materials and preparation

[0072] 1.3.1 Experimental materials

[0073] Salvia miltiorrhiza samples were collected from Magu Town, Hezhang County, Guizhou Province, with an average altitude of 1996 meters. This area is located in the northwest of Guizhou Province, at the location of the Guizhou roof, in the Wumeng Mountain area of the Yunnan-Guizhou Plateau. Its climate is warm temperate, cool in spring, dry in spring and wet in summer, with a frost-free period of 206 to 255 days. The lighting conditions in this area are good and the solar radiation is relatively high. The number of Salvia miltiorrhiza in the study area is not large, mainly artificial semi-wild cultivation. The original plants and medicinal materials were identified as Salvia miltiorrhiza Bge. of the Labiatae family by Associate Professor Yan Fulin of the Teaching and Research Section of Traditional Chinese Medicine Resources and Cultivation, Guizhou University of Traditional Chinese Medicine.

[0074] 1.3.2 Sample Preparation

[0075] The collected Salvia miltiorrhiza medicinal materials were spread out indoors to allow natural water loss, and the sediment and impurities were shaken off. About 180 kg of fresh Salvia miltiorrhiza roots were reduced in weight by half due to water loss and then piled up in a constant temperature and humidity oven at a temperature of 32 °C and a humidity of 68%. They were piled for 7 days to allow the internal moisture of the root strips to overflow, and a sterilized gunny sack was covered on the surface, and they were left to stand and sweat. The standard for the end of the assessment was that the outer skin was purple-red and the inside was purple-brown. During the "sweating" process of Salvia miltiorrhiza, samples were taken once at 8:30 am every day, for a total of 4 samples. After sampling, they were stored in liquid nitrogen for 20 minutes and then transferred to a -80 °C refrigerator for subsequent experimental analysis. The non-sweated samples were denoted as SmB-F, and the samples after sweating for 1, 3, 5, and 7 days were denoted as SmB-S1, SmB-S3, SmB-S5, and SmB-S7 respectively, with 3 replicates for each sample.

[0076] 2 Methods and Results

[0077] 2.1 Optimization of Color Development Conditions

[0078] 2.1.1 Investigation of the Dosage of Color Developer

[0079] Precisely weigh about 0.1 g of Salvia miltiorrhiza powder, extract it according to the best process in the literature, filter it, and select different amounts of color developer for experiments according to the literature. The absorbance was measured at 510 nm, and the results are shown in Table 3. Figure 1-2 .

[0080] Table 3 Results of the Investigation of the Dosage of Color Developer

[0081]

[0082] From Figure 1 , 2 it can be obtained that the optimal dosage of the color developer in this experiment is 2 mL of 10% sodium carbonate and 1 mL of Folin-Ciocalteu phenol.

[0083] 2.1.2 Investigation of Color Development Time

[0084] Precisely weigh about 0.1 g of Danshen powder, extract and filter it according to the optimal process. After adding each color reagent, let it stand for different times, and measure the absorbance at 765 nm. The results are shown in Table 4.

[0085] Table 4 Results of the investigation on the color development time

[0086]

[0087] As can be seen from Table 4, the optimal color development time is 5 minutes for 10% sodium carbonate color development followed by 80 minutes for Folin-Ciocalteu phenol color development.

[0088] 2.2 Optimization of the extraction process

[0089] 2.2.1 Investigation of the extraction method

[0090] Weigh 6 portions of about 0.1000 g of medicinal material powder, add 35 mL of 20% ethanol, extract by ultrasonic and reflux for 40 minutes, and filter. Color develop according to the color development method in item "2.1", and measure the absorbance value at 765 nm. The results are shown in Table 5.

[0091] Table 5 Results of the investigation on the extraction method

[0092]

[0093] 2.2.2 Investigation of the extraction solvent concentration

[0094] Precisely weigh about 0.1000 g of medicinal material powder, 9 portions, add ethanol with different concentrations, extract by ultrasonic for 40 minutes, filter, color develop and measure the absorbance. The results are shown in Table 6. Figure 3 。

[0095] Table 6 Results of the investigation on the extraction solvent concentration

[0096]

[0097] From Figure 3 it can be seen that when the solvent concentration is 20%, the extraction effect is better than that of 10% ethanol concentration.

[0098] 2.2.3 Investigation of the solid-liquid ratio

[0099] Precisely weigh about 0.1000 g of Danshen medicinal material powder, with other conditions unchanged, change the amount of 20% ethanol to 35, 45, 55, 65, 75 mL, extract by ultrasonic for 40 minutes, filter, and measure and calculate the content. The results are shown in Table 7. Figure 4 。

[0100] Table 7 Results of the investigation on the solvent usage

[0101]

[0102] Figure 4 It can be seen that the optimal solid-liquid ratio is 1:350 g / mL.

[0103] 2.2.4 Investigation of extraction time

[0104] Accurately weigh about 0.1000 g of Salvia miltiorrhiza powder passing through No. 2 sieve, add 35 mL of 20% ethanol, and perform ultrasonic treatment for 30 min, 40 min, 50 min, 60 min, and 70 min. Prepare three samples in parallel, filter, develop color, and measure the absorbance. The results are shown in Table 8. Figure 5 .

[0105] Table 8 Results of the investigation of extraction time

[0106]

[0107]

[0108] Figure 5 It can be obtained that the optimal extraction time is 40 min.

[0109] 2.2.5 Orthogonal test of extraction process

[0110] Use the optimal extraction method verified by the above experimental results to design the orthogonal design scheme as shown in Table 9, Table 10, and Table 11.

[0111] Accurately weigh 6 portions of medicinal material powder, each about 0.1000 g, perform extraction according to the scheme in "Table 9", filter, develop color, and measure the absorbance. The results of the orthogonal test are shown in Table 10.

[0112] Table 9 Factor level table of orthogonal test for extraction process

[0113]

[0114] Table 10 Results of orthogonal test

[0115]

[0116] Table 11 Variance analysis of regression model

[0117]

[0118]

[0119] The extraction conditions of total phenolic acids from Salvia miltiorrhiza were optimized by orthogonal design. As can be seen from Table 10, the influence of each factor on the extraction rate of total phenolic acids was as follows: solvent concentration > solid-liquid ratio > extraction time, and the solvent concentration had a significant correlation. The extraction rate of total phenolic acids was the highest when the solvent concentration was 20%, the solid-liquid ratio was 1:350 g / mL, and the ultrasonic extraction time was 40 min, that is, the A2B1C2 combination. The total phenolic acids contained in each gram of Salvia miltiorrhiza extracted were 35.12 mg.

[0120] 2.2.6 Process experiment verification

[0121] Precisely weigh about 0.1 g of medicinal material powder, a total of 3 portions, extract under the optimal extraction conditions, develop color and measure the absorbance. The results are shown in Table 12.

[0122] Table 12 Results table of process verification

[0123]

[0124] The results of extracting total phenolic acids by the optimal process had little difference, indicating that this process was stable and feasible.

[0125] 3 Summary

[0126] From the above experimental results, it can be seen that the absorbance of total phenolic acids from Salvia miltiorrhiza measured by the Folin-Ciocalteu colorimetric method and ultraviolet spectrophotometry was good, proving that this method was simple and easy to operate and beneficial to the experiment.

[0127] As can be seen from Table 11, the results of this orthogonal experiment were consistent with the results of single-factor investigation, proving that the orthogonal experiment method could be used to optimize the extraction process of total phenolic acids from Salvia miltiorrhiza.

[0128] II. Determination of the content and antioxidant activity of total phenolic acids from Salvia miltiorrhiza

[0129] 1 Instruments and materials

[0130] 1.1 Instruments

[0131] The required instruments are shown in Table 13.

[0132] Table 13 Instruments for experiments

[0133]

[0134]

[0135] 1.2 Reference substances and reagents

[0136] The required reference substances and reagents are shown in Table 14.

[0137] Table 14 Reference substances and reagents

[0138]

[0139] 1.3 Materials and Preparation

[0140] The source of the samples is the same as the samples under item 1.3 in the first part.

[0141] 2 Methods and Results

[0142] 2.1 Chromogenic Conditions

[0143] Pipette 1 mL of the test solution into a 10-mL test tube, add 1 mL of distilled water and 1 mL of 10% sodium carbonate. After waiting for 5 min, add 0.6 mL of Folin-Ciocalteu reagent. Measure the absorbance at 510 nm after 75 min of color development.

[0144] 2.2 Preparation of Reference Solution

[0145] Prepare a 0.1 mg / mL salvianolic acid B reference solution with 20% ethanol.

[0146] 2.3 Preparation of Test Solution

[0147] Prepare according to the optimal extraction conditions in item 2.2 of the first part.

[0148] 2.4 Methodological Investigation

[0149] 2.4.1 Investigation of Linear Relationship

[0150] Precisely pipette the salvianolic acid B reference solution, dilute it to different concentrations with 20% ethanol. Take 1 mL of each diluted solution for color development and measure the absorbance. The abscissa is the mass concentration of salvianolic acid B (mg / mL), and the absorbance is the ordinate. The linear regression equation is: Y = 30.627X + 0.0404, R 2 = 0.9992. The results show that there is a good linear relationship between salvianolic acid B and the absorbance in the concentration range of 0.0067 - 0.0233 mg·mL -1 . The results are shown in Table 15 and Figure 6 .

[0151] Table 15 Results of Standard Curve Determination

[0152]

[0153] 2.4.2 Precision Test

[0154] Precisely transfer 1 mL of the reference solution and prepare six parallel samples. Measure the absorbance at a wavelength of 510 nm after color development. The experimental results show that the precision of the instrument is good. The detailed data are shown in Table 16.

[0155] Table 16 Results of Precision Test

[0156]

[0157] 2.4.3 Stability test

[0158] Approximately 0.1 g of Danshen samples from the same batch were used to prepare the solution according to the test sample preparation method, and the color reaction was carried out under the conditions of Section 2.1 of the first part. The absorbance was measured every 20, 30, and 60 minutes respectively, and the results were summarized in Table 17. The results showed that the test samples had good stability within 60 minutes.

[0159] Table 17 Results of stability test

[0160]

[0161]

[0162] 2.4.4 Repeatability test

[0163] Medicinal material samples from the same batch were used to prepare the samples to be tested, and 6 groups of parallel operations were carried out. The solution of the sample to be tested was accurately pipetted, and the absorbance was measured at a wavelength of 510 nm to calculate its content. The experimental results showed that the method had good repeatability. The specific data are shown in Table 18.

[0164] Table 18 Results of repeatability test

[0165]

[0166] 2.4.5 Spiked recovery test

[0167] During the preparation of the samples, 6 portions of about 0.0500 g of Danshen medicinal material powder were taken and 0.685 mg of the reference substance was added. Subsequently, the color development was carried out according to the conditions of item 2.1 and the recoveries of each group were measured at 510 nm. Finally, based on the experimental data, the recovery data of each group of reference substances were obtained. The details are shown in Table 19. After analysis, the accuracy and applicability of the method were relatively high.

[0168] Table 19 Results of spiked recovery test

[0169]

[0170] 2.5 Determination of sample content

[0171] Approximately 0.1000 g of Danshen powder from each batch was accurately weighed, and three portions were prepared in parallel according to the test sample preparation method. The color development was carried out, and the absorbance value was measured at 510 nm to calculate its total phenolic acid content. The results are shown in Table 20. Figure 7 、 8 、9、10.

[0172] Table 20 Results of sample content determination

[0173]

[0174]

[0175] Note: The weight loss conditions are 1 / 2, 1 / 3, and 1 / 4 of the 1st to 7th day of sweating.

[0176] In this experiment, the total phenolic acid content of 22 batches of processed Salvia miltiorrhiza products was determined. The determination results are shown in Table 20. The order of the total phenolic acid content in each processing group of Salvia miltiorrhiza is: sweating for 5 days (weight loss 1 / 3) > sweating for 4 days (weight loss 1 / 4) > sweating for 4 days (weight loss 1 / 3) > sweating for 5 days (weight loss 1 / 4) > sweating for 2 days (weight loss 1 / 4) > sweating for 5 days (weight loss 1 / 2) > sweating for 3 days (weight loss 1 / 3) > sweating for 1 day (weight loss 1 / 4) > sweating for 7 days (weight loss 1 / 2) > sweating for 1 day (weight loss 1 / 3) > sweating for 3 days (weight loss 1 / 4) > sweating for 4 days (weight loss 1 / 2) > sweating for 2 days (weight loss 1 / 3) > sweating for 2 days (weight loss 1 / 2) > drying at 60°C > sweating for 3 days (weight loss 1 / 2) > sweating for 1 day (weight loss 1 / 2) > freeze-drying > air-drying > sun-drying > drying at 80°C > drying at 70°C. Among them, the total phenolic acid content is the highest in sweating for 5 days (weight loss 1 / 3), with a total phenolic acid content of 37.60 mg / g; the content is the lowest in drying at 70°C, which is 17.80 mg / g.

[0177] 2.6 Antioxidant research

[0178] 2.6.1 Determination of ABTS free radical scavenging rate

[0179] According to the improvement of the literature research method, the Salvia miltiorrhiza sample powder was extracted with 20% ethanol at different concentrations, including extraction schemes of 1:300, 1:500, 1:700, 1:900, and 1:1100, and the solution was prepared within 40 min. In each extraction scheme, 1 mL of the test solution was mixed with 5 mL of ABTS stock solution, and the reaction was carried out for 10 min under dark conditions at room temperature. The absorbance value at 734 nm was measured (denoted as A1). At the same time, ethanol was used to replace the sample solution for determination to obtain the absorbance value A0. The relevant results are shown in Table 21, and the calculation formula is as follows: RSA ABTS (%) = (A0 - A1) / A0 × 100.

[0180] Table 21 Determination of ABTS free radical scavenging rate

[0181]

[0182]

[0183] The scavenging ability of the total phenolic acid extract of Salvia miltiorrhiza on ABTS is shown in Table 21 and Figure 11, the ability of the total phenolic acids extract from Salvia miltiorrhiza Bunge to scavenge ABTS free radicals within the solid-liquid ratio range of 1:300 to 1:1100 increases with the increase in concentration (14% - 73%). Drying at 70°C and 80°C shows slower growth and lower maximum scavenging rates compared to other processing methods, demonstrating weaker ABTS free radical scavenging ability. Within the solid-liquid ratio of 1:500, the decline in the ability to scavenge ABTS is relatively large. The scavenging rate of sweating for 4 days at 1 / 2 is the highest, reaching 73.51%, while the scavenging rate of drying at 70°C is the lowest, at 55.88%.

[0184] 2.6.2 Determination of ·OH Free Radical Scavenging Rate

[0185] Test samples were prepared at different ratios of 1:300, 1:500, 1:700, 1:900, and 1:1100. 2.0 mL of the test sample, H2O2, FeSO4, and salicylic acid solution were added to a test tube and mixed, and maintained at 37°C. The color developed for 30 min, and the absorbance A1 was measured at 510 nm under blue light as the experimental result. Among them, the absorbance value A0 was obtained by replacing the test sample with distilled water, and the absorbance value A2 was obtained by replacing the salicylic acid solution with distilled water. See the results shown in Table 22, and the calculation formula is as follows: ·OH scavenging rate (%) = [A0 - (A1 - A2)] / A0 × 100.

[0186] Table 22 Determination of Hydroxyl Free Radical Scavenging Rate

[0187]

[0188]

[0189] The ability of the total phenolic acids extract from Salvia miltiorrhiza Bunge to scavenge ·OH free radicals is shown in Table 22 and Figure 12 , the ability of the total phenolic acids extract from Salvia miltiorrhiza Bunge to scavenge ·OH free radicals within the solid-liquid ratio range of 1:300 to 1:1100 increases with the increase in concentration (14% - 78%). Drying at 80°C shows slower growth and lower maximum scavenging rates compared to other processing methods, demonstrating weaker ·OH free radical scavenging ability. Within the solid-liquid ratio of 1:900, the decline in the ability to scavenge ·OH is relatively large. The scavenging rate of sweating for 4 days at 1 / 4 is the highest, at 78.76%, while the scavenging rate of drying at 80°C is the lowest, at 66.17%.

[0190] 2.6.3 IC of Scavenging Rate 50 Value

[0191] Using SPSS software, a regression model of concentration and scavenging rate was established by regression analysis. Substituting the scavenging rate into the model equation, the corresponding concentration value can be obtained when the inhibition rate is 50%. See Table 23.

[0192] Table 23 IC of Each Scavenging Rate 50Calculation results

[0193]

[0194]

[0195] 2.6.4 Determination of total reducing power

[0196] Modified according to the method in the consulted resources. Prepare test samples at ratios of 1:300, 1:500, 1:700, 1:900, and 1:1100. Mix 2 mL of 0.2 mol / L phosphate buffer solution with pH = 6.6 and 2 mL of 1% potassium ferricyanide solution in a test tube. After a 20-minute water bath at 50 °C, add 2 mL of 10% trichloroacetic acid and mix well. Then centrifuge at 5000 r / min for 10 minutes. Take 2 mL of the supernatant, and successively add 2 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution. After 10 minutes of reaction treatment, zero with distilled water and measure the absorbance value at a visible wavelength of 700 nm. Measure in parallel 3 times, and the results are shown in Table 24.

[0197] Table 24 Determination of total reducing power

[0198]

[0199]

[0200] The reducing power of the total phenolic acid extract from Salvia miltiorrhiza is shown in Table 24 and Figure 13 , as the concentration ratio increases, the reducing power of its extract also increases, and the increase amplitude is relatively large. The reducing power of sweating for 2 days at 1 / 4 is the best compared with other processing methods, with a maximum absorbance value of 0.923, showing strong antioxidant ability. In addition, the reducing power of sweating for 2 days at 1 / 3 is the smallest, which is 0.209.

[0201] 2.6.5 Correlation analysis of content and activity

[0202] Using the SPSSPRO website for analysis, with the total phenolic acid content as the mother sequence and the oxidation scavenging rates (scavenging rates of the maximum concentration group) and absorbance indicators as the characteristic sequences, the data were homogenized, and the correlation degree between the characteristic sequences and the mother sequence was calculated. The results are shown in Table 25. As can be seen from the table, the processing methods with a greater impact on the ability to scavenge ABTS free radicals, ranked from largest to smallest, are: sweating for 3 days at 1 / 4 > drying at 60°C > sweating for 3 days at 1 / 2 > sweating for 4 days at 1 / 3 > sweating for 3 days at 1 / 3 > sweating for 2 days at 1 / 2 > sweating for 4 days at 1 / 2 > sweating for 1 day at 1 / 4 > sweating for 2 days at 1 / 3 > freeze-drying > sweating for 1 day at 1 / 2 > sweating for 7 days at 1 / 2 > sweating for 5 days at 1 / 4 > sweating for 4 days at 1 / 4 > sweating for two days at 1 / 4 > sweating for 5 days at 1 / 2 > sweating for 1 day at 1 / 3 > sun-drying > drying at 80°C > drying at 70°C. The processing methods with a greater impact on the ability to scavenge ·OH free radicals, ranked from largest to smallest, are: sweating for 3 days at 1 / 4 > drying at 60°C > sweating for 3 days at 1 / 2 > sweating for 2 days at 1 / 2 > sweating for 2 days at 1 / 3 > sweating for 1 day at 1 / 4 > sweating for 4 days at 1 / 2 > sweating for 7 days at 1 / 2 > sweating for 5 days at 1 / 2 > sweating for 1 day at 1 / 2 > sweating for 2 days at 1 / 4 > sweating for 4 days at 1 / 4 > freeze-drying > sweating for 1 day at 1 / 3 > sweating for 4 days at 1 / 3 > sweating for 5 days at 1 / 4 > sweating for 3 days at 1 / 3 > shade-drying > sweating for 5 days at 1 / 3 > sun-drying > drying at 80°C > drying at 70°C. The ranking of the absorbance measurements of the total reducing power is: sweating for 3 days at 1 / 2 > sweating for 2 days at 1 / 4 > sweating for 3 days at 1 / 4 > sweating for 4 days at 1 / 2 > drying at 60°C > sweating for 1 day at 1 / 3 > sweating for 2 days at 1 / 3 > sweating for 1 day at 1 / 4 > sweating for 7 days at 1 / 2 > sweating for 2 days at 1 / 2 > freeze-drying > sweating for 5 days at 1 / 4 > sweating for 5 days at 1 / 2 > sweating for 3 days at 1 / 3 > sweating for 4 days at 1 / 3 > sweating for 1 day at 1 / 2 > sweating for 4 days at 1 / 4 > shade-drying > sun-drying > sweating for 5 days at 1 / 3 > drying at 80°C > drying at 70°C.

[0203] The total phenolic acid content in the Danshen processing group is in the order of sweating for 5 days at 1 / 3 > sweating for 4 days at 1 / 4 > sweating for 4 days at 1 / 3 > sweating for 5 days at 1 / 4 > sweating for 2 days at 1 / 4 > sweating for 5 days at 1 / 2 > sweating for 3 days at 1 / 3 > sweating for 1 day at 1 / 4 > sweating for 7 days at 1 / 2 > sweating for 1 day at 1 / 3 > sweating for 3 days at 1 / 4 > sweating for 4 days at 1 / 2 > sweating for 2 days at 1 / 3 > sweating for 2 days at 1 / 2 > drying at 60°C > sweating for 3 days at 1 / 2 > sweating for 1 day at 1 / 2 > freeze-drying > shade-drying > sun-drying > drying at 80°C > drying at 70°C.

[0204] Table 25 Results of the correlation between the total phenolic acid content and the free radical scavenging ability and reducing ability of 22 Danshen processed samples

[0205]

[0206] 2.6.6 Correlation analysis of content and activity

[0207] The results of the correlation analysis of the Person method showed that the total phenolic acid content of Salvia miltiorrhiza was positively correlated with the ABTS and ·OH scavenging rates and the total reducing power determination, with R 2 being 0.744, 0.619, and 0.382 respectively. Among them, the correlation between the total phenolic acid content and ABTS and ·OH was extremely significant (p < 0.01). The results indicated that phenolic acid substances in Salvia miltiorrhiza were important components for its antioxidant activity.

[0208] Table 26 Pearson correlation analysis of the total phenolic acid content with the ABTS and ·OH scavenging rates and the total reducing power determination

[0209]

[0210] ﹡﹡ At the 0.01 level (two-tailed), the correlation was extremely significant

[0211] ﹡ At the 0.05 level (two-tailed), the correlation was significant

[0212] 3 Summary

[0213] Analysis of the data obtained in Table 20 showed that SPSS analysis indicated significant differences (p < 0.01) between the contents of the groups of dried in the shade, dried in the sun, freeze-dried, dried at 60°C, dried at 70°C, dried at 80°C, sweating for 1 day 1 / 2, sweating for 2 days 1 / 4, and sweating for 5 days 1 / 3. Among them, there was no significant difference (p = 0.34) between sweating for 3 days 1 / 2 and sweating for 3 days 1 / 4, no significant difference (p = 0.57) between sweating for 5 days 1 / 2 and sweating for 5 days 1 / 3, no significant difference (p = 0.06) between sweating for 5 days 1 / 4 and sweating for 3 days 1 / 2, and no significant difference among the five groups of sweating for 1 day 1 / 4, sweating for 2 days 1 / 3, sweating for 2 days 1 / 4, sweating for 4 days 1 / 2, and sweating for 4 days 1 / 3, and the five groups had no statistical significance. There were significant differences (p < 0.05) among the remaining groups.

[0214] From the experimental results of the antioxidant research in 2.6, it can be seen that the group of sweating for 3 days (weight loss 1 / 4) had the greatest impact on the scavenging ability of ABTS and ·OH free radicals by the total amount of phenolic acid substances, and the drying at 70°C had the least impact; the group of sweating for 3 days (weight loss 1 / 2) had the largest value for the total reducing power determination by the total amount of phenolic acid, and the value determined by drying at 70°C was the smallest. The results revealed that the level of the total phenolic acid content of the processed products of different processing methods of Salvia miltiorrhiza theoretically determined the magnitude of its antioxidant activity ability. The inconsistency between the level of the total phenolic acid content and the antioxidant ability in some groups of the samples in each experimental group might be caused by experimental operation errors.

[0215] The ability to affect ABTS and ·OH free radicals was the greatest after sweating for 3 days (weight loss of 1 / 4), and the reducing ability was the best after sweating for 3 days (weight loss of 1 / 2). Literature research shows that danshen generates heat by itself during the "sweating" process, the temperature rises, creating favorable conditions for the activities of enzymes in cells and promoting the formation of phenolic acid components. [8-9] . The antioxidant ability was weak when dried at 70°C and 80°C. The possible reason is that too high a temperature led to a decrease in the water content in the danshen medicinal materials, destroying the cell survival environment and enzyme activity, resulting in a decrease in the content of phenolic acid substances generated. Figure 8 As can be seen, the longer the sweating time, the lower the total phenolic acid content, and its scavenging rate and reducing ability decrease. It is speculated that the possible reason is that the medicinal materials are contaminated by microorganisms after too long sweating time.

[0216] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope claimed by the present invention.

[0217] References

[0218] [1] National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China [M]. Beijing: China Medical Science and Technology Press, 2015: 77.

[0219] [2] Li Xiang, Wu Leihong. Network pharmacology study on the main active components of compound danshen formula [J]. China Journal of Chinese Materia Medica, 2011, 36(21): 2912 - 2915.

[0220] [3] Wang Yan, Wen Xinbao, Qin Cuiping, etc. Study on the in vitro antioxidant activity of danshen extracts [J]. Acta Agriculturae Boreali - occidentalis Sinica, 2011, 20(11): 160 - 163.

[0221] [4] Li Min, Zhou Jing, Yu Li. Optimization of the extraction process of tanshinone ⅡA [J]. Chinese Traditional Patent Medicine, 2018, 40(3): 741 - 744.

[0222] [5] Xiong Jiawei, Ge Songlan, Ma Lei. Study on the extraction and purification process of tanshinone IIA and salvianolic acid B in danshen [J]. Natural Product Research and Development, 2017, 29: 1396 - 1402.

[0223]

[0224] [6] Ren Zhihui, Su Huixia, Bai Yanliu. Study on the integrated extraction process of fat - soluble and water - soluble components of danshen [J]. Chinese Journal of Information on Traditional Chinese Medicine, 2009, 16(3): 54 - 56.

[0225] ​

[0226] [7] He Shan, Fu Xianjun, Zhang Ling, et al. Study on the extraction process optimization of effective components of Salvia miltiorrhiza by response surface methodology and its in vitro antioxidant activity [J]. Chinese Archives of Traditional Chinese Medicine

[0227] , 2021, 39(01): 28 - 32 + 266—267.

[0228] [8] Duan Jin'ao, Su Shulan, Yan Hui, et al. Discussion on the "sweating" process of crude drug preliminary processing and its enzymatic reaction and chemical transformation mechanism [J]. Chinese Traditional and Herbal Drugs

[0229] , 2013, 449(10): 1219—1225.

[0230] [9] Zhou Tongshui. The main active component salvianolic acid B of Salvia miltiorrhiza is a product induced by post - harvest drying stress [J]. Modern Chinese Medicine, 2013, 15(3): 211—218.

Claims

1. An extraction process of total phenolic acids from Salvia miltiorrhiza, characterized in that, The extraction process includes the following steps: Step 1: Weigh Danshen powder precisely, sieve it through a No. 2 sieve, and reserve it for use. Step 2: Add 10 - 30% ethanol solution according to a solid-liquid ratio of 1:350 - 750 g / mL, perform ultrasonic extraction for 30 - 70 min, filter, and collect the filtrate to obtain the Danshen total phenolic acids extraction solution.

2. The extraction process of total phenolic acids from Salvia miltiorrhiza Bunge as claimed in claim 1, wherein, The solid-liquid ratio described in Step 2 is 1:350 - 450 g / mL.

3. The extraction process of total phenolic acids of Salvia miltiorrhiza Bunge according to claim 2, characterized in that, The solid-liquid ratio described in Step 2 is 1:350 g / mL.

4. The extraction process of total phenolic acids from Salvia miltiorrhiza Bunge according to claim 1, characterized in that, The ethanol concentration described in Step 2 is 10 - 20%.

5. The extraction process of total phenolic acids from Salvia miltiorrhiza Bunge according to claim 4, wherein, The ethanol concentration described in Step 2 is 20%.

6. The extraction process of total phenolic acids from Salvia miltiorrhiza Bunge as claimed in claim 1, wherein The ultrasonic extraction time described in Step 2 is 30 - 50 min.

7. The extraction process of total phenolic acids from Salvia miltiorrhiza Bunge according to claim 6, characterized in that, The ultrasonic extraction time described in Step 2 is 40 min.

8. Use of the Danshen total phenolic acids obtained by the extraction process of the Danshen total phenolic acids extraction process according to any one of claims 1 - 7 in the preparation of antioxidant drugs or health foods.

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