A quality detection method for Thirteen-ingredient stasis-removing mixture

Abstract

The present invention discloses a quality detection method for a thirteen-ingredient stasis-removing mixture. The thirteen-ingredient stasis-removing mixture is prepared by decocting thirteen traditional Chinese medicines, including wine-processed rhubarb, bupleurum, safflower, ligusticum chuanxiong, peach kernel, angelica, and liquorice, with water. The quality detection method includes thin-layer chromatography identification of rhubarb in the mixture and an HPLC content determination method for free chrysophanol. The thin-layer chromatography identification method for rhubarb in the mixture established by the present invention has strong specificity, clear spots, and good tolerance; the established content determination method can effectively determine the content of free chrysophanol in the mixture. The quality detection method of the present invention is simple to operate, has strong specificity, good reproducibility, and is stable and reliable. Using this method, the thirteen-ingredient stasis-removing mixture can be effectively tested for quality, thereby controlling the quality of the thirteen-ingredient stasis-removing mixture and ensuring its therapeutic effect. The present invention also provides a preferred preparation process for the thirteen-ingredient stasis-removing mixture, which can be used to standardize the extraction and preparation of the thirteen-ingredient stasis-removing mixture.

Description

Technical Field

[0001] The present invention relates to the technical field of quality inspection of traditional Chinese medicine, and in particular to a quality inspection method of Thirteen-Ingredients Blood-Removing Mixture, a medicine for treating pleural effusion caused by rib fracture. Background Art

[0002] Thirteen-Ingredient Blood-Removing Mixture was developed from a proven formula by veteran Chinese medicine practitioners at Baise City Traditional Chinese Medicine Hospital. It clears blood stasis, promotes blood circulation, and relieves swelling and pain. It is primarily used to treat pleural effusions caused by rib fractures. The mixture consists of thirteen herbs: wine-processed rhubarb, bupleurum, safflower, ligusticum chuanxiong, peach kernel, angelica, and licorice. The formula prioritizes wine-processed rhubarb to clear blood stasis and promote downward movement, while bupleurum soothes the liver and promotes qi, guiding other herbs into the liver meridian. Together, these two herbs serve as the main ingredients, one ascending and one descending, to dissipate stasis in the hypochondrium. Peach kernel and safflower promote blood circulation, dissipate stasis, and relieve swelling and pain, and are collectively referred to as assistant herbs. Angelica sinensis nourishes and invigorates blood circulation, helping the other herbs dissipate stasis and resolve stagnation, while it also clears heat and moistens dryness. Chuanxiong promotes blood circulation and dissipates stasis, while Eupolyphaga sinensis dissolves blood and removes stasis. Corydalis yanhusuo promotes qi and relieves pain. Radix Trichosanthis, Poria, Rhizoma Alismatis, and Platycodon grandiflorum promote diuresis and reduce swelling, regulating the waterways, and are collectively referred to as adjuvant herbs. Licorice relieves urgency and relieves pain, harmonizing the other herbs, and is referred to as the guiding herb.

[0003] In this prescription, rhubarb is the main herb, known for its purgative and detoxifying properties, clearing away heat and purging fire, cooling blood and detoxifying, and removing blood stasis and relieving menstruation. The Chinese Pharmacopoeia lists rhubarb as the dried roots and rhizomes of Rheum palmatum L., Rheum tanguticum Maxim. ex Bal£., or Rheum officinale Bal£., all of the Polygonaceae family. Anthraquinones are its primary pharmacologically active ingredients, existing in both free and bound forms, including emodin and chrysophanol. The auxiliary herb, safflower, is the dried flower of Carthamus tinctorius L., a member of the Asteraceae family. Traditional Chinese Medicine considers safflower to be pungent and warming in nature. It enters the heart and liver meridians, promoting blood circulation and relieving pain. Literature review revealed that the active ingredient in safflower is hydroxysafflower yellow A, which inhibits blood-brain barrier damage and reduces nerve cell damage. The ingredients used in the Thirteen-Ingredients Blood-Removing Decoction are closely related to the efficacy of the preparation.

[0004] Thirteen-ingredient stasis-removing mixture has the effects of clearing away blood stasis, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain. At present, the preparation of Thirteen-ingredient stasis-removing mixture mainly adopts the water decoction method, but there is no standardized decoction standard, resulting in uneven efficacy; and there is currently no quality inspection method related to this drug, which makes it difficult to monitor the quality of the drug and ensure the quality and efficacy of the preparation, which limits the clinical research and application prospects of Thirteen-ingredient stasis-removing mixture. Summary of the Invention

[0005] The present invention aims to provide a quality testing method for Thirteen Ingredients Blood-Removing Mixture, a drug used to treat pleural effusion caused by rib fractures, thereby facilitating quality control of the Thirteen Ingredients Blood-Removing Mixture and ensuring its efficacy. The present invention also provides a preferred preparation process for the Thirteen Ingredients Blood-Removing Mixture, which can be used to standardize the extraction and preparation of the Thirteen Ingredients Blood-Removing Mixture.

[0006] The technical solution adopted in the present invention is as follows:

[0007] A quality inspection method for a thirteen-ingredient stasis-removing mixture, wherein the thirteen-ingredient stasis-removing mixture is prepared from the following raw materials in parts by weight: 12 parts of rhubarb (processed with wine); 10 parts of chuanxiong; 9 parts of peach kernel; 9 parts of safflower; 12 parts of bupleurum; 9 parts of trichosanthes; 9 parts of angelica; 6 parts of liquorice; 12 parts of corydalis; 9 parts of stem borer; 15 parts of tuckahoe; 12 parts of oriental rhizome; 12 parts of platycodon;

[0008] The preparation method of the thirteen-ingredient stasis-removing mixture is as follows: raw medicinal materials are taken according to weight, the raw medicinal materials except rhubarb are firstly taken and boiled in water, rhubarb is added later, water is added and boiled twice, and the decoctions obtained are combined to obtain the mixture.

[0009] The quality detection method includes thin layer chromatography identification of rhubarb in the mixture and determination of the content of free chrysophanol; wherein the thin layer chromatography identification method of rhubarb in the mixture includes the following steps:

[0010] (1) Preparation of reference solution: Weigh 1 mg of chrysophanol reference substance accurately, place in a 10 ml volumetric flask, add methanol to the scale line, weigh, ultrasonicate for 30 min, and add methanol to the weight to make a chrysophanol reference solution containing 0.1 mg of chrysophanol reference substance per 1 ml, let it stand, and take the supernatant as the reference solution;

[0011] (2) Preparation of control medicinal material solution: Take 1 g of rhubarb control medicinal material, weigh it accurately, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 min, add methanol to the weight, let it stand, and take the supernatant as the control medicinal material solution to prepare a control medicinal material solution containing 0.1 g of the control medicinal material per 1 ml;

[0012] (3) Preparation of test solution: Take an appropriate amount of the decoction of the mixture, evaporate it in a water bath to obtain the extract, grind it into powder, weigh 2 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 min, replenish the weight with methanol, let it stand, and take the supernatant as the test solution to prepare a test solution containing 0.2 g of the test extract per 1 ml;

[0013] (4) Preparation of negative sample solution: Weigh the other medicinal materials without rhubarb according to the prescription ratio, prepare the negative sample in the same way as the preparation method of Thirteen Flavors to Remove Stasis Mixture, and then prepare the negative sample solution according to the preparation method of (3) test solution;

[0014] (5) Identification: According to the thin layer chromatography test, 10 μl of each of the reference solution, control medicinal material solution, test solution, and negative sample solution were taken and spotted on the same silica gel G plate. The plate was developed with a volume ratio of cyclohexane: ethyl acetate: formic acid = 7:3:0.2. The plate was taken out, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the control medicinal material and the reference sample, and there was no interference with the negative sample at the same position.

[0015] The method for determining the content of free chrysophanol in the mixture comprises the following steps:

[0016] (1) Chromatographic conditions: The chromatographic column was a thermos C18 column, 250 mm × 4.6 mm, 5 μm; the mobile phase was a methanol-0.1% phosphoric acid solution with a volume ratio of 75:25, isocratic elution, a flow rate of 1.0 ml / min, a detection wavelength of 254 nm, a column temperature of 25°C, and an injection volume of 10 μl.

[0017] (2) Preparation of reference solution: Weigh 8.16 mg of chrysophanol reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add methanol to the scale line, weigh it, sonicate for 30 min, replenish the weight with methanol, filter it through a 0.45 μm microporous filter membrane, take the filtrate, and prepare a reference solution containing 163.2 μg of chrysophanol per 1 ml. Set aside and dilute it with methanol to prepare standard solutions of different concentrations when used;

[0018] (3) Preparation of test solution: Take an appropriate amount of the decoction of the mixture, evaporate it in a water bath to obtain the extract, grind it into powder, weigh 0.8 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 min, and replenish the weight of methanol to make a test solution containing 0.08 g of the extract per 1 ml. Filter it through a 0.45 μm microporous membrane, take the filtrate and set aside;

[0019] (4) HPLC detection: Take 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, measure according to the chromatographic conditions, record the chromatogram, and calculate.

[0020] The above-mentioned quality inspection method of the Thirteen-Ingredient Blood-Removing Mixture, the specific preparation method of the Thirteen-Ingredient Blood-Removing Mixture is: take each raw medicinal material according to weight, first take the other raw medicinal materials except rhubarb and mix them, add 8 times the total weight of the medicinal materials in water and boil for 65-85 minutes, after adding rhubarb, boil for another 10-15 minutes, filter, add 8 times the amount of water to the residue and boil for 75-100 minutes; mix the decoctions obtained from the two decoctions to obtain the mixture.

[0021] Preferably, the preparation method of the thirteen-ingredient stasis-removing mixture is as follows: take each raw medicinal material according to weight, first take the other raw medicinal materials except rhubarb and mix them, add 8 times the total weight of the medicinal materials in water and boil for 80 minutes, add rhubarb, boil for another 10 minutes, filter, add 8 times the amount of water to the residue and boil for 90 minutes; mix the decoctions obtained from the two decoctions to obtain the mixture.

[0022] The beneficial effects of the present invention are:

[0023] 1. The quality detection method of the thirteen-flavor stasis-removing mixture established by the present invention includes thin-layer chromatography identification of rhubarb in the mixture and determination of the content of free chrysophanol; wherein, the thin-layer chromatography identification method of rhubarb in the mixture established is reasonable and simple, has strong specificity, clear spots, and good tolerance; the content determination method established can effectively separate the chrysophanol component, and the specificity, linearity, repeatability, recovery rate, and stability of the method are all good and meet the requirements of the Chinese Pharmacopoeia. The quality detection method established by the present invention is simple to operate, has strong specificity, good reproducibility, and is stable and reliable. The quality detection method can be used to effectively detect the quality of the thirteen-flavor stasis-removing mixture, thereby controlling the quality of the thirteen-flavor stasis-removing mixture and ensuring its therapeutic effect. The quality detection method established by the present invention provides a reference for the research on the treatment standard of the thirteen-flavor stasis-removing mixture and also provides a theoretical basis for subsequent research.

[0024] 2. The present invention studies the extraction process of thirteen flavors of stasis-removing mixture by adopting orthogonal test, optimizes the decoction condition of water, takes decoction number, water addition amount, decoction time as investigation factor, and the content of cream yield and hydroxysafflor yellow A as investigation index, selects the optimal decoction process, that is, the prescription adds 8 times of water, decocts 2 times, and decocts under the decoction process of 90 min each time, the cream yield and hydroxysafflor yellow A content are both high, indicating that the decoction process is reasonable, stable and feasible. The preferred preparation process of the thirteen flavors of stasis-removing mixture provided by the present invention can be used for standardizing the extraction preparation of the thirteen flavors of stasis-removing mixture. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 HPLC chromatogram of the test solution of Thirteen Flavors to Remove Stasis Mixture;

[0026] Figure 2HPLC chromatogram of hydroxysafflor yellow A reference solution;

[0027] Figure 3 HPLC chromatogram of blank solvent (25% methanol);

[0028] Figure 4 Linear relationship diagram of hydroxysafflor yellow A;

[0029] Figure 5 L9 (3 4 ) Orthogonal test factor effect curve;

[0030] Figure 6 Thin layer chromatograms of different extractants, in the figure, 1, rhubarb reference medicinal material, 2, chrysophanol reference, 3, methanol-extracted test sample, 4, anhydrous ethanol-extracted test sample, 5, negative sample;

[0031] Figure 7 Thin layer chromatograms under different developing solvents, in the figure, a, petroleum ether (60-90): ethyl formate: formic acid (15:5:1, v / v / v); b, cyclohexane: ethyl acetate: formic acid (7:3:0.2, v / v / v), 1, rhubarb reference material; 2, chrysophanol reference; 3, methanol-extracted test sample; 4, negative sample;

[0032] Figure 8 Thin layer chromatograms under different inspection conditions, in the figure, a, 365 nm, b, 254 nm, 1, rhubarb reference material, 2, chrysophanol reference substance, 3, methanol-extracted test sample, 4, negative sample;

[0033] Figure 9 Thin layer chromatograms of different sample amounts, in the figure, 1, chrysophanol reference, 2, test sample 2 μL, 3, test sample 6 μL, 4, test sample 10 μL;

[0034] Figure 10 Thin layer chromatograms of different spotting methods, in the figure, 1, dot-spotted test sample, 2, chrysophanol reference substance, 3, strip-spotted test sample;

[0035] Figure 11 Thin layer chromatograms of silica gel G plates from different brands. In the figure, a, Haiyang brand; b, Huanghai brand, 1, rhubarb reference material, 2, chrysophanol reference substance, 3, methanol-extracted test sample, 4, negative sample;

[0036] Figure 12 HPLC profile of reference product;

[0037] Figure 13 HPLC profile of the test sample;

[0038] Figure 14HPLC profile of negative sample;

[0039] Figure 15 Methanol HPLC chart;

[0040] Figure 16 Standard curve of chrysophanol. DETAILED DESCRIPTION

[0041] Experimental Example 1

[0042] 1 Experimental Materials

[0043] 1.1 Prescription and medicinal materials

[0044] Prescription for Thirteen-Ingredient Blood-Removing Mixture: 12g of wine-processed rhubarb (added last), 10g of Chuanxiong, 9g of peach kernel, 9g of safflower, 12g of bupleurum, 9g of radix trichosanthis, 9g of angelica, 6g of liquorice, 12g of Corydalis yanhusuo, 9g of Scolopendra sinensis, 15g of Poria, 12g of Alisma orientalis, and 12g of Platycodon grandiflorum.

[0045] In order to facilitate calculation and the amount of water to be added, the total amount of the prescription is set at 100 g, so the actual amount of safflower used in the prescription is about 6.6 g.

[0046]

[0047] The above medicinal materials were purchased from Guilin Bisheng Pharmaceutical Co., Ltd. (batch number: 210329), and are included in the "Chinese Pharmacopoeia" (2020 edition, Part 1), with quality inspection reports attached.

[0048] 1.2 Reagents and Materials

[0049]

[0050] 1.3 Instruments

[0051]

[0052] 2 Experimental methods

[0053] 2.1 Preparation of Thirteen-Ingredients-Removing-Yu Mixture

[0054] Prescription: Wine-processed rhubarb (added later), bupleurum, ligusticum chuanxiong, peach kernel, safflower, radix trichosanthis, angelica, licorice, yanhusuo, earthworm, poria, oriental water plantain, platycodon.

[0055] 2.1.1 Preparation method: Combine 100 g of the following herbs: bupleurum, ligusticum chuanxiong, peach kernel, safflower, radix trichosanthis, angelica sinensis, liquorice, corydalis yanhusuo, earthworm, tuckahoe, rhizome of orientalis, and platycodon grandiflorum according to the prescribed ratio. Based on the previously optimized extraction process, first mix the other herbs except rhubarb, add 8 times the amount of water as the total weight of the herbs, and boil for 80 min. After adding rhubarb, boil for another 10 min, filter, and add 8 times the amount of water as the total weight of the herbs to the residue and boil for 90 min. The decoctions obtained from the two decoctions are combined to obtain the final product.

[0056] 2.2 Optimization of the extraction process

[0057] 2.2.1 Orthogonal experimental design: With reference to previous literature, factors such as the medicinal properties, medicinal parts, and texture of traditional Chinese medicine were considered, and with reference to relevant water decoction management regulations, the effects of factors such as the number of decoctions, amount of water added, and decoction time on the water decoction process were investigated.

[0058] 2.2.1.1 Decoction Time: This experiment tested decoction times of 30, 60, and 90 minutes. Water Amount: This experiment tested water amounts of 6, 8, and 10 times the prescribed amount of medicinal material. Number of Decoctions: This experiment tested decoctions of 1, 2, and 3 times.

[0059] 2.2.1.2 Indicator selection

[0060] The yield of paste is often used to assess the decoction process of traditional Chinese medicines, as it is closely related to the quality and cost of the medicinal materials. However, the yield of paste cannot be used as the sole indicator of decoction process. Experiments can also be used to determine the content of representative ingredients in the prescribed medicinal materials to screen the decoction process and ensure the quality and efficacy of the finished traditional Chinese medicine preparation.

[0061] Hydroxysafflor yellow A (HSYA), a single chalcone glycoside compound, is a pharmacologically active, water-soluble ingredient in safflower. It inhibits platelet aggregation and release induced by platelet activating factor (PAF), and competitively inhibits the binding of PAF to platelet receptors. HSYA has a well-established HPLC assay method in the Chinese Pharmacopoeia (2020 Edition, Part I) and related literature. It can be used as an indicator for the determination of the Thirteen-Ingredient Blood-Removing Mixture, with a weighting factor of 70%. The yield of the paste indirectly measures the number of active chemical components, with a weighting factor of 30%. The comprehensive scoring of the orthogonal evaluation indicators is as follows:

[0062]

[0063] 2.2.1.3 Orthogonal experiment

[0064] Weigh all the medicinal materials according to the prescription in "2.1.1". With the number of decoctions (factor A), the amount of water added (factor B), and the decoction time (factor C) as the investigation factors, and the paste yield and hydroxysafflor yellow A content as indicators, the L9 (3 4 ) were conducted by adding the appropriate amount of ultrapure water, heating and decocting, combining the decoctions, filtering the decoction through a 120-mesh sieve, cooling, and concentrating it before measuring the paste yield and hydroxysafflor yellow A content. The factor levels are shown in Table 4.

[0065]

[0066] 2.2.2 Determination of paste yield

[0067] Accurately measure 190 ml of each of the nine medicinal solutions (combined decoctions) prepared under the orthogonal test table in Table 9. Place the solution in a clean, dried ceramic evaporating dish to a constant weight. Evaporate to dryness in a 70°C water bath. Dry the dish in a 70°C forced air drying oven for 3 hours. Remove the dish and cool it in a desiccator for 0.5 hours. Quickly weigh the mass to obtain m, and calculate the paste yield. The calculation formula is as follows:

[0068]

[0069] 2.2.3 Determination of hydroxysafflor yellow A by HPLC

[0070] 2.2.3.1 Chromatographic conditions: Thermos C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol: acetonitrile: 0.7% phosphoric acid (26:2:72, v / v / v); flow rate: 1 ml / min; column temperature: 25°C; detection wavelength: 403 nm; injection volume: 10 μl.

[0071] 2.2.3.2 Preparation of Reference Solution: Weigh an appropriate amount of Hydroxysafflor Yellow A Reference Standard accurately into a 25ml volumetric flask. Add 25% methanol to the mark and shake well to prepare a Hydroxysafflor Yellow A Reference Standard Solution with a concentration of approximately 0.06384 mg / ml. Store in a sealed container until ready for use. This solution can also be used as a stock solution in spike-in recovery experiments.

[0072] 2.2.3.3 Preparation of Test Solution: Since the content of the target component does not reach the detection limit when the mixture is directly used for content determination, it is concentrated into an extract for content determination. Accurately weigh approximately 1 g of the extract and place it in a 100 ml stoppered conical flask. Accurately add 50 ml of 25% methanol and weigh the solution. Ultrasonicate (300 W, 50 kHz) for 40 min. Allow to cool and weigh again. Make up the lost weight with 25% methanol. Shake well and filter through a 0.45 μm microporous filter membrane. The filtrate is the test solution.

[0073] 2.2.3.4 Preparation of blank solvent: Take the solvent (25% methanol) under the preparation of reference solution in "2.2.3.2" and filter it through a 0.45μm microporous filter membrane. Take the filtrate as the blank solvent.

[0074] 2.2.3.5 System suitability test: Take 10 μl each of the test solution of Thirteen-Ingredients Zhuyu Mixture in item “2.2.3.3”, the reference solution of Hydroxysafflor Yellow A in item “2.2.3.2” and the blank solvent prepared in item “2.2.3.4”, inject the samples, and determine according to the chromatographic conditions in item “2.2.3.1”, and record the chromatographic results respectively.

[0075] 2.2.3.5 Linear Relationship

[0076] Weigh an appropriate amount of hydroxysafflor yellow yellow A reference standard accurately and place it into a 25 ml volumetric flask. Add 25% methanol to prepare a 0.1596 mg / ml hydroxysafflor yellow A reference standard stock solution. Accurately measure 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, and 6 ml of the reference standard into 10 ml volumetric flasks, dilute to the mark with 25% methanol, and shake well to prepare a series of hydroxysafflor yellow A reference standard solutions (0.01596 mg / ml, 0.03192 mg / ml, 0.04788 mg / ml, 0.06384 mg / ml, 0.0798 mg / ml, and 0.09576 mg / ml, respectively). Inject 10 μl of each reference standard solution and determine the peak area of ​​hydroxysafflor yellow A according to the chromatographic conditions in "2.2.3.1." The standard curve was drawn with the concentration of the hydroxysafflor yellow A reference solution as the abscissa and the peak area as the ordinate.

[0077] 2.2.3.6 Methodological Review

[0078] 2.2.3.6.1 Precision experiment: Pipette a 0.01596 mg / ml hydroxysafflor yellow A reference solution, then repeat the injection six times according to the chromatographic conditions in “2.2.3.1” and record the peak area.

[0079] 2.2.3.6.2 Stability test: Accurately weigh about 1 g of the extract, extract with 25% methanol according to the operation in "2.2.3.3". After standing at room temperature for 0, 2, 4, 8, and 12 hours, respectively, determine according to the chromatographic conditions in "2.2.3.1", inject the sample, and record the peak area.

[0080] 2.2.3.6.3 Repeatability test: Take 6 portions of 100 g of the medicinal material prescribed in "2.1" and weigh them accurately; extract according to the method in "2.2.3.3", and measure according to the chromatographic conditions in "2.2.3.1" and record the peak area.

[0081] 2.2.3.6.4 Sample Recovery Test: Weigh accurately six portions of a decoction extract of a known concentration of medicinal materials. Add 10 ml of a 0.06384 mg / ml hydroxysafflor yellow A reference solution to each portion. Prepare the test solution according to "2.2.3.3." Filter through a 0.45 μm microporous membrane. Then, perform the sample injection and determination according to the chromatographic conditions specified in "2.2.3.1."

[0082] 2.3 Identification of rhubarb in prescriptions by thin layer chromatography

[0083] 2.3.1 Solution preparation

[0084] 2.3.1.1 Preparation of reference solution: Weigh 1 mg of chrysophanol reference substance accurately and place it in a 10 ml volumetric flask. Add methanol to the scale line and weigh it. Ultrasonicate for 30 min and make up the weight of methanol to prepare a chrysophanol reference solution containing approximately 0.1 mg of chrysophanol reference substance per 1 ml. Let it stand and take the supernatant as the reference solution.

[0085] 2.3.1.2 Preparation of control medicinal material solution: Take 1 g of rhubarb control medicinal material, weigh accurately, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh, ultrasonicate for 30 minutes, replenish the weight with methanol, let it stand, and take the supernatant as the control medicinal material solution to prepare a control medicinal material solution containing approximately 0.1 g of the control medicinal material per 1 ml.

[0086] 2.3.1.3 Preparation of test solution: Measure an appropriate amount of decoction liquid, evaporate it in a water bath to obtain an extract, grind it finely, weigh 2 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, sonicate for 30 min, replenish the weight with methanol, let it stand, and take the supernatant as the test solution to prepare a test solution containing approximately 0.2 g of the test sample extract per 1 ml.

[0087] 2.3.1.4 Preparation of negative sample solution: Weigh the other medicinal materials (excluding rhubarb) according to the prescription ratio and prepare the negative sample according to "2.1.1". Prepare the negative sample solution according to "2.3.1.3".

[0088] 2.3.2 Investigation of thin layer chromatography conditions

[0089] 2.3.2.1 Examination of Extraction Solvents: The main purpose was to investigate the differences in the degree of extraction of active ingredients by different extraction solvents. Two extraction solvents were used for this study: Extraction Solvent 1: An appropriate amount of the decoction was measured and evaporated in a water bath to obtain an extract. The extract was ground into powder. 2 g of the extract was weighed accurately and placed in a 10 ml volumetric flask. Methanol was added to the mark, weighed, and ultrasonicated for 30 min. The methanol was refilled and allowed to stand. The supernatant was used as the test solution, and a test solution containing approximately 0.2 g of the test sample extract per 1 ml was prepared. Extraction Solvent 2: An appropriate amount of the decoction was measured and evaporated in a water bath to obtain an extract. The extract was ground into powder. 2 g of the extract was weighed accurately and placed in a 10 ml volumetric flask. Anhydrous ethanol was added to the mark, weighed, and ultrasonicated for 30 min. The methanol was refilled and allowed to stand. The supernatant was used as the test solution, and a test solution containing approximately 0.2 g of the test sample extract per 1 ml was prepared.

[0090] Developing solvent: cyclohexane:ethyl acetate:formic acid (7:3:0.2, V / V / V)

[0091] 2.3.2.2 Developing Solvent Evaluation: This study primarily examined the differences in the separation effects of different developing solvents on the test samples. Two developing solvents were used in this experiment. Development Condition 1: Cyclohexane: Ethyl Acetate: Formic Acid (7:3:0.2, V / V / V), room temperature, development, removal, air drying, and observation under 365 nm UV light. Development Condition 2: Petroleum Ether (60-90%): Ethyl Formate: Formic Acid (15:5:1, V / V / V), room temperature, development, removal, air drying, and observation under 365 nm UV light.

[0092] 2.3.2.3 Examination of different inspection conditions: 10 μl each of the control medicinal material solution, reference substance solution, test solution, and negative sample solution were aspirated and developed in cyclohexane:ethyl acetate:formic acid (7:3:0.2, V / V / V). The samples were observed under the following conditions: Inspection condition 1: After development and drying, the samples were placed under a 254 nm ultraviolet lamp for observation; Inspection condition 2: After development and drying, the samples were placed under a 365 nm ultraviolet lamp for observation.

[0093] 2.3.2.4 Tolerance test: According to the requirements of the 2020 edition of the "Chinese Pharmacopoeia" for TLC identification, the thin layer chromatography behavior of conditions such as sample amount, sample spotting method, and thin layer plates of different brands were investigated.

[0094] 2.3.2.4.1 Investigation of sample spotting amount: Compare the chromatographic effects of different sample spotting amounts as follows: aspirate 10 μl of the reference solution, 2 μl, 6 μl, and 10 μl of the test solution, spot them on the same silica gel plate, develop them in cyclohexane: ethyl acetate: formic acid (7:3:0.2, V / V / V), dry them, and observe them under a UV lamp at 365 nm.

[0095] 2.3.2.4.2 Investigation of spotting method: Compare the chromatographic effects of strip spotting method and dot spotting method as follows: aspirate 10 ml of reference solution and test solution respectively, spot them on the same silica gel G plate in dot and strip spotting methods at room temperature, develop them in cyclohexane:ethyl acetate:formic acid (7:3:0.2, V / V / V), dry them, and observe them under ultraviolet light (365 nm).

[0096] 2.3.2.4.3 Investigation of different brands of thin layer plates: The separation effects of different brands of silica gel G plates were compared. Silica gel G plates (specifications 50 mm × 100 mm) from Qingdao Ocean Chemical Plant Branch (Ocean brand) and Yantai Jiangyou Silica Gel Development Co., Ltd. (Huanghai brand) were used for thin layer chromatography comparison. The method was as follows: 10 μl each of the control medicinal material, reference substance solution, test substance solution and negative sample solution were taken and developed on the silica gel G plates of Ocean brand and Huanghai brand, respectively, under the conditions of cyclohexane:ethyl acetate:formic acid (7:3:0.2, V / V / V). The plates were taken out, dried and observed under ultraviolet light (365 nm).

[0097] 2.4 Determination of free chrysophanol by HPLC

[0098] 2.4.1 Chromatographic conditions: A thermos C18 column (250 mm × 4.6 mm, 5 μm) was used, with a mobile phase of methanol:0.1% phosphoric acid (75:25, v / v) and an isocratic elution rate of 1.0 ml / min; the detection wavelength was 254 nm; the column temperature was 25°C; and the injection volume was 10 μl.

[0099] 2.4.2 Preparation of solution:

[0100] 2.4.2.1 Preparation of Reference Solution: Weigh 8.16 mg of chrysophanol reference substance accurately into a 50 ml volumetric flask. Add methanol to the mark and weigh. Ultrasonicate for 30 min. Replenish the volume with methanol. Filter through a 0.45 μm microporous membrane. Prepare the filtrate to prepare a reference solution containing 163.2 μg of chrysophanol per ml. Set aside. Dilute with methanol to prepare standard solutions of varying concentrations for use.

[0101] 2.4.2.2 Preparation of test solution: Prepare Thirteen-ingredient stasis-removing mixture according to the preparation method under "2.1.1". Measure an appropriate amount of decoction, evaporate in a water bath to obtain an extract, grind it finely, weigh 0.8 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, and sonicate for 30 min. Replenish the weight of methanol to make a test solution containing approximately 0.08 g of the extract per 1 ml. Filter it through a 0.45 μm microporous membrane, take the filtrate and set aside.

[0102] 2.4.2.3 Preparation of negative sample solution: Weigh the other medicinal materials missing rhubarb according to the prescription ratio, prepare rhubarb negative sample according to the preparation method under "2.1.1", and prepare negative sample solution according to the preparation method under "2.4.2.2" and set aside.

[0103] 2.4.2.4 Preparation of blank solvent: Take an appropriate amount of methanol and filter it through a 0.45 μm microporous filter membrane. Take the filtrate and set aside.

[0104] 2.4.3 System suitability test: Accurately measure 10 μl each of the reference solution, test solution, negative sample solution, and chromatographic methanol. Determine the peak area according to the chromatographic conditions under "2.4.1" and record the chromatogram.

[0105] 2.4.4 Single-factor experiment

[0106] 2.4.4.1 Extraction solvent: The main purpose is to investigate the differences in the degree of extraction of chrysophanol by different extraction solvents. This experiment uses three extraction solvents for investigation, and the method is as follows: take three portions of 0.8 g of the extract of Shisanwei Zhuyu Mixture, accurately weigh them, and place them in three 10 ml volumetric flasks respectively, add methanol, methanol: water (80:20, V / V), and methanol: water (60:40, V / V) to the scale line, weigh them, ultrasonicate for 30 minutes, make up the weight with methanol, filter through a 0.45 μm microporous filter membrane, take the filtrate, and determine the peak area according to the chromatographic conditions under "2.4.1". Inject each group twice, and record the peak area.

[0107] 2.4.4.2 Sampling volume: The main purpose is to investigate the effects of different sampling volumes on the experimental results. This experiment uses three concentrations for investigation, and the method is as follows: weigh 0.4 g, 0.8 g, and 1.2 g of Thirteen-ingredient Yu-removing Extract, accurately weigh, and place them in three 10 ml volumetric flasks respectively, add methanol to the scale line, weigh, ultrasonicate for 30 min, replenish the weight with methanol, filter through a 0.45 μm microporous filter membrane, take the filtrate, and determine the peak area according to the chromatographic conditions under "2.4.1". Each group is sampled twice, and the peak area is recorded.

[0108] 2.4.4.3 Extraction time: The main purpose is to investigate the difference in the degree of extraction of effective ingredients at different extraction times. This experiment used three different extraction times for investigation. The method is as follows: Take three portions of 0.8 g of the extract of Shisanwei Zhuyu Mixture, accurately weigh them, and place them in three 10 ml volumetric flasks respectively. Add methanol to the scale line, weigh them, and ultrasonicate them for 10 minutes, 20 minutes, and 30 minutes respectively. Methanol is used to replenish the weight, and the mixture is filtered through a 0.45 μm microporous filter membrane. The filtrate is taken and the peak area of ​​chrysophanol is determined according to item "2.4.1". Each group is sampled twice.

[0109] 2.4.5 Standard curve: Prepare reference solutions of 16.32 μg / ml, 8.16 μg / ml, 6.528 μg / ml, 3.264 μg / ml, and 1.632 μg / ml, respectively. Determine the peak area according to the chromatographic conditions under “2.4.1”. Use the injection concentration of chrysophanol as the horizontal axis and the peak area as the vertical axis to draw a standard curve.

[0110] 2.4.6 Precision experiment: Accurately measure 400 μl of chrysophanol reference solution and place it in a 10 ml volumetric flask. Add methanol to the scale line and weigh it. Ultrasonicate for 30 min, add methanol to make up the weight, filter through a 0.45 μm microporous filter membrane, take the filtrate, and determine the peak area according to the chromatographic conditions under "2.4.1". Measure it 6 times in a row and record the peak area.

[0111] 2.4.7 Stability test: Take the same test sample solution and place it at room temperature for 0, 1, 2, 4, 8, and 12 hours. Then determine the peak area according to the chromatographic conditions under "2.4.1". Inject each group twice and record the peak area.

[0112] 2.4.8 Repeatability experiment: Weigh 0.8 g of the extract of Thirteen-ingredient Yu-removing Mixture, a total of 6 portions, accurately weighed, and placed in 6 10 ml volumetric flasks respectively. Methanol was added to the scale line, weighed, ultrasonicated for 30 minutes, and methanol was added to the weight. Filtered through a 0.45 μm microporous membrane, took the filtrate, and determined the peak area according to the chromatographic conditions under "2.4.1". Each group was sampled twice, and the peak area was recorded.

[0113] 2.4.9 Sample addition and recovery experiment: Take 6 portions of 0.4 g of the extract of Thirteen Flavors to Remove Yu Mixture with known content, weigh them accurately, and place them in 6 10 ml volumetric flasks respectively. Accurately measure the reference solution containing 26.01 μg of chrysophanol and add it to the above 6 portions of test sample extract respectively. Add methanol to the scale line, weigh them, sonicate for 30 min, make up the weight with methanol, filter through a 0.45 μm microporous membrane, determine the peak area according to the chromatographic conditions under "2.4.1", and calculate the recovery rate and RSD.

[0114] 3 Experimental Results

[0115] 3.1 Prescription and process optimization results

[0116] 3.1.1 HPLC system suitability results Figure 1-3 It can be seen that there are no other interfering peaks before and after the elution of hydroxysafflor yellow A, and the separation degree from the adjacent peaks is greater than 1.5. The theoretical plate number calculated based on the hydroxysafflor yellow A peak is greater than 3000, and the retention time of the test solution and the hydroxysafflor yellow A reference solution is about 13 min.

[0117] 3.1.2 Standard curve The standard curve of hydroxysafflor yellow A is as follows Figure 4 As shown, the regression equation is y=35746x-103.22, and R=0.9995. The experimental results show that hydroxysafflor yellow A has a good linear relationship with the peak area in the concentration range of 0.01596-0.09576 mg / ml.

[0118] 3.1.3 Methodological Review

[0119] 3.1.3.1 Precision The instrument precision results are shown in Table 5. The calculated RSD was 0.41%, indicating good instrument precision.

[0120]

[0121] 3.1.3.2 Stability As shown in Table 6, the RSD of the peak area of ​​hydroxysafflor yellow A within 4 h was 1.91%, indicating that the Thirteen Flavors Zhuyu Mixture sample had good stability within 4 h; the RSD of the peak area within 12 h was 2.44%, indicating that its stability within 12 h was poor and should be tested immediately after preparation.

[0122]

[0123] 3.1.3.3 Repeatability The repeatability results are shown in Table 7. The RSD of the peak area of ​​hydroxysafflor yellow A was 3.22%, indicating that the repeatability of this experimental method was acceptable.

[0124]

[0125] 3.1.3.4 Sample recovery The sample recovery results are shown in Table 8. The average recovery of hydroxysafflor yellow A was 101.39% with an RSD of 2.77%.

[0126]

[0127] 3.1.4 Orthogonal test and results

[0128] L9(3 4) The results of the orthogonal test are shown in Table 9, L9 (3 4 )Orthogonal test factor effect curve diagram is shown in Figure 5 .

[0129]

[0130]

[0131] Result analysis: According to the range and Figure 5 The orthogonal experimental factor effect curve shows the order of influence of the factors: number of decoctions (A) > decoction time (C) > amount of water added (B). Comparison of the F ratio and critical F value in the variance analysis in Table 10 indicates that the number of decoctions and decoction time have significant effects. The number of decoctions (A) and decoction time (C) have a significant impact on the content of hydroxysafflor yellow A and the yield of the paste, while the amount of water added (B) has no significant effect. In summary, the optimal extraction process for Thirteen Flavors to Remove Stasis Mixture is A2B2C3, namely, adding 8 times the amount of water and decocting twice, each time for 90 minutes.

[0132] 3.1.5 Verification experiment of extraction process

[0133] The results of the verification test showed that the paste yield and hydroxysafflor yellow A content were high under the process conditions, indicating that the optimized decoction process conditions were reasonable and feasible.

[0134]

[0135] 3.2 Results of thin layer chromatography conditions

[0136] 3.2.1 Results of the investigation on the extraction solvent: Figure 6 As shown, the test sample chromatogram shows spots of the same color at corresponding positions in the control herb and reference sample chromatograms, and there is no interference from the negative sample at the same position. Comparing the expanded result charts, it can be seen that methanol is more selective for extracting rhubarb than anhydrous ethanol. Therefore, methanol was used as the extraction solvent in the preparation of the following test samples.

[0137] 3.2.2 Results of the development agent inspection: The results of the development agent inspection are as follows: Figure 7 As shown in Figure b, the spots are clearer and the separation between spots is relatively good, so the subsequent conditions are all investigated using cyclohexane: ethyl acetate: formic acid (7:3:0.2, V / V / V) as the development system.

[0138] 3.2.3 Results of different inspection conditions: The experimental results are shown in Figure 8The characteristic rhubarb spots were observed under 365 nm and 254 nm UV light after drying. Chromatographic results showed that the spots were clearer under 365 nm UV light and had no interference with negative samples. Therefore, the optimal inspection condition is to unfold and dry the sample and then observe it under 365 nm UV light.

[0139] 3.2.4 Results of tolerance test

[0140] 3.2.4.1 Results of spotting volume: The experimental results show that the spots with a spotting volume of 10 μl are clearer and brighter, so 10 μl was selected as the spotting volume in the following experiments. Figure 9 .

[0141] 3.2.4.2 Results of spotting method: The experimental results show that the strip spotting method has better separation effect than the dot spotting method, and the spots are clearer. Finally, the strip spotting method was selected. Figure 10 .

[0142] 3.2.4.3 Results of the investigation of different brands of thin layer plates: The experimental results show that both brands of silica gel G plates can obtain good chromatographic effects, with clear spots, good separation effects and similar relative transfer values, indicating that silica gel G plates produced by different manufacturers have little effect on the thin layer identification of rhubarb. Figure 11 .

[0143] 3.3 Determination of free chrysophanol by HPLC

[0144] 3.3.1 HLPC system adaptability conditions From the chromatographic data, it can be seen that under the chromatographic conditions, the peak of chrysophanol can be well separated from other peaks. The peak of chrysophanol appears around 14 minutes without interference; the negative sample has no interference peak; and the solvent has no interference peak. Figure 12-15 .

[0145] 3.3.2 Single-factor experimental results

[0146] 3.3.2.1 Extraction Solvents The results of the extraction solvent investigation are shown in Table 12. The experimental results show that methanol has the highest content of chrysophanol among the three solvents, so methanol was selected as the extraction solvent.

[0147]

[0148] 3.3.2.2 Sampling Size The results of the sampling size investigation are shown in Table 13. The experimental results show that a sampling size of 0.8 g is more complete for extraction, so 0.8 g of sample is selected as the sampling size.

[0149]

[0150] 3.3.2.3 Extraction time The results of the extraction time investigation are shown in Table 14. The experimental results show that the ultrasonic time has no significant effect on the degree of extraction of chrysophanol components. In order to achieve complete extraction, the ultrasonication time of 30 min was selected as the extraction method.

[0151]

[0152] 3.3.3 Standard curve Figure 16 The calculated regression equation was Y=59.366X-30.522, R=0.9997, and the results showed that chrysophanol had a good linear relationship in the range of 1.632 μg / ml-16.32 μg / ml.

[0153] 3.3.3 Precision Experiment The results of the precision experiment are shown in Table 15. The RSD value of 0.24% for the six experiments was calculated to be less than 2%, indicating that the instrument had good precision.

[0154]

[0155] 3.3.4 Stability Test The results of the stability test are shown in Table 16. The calculated RSD value is 0.23%, indicating that the test solution has good stability within 12 h.

[0156]

[0157] 3.3.5 Repeatability experiment The results of the repeatability experiment are shown in Table 17. The RSD value of chrysophanol content was calculated to be 1.804%, indicating that the method has good repeatability.

[0158]

[0159] 3.3.6 Sample recovery experiment The results of the sample recovery experiment are shown in Figure 18. The final average recovery rate of chrysophanol was 104.17%, and the RSD value was 1.91%, indicating that the recovery rate of this method was good.

[0160]

[0161] 4 Discussion

[0162] Design L9(3 4 ) Orthogonal experiment was used to screen the optimal prescription process of Thirteen-ingredients-Removing Stasis Mixture. The number of decoctions (A), amount of water added (B), and decoction time (C) were used as investigation factors, and the ointment yield and hydroxysafflor yellow A content were used as investigation indicators. The optimal decoction process was selected as A2B2C3, that is, when the prescription was added with 8 times the amount of water, decocted twice, and decocted for 90 min each time, the ointment yield and hydroxysafflor yellow A content were both high, indicating that the decoction process was reasonable, stable and feasible.

[0163] In the thin-layer chromatography (TLC) identification of rhubarb in the Thirteen-Ingredients-Removing-Yu Mixture, good separation was achieved using a developing solvent consisting of cyclohexane:ethyl acetate:formic acid (7:3:0.2, v / v / v). This effect was evident under ultraviolet light (365 nm). The chromatogram showed identical spots of color at corresponding positions on the chromatograms for the test sample, the control herb, and the reference substance, and no interference was observed with the negative sample at the same position. This demonstrates that the established TLC method for rhubarb is reasonable, simple, highly specific, and well tolerated, and can be used as an identification method for rhubarb in the Thirteen-Ingredients-Removing-Yu Mixture.

[0164] This experiment ultimately determined the optimal HPLC conditions for the determination of free chrysophanol in Thirteen Ingredients Zhuyu Mixture: a thermos C18 column (250 mm × 4.6 mm, 5 μm), a mobile phase of methanol:0.1% phosphoric acid (75:25, v / v), isocratic elution at a flow rate of 1.0 mL / min, detection wavelength at 254 nm, column temperature at 25°C, injection volume of 10 μl, sample volume of 0.8 g, methanol as the extraction solvent, and ultrasonic extraction for 30 minutes. The results showed that the established method effectively separated chrysophanol components. The method exhibited excellent specificity, linearity, reproducibility, recovery, and stability, and complied with the requirements of the Chinese Pharmacopoeia. Therefore, this method was applied to the determination of free chrysophanol in Thirteen Ingredients Zhuyu Mixture.

[0165] Example 2

[0166] A quality inspection method for a thirteen-ingredient stasis-removing mixture is disclosed. The thirteen-ingredient stasis-removing mixture is prepared from the following Chinese medicinal raw materials in parts by weight: 12g of rhubarb (processed with wine), 10g of chuanxiong, 9g of peach kernel, 9g of safflower, 12g of bupleurum, 9g of trichosanthes root, 9g of angelica, 6g of liquorice, 12g of corydalis, 9g of earthworm, 15g of poria, 12g of oriental dwarfism, and 12g of platycodon. The preparation method of the thirteen-ingredient stasis-removing mixture comprises the following steps: taking the raw medicinal materials in parts by weight, first mixing the raw medicinal materials except the rhubarb, adding 8 times the total weight of water of the medicinal materials, and decocting for 80 minutes; after adding the rhubarb, decocting for another 10 minutes, filtering, and adding 8 times the amount of water to the residue and decocting for 90 minutes; and mixing the decoctions obtained from the two decoctions to obtain the mixture.

[0167] The quality detection method includes thin layer chromatography identification of rhubarb in the mixture and determination of the content of free chrysophanol; wherein the thin layer chromatography identification method of rhubarb in the mixture includes the following steps:

[0168] (1) Preparation of reference solution: Weigh 1 mg of chrysophanol reference substance accurately, place in a 10 ml volumetric flask, add methanol to the scale line, weigh, ultrasonicate for 30 min, and add methanol to the weight to make a chrysophanol reference solution containing 0.1 mg of chrysophanol reference substance per 1 ml, let it stand, and take the supernatant as the reference solution;

[0169] (2) Preparation of control medicinal material solution: Take 1 g of rhubarb control medicinal material, weigh it accurately, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 min, add methanol to the weight, let it stand, and take the supernatant as the control medicinal material solution to prepare a control medicinal material solution containing 0.1 g of the control medicinal material per 1 ml;

[0170] (3) Preparation of test solution: Take an appropriate amount of the decoction of this mixture, evaporate it in a water bath to obtain the extract, grind it into powder, weigh 2 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 minutes, replenish the weight with methanol, let it stand, and take the supernatant as the test solution to prepare a test solution containing 0.2 g of the test extract per 1 ml;

[0171] (4) Preparation of negative sample solution: Weigh the other medicinal materials except rhubarb according to the prescription ratio, prepare the negative sample in the same way as the preparation method of Thirteen Flavors to Remove Stasis Mixture, and then prepare the negative sample solution according to the preparation method of the test solution in step (3);

[0172] (5) Identification: According to the thin layer chromatography test, 10 μl of each of the reference solution, control medicinal material solution, test solution, and negative sample solution were taken and spotted on the same silica gel G plate. The plate was developed with a volume ratio of cyclohexane: ethyl acetate: formic acid = 7:3:0.2. The plate was taken out, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the control medicinal material and the reference sample, and there was no interference with the negative sample at the same position.

[0173] The method for determining the content of free chrysophanol in the mixture comprises the following steps:

[0174] (1) Chromatographic conditions: The chromatographic column was a thermos C18 column, 250 mm × 4.6 mm, 5 μm; the mobile phase was a methanol-0.1% phosphoric acid solution with a volume ratio of 75:25, isocratic elution, a flow rate of 1.0 ml / min, a detection wavelength of 254 nm, a column temperature of 25°C, and an injection volume of 10 μl.

[0175] (2) Preparation of reference solution: Weigh 8.16 mg of chrysophanol reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add methanol to the scale line, weigh it, sonicate for 30 min, replenish the weight with methanol, filter it through a 0.45 μm microporous filter membrane, take the filtrate, and prepare a reference solution containing 163.2 μg of chrysophanol per 1 ml. Set aside and dilute it with methanol to prepare standard solutions of different concentrations when used;

[0176] (3) Preparation of test solution: Take an appropriate amount of the decoction of the mixture, evaporate it in a water bath to obtain the extract, grind it into powder, weigh 0.8 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 min, and replenish the weight of methanol to make a test solution containing 0.08 g of the extract per 1 ml. Filter it through a 0.45 μm microporous membrane, take the filtrate and set aside;

[0177] (4) HPLC detection: Take 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, measure according to the chromatographic conditions, record the chromatogram, and calculate.

Claims

1. A quality testing method for Thirteen-Ingredients-Removing-Yu Mixture, characterized by: The Thirteen-Ingredient Blood-Removing Mixture is prepared from the following raw materials in parts by weight: 12 parts of Rhubarb; 10 parts of Chuanxiong; 9 parts of Peach Kernel; 9 parts of Carthamus tinctorius; 12 parts of Bupleurum; 9 parts of Radix Trichosanthis; 9 parts of Angelica sinensis; 6 parts of Licorice; 12 parts of Corydalis; 9 parts of Eupolyphaga sinensis; 15 parts of Poria cocos; 12 parts of Rhizoma Alismatis; 12 parts of Platycodon grandiflorum; The preparation method of the Thirteen-ingredient Blood-Removing Mixture is as follows: taking the raw medicinal materials according to their weight, first taking the raw medicinal materials except rhubarb, adding water and boiling, then adding rhubarb, boiling twice, and combining the decoctions to obtain the mixture; The quality detection method includes thin layer chromatography identification of rhubarb in the mixture and determination of the content of free chrysophanol; wherein the thin layer chromatography identification method of rhubarb in the mixture includes the following steps: (1) Preparation of reference solution: Weigh 1 mg of chrysophanol reference substance accurately, place in a 10 ml volumetric flask, add methanol to the scale line, weigh, ultrasonicate for 30 min, and add methanol to the weight to make a chrysophanol reference solution containing 0.1 mg of chrysophanol reference substance per 1 ml, let it stand, and take the supernatant as the reference solution; (2) Preparation of control medicinal material solution: Take 1 g of rhubarb control medicinal material, weigh it accurately, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 min, add methanol to the weight, let it stand, and take the supernatant as the control medicinal material solution to prepare a control medicinal material solution containing 0.1 g of the control medicinal material per 1 ml; (3) Preparation of test solution: Take an appropriate amount of the decoction of this mixture, evaporate it in a water bath to obtain the extract, grind it into powder, weigh 2 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 minutes, replenish the weight with methanol, let it stand, and take the supernatant as the test solution to prepare a test solution containing 0.2 g of the test extract per 1 ml; (4) Preparation of negative sample solution: Weigh the other medicinal materials except rhubarb according to the prescription ratio, prepare the negative sample in the same way as the preparation method of Thirteen Flavors to Remove Stasis Mixture, and then prepare the negative sample solution according to the preparation method of the test solution in step (3); (5) Identification: According to the thin layer chromatography test, 10 μl of each of the reference solution, reference medicinal material solution, test solution, and negative sample solution were taken and spotted on the same silica gel G plate. The plate was developed with a volume ratio of cyclohexane: ethyl acetate: formic acid = 7:3:0.

2. The plate was taken out, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal material and reference sample, and there was no interference with the negative sample at the same position. The method for determining the content of free chrysophanol in the mixture comprises the following steps: (1) Chromatographic conditions: The chromatographic column was a thermos C18 column, 250 mm × 4.6 mm, 5 μm; the mobile phase was a methanol-0.1% phosphoric acid solution with a volume ratio of 75:25, isocratic elution, a flow rate of 1.0 ml / min, a detection wavelength of 254 nm, a column temperature of 25°C, and an injection volume of 10 μl; (2) Preparation of reference solution: Weigh 8.16 mg of chrysophanol reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add methanol to the scale line, weigh it, sonicate for 30 min, replenish the weight with methanol, filter it through a 0.45 μm microporous filter membrane, take the filtrate, and prepare a reference solution containing 163.2 μg of chrysophanol per 1 ml. Set aside and dilute it with methanol to prepare standard solutions of different concentrations when used; (3) Preparation of test solution: Take an appropriate amount of the decoction of the mixture, evaporate it in a water bath to obtain the extract, grind it into powder, weigh 0.8 g of the extract, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to the scale line, weigh it, ultrasonicate it for 30 min, and replenish the weight of methanol to make a test solution containing 0.08 g of the extract per 1 ml. Filter it through a 0.45 μm microporous membrane, take the filtrate and set aside; (4) HPLC detection: Take 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, measure according to the chromatographic conditions, record the chromatogram, and calculate.

2. The quality detection method of the Thirteen-Ingredients-Removing-Yu Mixture according to claim 1, wherein: The preparation method of the thirteen-ingredient stasis-removing mixture is as follows: taking each raw medicinal material according to weight, first taking the other raw medicinal materials except rhubarb and mixing them, adding 8 times the total weight of water of the medicinal materials and decocting for 65-85 minutes, adding rhubarb, and then decocting for another 10-15 minutes, filtering, and adding 8 times the amount of water to the residue and decocting for 75-100 minutes; mixing the decoctions obtained from the two decoctions to obtain the mixture.

3. The quality detection method of the Thirteen Flavors Removing Yu Mixture according to claim 2, wherein: The preparation method of the thirteen-ingredient stasis-removing mixture is as follows: taking each raw medicinal material according to weight, first taking the other raw medicinal materials except rhubarb and mixing them, adding 8 times the total weight of water of the medicinal materials and decocting for 80 minutes, after adding rhubarb, decocting for another 10 minutes, filtering, adding 8 times the amount of water to the residue and decocting for 90 minutes; mixing the decoctions obtained from the two decoctions to obtain the mixture.

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