Quality control evaluation method for lipid-lowering and weight-losing efficacy of salvia miltiorrhiza medicinal material based on biological activity detection

Abstract

The invention provides a quality control evaluation method for lipid-lowering and weight-losing efficacy of a salvia miltiorrhiza medicinal material based on biological activity detection, which comprises the following steps: respectively obtaining a salvia miltiorrhiza medicinal material extract solution and a lipase solution, mixing the salvia miltiorrhiza medicinal material extract solution and the lipase solution, incubating twice to obtain a sample reaction solution, measuring the fluorescence absorbance of the sample reaction solution, and calculating the weight-losing efficacy of the salvia miltiorrhiza medicinal material. The absorbance is used as an administration group; setting a blank reaction solution, and measuring the fluorescence absorbance of the blank reaction solution as the absorbance of a blank group; according to the absorbance of the administration group and the absorbance of the blank group, calculating to obtain the lipase inhibition rate of the salvia miltiorrhiza extract; and controlling and evaluating the quality of the lipid-lowering and weight-losing efficacy of the salvia miltiorrhiza medicinal material according to the lipase inhibition rate. According to the method, the lipase inhibitory activity of the active component of the salvia miltiorrhiza is used as one of quality evaluation indexes of the salvia miltiorrhiza medicinal material, a traditional Chinese medicine quality biological determination method based on the medicine effect is perfectly established, the quality control and quality evaluation of the biological activity of the salvia miltiorrhiza sample can be realized, and the quality control problem of the salvia miltiorrhiza sample is effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine extraction and quality control, and particularly relates to a quality control and evaluation method for the lipid-lowering and weight-loss effects of Salvia miltiorrhiza based on bioactivity detection. Background Art

[0002] Salvia miltiorrhiza is the dried root and rhizome of the plant Salvia miltiorrhiza Bge. of the Labiatae family. It was listed as a top-grade herb in "Shennong's Herbal Classic" and has the effects of promoting blood circulation to remove blood stasis, dredging meridians to relieve pain, clearing the heart to remove vexation, and cooling blood to eliminate carbuncles. Salvia miltiorrhiza contains a variety of chemical components, mainly including tanshinones, salvianolic acids, volatile oils, and inorganic elements. Taking the lipid-soluble component tanshinones and the water-soluble component phenolic acids as representatives, the lipid-soluble components include a variety of phenanthraquinone derivatives, such as tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone, etc. The current quality control method for traditional Chinese medicine is mainly to determine one or two chemical components contained therein. The determined chemical components are called index components, and the quality of the traditional Chinese medicine is judged or evaluated by qualitative or quantitative determination of the index components. In the 2020 edition of the "Chinese Pharmacopoeia", the total amount of tanshinone IIA, cryptotanshinone, and tanshinone I is mainly used as the index component under Salvia miltiorrhiza, and the content of cryptotanshinone and tanshinone IIA is used as the index component for tanshinone extract.

[0003] At the present stage, the quality control and evaluation of Chinese patent medicines mainly rely on the qualitative and quantitative analysis of chemical components. With the continuous deepening of the research on traditional Chinese medicine, this mode has gradually shown limitations in the quality control and evaluation system of Chinese patent medicines. Using the content of individual or partial index components as an index of the quality standard can control the content of the index components, but it only controls the content of one or some of the components, making it difficult to ensure the stability of all components and the consistency of clinical efficacy, and even more difficult to reflect the overall biological effects of Chinese patent medicines.

[0004] As a quality analysis method based on bioactivity detection, the bioactivity determination method is expected to become a quality control and evaluation method that truly conforms to the traditional theory of traditional Chinese medicine, reflects the clinical application characteristics of Chinese patent medicines, and ensures the safety and effectiveness of Chinese patent medicines. The "Guidelines for Bioactivity Determination of Traditional Chinese Medicines" in General Principles 9105 of the Fourth Part of the 2020 Edition of the "Chinese Pharmacopoeia" clearly states that "the sources of traditional Chinese medicine materials are extensive and variable, and the preparation processes are complex, making the quality control of traditional Chinese medicine preparations relatively difficult. In addition, traditional Chinese medicine contains a variety of active ingredients and has multiple pharmacological effects. Therefore, only controlling a few components cannot fully control its quality and reflect clinical efficacy. In order to enable the quality standards of traditional Chinese medicine to better ensure the safety and effectiveness of each batch of drugs in clinical use, it is necessary to add bioactivity determination on the basis of the existing content determination to comprehensively evaluate its quality". Summary of the Invention

[0005] Lipase is an important enzyme in the body, mainly responsible for decomposing the fat in food into free fatty acids and glycerol for intestinal absorption. Lipase inhibitors reduce the digestion and absorption of fat by inhibiting the activity of lipase, thereby reducing the fat content in the blood. By evaluating the effect of lipase inhibitors in in vitro experiments, compounds with potential weight loss effects can be effectively screened. Common in vitro experimental methods include reacting pancreatic lipase with substrates (such as oils and fats) to observe whether lipase inhibitors can reduce the degree of fat hydrolysis. For example, Orlistat is a clinically applied lipase inhibitor that can effectively reduce fat absorption and thus achieve weight loss effects. Based on this, the present invention provides a quality control evaluation method for the lipid-lowering and weight-loss effects of Salvia miltiorrhiza herbs based on bioactivity detection. By using the lipase inhibitory bioactivity of all active ingredients in Salvia miltiorrhiza herbs as one of the quality evaluation indicators, the quality control standards of traditional Chinese medicine Salvia miltiorrhiza samples can be evaluated more objectively and comprehensively, thereby solving the problems of one-sided and single evaluation criteria, poor consistency, and inability to accurately reflect the quality and efficacy of Salvia miltiorrhiza samples in the quality control of Salvia miltiorrhiza samples.

[0006] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a quality control evaluation method for the lipid-lowering and weight-loss effects of Salvia miltiorrhiza herbs based on bioactivity detection, including:

[0008] S1, Extract the Salvia miltiorrhiza herbs to obtain a Salvia miltiorrhiza extract; dissolve the Salvia miltiorrhiza extract with a first solution to prepare a Salvia miltiorrhiza extract solution;

[0009] S2, Dissolve lipase to obtain a lipase solution;

[0010] S3, Mix the Salvia miltiorrhiza extract solution and the lipase solution, incubate for the first time, and then add a second solution for the second incubation to obtain a sample reaction solution, and measure its fluorescence absorbance as the absorbance of the administration group;

[0011] S4, Replace the lipase solution with an equal amount of the first solution, repeat S3 to obtain a blank reaction solution, and measure its fluorescence absorbance as the absorbance of the blank group;

[0012] S5, Calculate the lipase inhibition rate of the Salvia miltiorrhiza extract according to the absorbance of the administration group and the absorbance of the blank group;

[0013] S6, Control and evaluate the quality of the lipid-lowering and weight-loss effects of the Salvia miltiorrhiza herbs according to the lipase inhibition rate;

[0014] The first solution includes a tris (hydroxymethyl) aminomethane buffer with a pH value of 8.4, 0.5 - 1.5 μg / ml of co-lipase, and 0.5 - 1 mg / ml of sodium deoxycholate;

[0015] The second solution includes an acetate buffer with a pH of 4.0, 30 - 40 mg / ml of sodium taurodeoxycholate, 0.01 - 0.02 mg / ml of calcium chloride, and 0.2 - 0.4 mg / ml of DGGR.

[0016] Preferably, the solvent in the lipase solution is the first solution or water; and / or, the lipase concentration in the lipase solution is 8 - 12 mg / ml.

[0017] Preferably, the first solution includes a tris (hydroxymethyl) aminomethane buffer with a pH value of 8.4, 0.9 - 1.1 μg / ml of co-lipase, and 0.7 - 0.8 mg / ml of sodium deoxycholate.

[0018] Preferably, in the danshen extract solution, the concentration of the danshen extract is 0.03 - 1.5 mg / ml.

[0019] Preferably, the concentration of the danshen extract is 0.03 - 1.0 mg / ml.

[0020] Preferably, the second solution includes an acetate buffer with a pH of 4.0, 36 - 38 mg / ml of sodium taurodeoxycholate, 0.01 - 0.02 mg / ml of calcium chloride, and 0.3 - 0.4 mg / ml of DGGR.

[0021] Preferably, the calculation formula for the lipase inhibition rate is as follows: Inhibition rate (%) = [1 - (absorbance value of the administration group / absorbance value of the blank group)] × 100%.

[0022] Preferably, the volume ratio of the danshen extract solution to the lipase solution is 8:1 to 15:1, more preferably 10:1 to 12:1; and / or, the volume ratio of the danshen extract solution to the second solution is 8:1 to 15:1, more preferably 10:1 to 12:1.

[0023] Preferably, the time for the first incubation is 0.5 - 1.5 h; the time for the second incubation is 0.2 - 0.5 h; and / or, the incubation temperature is 30 - 50 °C, more preferably 35 - 40 °C.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] The present invention takes the bioactivity of the active ingredients extracted from Salvia miltiorrhiza Bunge against lipase inhibition as one of the quality evaluation indexes for its lipid-lowering and weight-loss effects, improves and establishes a biological assay method for the quality of traditional Chinese medicine based on pharmacodynamic effects, can achieve better evaluation of the biological activity quality control and quality of Salvia miltiorrhiza samples, and can provide new technical conditions for the quality control and quality evaluation research of other traditional Chinese medicines with unclear pharmacodynamic substances, and can effectively solve the quality control problem of Salvia miltiorrhiza samples. The method of the present invention has good reproducibility and can be used for the biological activity determination of Salvia miltiorrhiza samples in terms of lipid-lowering and weight-loss, so as to achieve the purpose of comprehensively evaluating and controlling product quality, which is an innovation in the quality control and evaluation method of traditional Chinese medicine. The evaluation method of the present invention is easy to operate, has strong practicability and wide application range, and is an innovation in the quality control and evaluation of traditional Chinese medicine. The present invention can not only be used to evaluate the quality of Salvia miltiorrhiza samples, but also be used as an evaluation index for its process optimization, which is of great significance for the monitoring and management of its production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the influence curve of the Salvia miltiorrhiza extract obtained by reflux extraction of Salvia miltiorrhiza from different producing areas in Example 1 of the present invention on lipase activity.

[0027] Figure 2 It is the influence curve of the Salvia miltiorrhiza extract obtained by different extraction methods in Example 2 of the present invention on lipase activity.

[0028] Figure 3 It is the comparison of the inhibitory activities of different active ingredients of Salvia miltiorrhiza on lipase in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0029] In the description of the present invention, it should be noted that for those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0030] Moreover, when a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value in the specified range is covered within the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and are also covered within the present invention, subject to any explicit exclusion limits in the specified range. When the specified range includes one or both of the limits, the ranges excluding either or both of the included limits are also included in the present invention.

[0031] In addition, it should be further noted that the Salvia miltiorrhiza Bunge from Changyi, Shandong used in Examples 1 to 3 of the present invention are 3 different batches, so Figures 1 to 3 there are certain differences in the results.

[0032] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and do not represent limitations.

[0033] Example 1

[0034] This example compared the lipase inhibitory activities of Salvia miltiorrhiza samples from different regions obtained by the reflux extraction method, as follows:

[0035] 1. Extraction of Salvia miltiorrhiza samples

[0036] Respectively take Salvia miltiorrhiza samples from Changyi, Binzhou, Xintai, Linqu in Shandong, Zhongjiang in Sichuan, Yunnan, and Henan (all are commercially available products), crush them without sieving, and weigh about 100 g each. Place the samples in a round-bottom flask, add 1000 ml of 95% ethanol, reflux for 60 minutes, repeat the extraction twice, combine the filtrates, concentrate, and dry in a blast dryer at 70 °C until dry.

[0037] 2. Detection method for lipase inhibitory activity

[0038] (1) Reagent preparation

[0039] a. Solution 1:

[0040] pH 8.4 Tris buffer (Tris 6.055 mg / ml, Tris.HCl 7.882 mg / ml), 1 μg / ml co-lipase, 0.746 mg / ml sodium deoxycholate.

[0041] b. Solution 2:

[0042] Acetate buffer (pH 4.0, containing anhydrous sodium acetate 1.476 mg / ml, glacial acetic acid 1.081 mg / ml), 37.56 mg / ml taurodeoxycholic acid, 0.0147 mg / ml calcium chloride, 0.361 mg / ml DGGR (dissolved in absolute ethanol to form an 8 mg / ml stock solution).

[0043] c. Lipase solution:

[0044] Prepare a stock solution with a concentration of 10 mg / ml using Tris buffer or deionized water, aliquot and store frozen at -20 °C for later use.

[0045] (2) Preparation of sample solution

[0046] Weigh 10 mg of Danshen samples, dissolve them in Solution 1 to make a 1 mg / ml solution, and then serially dilute it by a factor of 2 to obtain 6 sample solutions with different concentration gradients: 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, and 1.5625 μg / ml.

[0047] (3) Detection of lipase inhibitory activity

[0048] Add 170 μl of the medicinal liquid of each concentration prepared with Solution 1 to each well of a 96-well plate. On this basis, for the dosing wells: add 15 μl of 10 mg / ml Lipase solution to each well, and for the blank wells: add 15 μl of Tris buffer to each well. After incubating in the dark at 37 °C for 1 h, add 15 μl of Solution 2 to each well. There are 6 replicates for each sample solution of each concentration, and incubate at 37 °C for 20 min. Measure the fluorescence value at 530 nm / 590 nm.

[0049] Inhibition rate (%) = [1 - (OD value of dosing well / OD value of blank well)] × 100%.

[0050] It can be seen from Figure 1 that the lipase inhibitory activity of Danshen samples from Shandong region is significantly better than those from Zhongjiang in Sichuan, Yunnan, and Henan, and the activity of samples from Changyi in Shandong is the best.

[0051] Example 2

[0052] This example compared the lipase activities of different extraction methods of Danshen samples, as follows:

[0053] 1. Extraction of Danshen samples

[0054] Reflux extraction method: Take the unfiltered and crushed Danshen samples from Changyi in Shandong, add 10 times the amount of 95% ethanol, reflux for 60 min, extract twice, combine the filtrates, concentrate, and dry to constant weight at 70 °C under forced air drying.

[0055] Ultrasonic extraction method: Take the unfiltered and crushed Danshen samples, add 10 times the amount of 95% ethanol, ultrasonically extract for 30 minutes, filter, add 10 times the amount of 95% ethanol to the residue and ultrasonically extract for 30 minutes, combine the filtrates, concentrate, and dry to constant weight at 70 °C under forced air drying.

[0056] Supercritical extraction method: Take the unfiltered and crushed Danshen samples, use 95% medicinal alcohol as the entrainer with a ratio of 1:1, extraction temperature of 45 °C, pressure of 25 Mpa, concentrate, and dry to constant weight at 70 °C under hot air circulation drying.

[0057] The above samples were measured according to the lipase activity detection method in Example 1. Weigh 10 mg of the Salvia miltiorrhiza sample, prepare a 1 mg / ml stock solution with Solution 1, dilute the stock solution 1000-fold, and then perform serial-fold dilution to obtain 6 sample solutions with different concentration gradients: 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, and 0.03125 μg / ml. The lipase activity was detected according to the method described in item (3) of Example 1, and the results are as Figure 2 shown. As can be seen from Figure 2 , the activity of the supercritical extraction sample is significantly higher than that of the reflux extraction method and the ultrasonic extraction method. It can be seen that there are differences in the lipase inhibitory activities of the active ingredients in the Salvia miltiorrhiza samples obtained by different extraction methods. Therefore, this result can also be used as one of the bases for evaluating the effects of different extraction methods of Salvia miltiorrhiza. It should be noted that the methods for extracting the active ingredients from Salvia miltiorrhiza herbs are not limited to the aforementioned three methods.

[0058] In addition, this result can also show that evaluating the quality of Salvia miltiorrhiza herbs by measuring the lipase inhibitory activity of the active ingredients in the Salvia miltiorrhiza samples may be a more effective method for its quality control.

[0059] Example 3

[0060] Taking Salvia miltiorrhiza from Changyi, Shandong as the experimental object, the lipase inhibitory activities of the supercritical extraction sample in Example 2 were compared with the four main components in Salvia miltiorrhiza, namely tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone.

[0061] The above samples were measured according to the lipase activity detection method in Example 1. Weigh 10 mg of the Salvia miltiorrhiza sample, prepare a 1 mg / ml stock solution with Solution 1, dilute the stock solution 1000-fold, and then perform serial dilution to obtain 6 sample solutions with different concentration gradients: 1 μg / ml, 0.4 μg / ml, 0.16 μg / ml, 0.064 μg / ml, 0.0256 μg / ml, and 0.01024 μg / ml; prepare solutions of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone with concentrations of 4 μg / ml, 1.6 μg / ml, 0.64 μg / ml, 0.256 μg / ml, and 0.1024 μg / ml respectively. The lipase activity was detected according to the method described in item (3) of Example 1, and the activities were compared. The results are as Figure 3 shown. As can be seen from Figure 3 , even when the concentrations of the solutions of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone are increased, the lipase inhibitory activity of the supercritical extraction sample is still significantly better than that of any one of the main components in Salvia miltiorrhiza. Therefore, it is not advisable to evaluate the quality of Salvia miltiorrhiza with a single component. The lipase inhibitory activity is a more comprehensive and accurate evaluation method for the lipid-lowering and weight-loss quality of Salvia miltiorrhiza herbs.

[0062] In summary, the quality control and evaluation method for the lipid-lowering and weight-loss effects of Salvia miltiorrhiza established in the present invention is simple to operate, highly stable, sensitive and reliable, and feasible, which can provide guarantee for the integrity of its pharmacodynamic effects and the controllability of its quality.

[0063] The above-described embodiments merely represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A quality control evaluation method for the lipid-lowering and weight-loss efficacy of Danshen medicinal materials based on biological activity detection, characterized in that: include: S1, extracting Danshen medicinal materials to obtain Danshen extract; dissolving the salvia miltiorrhiza extract with the first solution to prepare a salvia miltiorrhiza extract solution; S2, dissolving the lipase to obtain a lipase solution; S3, mixing the Danshen extract solution and the lipase solution, incubating for the first time, and then adding the second solution for a second incubation to obtain a sample reaction solution, and measuring the fluorescence absorbance thereof as the absorbance of the drug administration group; S4, replacing the lipase solution with an equal amount of the first solution, repeating S3 to obtain a blank reaction solution, and measuring its fluorescence absorbance as the absorbance of the blank group; S5, calculating the lipase inhibition rate of the Salvia miltiorrhiza extract according to the absorbance of the drug administration group and the absorbance of the blank group; S6, controlling and evaluating the quality of the lipid-lowering and weight-loss efficacy of the Danshen medicinal material according to the lipase inhibition rate; The first solution includes a tris-based buffer with a pH value of 8.4, 0.5-1.5 μg / ml co-lipase, and 0.5-1 mg / ml sodium deoxycholate; The second solution includes pH 4.0 acetate buffer, 30-40 mg / ml sodium taurodeoxycholate, 0.01-0.02 mg / ml calcium chloride, and 0.2-0.4 mg / ml DGGR.

2. The method according to claim 1, characterized in that The solvent in the lipase solution is the first solution or water; and / or the lipase concentration in the lipase solution is 8-12 mg / ml.

3. The method according to claim 1 or 2, characterized in that: The first solution includes a tris-based buffer with a pH value of 8.4, 0.9-1.1 μg / ml co-lipase, and 0.7-0.8 mg / ml sodium deoxycholate.

4. The method according to claim 1, characterized in that In the salvia miltiorrhiza extract solution, the concentration of the salvia miltiorrhiza extract is 0.03-1.5 mg / ml.

5. The method according to claim 4, characterized in that The concentration of the salvia miltiorrhiza extract is 0.03-1.0 mg / ml.

6. The method according to claim 1, characterized in that The second solution includes pH 4.0 acetate buffer, 36-38 mg / ml sodium taurodeoxycholate, 0.01-0.02 mg / ml calcium chloride, and 0.3-0.4 mg / ml DGGR.

7. The method according to claim 1, characterized in that The calculation formula of the lipase inhibition rate is as follows: inhibition rate (%) = [1-(absorbance value of the drug administration group / absorbance value of the blank group)] × 100%.

8. The method according to any one of claims 1 to 4, characterized in that: The volume ratio of the Salvia miltiorrhiza extract solution to the lipase solution is 8:1-15:1, preferably 10:1-12:1; and / or the volume ratio of the Salvia miltiorrhiza extract solution to the second solution is 8:1-15:1, preferably 10:1-12:

1.

9. The method according to claim 7, characterized in that: The first incubation time is 0.5-1.5 h; the second incubation time is 0.2-0.5 h; and / or the incubation temperature is 30-50° C., preferably 35-40° C.

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