Preparation and quality control method of salvia miltiorrhiza standard extract

Abstract

The invention relates to a preparation method and a quality control method of a salvia miltiorrhiza standard extract containing salvianolic acid, tanshinone and polysaccharide components. The preparation method of the salvia miltiorrhiza standard extract specifically comprises the following steps: (1) taking salvia miltiorrhiza decoction pieces, adding 10 times of 80% ethanol, performing reflux extraction for 3 times, 1 hour each time, and combining filtrates; (2) adding 9 times of pure water into decoction dregs, decocting for 3 times, 1.5 hours each time, and combining filtrates; and (3) concentrating the combined filtrate at 60 DEG C under reduced pressure to a certain volume, transferring to a freeze-drying machine, freeze-drying, and crushing to obtain the traditional Chinese medicine composition. The invention simultaneously establishes a fingerprint spectrum of the salvia miltiorrhiza standard extract and a method for determining the content of tanshinone, salvianolic acid and polysaccharide components in the salvia miltiorrhiza standard extract, and establishes a method for determining the molecular weight of the polysaccharide components in the salvia miltiorrhiza standard extract and analyzing the monosaccharide composition and content in the salvia miltiorrhiza standard extract, so as to control the quality of the salvia miltiorrhiza standard extract.

Description

Technical Field

[0001] The invention belongs to the field of deep processing and modern application of traditional Chinese medicine, and specifically relates to a preparation and quality control method of a salvia miltiorrhiza standard extract containing salvianolic acids, tanshinones and polysaccharide components. Background Art

[0002] Danshen is the dried root and rhizome of Salvia miltiorrhiza Bge., a plant of the Lamiaceae family. It was first recorded in Shennong's Herbal Classic and has the effects of promoting blood circulation and removing blood stasis, relieving pain through menstruation, clearing the heart and eliminating vexation, cooling blood and eliminating carbuncle (China Pharmacopoeia Committee, Pharmacopoeia of the People's Republic of China: Volume 1 [M], Beijing, China Medical Science and Technology Press, 2020, 79). Danshen is mainly produced in Shandong, Sichuan, Hebei, Shanxi, Henan, Anhui and other provinces, and is distributed in most parts of the country. Danshen has significant therapeutic effects and is in great demand. Wild resources are limited, and currently it mainly relies on artificial cultivation to meet demand. The quality of Danshen medicinal materials from different origins varies, and the content of chemical components is also different. The standard extract of traditional Chinese medicine has the characteristics of stable quality, which has a positive effect on stabilizing the quality of Danshen. Standard extracts of traditional Chinese medicine refer to a kind of traditional Chinese medicine products that are extracted and processed by modern scientific and technological means, and have a relatively clear basis for efficacy and strict quality standards. They can be used as raw materials for traditional Chinese medicine preparations (Sun Chengjing, Research on the effective parts of phenolic acids and flavonoids in the stems and leaves of Salvia miltiorrhiza and their effects on improving microcirculatory disorders [D], Nanjing University of Chinese Medicine, 2020), and are widely used in the fields of medicine, health care products and beauty. At present, the research on the chemical composition of Salvia miltiorrhiza mainly focuses on tanshinone and tanshinone, which are also considered to be the main active ingredients of Salvia miltiorrhiza. Polysaccharides have multiple biological activities, and their pharmacological effects are gradually being valued by people. Some traditional Chinese medicines use the content of polysaccharides as one of their quality control standards, such as Yunzhi, Ganoderma lucidum, Polygonatum odoratum, Dendrobium, etc. This can not only reveal the role of polysaccharides in traditional Chinese medicine, but also provide quality assurance for the research, development and application of polysaccharides in traditional Chinese medicine. Existing studies have shown that salvia miltiorrhiza polysaccharides have anti-inflammatory, antioxidant, anti-tumor, organ protection and other pharmacological effects, which are closely related to the various effects of salvia miltiorrhiza (Jing Yongshuai et al., Research Progress on Content Determination, Structural Characterization and Pharmacological Activity of Salvia miltiorrhiza Polysaccharides [J], China Pharmacy, 2020, 31 (16), 2037-2042). To this end, the present invention develops a salvia miltiorrhiza standard extract containing salvianolic acids, tanshinones and polysaccharides, and establishes a quality control method thereof, so as to ensure the scientific use of salvia miltiorrhiza and promote the development of the salvia miltiorrhiza industry. Summary of the invention

[0003] The purpose of the present invention is to provide a standard extract of Salvia miltiorrhiza containing three main components: salvianolic acids, tanshinones and polysaccharides, determine its preparation method and establish a quality control method, so as to provide a material basis for fully developing and utilizing Salvia miltiorrhiza and promoting its modern application.

[0004] In order to achieve the purpose of the above invention, the present invention provides the following technical solutions:

[0005] The present invention provides a standard extract of Salvia miltiorrhiza containing salvianolic acids, tanshinones and polysaccharides, and the preparation steps are as follows:

[0006] Take Danshen decoction pieces, add ethanol to reflux extraction, collect the filtrate, take the residue and add pure water to decoct, combine the extracts and concentrate under reduced pressure, freeze-dry and then crush to obtain the product.

[0007] The ethanol extraction solvent is 0-95% ethanol, preferably 80% ethanol.

[0008] Wherein, 6 to 10 times the amount of 80% ethanol is added for extraction 1 to 3 times, each time for 1 to 2 hours, preferably 10 times the amount of 80% ethanol is added for extraction 3 times, each time for 1 hour.

[0009] The extraction is performed 1 to 3 times with 9 to 15 times amount of pure water, each time for 1 to 2 hours. Preferably, the extraction is performed 3 times with 9 times amount of pure water, each time for 1.5 hours.

[0010] The quality control method of the standard extract of Danshen miltiorrhiza comprises constructing a characteristic spectrum of the standard extract of Danshen miltiorrhiza ..., and the steps are as follows:

[0011] Step A. Preparation of test solution: Take 15 batches of Salvia miltiorrhiza extract, add solvent and ultrasonically dissolve, and filter to obtain;

[0012] Step B. Preparation of reference substance solution: Take each reference substance and dissolve it in a solvent;

[0013] Step C. taking the test solution and the reference solution respectively, and measuring them by high performance liquid chromatography to obtain corresponding characteristic spectra;

[0014] Step D: identifying some common peaks in the spectrum, and constructing a characteristic spectrum of the standard Salvia miltiorrhiza extract according to the spectrum characteristics of each batch of Salvia miltiorrhiza extract.

[0015] The characteristic fingerprint spectrum of the standard extract of Salvia miltiorrhiza includes 16 common peaks.

[0016] The method includes determining the content of the main compounds in the standard extract of Danshen, and the steps are as follows:

[0017] Step A. Preparation of reference solution: a. Accurately weigh 8 reference substances of salvianolic acid components and prepare reference substance stock solution with 80% methanol. b. Accurately weigh 4 reference substances of tanshinone components and prepare reference substance stock solution with methanol;

[0018] Step B. Preparation of test solution: a. Take each batch of Danshen extract and dissolve it in 80% methanol (for determination of salvianolic acid components); b. Take each batch of Danshen extract and dissolve it in methanol (for determination of tanshinone components);

[0019] Step C. Take the test solution and the reference solution respectively, measure them by high performance liquid chromatography, obtain the corresponding chromatographic peak area, and calculate the corresponding concentration according to the standard curve.

[0020] The total content of eight main salvianolic acid components in the standard salvia miltiorrhiza extract is between 2.87% and 16.06%, and the content of four main tanshinone components is between 0.17% and 1.09%.

[0021] The method comprises determining the total content of three components in the standard Danshen extract, and the steps are as follows:

[0022] Step A. Preparation of reference solution: Accurately weigh salvianolic acid B and tanshinone II A , glucose reference substance, add respective solvents to dissolve;

[0023] Step B. Preparation of test solution: Take the Salvia miltiorrhiza extract, add a solvent to dissolve it, and filter it;

[0024] Step C. Content determination: a. Determination of total tanshinone: the sodium nitrite-aluminum nitrate method is used to determine its content, and the detection wavelength is 496nm; b. Determination of total tanshinone: the total tanshinone content is directly determined, and the detection wavelength is 268nm; c. Determination of total danshen polysaccharides: the test solution is precipitated with 70% ethanol overnight, and the protein is removed by Sevage reagent, and the content is determined by phenol-sulfuric acid method, and the detection wavelength is 490am.

[0025] The total salvianolic acid content in the standard salvia miltiorrhiza extract is between 5.30% and 14.02%, the total tanshinone content is between 3.84% and 5.95%, and the total polysaccharide content is between 2.04% and 3.17%.

[0026] The method includes analyzing the molecular weight distribution of polysaccharides in the standard extract of Danshen, and the steps are as follows:

[0027] Step A. Preparation of reference solution: Accurately weigh the dextran reference of each molecular weight and add the mobile phase to dissolve it;

[0028] Step B. Preparation of test solution: Take Danshen extract, add pure water to dissolve by ultrasonic, precipitate the solution with alcohol, remove protein by Sevage method, and spin dry to obtain Danshen polysaccharide. Weigh 5.00 mg of Danshen polysaccharide, add mobile phase to dissolve by ultrasonic.

[0029] Step C. Take the test solution and the reference solution respectively, measure them by high performance liquid chromatography, obtain the corresponding chromatographic retention time, and calculate the corresponding molecular weight distribution according to the standard curve.

[0030] In the standard extract of Danshen, the molecular weight distribution of polysaccharides of DS2 to DS15 is clearly divided into three parts: high, medium and low, while DS1 contains only two parts, medium and low, and their molecular weight distribution ranges are 3.94×10 5 ~4.50×10 5 Da, 6.69×10 3 ~2.00×10 4 Da, 1.20×10 3 ~1.28×10 3 Da, and 6.69×10 3 ~2.00×10 4 The polysaccharides in the Da range account for the largest proportion, accounting for 61.69% to 77.71% of the total polysaccharides.

[0031] The method also includes analyzing the monosaccharide composition and content of Danshen polysaccharide in Danshen standard extract, and the steps are as follows:

[0032] Step A. Preparation of reference solution: Accurately weigh each monosaccharide reference substance and dissolve it in pure water;

[0033] Step B. Preparation of Danshen polysaccharide: taking Danshen extract, adding pure water to dissolve by ultrasonication, and then subjecting to alcohol precipitation, centrifugation, protein removal, and freeze concentration to obtain Danshen crude polysaccharide;

[0034] Step C. Derivatization of monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP): Precisely weigh the salvia miltiorrhiza polysaccharide, hydrolyze it into monosaccharides under acidic conditions, and spin dry the solvent. Redissolve the hydrolyzed salvia miltiorrhiza polysaccharide sample in pure water, derivatize it with PMP under alkaline conditions, and then perform HPLC detection.

[0035] The standard extract of Salvia miltiorrhiza contains mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose. Among them, the content of mannose is the lowest among the 6 monosaccharides, the content of galactose in the standard extract of Salvia miltiorrhiza batches S1-S8 and S10 is the highest, and the content of galacturonic acid is the highest in S9, S11-S15. In all Salvia miltiorrhiza samples, the content of galacturonic acid, glucose and galactose is higher than other monosaccharides as a whole.

[0036] The beneficial effects of this technical solution are as follows:

[0037] (1) The method of the present invention provides a method for preparing a standard extract of Salvia miltiorrhiza based on alcohol extraction followed by water extraction. By reasonably controlling the process parameters during the alcohol extraction and water extraction, such as extraction time, number of extractions and solvent amount, the yield of the extract and the content of its effective components, such as salvianolic acids, tanshinones and polysaccharides, can be controlled, thereby ensuring the stability of the Salvia miltiorrhiza extract and providing accurate and stable data for the subsequent development and utilization of the standard extract of Salvia miltiorrhiza.

[0038] (2) The present invention also establishes a method for determining the content of total salvianolic acid, total tanshinone and total danshen polysaccharide in the standard extract of danshen, using salvianolic acid B, tanshinone II and total danshen polysaccharide as the A With glucose as reference substances, the contents of total salvianolic acid, total tanshinone and total danshen polysaccharides in the standard extract of Danshen were determined by the calculation formula of the standard curve. It can be used to determine the content of the index components when optimizing the preparation process of the standard extract of Danshen, and to control the quality of the standard extract of Danshen.

[0039] (3) The present invention has established an exclusive quality control method for this standard extract of Danshen. Fifteen batches of Danshen pieces from different origins or batches were collected and prepared into Danshen extracts. A characteristic spectrum was established, in which 16 common peaks were obtained, and 12 of them were identified.

[0040] (4) The present invention also establishes the content of danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone II in the standard extract of danshen A The content determination method can be used for the quality control of Danshen standard extract.

[0041] (5) Regarding the quality control of polysaccharide components, the present invention established a method for determining the molecular weight of polysaccharides using HPLC-GPC.

[0042] (6) Regarding the quality control of polysaccharide components, the present invention also established a method for determining the composition and content of monosaccharides in polysaccharides by HPLC-PMP.

[0043] In summary, the present invention provides a preparation method and quality control method of a standard extract of Danshen containing salvianolic acid, tanshinone and polysaccharide components, and the results of the process stability experiment and the content determination methodology are good, which can be used for the development and application of Danshen products. In addition, compared with other Danshen extract preparation processes and quality control standards, the present invention comprehensively considers the extraction of three types of components, namely, salvianolic acid, tanshinone and Danshen polysaccharide, to make fuller use of Danshen medicinal materials, save actual production costs and increase income. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Fingerprint of the standard extract of Salvia miltiorrhiza

[0045] Figure 2 HPLC chromatogram of mixed reference substance

[0046] Figure 3 The reference fingerprint of Danshen standard extract

[0047] Figure 4 HPLC chromatograms of Danshen sample solution (A) and 8 salvianolic acid reference solutions (B)

[0048] Figure 5 HPLC chromatograms of Danshen sample solution (A) and four tanshinone reference substance solutions (B)

[0049] Figure 6 HPLC-GPC chromatogram of Danshen polysaccharide

[0050] Figure 7 This is the chromatogram of the HPLC-PMP sample of Danshen polysaccharide

[0051] Figure 8 This is the chromatogram of HPLC-PMP mixed reference substance of Danshen polysaccharide DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with embodiments.

[0053] Example 1: Determination of the content of each component of Salvia miltiorrhiza by spectrophotometry

[0054] 1. Test solution preparation

[0055] 1.1 Preparation of reference solution

[0056] Accurately weigh the reference substance of salvianolic acid B, add 80% methanol to prepare a 0.12 mg / mL reference substance stock solution; accurately weigh tanshinone II A For the reference substance, add methanol to prepare a 0.10 mg / mL reference substance stock solution; accurately weigh the glucose reference substance and add pure water to prepare a 1.03 mg / mL reference substance stock solution.

[0057] 1.2 Preparation of color developer solution

[0058] Accurately weigh 5.00 g of sodium nitrite and 10.00 g of aluminum nitrate, dilute to 100 mL with pure water respectively, and completely dissolve by ultrasonication to prepare 5.0% sodium nitrite solution and 10.0% aluminum nitrate solution;

[0059] Accurately weigh 8.32 g of sodium hydroxide and prepare a sodium hydroxide solution with a final concentration of 1 mol / L using pure water;

[0060] Accurately weigh 5.00 g of phenol, dilute to 100 mL with pure water, heat in a water bath to aid dissolution, and completely dissolve by ultrasound to prepare a 5.0% phenol solution.

[0061] 1.3 Preparation of test solution

[0062] Preparation of test solution for determination of total salvianolic acid: accurately weigh 0.2000 g of Salvia miltiorrhiza extract into a conical flask, add 40 mL of 80% methanol, weigh, ultrasonicate at 80°C for 30 min, cool, weigh again to make up the weight, centrifuge at 12000 rpm for 8 min, and take the supernatant.

[0063] Preparation of test solution for determination of total tanshinone: accurately weigh 0.5000 g of danshen extract into a conical flask, add 40 mL of methanol, weigh, ultrasonicate at 70°C for 30 min, cool, weigh again to make up the weight, centrifuge at 12000 rpm for 8 min, and take the supernatant.

[0064] Preparation of test solution for determination of total polysaccharides: accurately weigh 0.2000 g of Salvia miltiorrhiza extract into a conical flask, add 40 mL of pure water, ultrasonicate at 70°C for 30 min, cool, weigh again to make up the weight, centrifuge at 12000 rpm for 8 min, and take the supernatant.

[0065] 2. Content determination method

[0066] Determination of total salvianolic acid: accurately pipette 0.3 mL of total salvianolic acid test solution into a 10 mL volumetric flask, operate in a dark environment, add 0.5 mL of 5% sodium nitrite solution in sequence, mix well, then add 0.5 mL of 10% aluminum nitrate solution, mix well, react for 5 minutes, add 1 mol / L sodium hydroxide solution to the scale, shake well, measure the absorbance at 496 nm, read the concentration against the standard curve, and calculate the total salvianolic acid content;

[0067] Determination of total tanshinone: accurately pipette 0.2 mL of total tanshinone test solution into a 10 mL volumetric flask, operate in a dark environment, dilute to the mark with methanol, shake well, measure the absorbance at 268 nm, read the concentration against the standard curve, and calculate the total tanshinone content;

[0068] Determination of total salvia miltiorrhiza polysaccharide: Accurately pipette 1mL of total salvia miltiorrhiza polysaccharide test solution into a 15mL centrifuge tube, add 95% ethanol to a final concentration of 70%, mix well, let stand for 12 hours overnight, centrifuge at 4000r / min for 10min, and dissolve the precipitate with 1mL of pure water. Add 2mL of Sevage reagent (chloroform: n-butanol = 4:1) to the solution, mix well, and centrifuge at 4000r / min for 10min. Pipet 0.4mL of supernatant, add water to 2mL, and mix well. Add 1mL of 5% phenol, mix well, quickly add 5mL of sulfuric acid, mix well, let stand for 5min, boil in water bath for 15min, cool to room temperature, measure absorbance at 490nm, read the concentration against the standard curve, and calculate the total salvia miltiorrhiza polysaccharide content.

[0069] 3 Methodological investigation

[0070] The results of the linear relationship investigation are shown in Table 1. Within their respective concentration ranges, the linear relationships of the compounds were good, and R 2 The values ​​were all greater than 0.9990. The results of precision, stability, repeatability and sample recovery test showed that the method was stable and reliable, and the results were accurate.

[0071] Table 1 Results of investigation on linear relationship of each component

[0072]

[0073] Example 2: Preparation process of Danshen standard extract

[0074] 1. Determine the weight coefficient of each indicator by using the analytic hierarchy process (AHP)

[0075] The extraction rate of total salvianolic acid, total tanshinone, total salvia miltiorrhiza polysaccharide and the quality of the extract were selected as evaluation indicators. According to the pharmacological effects of various components in salvia miltiorrhiza, relevant literature was consulted, and their quality was quantified as weight indicators to determine the order: total tanshinone extraction rate = total salvianolic acid extraction rate > total salvia miltiorrhiza polysaccharide extraction rate > extract quality. The judgment priority matrix of paired comparison was constructed to give relative scores to each indicator. The priority matrix of paired comparison of indicators is shown in Table 2.

[0076] Table 2 Judgment priority matrix for pairwise comparison of indicators

[0077]

[0078] Comprehensive score value = total tanshinone extraction rate / maximum tanshinone extraction rate × 0.3889 × 100 + total salvianolic acid extraction rate / maximum salvianolic acid extraction rate × 0.3889 × 100 + total salvia miltiorrhiza polysaccharide extraction rate / maximum salvia miltiorrhiza polysaccharide extraction rate × 0.1535 × 100 + extract mass / maximum extract mass × 0.0687 × 100.

[0079] 2. Selection of the best extraction solvent

[0080] Weigh 20.00g of Danshen slices, add 10 times of pure water and (20%, 40%, 50%, 60%, 80%, 95%) ethanol reflux extraction 3 times, each time for 1h, repeat extraction 3 times with each solvent, filter, and combine the filtrate. The extract was concentrated under reduced pressure and freeze-dried, and the dried extract was weighed and the total tannic acid, total tanshinone, total Danshen polysaccharide extraction rate and dry paste weight were determined by colorimetry, and the comprehensive score was calculated. According to the extraction results of each solvent and the extraction method of first alcohol extraction and then water extraction was investigated, the comprehensive score was calculated, and the results are shown in Table 3.

[0081] Table 3 Results of extracting each component with different solvents

[0082]

[0083] The results showed that according to the analysis of the extraction of various components by different solvents, the comprehensive score of Danshen slices extracted with 80% ethanol first and then the residue extracted with pure water was the highest, which was 99.96, and the extraction effect was the best.

[0084] 3 Orthogonal test

[0085] 3.180% ethanol extraction orthogonal experiment

[0086] Weigh 9 portions of Danshen decoction pieces and press L 9 (3 4 ) The orthogonal experimental table setting method was used for reflux extraction. The factor level table is shown in Table 4, the orthogonal test results are shown in Table 5, and the variance analysis results of SPSS 25.0 software are shown in Table 6.

[0087] Table 4 Factors and levels

[0088]

[0089] Table 5 Ethanol extraction orthogonal experimental design table and results

[0090]

[0091] Table 6 Results of variance analysis

[0092]

[0093] The results showed that the order of influence of the three factors was: B>C>A, and the number of extractions and the amount of solvent were significant factors. Therefore, the optimal extraction process was determined to be A. 1 B 3 C 3 That is, add 10 times the amount of 80% ethanol and reflux extract 3 times, each time for 1 hour.

[0094] 3.2 Orthogonal test of water extraction process

[0095] After extracting Danshen with the above-mentioned ethanol optimal extraction process, the residue was washed with pure water at 1 L 9 (34) The orthogonal experimental table was used for decoction. The factor level table is shown in Table 7, the water extraction AHP comprehensive analysis results are shown in Tables 8 and 9, and the water extraction Salvia miltiorrhiza polysaccharide orthogonal test results are shown in Tables 10 and 11.

[0096] Table 7 Factors and levels

[0097]

[0098] Table 8 Water extraction orthogonal experimental design table and AHP analysis results

[0099]

[0100]

[0101] Table 9 AHP variance analysis results

[0102]

[0103] Table 10 Orthogonal experimental design table and results of water extraction of Salvia miltiorrhiza polysaccharide

[0104]

[0105]

[0106] Table 11 Results of variance analysis

[0107]

[0108] The results showed that the order of influence of the AHP comprehensive analysis of water extraction factors was: B>A>C, and there was no significant factor; the results of water extraction of Salvia miltiorrhiza polysaccharide showed that the order of influence of the three factors was: B>A>C, which was the same as above, and the extraction time and extraction times were significant influencing factors. Considering the extraction rate of Salvia miltiorrhiza polysaccharide and the actual production situation, the optimal water extraction process was determined to be: A 2 B 3 C 1 , that is, add 9 times the amount of pure water and extract 3 times, each time for 1.5 hours.

[0109] 4. Process Validation

[0110] Take 20.00g of Danshen slices and place them in a round-bottom flask, add 10 times the amount of 80% ethanol, reflux extraction 3 times, each time for 1 hour, and filter the alcohol extract through gauze while hot. Add 9 times the amount of pure water to the residue and decoct it 3 times. Each time, boil it with high heat first and then keep it at a low boil for 1.5 hours. Filter the decoction through gauze while hot, and combine the alcohol extract and the water extract to obtain the Danshen extract. The combined filtrate is concentrated to a certain volume under reduced pressure at 60°C, transferred to a vacuum freeze dryer, and freeze-dried. Weigh and crush the freeze-dried sample to obtain the Danshen standard extract, and repeat the extraction 3 times. The dry paste weight and extraction rate of each component of three batches of extracts were determined. The average dry paste weight was 12.19 g, the paste yield was 60.94%, and the RSD value was 1.13%; the average extraction rate of total salvianolic acid was 4.67%, and the RSD value was 1.89%; the average extraction rate of total tanshinone was 1.33%, and the RSD value was 1.70%; the average extraction rate of total polysaccharides was 1.50%, and the RSD value was 1.75%.

[0111] Example 3: This example involves the quality control of the standard extract of Danshen. 15 batches of Danshen decoction pieces were used to prepare the standard extract of Danshen, and its characteristic spectrum was established.

[0112] 1 Preparation of Salvia miltiorrhiza standard extract

[0113] 1.1 Salvia miltiorrhiza sample information

[0114] The source information of Danshen decoction pieces is shown in the table below:

[0115] Table 12 Source information of Danshen decoction pieces

[0116]

[0117]

[0118] 1.2 Paste yield

[0119] The freeze-dried Salvia miltiorrhiza extract was quickly transferred to a dryer and weighed to obtain the mass of the dry paste. The paste rate was calculated based on the mass of the initial slices weighed.

[0120] The formula for calculating the paste yield = dry paste mass / dry paste mass*100.

[0121] The results of the paste yield of 15 batches of Danshen slices are shown in the following table:

[0122] Table 13 Quality and yield of each batch of Danshen extract

[0123]

[0124] It can be seen from the above table that the yield of the 15 batches of standard Danshen extracts fluctuates in the range of 51.05% to 65.58%, with an average of 58.64% and a SD of 4.70%.

[0125] 2 Fingerprint

[0126] 2.1 Solution preparation

[0127] Preparation of reference substance solution: Accurately weigh appropriate amount of each reference substance, add 80% methanol and ultrasonically dissolve it to obtain the reference substance stock solution.

[0128] Preparation of test solution: Accurately weigh 0.2000 g of each Salvia miltiorrhiza extract powder and place it in a conical flask, add 50 mL of 80% methanol, weigh, ultrasonicate for 30 min, cool to room temperature, weigh, make up for the weight loss with 80% methanol, shake well, filter through a 0.22 μm microporous filter membrane, and the filtrate is used as the Salvia miltiorrhiza test solution.

[0129] 2.2 Chromatographic conditions

[0130] The mobile phase was 0.05% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution (0-10 min, 8%-20% B; 10-15 min, 20%-25% B; 15-20 min, 25% B; 20-27 min, 25%-28% B; 27-30 min, 28%-30% B; 30-35 min, 30% B; 35-43 min, 30%-35% B; 43-47 min, 35%-55% B; 47-57min, 55%-65% B; 57-67min, 65%-70% B; 67-77min, 70%-100% B; 77-80min, 100% B; 80-95min, 100%-8% B; 95-100min, 8% B), flow rate: 1mL / min, injection volume 10μL, column temperature 25℃, detection wavelength 279nm.

[0131] 2.3 Methodology

[0132] The results of precision, repeatability and stability tests showed that the RSD values ​​of relative retention time and relative peak area of ​​the common peaks were both less than 3%, indicating that the method was stable and reliable.

[0133] 2.4 Establishment of fingerprint

[0134] Prepare the test sample solutions of 15 batches of Danshen extracts according to the method under "2.1", perform HPLC analysis according to the chromatographic conditions under "2.2", and record the chromatographic data. Import the fingerprint data of these 15 batches of Danshen test samples into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Edition)", set the reference spectrum (S14), use the median method, the time window width is 0.1, and after multi-point correction, automatically match the spectrum peaks to generate a superimposed spectrum, such as Figure 1 The characteristic spectrum results showed 16 common peaks, 12 of which were identified by the reference substance, as shown in Figure 2 As shown, the relative retention time and relative peak area results are shown in Table 14 and Table 15, and the similarity evaluation results are shown in Table 16.

[0135]

[0136]

[0137]

[0138] Example 4: This example involves the quality control of Danshen standard extract. 15 batches of Danshen decoction pieces were used to prepare Danshen standard extract, and an HPLC method for determining the content of salvianolic acids and tanshinone components was established.

[0139] 1. Solution Preparation

[0140] 1.1 Preparation of reference solution

[0141] Accurately weigh danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid A reference substances, and prepare reference substance stock solutions with a mass concentration of 1.00 mg / mL using 80% chromatographic methanol; accurately weigh dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone II A For the reference substance, use chromatographic methanol to prepare reference substance stock solutions with mass concentrations of 0.20, 1.00, 0.125, and 1.00 mg / mL, respectively.

[0142] 1.2 Preparation of test solution

[0143] Take 0.2000g of the extract powder from each batch, accurately weigh it, put it in a conical flask, accurately add 50mL of 80% methanol, seal it, weigh it, ultrasonicate it for 30min, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate for the determination of tanshinone components; take 0.5000g of the extract powder from each batch, accurately weigh it, put it in a conical flask, accurately add 50mL of methanol, seal it, weigh it, ultrasonicate it for 30min, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate for the determination of tanshinone components.

[0144] 2 Chromatographic conditions

[0145] Column: Agela C 18Chromatographic column (250mm×4.6mm, 5μm); mobile phase is 0.05% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution (0-5min, 5%-10% B; 5-15min, 10%-22.5% B; 15-25min, 22.5% B; 25-35min, 22.5%-23% B; 35-46min, 23%-25% B; 46-50min, 25%-30% B; 50-50.01min, 30%-5% B; 50.01-53min, 5% B); flow rate: 1.0mL / min; column temperature: 25℃; detection wavelength: 279nm, injection volume: 10μL (for determination of salvianolic acid components), chromatogram see Figure 4 The mobile phase was 0.02% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution (0-8 min, 63% B; 8-22 min, 63%-88% B; 22-22.5 min, 88%-63% B; 22.5-25 min, 63% B); flow rate: 1.0 mL / min; column temperature: 20°C; detection wavelength: 270 nm, injection volume: 10 μL (for the determination of tanshinone components), chromatogram see Figure 5 .

[0146] 3 Methodological investigation

[0147] The results of the linear relationship investigation are shown in Table 17. Within their respective concentration ranges, the linear relationships of the compounds are good, and R 2 The values ​​were all greater than 0.9990. The results of precision, stability, repeatability and sample recovery test showed that the method was stable and reliable, and the results were accurate.

[0148] Table 17 Results of investigation on linear relationship of each component

[0149]

[0150]

[0151] 4. Content determination

[0152] According to the method under "1.1", 15 batches of test solutions of Salvia miltiorrhiza extract were prepared and injected according to the chromatographic conditions under "2". The contents of salvianolic acid and tanshinone components in each batch of Salvia miltiorrhiza extract were calculated. The results are shown in Tables 18 and 19.

[0153]

[0154]

[0155] Example 5: This example involves the quality control of the standard extract of Danshen. 15 batches of Danshen decoction pieces were used to prepare the standard extract of Danshen, and the contents of total salvianolic acid, total tanshinone and total polysaccharide were determined.

[0156] Fifteen batches of prepared Salvia miltiorrhiza extracts were taken and the contents of total salvianolic acid, total tanshinone and total polysaccharides were determined according to the method under “Example 1: Spectrophotometric determination of components of Salvia miltiorrhiza”. The results are shown in Table 20.

[0157] Table 2015 batches of total salvia miltiorrhiza polysaccharide content in salvia miltiorrhiza extract (%)

[0158]

[0159] The results of content determination show that the content of total salvianolic acid components is between 5.30% and 14.02%, with an average of 10.12%. Taking 80% of the average value as the limit, the total salvianolic acid content is not less than 8.10%; the content of total tanshinone components is between 3.84% and 5.95%, with an average of 4.66%. Taking 80% of the average value as the limit, the total tanshinone content is not less than 3.73%; the content of total polysaccharides is between 2.04% and 3.17%, with an average of 2.62%. Taking 80% of the average value as the limit, the total polysaccharide content is not less than 2.10%.

[0160] Example 6: This example involves the quality control of Danshen standard extract. 15 batches of Danshen decoction pieces were used to prepare Danshen standard extract, and the molecular weight of Danshen polysaccharide was determined.

[0161] 1. Solution Preparation

[0162] 1.1 Preparation of reference solution

[0163] Accurately weigh 5.00 mg of each standard substance and dissolve it in 1 mL of mobile phase solution to prepare a 5 mg / mL reference solution.

[0164] 1.2 Preparation of test solution

[0165] Weigh 0.4500g of Danshen extract powder accurately into a 150mL conical flask, add 30mL pure water, ultrasonicate at 70℃ for 30min, cool, and weigh again to make up the weight. Transfer the solution to a 50mL EP tube, centrifuge at 4000r / min for 15min, take the supernatant and add 95% ethanol to a final concentration of 70%, mix, let stand for 12h, centrifuge at 4000r / min for 15min, dissolve the precipitate with 3mL pure water, add 6mL of Sevage reagent (chloroform: n-butanol = 4:1) to the solution, mix, centrifuge at 4000r / min for 15min. Take the supernatant and spin dry the solvent to obtain Danshen polysaccharide.

[0166] Weigh 5.00 mg of sample accurately, dissolve in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, ultrasonicate for 10 min, centrifuge at 12000 rpm for 10 min, aspirate the supernatant, and filter with a 0.22 μm water microporous membrane.

[0167] 2 Chromatographic conditions

[0168] Mobile phase: 0.5 mol / L NaCl solution; chromatographic column: BRT105-103-101 series gel column (8×300 mm); flow rate: 0.7 mL / min; column temperature: 40°C; injection volume: 100 μL.

[0169] 3 Linear Relationship

[0170] The standard product was analyzed by the above chromatographic method, the retention time was obtained, and the LgMw-RT standard curve was drawn. The results showed that the Mw was in the range of 7380 to 755900 Da, and the calibration curve equation was: Y = -0.1179X + 9.1556, R 2 =0.9947, good linear relationship.

[0171] 4 Molecular weight determination

[0172] The sample was analyzed according to the above chromatographic method to obtain the chromatogram and retention time. The molecular weight can be obtained by substituting the retention time into the formula. The results are shown in Table 21 and the chromatogram is shown in Figure 6 .

[0173] Table 21 Determination results of molecular weight of polysaccharides in 15 batches of standard extracts of Salvia miltiorrhiza

[0174]

[0175]

[0176] The results show that the molecular weight distribution of polysaccharides in the standard extracts of Danshen miltiorrhiza miltiorrhiza DS2 to DS15 is clearly divided into three parts: high, medium and low, while DS1 contains only two parts, medium and low, and their molecular weight distribution ranges are 3.94×10 5 ~4.50×10 5 Da, 6.69×10 3 ~2.00×10 4 Da, 1.20×10 3 ~1.28×10 3 Da, and 6.69×10 3 ~2.00×10 4 The polysaccharides in the Da range account for the largest proportion, accounting for 61.69% to 77.71% of the total polysaccharides.

[0177] Example 7: This example involves the quality control of Danshen standard extract. 15 batches of Danshen decoction pieces were used to prepare Danshen standard extract, and the monosaccharide composition and content analysis of Danshen polysaccharide were determined.

[0178] 1. Solution Preparation

[0179] 1.1 Preparation of reference solution

[0180] Weigh 0.52, 0.55, 0.58, 1.61, 1.50, and 2.05 mg of mannose, rhamnose, arabinose, galacturonic acid, glucose, and galactose reference substances, respectively, and add pure water to prepare a mixed reference substance stock solution.

[0181] 1.2 Preparation of Salvia miltiorrhiza polysaccharide

[0182] Weigh 0.45g of Danshen extract powder accurately into a 150mL conical flask, add 30mL of pure water, ultrasonicate at 70℃ for 30min, cool, and weigh again to make up the weight. Transfer the solution to a 50mL EP tube, centrifuge at 4000r / min for 15min, take the supernatant and add 95% ethanol to a final concentration of 70%, mix, let stand for 12h, centrifuge at 4000r / min for 15min, dissolve the precipitate with 3mL of pure water, add 6mL of Sevage reagent (chloroform: n-butanol = 4:1) to the solution, mix, centrifuge at 4000r / min for 15min. Take the supernatant into a 1.5mL EP tube, spin dry the solvent to obtain Danshen crude polysaccharide.

[0183] 2 Derivatization of Salvia miltiorrhiza polysaccharide

[0184] Weigh 5.00 mg of Danshen polysaccharide accurately, add 1 mL of 2 mol / L trifluoroacetic acid, seal the glass tube and place it at 100°C for hydrolysis for 6 hours, spin dry the solvent, add 0.5 mL of methanol and dry it to remove the residual trifluoroacetic acid. Add 0.2 mL of pure water to the hydrolyzed Danshen polysaccharide sample, filter, add 200 μL of 0.6 mol / L sodium hydroxide, then add 400 μL of PMP-methanol solution, vortex mix, react at 70°C for 1 hour, cool and add 400 μL of 0.3 mol / L hydrochloric acid to neutralize. Add 1.2 mL of chloroform to the neutralized solution, extract twice, and remove the excess PMP.

[0185] 3 Chromatographic conditions

[0186] Column: Agela C 18 Chromatographic column (250mm×4.6mm, 5μm); mobile phase: acetonitrile-0.5mol / L potassium dihydrogen phosphate solution, gradient elution (0-40min, 17%-33% acetonitrile; 40-45min, 33%-17% acetonitrile; 45-47min, 17% acetonitrile); detection wavelength: 250nm; column temperature: 30℃; flow rate: 1mL / min.

[0187] 4 Methodological investigation

[0188] The results of the linear relationship investigation are shown in Table 22. Within their respective concentration ranges, the linear relationships of the compounds are good, R 2 The values ​​were all greater than 0.9950. The results of precision, stability, repeatability, and sample recovery test showed that the method was stable and reliable, and the results were accurate. The specificity chromatogram is shown in Figure 7 and Figure 8 .

[0189] Table 22 Results of investigation on linear relationship of each component

[0190]

[0191] 5. Measurement results

[0192] Table 23 Molar ratio of monosaccharides in 15 batches of Salvia miltiorrhiza polysaccharides

[0193]

[0194] From the data in Table 23, it can be seen that the composition of monosaccharides in 15 batches of Salvia miltiorrhiza samples is the same, all of which contain mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose. Among them, mannose has the lowest content among the 6 monosaccharides, the content of galactose in the standard extracts of Salvia miltiorrhiza batches S1-S8 and S10 is the highest, and the content of galacturonic acid is the highest in S9, S11-S15. In all Salvia miltiorrhiza samples, the content of galacturonic acid, glucose and galactose is higher than that of other monosaccharides as a whole.

Claims

1. A method for preparing a standard extract of Salvia miltiorrhiza, characterized in that: Take Danshen decoction pieces, add ethanol to reflux extraction, filter, and set aside the residue; add pure water to the residue and boil, filter, and discard the residue; combine all the filtrates, reduce the pressure and concentrate the filtrates, freeze-dry in a vacuum, and grind to obtain the product.

2. The preparation method according to claim 1, characterized in that: The ethanol extraction concentration is selected to be 0-95% ethanol, preferably 80% ethanol.

3. The preparation method according to claims 1 and 2, characterized in that: First add 6 to 10 times the amount of 80% ethanol to extract 1 to 3 times, each time for 1 to 2 hours, then add 9 to 15 times the amount of pure water to extract 1 to 3 times, each time for 1 to 2 hours.

4. The preparation method according to claims 1 to 3, characterized in that: Add 80% ethanol in an amount 10 times that of the Danshen slices, extract 3 times, each time for 1 hour; then add pure water in an amount 9 times that of the Danshen slices, extract 3 times, each time for 1.5 hours.

5. The quality control method of the standard extract of Danshen according to claims 1 to 4, characterized in that: (1) Establish the characteristic fingerprint of the standard extract of Salvia miltiorrhiza; (2) Establish a method for determining the content of the standard extract of Salvia miltiorrhiza; (3) Establish a method for determining the molecular weight and monosaccharide composition of the polysaccharide in the standard extract of Salvia miltiorrhiza.

6. The quality control method of the standard extract of Danshen according to claim 5, characterized in that: (1) Establish an HPLC fingerprint of standard Salvia miltiorrhiza extract; (2) Establish an HPLC content determination method for tanshinone and salvianolic acid; (3) Establish an UV-visible spectrophotometric content determination method for total salvianolic acid, total tanshinone and total polysaccharides; (4) Establish an HPLC molecular weight determination method for polysaccharide components and a monosaccharide composition determination method.

7. The HPLC fingerprint of the standard extract of Salvia miltiorrhiza according to claim 6 (1), characterized in that: There are 16 common peaks, including tanshinone, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone II A characteristic peaks.

8. The method for determining the contents of salvianolic acids and tanshinones by high performance liquid chromatography according to item (2) of claim 6, characterized in that: In the standard extract of Danshen, the total content of 8 compounds including danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid A is between 2.87% and 16.06%, and the total content of dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone II is between 1.07% and 2.08%. A The total content of these four compounds is between 0.17% and 1.09%.

9. The method for determining the content of total salvianolic acid, total tanshinone and total polysaccharides by UV-visible spectrophotometry according to item (3) of claim 6, characterized in that: In the standard Danshen extract, the content of total salvianolic acid components is between 5.30% and 14.02%, the content of total tanshinone components is between 3.84% and 5.95%, and the content of total Danshen polysaccharides is between 2.04% and 3.17%.

10. The method for establishing a high performance liquid chromatography molecular weight determination method for polysaccharide components according to claim 6 (4), characterized in that: The molecular weight distribution ranges of the high, medium and low parts are 3.94×10 5 ~4.50×10 5 Da, 6.69×10 3 ~2.00×10 4 Da, 1.20×10 3 ~1.28×10 3 Da, and 6.69×10 3 ~2.00×10 4 Polysaccharides in the Da range account for the largest proportion, accounting for 61.69% to 77.71% of the total polysaccharides; the monosaccharide composition of the polysaccharide components in the standard extract of Salvia miltiorrhiza is characterized by: they all contain mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose, and the content of mannose is the lowest among the six monosaccharides. In all Salvia miltiorrhiza samples, the contents of galacturonic acid, glucose and galactose are generally higher than other monosaccharides.

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