CN1799591A - Preparation method of 'Xue Fu Zhu Yu' capsule and quality standard thereof - Google Patents

Abstract

The present invention relates to the technical field of medical preparations (International Patent Classification No. 35/79) derived from plants containing reaction products of unknown structure, in particular, a kind of Xuefu Zhuyu made from extracts of Chinese herbal medicines The preparation method of the capsule and its quality standard. The identification items of peach kernel, citrus aurantium and licorice are set in the quality standard, and each capsule contains paeoniflorin (C ₂₃ H ₂₈ O ₁₁ ), which shall not be less than 2.20 mg quantitative indicators and inspection methods, so as to improve product quality and Determine the best process route and process conditions, so that the Xuefu Zhuyu Capsule has the characteristics of small dosage, fast absorption in the body, and convenient administration.

Description

A preparation method and quality standard of Xuefu Zhuyu Capsules

technical field

The present invention relates to the technical field of medical preparations (International Patent Classification No. 35/79) derived from plants containing reaction products of unknown structure, in particular, a kind of Xuefu Zhuyu made from extracts of Chinese herbal medicines The preparation method of the capsule and its quality standard.

Background technique

Xuefu Zhuyu Jiaonang is a classic Chinese patent medicine, the Ministry of Health standard WS ₃ -B-0928-91 records the prescription and quality standards:

Peach kernel (stir-fried) Angelica citrus aurantium (stir-fried with bran) Chuanxiong

Bupleurum safflower

Rehmannia glutinosa Platycodon grandiflorum Licorice

【Properties】This product is a capsule, and the content is a brown powder; the gas is pungent, and the taste is slightly bitter.

[Identification] (1) Take this product and observe under a microscope: the stone cells are yellow, shell-shaped, thick on one side, and finely layered. The parenchyma cells are spindle-shaped, with slightly thicker walls and very fine oblique staggered textures. The tubules contain yellow or brownish-yellow discharge. The diameter of the threaded conduit is 8-23 μm, and the thickened wall is interconnected, resembling a mesh-like threaded conduit, and calcium oxalate crystals exist in the parenchyma in sheets.

(2) Take 5g of this product, add 20ml of methanol, soak in cold overnight, filter, and concentrate the filtrate to 2ml as the test solution. Take another hesperidin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the thin-layer chromatography (Appendix VIB) test, draw 2 μl of each of the above two solutions, respectively spot on the same silica gel G thin-layer plate with sodium carboxymethylcellulose as the binder, and mix with ethyl acetate-ethanol-water (8:2:1) was used as the developer, developed, taken out, dried in the air, sprayed with 5% magnesium acetate methanol solution, and inspected under ultraviolet light (365nm) after the methanol evaporated. In the chromatogram of the test product, at the position corresponding to the chromatogram of the reference product, there are fluorescent spots of the same color.

【Check】Should comply with the relevant provisions of the capsule (Appendix IL).

【Content Determination】Determination according to high performance liquid chromatography (Appendix VID).

Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as filler; acetonitrile-water-glacial acetic acid (16:84:1) was used as mobile phase; detection wavelength was 230nm. The number of theoretical plates should not be less than 2500 based on the peak of paeoniflorin. The degree of separation should meet the requirements.

Preparation of reference substance solution Take 2 mg of paeoniflorin reference substance dried at 80°C to constant weight, accurately weigh it, put it in a 10ml volumetric flask, add absolute ethanol to the mark, shake well, and obtain (each 1ml contains 0.2 mg of paeoniflorin). mg).

Preparation of the test solution Take the content of this product under the item of loading difference, accurately weigh, mix, weigh about 3g, accurately weigh, put in a conical flask, add absolute ethanol 50ml accurately, seal, Weigh, reflux and extract in a water bath for 4 hours, then ultrasonically extract for 1 hour, take it out, let it cool, weigh it, make up for the lost amount of absolute ethanol, filter, take 25ml of the continued filtrate and concentrate it on a water bath to 2ml. And transfer to 10ml measuring bottle, add absolute ethanol to the mark, shake well, as the test solution.

Determination method Precisely draw 20μl each of the reference solution and the test solution, inject it into the liquid chromatograph, measure, and calculate the content of paeoniflorin in the test according to the external standard method.

Each capsule of this product contains paeoniflorin (C ₂₃ H ₂₈ O ₁₁ ) not less than 0.24mg.

[Functions and Indications] Promoting blood circulation and removing blood stasis, promoting qi circulation and relieving pain. Used for blood stasis internal resistance, chest pain or headache, internal heat and depression, insomnia and dreaminess, palpitations, irritability and anger.

[Usage and Dosage] Take orally, 6 capsules at a time, 2 times a day, one month as a course of treatment.

[Note] Do not eat spicy or cold food. Pregnant women should not take it.

[Specification] 0.4g per capsule

【Storage】Sealed, store in a cool and dry place.

The original preparation process: crush 100g of peach kernels, angelica, red peony root, citrus aurantii, Chuanxiong, and Bupleurum into fine powder, sieve, mix well, add 100g of other five flavors such as safflower and peach kernels to decoct three times, filter, and combine the filtrates , concentrated into a thick paste with a relative density of 1.15-1.25 (65-70°C), mixed with the above-mentioned powder, made into granules, dried, crushed, sieved, and packed into capsules.

In addition, the application date is 2001.1.5, and the publication number is CN1362075A "Nano blood stasis preparation drug and its preparation method" patent

Record the prescription:

10-50 parts of nano peach kernel 8-60 parts of nano angelica 10-70 parts of nano citrus aurantium 8-60 parts of nano chuanxiong

Nano bupleurum 10-60 parts Nano safflower 8-60 parts Nano achyranthes bidentata 8-50 parts Nano red peony 8-60 parts

Nano Rehmanniae 10-60 parts Nano Bellflower 5-50 parts Nano Licorice 5-30 parts

It records the preparation method, function and indications of the nano-Xuefu Zhuyu preparation: it can be used for the treatment of headache, dizziness, sequelae of brain injury, coronary heart disease and angina pectoris.

Pharmacology and Clinical Practice of Traditional Chinese Medicine, 1990, 6(6), pp. 1-4, Peng Kang and Zheng Youshun, "Research on Disassembled Prescription of Xuefu Zhuyu Decoction—Pharmacological Effect on Microcirculation" proves that it can improve microcirculation, Increased blood pressure caused by increased microvascular resistance has a certain inhibitory effect.

The Xuefu Zhuyu capsule quality standards disclosed above still have identification items, identification means, and content determination methods that are not perfect. The cost is high, the preparation method of the original capsule dosage form, the dosage is 6 capsules each time, the dosage is large, the absorption in the body is slow, and it is inconvenient to take. It does not meet the standards of three small, three effective and five convenient.

Contents of the invention

The purpose of the present invention is to provide a preparation method of Xuefu Zhuyu Capsules and its quality standards, and strive to make the Xuefu Zhuyu Capsules after the improved process meet the standards of three small, three effective and five convenient.

In order to strictly control the quality of drugs and ensure the curative effect, quality standards have been formulated

1. In the study of qualitative identification of this product, the qualitative identification of peach kernel, citrus aurantium, licorice, achyranthes bidentata, and platycodon root in the prescription was designed. First, the method reported in the literature was used to test. The separation was still poor even though the developer was changed. Different sample processing methods and expansion systems with different polarities were used for separation, especially Achyranthes bidentata and Platycodon grandiflora with large interference, overlapping spots at the same position as the reference substance, and negative interference. After improving the sample processing, spot plate identification was performed. The results showed that the medicinal flavors of peach kernel, licorice, and Fructus Fructus Citrus Fructus were well separated, and the same spots appeared on the same positions as the reference substance, and the negatives had no interference, and the reproducibility was good. Therefore, the TLC identification method of peach kernel, licorice and Citrus aurantii in the prescription was established.

2. In the design of quantitative analysis of this product, firstly, the component analysis experiment was carried out with the medicinal herb Radix Paeoniae Rubra in the prescription. According to literature reports, Radix Paeoniae Rubra contains paeoniflorin, so high performance liquid chromatography was used to determine the content of paeoniflorin. According to the pharmacopoeia method, the detection wavelength λ=230nm was selected for the test, and the methodological investigation was carried out. The sample pretreatment was extracted by using different ultrasonic times, and 50% methanol was selected for ultrasonic extraction for 10 minutes, and the active ingredients were basically extracted. Adjust the ratio of mobile phase to be: acetonitrile-water (16:84), flow velocity is 1.0ml/min, etc. chromatographic conditions, through the experiment investigation to precision, reproducibility, stability, recovery rate, determined that peony The content of the glycoside reference substance is determined by HPLC method, which has good reproducibility and can quickly and accurately detect the quality of the drug.

The technical scheme of the present invention is: the identification of peach kernels is added to the existing quality standards: take 1 g of the content of this product, add 20 ml of water to dissolve, add 20 ml of ether to extract twice, discard the ether solution, and extract the aqueous solution with 30 ml of ethyl acetate 2 times, collect the ethyl acetate solution and evaporate to dryness, add 2ml of methanol to obtain the test solution. Take another naringin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the test of thin-layer chromatography (Appendix VIB of "Chinese Pharmacopoeia" 2000 edition), draw 5-10 μl of each of the above two solutions, respectively spot on the same silica gel G thin-layer plate, and mix with chloroform-methanol-water (45:17 : 5) The lower layer liquid left for 12 hours was developed, taken out, dried, sprayed with aluminum trichloride methanol solution and inspected under ultraviolet light (365nm). In the chromatogram of the test product, the same bright fluorescent spots appear at the positions corresponding to the reference product.

Identification of Fructus Citrus Fructus: Take 1g of the contents of this product, add 15ml of methanol and sonicate for 10min to filter, evaporate the filtrate to a small volume under reduced pressure, add 0.5g of brick glue (100-160 mesh), mix well, evaporate to dryness, and add to a silica gel column On the upper layer (column diameter 1cm, column length 25cm, silica gel 100~160 mesh 5g, dry packing) the lower layer of chloroform:ethyl acetate:methanol:water (15:40:22:10) at 5~10°C for 12 hours was Eluate, elute, and collect 16-21ml of liquid to obtain the test solution. Take another amygdalin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the test of thin-layer chromatography (Appendix VIB of "Chinese Pharmacopoeia" 2000 edition), draw 5-10 μl of each of the above two solutions, spot them on the same silica gel G thin-layer plate, develop with eluent as developing agent, and take out , dry in the air, immediately spray with phosphomolybdic acid solution (2g of phosphomolybdic acid, add 20ml of water to dissolve, then slowly add 30ml of sulfuric acid and mix well.) Heat at 105°C for about 10 minutes to develop color. On the corresponding position of the control substance, the same dark blue spots appeared.

Identification of licorice: Take 2g of the content of this product, add 30ml of ether, sonicate for 10min, filter, discard the ether solution, add 30ml of methanol to the residue, sonicate for 10min, evaporate the filtrate to dryness under reduced pressure, add 40ml of water to dissolve, and dissolve with n-butyl Alcohol extraction 3 times, each time 20ml combined n-butanol solution, reduced pressure to dryness, added 5ml methanol to dissolve, that is, the test solution. Take another monoammonium glycyrrhizinate reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the test of thin-layer chromatography (Appendix VIB of "Chinese Pharmacopoeia" 2000 edition), draw 2 to 4 μl of each of the above two solutions, respectively spot on the same high-efficiency silica gel GF ₂₅₄ thin-layer plate, and use n-butanol-20% ammonia water -Methanol (5:2:1.5) was developed as a developer, taken out, dried, and inspected under a UV lamp (254nm). In the chromatogram of the test product, the same blue-black spots appeared at the corresponding positions of the reference product. .

The content determination method has been improved and the standard of paeoniflorin (C ₂₃ H ₂₈ O ₁₁ ) has been increased as follows

[Content Determination] Determination according to high performance liquid chromatography (Appendix VID)

Chromatographic conditions and system suitability test Octadecyl bonded silica gel was used as filler; acetonitrile-water (16:84) was used as mobile phase; detection wavelength was 230nm. The number of theoretical plates calculated based on the peak of paeoniflorin should not be less than 4300.

Preparation of reference substance solution Weigh an appropriate amount of paeoniflorin reference substance, weigh it accurately, add methanol to make a solution containing 0.1mg per 1ml, and get it.

Preparation of the test solution Take 0.3g of the content of this product, grind it finely, and weigh it accurately. Put 24ml of methanol into a 25ml measuring bottle, sonicate for 10min, let it cool, add methanol to the mark, shake well, filter through a microporous membrane, take the subsequent filtrate and put it in a brown bottle to obtain the product.

Determination method Precisely draw 10 μl each of the reference substance solution and the test solution, inject it into the liquid chromatograph, measure it, and obtain it.

Each capsule of this product contains paeoniflorin (C ₂₃ H ₂₈ O ₁₁ ), not less than 2.20mg.

Quality Standard Research Data

[identification]

1. Identification objects There are eleven herbs in this prescription, and the identification method is established for the five herbs of peach kernel, citrus aurantium, licorice, achyranthes bidentata, and platycodon grandiflorum. In the experimental research, different sample processing methods and different polarity expansion system separation chromatography were used respectively. Due to the fact that there are many medicinal flavors in the prescription, the components are complex, and the interference is large. Thin layer identification method. Compared with the original craft capsule, two qualitative identifications have been added.

2. Source of reference substance: All reference substances used in this product are purchased from China Institute for the Control of Biological Products

3. Identification method

1. Identification of peach kernels

Preparation of the test solution Take 1g of the content of this product, add methanol 15ml, sonicate for 10min, filter, evaporate the filtrate to a small volume under reduced pressure, add 0.5g of silica gel (100-160 mesh), mix well, evaporate to dryness, add On a silica gel column (column diameter 1cm, column length 25cm, silica gel 100-160 mesh 5g, dry packing) with chloroform-ethyl acetate-methanol-water (15:40:22:10) at 5-10°C for 12 hours The lower layer is the eluent, elute, and collect 16-21ml of liquid to obtain the test solution.

Preparation of reference substance solution Take amygdalin reference substance and add methanol to make a solution containing 1mg per 1ml as the reference substance solution. According to the test of thin-layer chromatography (Appendix VIB of "Chinese Pharmacopoeia" 2000 edition), draw 5-10 μl of each of the above two solutions, respectively spot on the same silica gel G thin-layer plate, and develop with eluent as developing agent.

Preparation of negative blank control solution According to the composition of the prescription, take the remaining medicinal flavors except peach kernels, make a negative sample according to the process requirements, weigh 1g, and operate according to the method under the preparation of the test solution to obtain a negative sample solution.

TLC:

Adsorbent Silica gel G plate (10×10cm) Qingdao Ocean Chemical Factory

Sample volume 5--10μl

Developing solvent Chloroform-ethyl acetate-methanol-water (15:40:22:10) 5～10℃ for 12 hours, the lower layer is the eluent

Spread distance 8cm

Results Take it out, dry it, and immediately spray it with phosphomolybdic acid solution (2g of phosphomolybdic acid, add 20ml of water to dissolve, then slowly add 30ml of sulfuric acid and mix well.) Heat at 105°C to develop color for about 10 minutes. In the chromatogram of the test product, On the position corresponding to the control substance, the same dark blue spots appear. Negative samples do not have this spot.

2. Identification of Citrus aurantium

Preparation of the test solution Take 1g of the product content, add 20ml of water to dissolve, add 20ml of ether to extract twice, discard the ether solution, extract the aqueous solution twice with 30ml of ethyl acetate, collect the ethyl acetate solution and evaporate to dryness, add 2ml Methanol is the test solution.

Preparation of reference substance solution Take naringin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution.

Preparation of negative blank control solution According to the composition of the prescription, take the rest of the medicinal flavors except Fructus Aurantii, make a negative sample according to the process requirements, weigh 1g, and operate according to the method under the preparation of the test solution to obtain a negative sample solution.

TLC:

Adsorbent Silica gel G plate (10×10cm) Qingdao Ocean Chemical Factory

Sample volume 5～10μl

Developing agent Chloroform-methanol-water (32:17:5) to develop the lower layer,

Spread distance 8cm

The results were taken out, dried in the air, sprayed with aluminum trichloride methanol solution and inspected under ultraviolet light (365nm). In the chromatogram of the test product, the same bright fluorescent spots appear at the positions corresponding to the reference product. Negative sample solution does not have this spot.

3. Identification of licorice

Preparation of the test solution: Take 2g of the content of this product, add 30ml of ether, sonicate for 10min, filter, discard the ether solution, add 30ml of methanol to the residue, sonicate for 10min, evaporate the filtrate to dryness under reduced pressure, add 40ml of water to dissolve, and use Extract 3 times with n-butanol, combine 20ml of n-butanol solution each time, depressurize to dryness, add 5ml of methanol to dissolve, and obtain the test solution.

Preparation of reference substance solution Take glycyrrhizinate monoammonium salt reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution.

Preparation of negative blank solution According to the composition of the prescription, take the rest of the medicinal flavors except licorice, make a negative sample according to the process requirements, weigh 1g, and operate according to the method under the preparation of the test solution to obtain a negative sample solution.

TLC:

Adsorbent High-efficiency silica gel GF ₂₅₁ thin-layer plate, (10×10cm) Qingdao Ocean Chemical Factory

Sample volume 2-5μl

Developing agent n-butanol-20% ammonia water-methanol (5:2:1.5)

Spread distance 8cm

Results Take it out, dry it, and inspect it under an ultraviolet lamp (254nm). In the chromatogram of the test product, the same blue-black spots appear at the positions corresponding to the reference product. Negative sample solution does not have this spot.

[Content Determination] Content determination object and ingredient selection basis: This product mainly contains paeoniflorin, paeoniflorin, which is exclusive in this prescription, is selected as the ingredient index to determine the content, which can better control the quality of the drug.

1. Instruments and reagents

HP1100 High Performance Liquid Chromatograph, Detector: G1314A, Als: 1313A, Colcomp: G1316A, Pump: G1310A

Methanol and acetonitrile were analytically pure and chromatographically pure

Paeoniflorin reference substance: purchased from China National Institute for the Control of Pharmaceutical and Biological Products

Xuefu Zhuyu Capsules: 3 batches, provided by the special group of Xuefu Zhuyu Capsules.

2. High performance liquid chromatography conditions

Chromatographic column: YWG-C ₁₈ (250×4.6mm) 5μm

Detection wavelength: λ=230nm Column temperature: 30°C

Mobile phase: acetonitrile-water (16:84),

Flow rate: 1.0ml/min

3. Determination of the purity of the reference substance paeoniflorin

Source: China Institute for the Control of Pharmaceutical and Biological Products

Purity: Prepare the reference substance as 0.92 mg/ml methanol solution, inject 11 μl, and inject 11 μl of methanol in parallel, record the chromatogram, calculate the content by peak area normalization method, and the content is 98.10%.

4. Calculation of reference substance, sample, negative liquid chromatogram and number of theoretical plates of paeoniflorin shall be carried out under the content determination item.

1. Liquid chromatogram of reference substance paeoniflorin

2. Negative liquid chromatogram

3. Liquid chromatogram of positive medicinal materials

4. The liquid chromatogram of the test product

5. Calculation of theoretical plate number of paeoniflorin: N=5.54×(t _R /W _1/2h ) ²

=5.54×(9.989/0.3589) ² =4291

The number of theoretical plates is 4291

5. Determination of content

(1) Method inspection

The method of sample extraction is based on the fact that paeoniflorin is a water-soluble component, and with reference to the determination method of Radix Paeoniae Rubra in Pharmacopoeia, the comparison of ultrasonic extraction for sample pretreatment and the selection of mobile phase were investigated.

1. Ultrasonic extraction test, adding solvent to the sample and then treating it with different ultrasonic time. Determination content see table 6

Table 6 Different ultrasonic time extraction content determination results time 10 minutes 20 minutes 30 minutes 40 minutes Content (mg/g) 8.285 8.275 8.350 8.340

The test results show that the contents measured by different ultrasonic extraction methods are basically similar, and the ultrasonic extraction is basically complete in 10 minutes. Therefore, the ultrasound time was selected as 10 minutes.

2. Selection of mobile phase: The selection of mobile phase refers to the determination method of Radix Paeoniae Rubra in Pharmacopoeia. The mobile phase is methanol-potassium dihydrogen phosphate 0.05M (40:65). It is also okay, but the sample chromatogram is not ideal, and the separation of the main peak is not good, so the mobile phase has been adjusted. After repeated tests, it is finally determined that the best mobile phase and the ratio are acetonitrile-water (16:84), and the separation of the main peak is relatively good. OK, stable.

3. Selection of detection wavelength Refer to the Pharmacopoeia method, the detection wavelength of paeoniflorin is λ=230nm, and the test is carried out. The result shows: Negative has no interference at the wavelength of 230nm, and the sample and the reference substance have the same maximum absorption peak, the result is ideal, so the detection wavelength is λ=230nm.

(2) System suitability experiment Chromatographic column YWG-C ₁₈ (250×4.6mm) 5 μm; mobile phase acetonitrile-water (16:84); detection wavelength λ=230nm. The number of theoretical plates calculated by paeoniflorin is not less than 4300

(3) Preparation of the test solution Take an appropriate amount of content and grind it finely, weigh 0.3g, and accurately weigh. Put it into a 25ml measuring bottle, add 24ml of 50% methanol precisely, ultrasonic for 10min, let it cool, add methanol to the mark, shake well, and filter through a microporous membrane to obtain the test solution.

(4) Preparation of positive solution Take 0.1 g of the coarse powder of Radix Paeoniae Rubra, weigh it accurately, and obtain the positive solution with the preparation method of Radix Radix Radix Paeoniae Alba.

(5) Preparation of reference substance solution Take paeoniflorin reference substance, add methanol to make 1ml solution containing 0.5072mg as reference substance stock solution.

(6) Preparation of negative solution Take Radix Paeoniae Rubra extract 23g, and prepare negative solution with the same method as (2).

(7) Determination of content

1. Investigation of linear relationship: draw reference substance stock solution 0.6, 0.8, 1.0, 1.2, 1.4, 1.6ml,

Put them into 5.0ml measuring bottles respectively, add methanol to the mark, shake well, and you get it. Inject 10 μl each, take the injection volume as the abscissa, and the peak area as the ordinate, draw a standard curve, and process to obtain a regression equation, see Table 7 and Figure 1.

Table 7 Paeoniflorin standard curve Concentration (μg) 0.60864 0.81152 1.01440 1.21728 1.42016 1.62304 Peak area 809.7949 1108.939 1400.748 1692.776 1950.427 2250.17

Results: There was a good linear relationship in the range of 0.60864～1.62304μg.

2. Blank test: take the prescription drug lacking Radix Paeoniae Rubra, and make a negative sample according to the sample preparation method. Process the solution of the test substance under the same content determination item, and measure the chromatogram to confirm that there is no interference

3. Stability test: The sample solution (040907) to be injected was left for different time, injected into a liquid chromatograph for measurement, and the peak area integral value was recorded. See Table 8

Table 8 Stability test results time (h) 0 4 8 12 twenty four Content (mg/g) 9.00 8.96 8.96 8.98 9.07

RSD=0.51%

4. Precision test: Take 10 μl of the sample solution (040907) under the content determination item, inject the sample five times, and record the peak area integral value respectively. The results are shown in Table 9

Table 9 precision test results Number of injections 1 2 3 4 5 Content (mg/g) 9.00 8.98 8.93 8.94 8.96

RSD=0.32%

5. Reproducibility test: According to the operation under the content determination method, 5 samples of 040907 were accurately weighed for content determination. The results are shown in Table 10

Table 10 Reproducibility test results Injected NO 1 2 3 4 5 Content (mg/g) 9.00 8.98 8.98 8.99 8.99

RSD=0.08%

6. Recovery rate test: Take 0.15g of the 040907 batch of samples, weigh them accurately, add 1.268mg of the reference substance to the finished product, process according to the method of preparing the test solution, and measure according to the content determination method. Content results are shown in Table 11

Table 11 Recovery rate test results

RSD=2.7%

7. Sample determination: according to the chromatographic conditions drawn up by the assay method, the test solution and the reference solution were respectively

10 μl was injected into a liquid chromatograph, and the content of paeoniflorin was determined. The assay results of three batches of samples are shown in Table 12

                                                                                           

The mean value of the content measured in this experiment is 8.03mg/g, which is equivalent to 0.24g of Radix Paeoniae Rubra in the prescription, and the content of Radix Radix Radix Paeoniae Alba in the converted prescription is 33.45mg/g. Its extraction rate is 75.7%. Considering the actual situation of the factory, it is stipulated that the content of this product should not be less than 4.80 mg/g in terms of paeoniflorin (C ₂₃ H ₂₈ O ₁₁ ). Not less than 2.20mg per capsule.

Xuefu Zhuyu (the original dosage form is capsule) is a traditional Chinese medicine compound preparation produced by Tianjin Hongrentang Pharmaceutical Factory. The drug has been widely used clinically and achieved good results. Due to the large amount of clinical use, process improvement has been carried out on Xuefu Zhuyu Capsules. In order to ensure and improve the quality of Xuefu Zhuyu Capsules, the technical solution of the present invention is: a preparation method of Xuefu Zhuyu Capsules, which is characterized in that: The weight ratio of Chinese herbal medicines as raw materials

Angelica 162g Peach kernel 216g Safflower 162g Achyranthes bidentata 162g

Rehmannia 162g Radix Paeoniae Rubra 108g Citrus aurantium 108g Bellflower 81g

Chuanxiong 81g Bupleurum 54g Licorice 54g,

The preparation method is: extract 11 medicinal herbs such as angelica with water for 1-3 times, use 6-10 times the water consumption, and filter for 0.5-1.5 hours each time, collect the filtrate, concentrate under reduced pressure to a relative density of 1.07-1.35, Measure at 60-70°C, leave it at room temperature, add ethanol to make the alcohol content 60-80%, stir well, let stand (5-25h) to precipitate, take the supernatant and concentrate under reduced pressure to a relative density of 1.30-1.50 , measured at 60-70°C, adding 10-15% of the starch extract, drying in a drying oven for 5-12h, T = 65-80°C, to obtain dry matter, dry paste rate 20-28%, adding dry matter weight 2%-8% silicon dioxide, packed into capsules, crushed through a 24-60 mesh sieve, each gram of capsule is equivalent to 3.10~3.38g of crude drug.

The preparation method of the Xuefu Zhuyu Capsules is characterized in that the following weight ratio Chinese medicinal materials are used as raw materials

Angelica 162g Peach kernel 216g Safflower 162g Achyranthes bidentata 162g

Rehmannia 162g Radix Paeoniae Rubra 108g Citrus aurantium 108g Bellflower 81g

Chuanxiong 81g Bupleurum 54g Licorice 54g

The preferred preparation method is: extract 11 medicinal herbs such as angelica with water twice, each time with 8 times the water consumption, each extraction time is 1h, filter, collect the filtrate, concentrate under reduced pressure to a relative density of 1.16-1.19, 60°C Determination, put it at room temperature after cooling, add ethanol to make the alcohol content 70%, stir evenly, let it stand for precipitation, take the supernatant and concentrate it under reduced pressure to a relative density of 1.35-1.43, measure at 60°C, add 15% extract The starch was dried in a drying oven for 9h, T=70-75°C, to obtain a dry matter, the dry paste rate was 23-25%, adding silicon dioxide with a weight of 6% of the dry matter, pulverized through a 40-mesh sieve, and packed into capsules. Made into 1000 capsules, each gram of capsule is equivalent to 3.375g of crude drug.

Research data on the production process of Xuefu Zhuyu Capsules:

The original dosage form is capsules, 6 capsules per serving. Due to the reason of dosage and the limitation of coverage in the market, it cannot meet the needs of the majority of patients. The design idea of improving the original capsule process to reduce the dosage is: conduct systematic research on the process, quality, pharmacology, etc. of Xuefu Zhuyu Capsules, in order to ensure and improve the curative effect of the improved capsules, and control them with quality standards. Process and drug efficacy are used as indicators to determine the best process route and process conditions, so that it has the characteristics of small dosage, fast absorption in the body, and convenient administration. Strive to make the Xuefu Zhuyu Capsules developed with improved technology meet the standards of three small, three effective and five convenient.

1. Prescription composition:

Angelica 162g Peach kernel 216g Safflower 162g Achyranthes bidentata 162g

Rehmannia 162g Radix Paeoniae Rubra 108g Citrus aurantium 108g Bellflower 81g

Chuanxiong 81g Bupleurum 54g Licorice 54g

2. Optimal preparation process

1. Extract 11 herbs such as angelica with water for 2 times, each time with 8 times the water consumption, and each extraction time is 1h, filter, collect the filtrate, concentrate under reduced pressure to a relative density of 1.16-1.19 (60°C), and wait for Cool at room temperature, add ethanol to make the alcohol content 70%, stir evenly, let it stand for precipitation, take the supernatant and concentrate it under reduced pressure to a relative density of 1.35-1.43 (60°C), add 15% starch extract, Dry in a drying oven for 9 hours (T=70-75°C) to obtain a dry product (dry paste rate 23-25%), add silicon dioxide with a weight of 6% of the dry product, crush it through a 40-mesh sieve, put it into a capsule, and prepare Into 1000 capsules. (Each gram of capsule is equivalent to 3.375g of crude drug). The best preparation process is shown in Fig. 2.

3. Research on the preparation process

(1) Process efficacy comparison

According to the effect of the drug on promoting blood circulation and removing blood stasis, the pharmacodynamic tests of the two models of hemorheology and carotid artery thrombosis were carried out for the above two processes. The result is as follows:

1. The test drugs were provided by the Xuefu Zhuyu Research Group of our hospital:

Process sample of Xuefu Zhuyu No. 1: light brown powder, each gram of powder is equivalent to 3.38 grams of crude drug, batch number 0513.

Process sample of Xuefu Zhuyu No. 2: dark brown powder, each gram of powder is equivalent to 2.87 grams of crude drug, batch number 0514.

Xuefu Zhuyu Capsules: dark brown powder, each gram of powder is equivalent to 1.69 grams of crude drug, and each capsule contains 0.675 grams of medicinal powder. Produced by Tianjin Hongrentang Pharmaceutical Factory. Lot number D03022.

Compound Danshen Tablets: Shijiazhuang Hualong Pharmaceutical Co., Ltd., batch number 040504.

10% polymer dextran injection: a product of the Institute of Hematology, Chinese Academy of Medical Sciences, with a molecular weight of 300,000.

Aspirin: white crystal, 99.5%, provided by Northwest Synthetic Pharmaceutical Factory.

2. Test animals Wistar mice, both male and female, weighing 200-250g; the above animals are provided by the animal department of the hospital, and the certificate of conformity is "Jindongzhizi No. 001".

3. Instrument BR2-500E cone-plate annual gauge: product of Tokyo Keiki Co., Ltd., Japan

BT87-3 Experimental In vivo Thrombosis Tester: Developed by Baotou Medical College

4. Test methods and results

1) Effects on rat hemorheology induced by polymer dextran

The above-mentioned healthy rats were selected and randomly divided into a normal group, a model control group, a sample group No. 2, a sample group No. 1, an original dosage group, and a positive drug compound Danshen tablet group according to sex and body weight. Xuefu Zhuyu No. 1 sample group, No. 2 sample group, and the original dosage form control group were administered by intragastric administration of 1.5, 3, and 6 g of crude drug/kg, and the positive drug control group was administered by intragastric administration of 2.0 g of drug powder/kg. , the administration volume was 1ml/100g, and the model control group and the normal control group were given an equal volume of 0.5% CMC once a day for 10 consecutive days. One hour after the last administration, the rats in each group were injected with sodium pentobarbital (40 mg/kg) and 1 ml/kg of 10% polymer dextran injection into the tail vein to form a hyperviscosity model. After 10 minutes of intravenous injection, blood was taken from the abdominal aorta, anticoagulated with 3.8% sodium citrate (the ratio of anticoagulant to whole blood was 1:9), and measured at four cutting speeds on a cone-plate viscometer. whole blood viscosity. The hematocrit and erythrocyte sedimentation rate were determined in the hematocrit tube. Centrifuge at 3000 rpm for 10 minutes, take plasma and measure plasma specific viscosity on a capillary viscometer. see table 1

The test results were statistically analyzed by means of two groups - t-value method for significance test. The results showed that compared with the normal control group, the model control group significantly increased the whole blood viscosity and plasma specific viscosity at each cutting speed, indicating that the modeling was successful. Compared with the model control group, the 6g crude drug/kg dosage group of Xuefu Zhuyu No. 1 sample significantly reduced the whole blood viscosity at each cutting speed, and significantly reduced the plasma specific viscosity. The original dosage form of Xuefu Zhuyu and the positive control drug Compound Danshen Tablet also had obvious effects. The Xuefu Zhuyu No. 2 sample only reduced the viscosity of whole blood at low shear rates. Each dose group had no significant effect on erythrocyte sedimentation rate and hematocrit.

2) Effect of Xuefu Zhuyu No. 1 and No. 2 samples on carotid thrombosis

The above-mentioned healthy rats were selected and randomly divided into normal group, No. 2 sample group, No. 1 sample group, original dosage form group, and positive drug aspirin control group according to sex and body weight. Xuefu Zhuyu No. 1 sample group, No. 2 sample group, and the original dosage group were administered by intragastric administration at 1.5, 3, and 6 g of crude drug/kg, once a day, for 10 consecutive days, and the positive drug control group was administered with 150 mg/kg of aspirin by intragastric administration Administration, once a day, for 5 consecutive days, the administration volume was 1ml/100g, and the normal control group was given equal volume of 0.5% CMC. One hour after the last administration, the rats in each group were intraperitoneally anesthetized with pentobarbital sodium (40 mg/kg), separated the carotid artery on one side by about 15 mm, and stimulated the electrodes and temperature sensor hooks with a BT-87-3 thrombosis instrument. On the carotid artery, continuously stimulate the blood vessel wall with a current of 1.5mA for 7 minutes. At the same time, the temperature sensor detects the change of the surface temperature of the blood vessel. Start to alarm time as thrombosis time (OT value), test result adopts two groups of mean statistical analysis t-value method to carry out significance test, see table 2

Table 1 Effect of Xuefu Zhuyu No. 1 and No. 2 process samples on the hemorheology of rats induced by polymer dextran (X±SD) group dose number of animals Whole blood viscosity (mpa.s) Plasma Specific Viscosity ESR Compaction (g crude drug/kg) (Only) 7.5S' 18.8S' 37.5S' 75S' (cm/h) (%) normal control 10 7.14±1.95 3.38±0.80 3.08±0.55 2.80±0.40 1.06±0.15 0.20±0.08 42.4±4.19 Model comparison 10 13.56±2.53^^ 7.32±1.66^^ 6.12±1.30^^ 5.29±1.06^^ 1.46±0.11^^ 0.21±0.10 45.6±4.09 Process No. 1 6 10 10.50±2.12** 5.71±1.23* 4.86±0.88* 4.24±0.66* 1.26±0.14** 0.22±0.09 43.7±3.34 3 8 10.95±1.46* 6.21±1.11 5.07±0.85 4.37±0.71* 1.34±0.14 0.25±0.08 43.5±3.16 1.5 8 12.30±2.33 7.41±1.57 5.55±1.35 4.70±1.25 1.38±0.06 0.24±0.11 43.3±3.62 Process No. 2 6 10 11.28±2.09* 6.00±1.44 5.04±1.00 4.44±0.88 1.31±0.18* 0.27±0.11 43.7±3.53 3 8 12.68±2.32 7.35±2.27 5.96±1.78 4.83±1.37 1.41±0.12 0.24±0.05 43.8±7.80 1.5 8 13.65±3.54 7.29±2.01 5.84±1.51 4.83±1.15 1.45±0.09 0.26±0.11 45.6±5.63 Prototype 6 10 10.14±3.07* 5.50±1.51* 4.84±1.02* 4.17±0.79* 1.24±0.14** 0.22±0.09 42.0±4.76 3 8 10.50±2.63* 5.46±1.45 4.40±0.85* 3.56±0.75** 1.31±0.11* 0.24±0.09 41.8±7.11 1.5 8 11.88±3.33 6.48±1.12 5.22±0.93 4.43±0.96 1.39±0.10 0.25±0.12 43.5±11.78 Compound Danshen Tablets 2g powder 8 10.80±2.70** 5.28±2.04* 4.53±1.59* 4.02±1.28* 1.25±0.21* 0.24±0.07 42.5±8.54

^ΔΔ : p<0.01 (compared with normal control group) *p<0.05, **p<0.01 (compared with model control group)

Table 2 Effect of Xuefu Zhuyu No. 1 and No. 2 process samples on carotid artery thrombosis (±SD) group Dose (g crude drug/kg) Number of animals (only) OT value (points) Normal Control No. 1 Process No. 1 Process No. 2 Process No. 2 Process No. 2 Process Original Dosage Original Dosage Original Dosage Aspirin 631.5631.5631.5150mg/kg 88888888888 16.29±4.9026.33±9.85*23.55±9.3018.57±4.6419.42±8.9920.50±4.5216.95±5.7628.40±5.87**22.62±6.25*18.64±4.3728.79±5.47**

*:: P<0.05 **:: P<0.01 (compared with normal control group)

The results showed that, compared with the normal control group, the 6g crude drug/kg dose group of Xuefu Zhuyu No. 1 sample significantly prolongs the formation time of carotid artery thrombosis in rats. The original dosage form of Xuefu Zhuyu and the positive control drug aspirin also have obvious effects. The sample of Xuefu Zhuyu No. 2 had no obvious effect.

Conclusion: Xuefu Zhuyu No. 1 process sample 6g crude drug/kg dosage group can significantly reduce the whole blood viscosity and plasma specific viscosity of rats with hyperviscosity at various cutting speeds. Significantly prolong the time of carotid artery thrombosis in rats. The 6g crude drug/kg dose group of Xuefu Zhuyu No. 2 sample group only reduced the blood rheology of rats with hyperviscosity at low shear rates, but had no significant effect on carotid artery thrombosis in rats.

(2) Research on extraction process

According to the above pharmacodynamic experiment results, and considering the actual operation of the factory's large-scale production, the No. 1 process route, namely the water extraction and alcohol precipitation process, was determined, and the water extraction and alcohol precipitation conditions were investigated for this process route.

1. Investigation of water extraction process: choose three factors of different dosage, extraction times, and extraction time, and three levels of test programs. The factor levels are shown in Table 3. Taking the paeoniflorin content as the component index, an orthogonal test was carried out to study the optimization of the water extraction process.

Table 3 Factor level table L ₉ (3 ³ ) level factor Water consumption A (times) Extraction time B(h) Extraction times C 123 6810 306090 123

Preparation of samples for orthogonal experiment: Weigh 9 parts of 100g medicinal materials with the prescription amount, and extract with different amounts of water, extraction time, and extraction times to obtain extracts. Carry out the pretreatment of sample and measure the content of paeoniflorin according to the content determination method, select the L ₉ (3 ³ ) orthogonal experiment table to test, the results are shown in Table 4

Table 4 water extraction process investigation L ₉ (3 ³ ) test table

R value: RC>RA>RB

It can be seen from Table 4 that the three factors all have a certain influence on the content of paeoniflorin. The degree of influence of each factor is in the order of C>A>B, where A factor A ₃ >A ₂ >A ₁ , B factor B ₃ >B ₂ >B ₁ , C factor C ₃ >C ₂ >C ₁ . Among them, K2 in data ABC is basically similar to K3 in ABC. Considering low energy consumption and cost, saving time and suitable for large-scale production, factors A ₂ , B ₂ , and C ₂ are adopted. Determine the water extraction process conditions: A ₂ B ₂ C ₂ , that is, use 8 times the amount of water to extract twice, and extract under the best conditions of 60 minutes each time. And carried out three batches of test verification, according to the above extraction method operation, determination of content. The results of calculating the transfer rate are shown in Table 5:

Table 5 Three batches of test transfer rate results Sample 1 Sample 2 Sample 3 Content transfer rate (%)X 76.77 82.9477.8 73.64

The verification results of three batches showed that the average content transfer rate was 77.8%. The improved process condition is stable, repeatable and operable.

2. Investigation of water extraction and alcohol precipitation process

After water extraction in the process, there are many impurities, and the dosage is too large, which directly affects the establishment of the dosage form. After water extraction, alcohol precipitation is selected, and the determination of the content is used as the investigation index. The prescription amount is 900g of medicinal materials, according to the water extraction process conditions Extraction, water extraction twice, each time for 1 hour, the water extract is concentrated to a different ratio with the medicinal material, the relative density is measured, and ethanol is added respectively to make it reach different concentrations of alcohol content, alcohol precipitation for different times, and its content is investigated. Select the L ₉ (3 ³ ) orthogonal test table for the test, the results are shown in Table 6, Table 7, and Table 8

Table 6 Factor level table factor level Concentrate Relative Density A Alcohol precipitation concentration (%) B Alcohol precipitation time (h)C 123 1.071.161.35 607080 51525

Table 7 L ₉ (3 ³ ) Test Calculation Table Results of Water Extraction and Alcohol Precipitation Process Investigation

R: A > B > C

Table 8 variance analysis table source of variance sum of square degrees of freedom mean square f factors Water Concentrate Density A Alcohol Precipitation Concentration B Extraction Time C Error Sum 7410.38326.2338.834120.5477565.997 22228 3705.19213.1174.41760.274 61.4720.2180.073 significant not significant not significant

The results of variance analysis showed that factor A had a significant impact in the orthogonal test, while factors B and C had no significant impact.

Orthogonal test results show that the relative density of water-extraction concentrate has certain influence on the content, R value shows that the relative density of water-concentrate is the main factor, and alcohol precipitation concentration and alcohol precipitation time are secondary factors. The degree of influence of each factor is in the order of RA>RB>RC, and the values of _A1 and A2 in factor A are basically similar, but A2 saves twice the alcohol consumption and cost of A1, so A2 is used. B ₂ is used for B factor, and the value of C factor is basically approximate. Considering the operability, C ₂ is used. Therefore, the conditions of A ₂ B ₂ C ₂ are determined, namely: extract the concentrated solution with water to a relative density of 1.16, alcohol precipitation to reach an alcohol content of 70%, and alcohol precipitation time of 15 hours.

Three batches of verifications have been carried out to the above-mentioned process, and the operation is carried out according to the above-mentioned extraction method. The results of the transfer rate of the measured content are as follows in Table 9

Table 9 Alcohol precipitation three batches verification content transfer rate results Sample 1 Sample 2 Sample 3 Content transfer rate (%)X 78.31 76.9576.5 74.43

As a result, the average content transfer rate was 76.5%. It shows that the experimental process conditions are reliable.

3. The results of investigation on the content transfer rate of dry matter

Extract according to the above-mentioned water extraction and alcohol precipitation process to obtain concentrated extracts, add auxiliary materials, and dry in an oven at a temperature of 70-75°C to obtain dry matter, determine the content, and the results of the content transfer rate of the three batches of tests are shown in Table 10.

Table 10 Dry content transfer rate investigation results No. Sample 1 Sample 2 Sample 3 average Content transfer rate% 73.79 73.19 71.21 72.73

The results show that the content transfer rate of the concentrated dry product of the extract is 73.4%, which is 97.7% of the content transfer rate of the water extraction and ethanol precipitation solution. It shows that this drying condition is feasible.

(3) Investigation of preparation molding process

The purpose of improving the process according to this product is to reduce the dosage, and the conditions for its preparation molding have been investigated:

1. Selection of the amount of starch added The above-mentioned process was selected according to the efficacy of the drug, and the process extract was prepared into a stable

The content of the capsule needs to be added with starch to make it better shaped, and 10%, 15%, and 30% of the starch content is added to the extract respectively, and the shape of the capsule is investigated in Table 11

Table 11 Adding the investigation of auxiliary material starch amount Amount of starch added % 10 15 30 Molding condition poor liquidity good liquidity good liquidity

The result shows that the content of the preparation is formed well with 15% and 30%, and the fluidity is also good. Considering the dosage, 15% starch is added to form the prepared content. However, in the stability test, it was found that the content had agglomeration, and the auxiliary material starch could not be added in consideration of the dosage of the dosage form. Therefore, a glidant silicon dioxide was added for investigation.

2. The choice of the amount of silicon dioxide added: add 2%, 4%, 6%, and 8% to the content respectively

Proportion of silicon dioxide, under the conditions of room temperature 25°C and humidity 45%, to measure the angle of repose to investigate the fluidity of its contents, the results are shown in Table 12

Table 12 Adds the Investigation of Different Amounts of Silica Amounts Amount of silica added % 2 4 6 8 Liquidity 68° 50° 40° 30°

The results show that the fluidity is good with the addition amount of 6% and 8%, and there is no agglomeration and moisture absorption phenomenon at room temperature, indicating stability. Considering various factors, it is feasible to select 6% as the amount of silica added.

(4) The basis for determining the dose after the process is changed

Dosage of Xuefu Zhuyuyuan Capsules: Take 6 capsules/time orally, twice a day (the dosage is equivalent to 4.05g crude drug each time). The dosage of this product is determined according to the dosage of the original Xuefu Zhuyu Capsules: the dosage of the Xuefu Zhuyu Capsules after the improved process: take 3 capsules/time, twice a day. (Each dose is equivalent to crude drug 4.05g). The oral dosage of the original Xuefu Zhuyu Capsules and the modified Xuefu Zhuyu Capsules contain the same amount of crude drug.

The improved Xuefu Zhuyu Capsules were compared with the original Xuefu Zhuyu Capsules, the results are shown in the table below:

Comparison of new technology capsules and original technology capsules

The above data show that the new capsule is superior to the original capsule, with reasonable process, controllable quality, advanced process, remarkable drug effect, small dose, and clinical dose is safe and effective.

Pharmacodynamic test of Xuefu Zhuyu:

Xuefu Zhuyu (the original dosage form is capsule) has been widely used clinically and achieved good results. Due to the large amount of clinical use, the process of Xuefu Zhuyu has been improved. We have conducted pharmacodynamic studies on the samples of the new process in three experimental models of hemorheology, platelet aggregation and microcirculation, and compared them with the original dosage form. . The result is as follows:

Hemorrheology test: select the above-mentioned healthy rats, both male and female, with a body weight of 200-250 g. Randomly divided into normal control group, model control group, Xuefuzhuyu 1.5, 3, 6g crude drug/kg dosage group, original dosage form (6g crude drug/kg) dosage group, positive drug compound salvia miltiorrhiza (3g medicinal powder/kg) according to gender and body weight. ) dose group, a total of 7 groups, each group is 10. Gastrointestinal administration, once a day, for 10 consecutive days, the volume of intragastric administration was 1ml/100g body weight, and the normal control group and model control group were given equal volume of 0.5% CMC by intragastric administration. One hour after the last administration, except for the normal control group, rats in other groups were injected with 10% polymer dextran injection 1ml/kg through the tail vein to form a hyperviscosity model. After 10 minutes of intravenous injection, blood was taken from the abdominal aorta, anticoagulated with 3.8% sodium citrate (the ratio of anticoagulant to whole blood was 1:9), and four cutting speeds were measured on the LG-R-80C viscometer whole blood and plasma specific viscosities. The hematocrit and erythrocyte sedimentation rate were determined in the hematocrit tube. The test results were statistically analyzed by two groups of means - t value method for significance test. Compared with the normal control group, the model control group significantly increased the whole blood viscosity, plasma specific viscosity and hematocrit at each cutting speed, and significantly decreased the erythrocyte sedimentation rate. Speed, indicating successful styling. Compared with the model control group, Xuefuzhuyu 1.5, 3, 6g crude drug/kg dose groups all significantly reduced the whole blood viscosity of hyperviscosity rats, and the 6g crude drug/kg dose also significantly reduced plasma specific viscosity and red blood cell Compaction. There was no significant difference in the erythrocyte sedimentation rate in each dose group. The original dosage form of Xuefu Zhuyu and the positive control drug Compound Danshen Tablet also have similar effects.

Effect test on the platelet aggregation function of "blood stasis" rats: select healthy male Wistar rats, weighing 250g-300g, and randomly divide them into 6 groups according to body weight, with 10 rats in each group. The Xuefu Zhuyu medication group was given 1.5, 3, and 6 g crude drug/kg by intragastric administration, the Xuefu Zhuyu original dosage group was given 6 g crude drug/kg by intragastric administration, and the positive drug group was given aspirin 150 mg/kg by intragastric administration. 1ml/100g body weight, the model control group was given equal volume of 0.5% CMC by intragastric administration, once a day for 10 consecutive days. Rats in each dosage group were subcutaneously injected with epinephrine hydrochloride 0.08ml/100g body weight of 1mg/ml 1 day before the test, twice in total with an interval of 6 hours, during which (about 3 hours) the rats were put into ice water to swim for 5 minutes , and then fasted (without water) overnight. One hour after intragastric administration on the next day, intraperitoneal anesthesia was performed with sodium pentobarbital (40 mg/kg), blood was collected from the abdominal aorta, and anticoagulated with 3.8% sodium citrate (the ratio of anticoagulant to whole blood was 1: 9) Centrifuge to prepare platelet-rich plasma (PRP) and platelet-poor plasma (PPP), and adjust PRP with PPP (the number of platelets is about 300,000/mm ³ ). Using turbidimetry, with ADP, AA, and collagen as inducers, the platelet aggregation test was carried out in a PAM-3 dual-channel platelet aggregation apparatus. According to the tracing curve, the maximum aggregation percentage of platelets was calculated, and the effect of removing blood stasis was observed. The results used the statistical analysis of the two groups - t value method to carry out the significance test, Xuefu Zhuyu 1.5, 3, 6g crude drug/kg dose group compared with the model control group, significantly inhibited the ADP, AA (except 1.5g crude drug/kg dosage group), collagen (except 1.5g crude drug/kg dosage group) induced platelet aggregation in "blood stasis" rats, and with the increase of dosage, the effect was enhanced, indicating that Xuefu Zhuyu has obvious anti-platelet aggregation effect. The original dosage form of Xuefu Zhuyu and the positive drug aspirin also have obvious inhibitory effect.

Effect test on auricular microcirculation of mice with microcirculation disorder: Select healthy Kunming mice, weighing 18-22g, and divide them into 7 groups randomly, with 11 mice in each group, both male and female. The experiment consisted of normal control group, model control group, Xuefu Zhuyu 3, 6, 12g crude drug/kg dose group, Xuefu Zhuyu original formulation 6g crude drug/kg dose group, and positive drug Compound Danshen Tablet 4g powder/kg dose group. Gastrointestinal administration, once a day, for 10 consecutive days, the volume of intragastric administration was 0.2ml/10g body weight, and the normal control group and model control group were administered intragastrically with the same volume of 0.5% CMC. One hour after the last administration, pentobarbital sodium 50 mg/kg was injected intraperitoneally, and after anesthesia, a drop of olive oil was dropped on the glass slide, and the left ear of the mouse was flattened and fixed. Observe and record the flow state of mouse auricle microvessels, the degree of red blood cell aggregation, and the number of capillary network intersections under a 50-fold microscope, as the values before modeling. Then tail vein injected 10% polymer dextran (except normal control group) 0.1ml/10g body weight, and recorded the microvessel flow state, red blood cell aggregation degree, and capillary network intersection of the same part after 5 minutes, 10 minutes, 20 minutes, and 30 minutes after injection. Points, compare the changes before and after the shape, and observe the effect of blood stasis. The significance test of the flow state and red blood cell aggregation degree was carried out by the rank order value method, and the significance test was carried out by the paired t-value method for the number of intersection points of the capillary network. Before molecular dextran modeling, there was no significant difference in blood flow, red blood cell aggregation, and capillary network intersections. After 5, 10, 20, and 30 minutes of modeling, blood flow slowed down significantly, red blood cell aggregation increased significantly, and the number of capillary network intersections significantly increased. Significant reduction (no significant difference in 30 minutes), indicating that microcirculation barriers have been formed, and the model has been replicated successfully. Compared with the model control group, there was no significant difference in the blood flow state, red blood cell aggregation degree, and capillary network intersection number in each dosage group before modeling. /kg and the original dosage form 6g crude drug/kg dose group can significantly reduce the blood flow level and red blood cell aggregation (5 minutes, 10 minutes and 20 minutes), and significantly increase the number of capillary network intersections (5 minutes, 10 minutes and 20 minutes) . Compound Danshen Tablets 4g powder/kg dosage group also had obvious effect.

Influence test on the conjunctival microcirculation of rabbits: select healthy big-eared white rabbits, both male and female, weighing 2-2.6kg, and randomly divide them into 7 groups according to sex and body weight, with 8 rabbits in each group. The experiment consisted of normal control group, model control group, Xuefu Zhuyu 0.75, 1.5, 3g crude drug/kg dosage group, Xuefu Zhuyu original dosage form 3g crude drug/kg dosage group, positive drug Compound Danshen Tablet 2g powder/kg dosage group. Gastrointestinal administration, once a day, for 7 consecutive days, the volume of intragastric administration was 5ml/kg body weight, and the normal control group and model control group were administered intragastrically with the same volume of 0.5% CMC. One hour after the last administration, intravenous injection of pentobarbital sodium 30mg/kg, after anesthesia, observe and record the red blood cell flow rate, flow state and capillary network exchange of rabbit conjunctival microvessels under a dissecting microscope. Points, as the value before modeling. Then ear edge vein injects 10% macromolecular dextran (except normal control group) 1ml/kg body weight, causes the microcirculation disturbance of domestic rabbit, records the erythrocyte flow velocity, flow state, capillary network intersection number of the same position microvessel after injection 20 minutes, compares Changes before and after modeling, observe the effect of blood stasis. The flow state was tested for significance using the rank order value method, and the flow velocity and capillary network intersections were tested for significance using the paired t-value method. Before sculpting, there was no significant difference in the blood flow state, flow rate, and number of capillary network intersections in conjunctival microvessels; 20 minutes after sculpting, the blood flow rate slowed down significantly, the level of flow state increased significantly, and the number of capillary network intersections decreased significantly, indicating that Microcirculation barriers have been formed, and the model has been replicated successfully. Compared with the model control group, there was no significant difference in the blood flow state, flow velocity, and number of capillary network intersections in the conjunctival microvessels of each administration group before modeling; The dosage form of 3g crude drug/kg dosage group can significantly increase the blood flow rate and the number of capillary network intersections, and significantly reduce the level of blood flow state. Positive drug Compound Danshen Tablets 2g powder/kg dosage group also had obvious effect.

Test Conclusions:

1. Xuefu Zhuyu 1.5, 3, 6g crude drug/kg dose group and the original dosage form (6g crude drug/kg) group all significantly reduced the whole blood viscosity of hyperviscosity rats, and the 6g crude drug/kg dose group also significantly reduced Plasma viscosity and hematocrit.

2. Xuefuzhuyu 1.5, 3, 6g crude drug/kg dose groups and the original dosage form group all significantly inhibited the "blood stasis" rats from ADP, AA (except 1.5g crude drug/kg dose group), collagen (except 1.5g crude drug /kg dose group) induced platelet aggregation.

3. Xuefu Zhuyu 6, 12g crude drug/kg dose group and the original dosage group all significantly reduced the level of microvascular blood flow state and the degree of red blood cell aggregation in the auricle of mice with microcirculation disturbance, and significantly increased the number of capillary network intersections.

4. Xuefuzhuyu 1.5, 3g crude drug/kg dose group and the original dosage group all significantly reduced the blood flow level of conjunctival microvessels in rabbits with microcirculation disorders, and significantly increased blood flow velocity and capillary network intersection points.

The above test results show that the new technology Xuefu Zhuyu has obvious effects of "activating blood and removing blood stasis". It is basically consistent with the function and indications of the medicine. It has the same effect as the original dosage form of Xuefu Zhuyu with the same dose. Dosage of Fuzhuyuyuan Capsules: Oral 6 capsules/time, twice a day, (each dose is equivalent to 4.05g crude drug). The dosage of this product is determined according to the dosage of the original Xuefu Zhuyu Capsules: the dosage of the Xuefu Zhuyu Capsules after the improved process: take 3 capsules/time, twice a day. (Each dose is equivalent to crude drug 4.05g). The oral dosage of the original Xuefu Zhuyu Capsules and the modified Xuefu Zhuyu Capsules contain the same amount of crude drug.

Beneficial effects of the invention: the identification items of peach kernel, citrus aurantium and licorice are set in the quality standard, and each capsule contains paeoniflorin (C ₂₃ H ₂₈ O ₁₁ ) and shall not be less than 2.20 mg quantitative index and inspection method, improving The quality of the product, and the determination of the best process route and process conditions, make Xuefu Zhuyu Capsules have the characteristics of small dosage, fast absorption in the body, and convenient administration.

Description of drawings

Figure 1 is a standard curve diagram of paeoniflorin.

Fig. 2 is the optimal process flow chart.

Detailed ways

Example 1 Improved Xuefu Zhuyu Capsule Quality Standard

1. Name: Xuefu Zhuyu Capsules

2. Prescription:

Peach kernel (stir-fried) Angelica citrus aurantium (stir-fried with bran) Chuanxiong

Bupleuri

Rehmannia glutinosa Platycodon grandiflorum Licorice

【Properties】This product is a capsule, and the content is a brown powder; the gas is pungent, and the taste is slightly bitter.

[Preparation method] Extract 11 medicinal herbs such as angelica with water for 1-3 times, use 6-10 times the amount of water, filter for 0.5-1.5 hours each time, collect the filtrate, and concentrate under reduced pressure to a relative density of 1.07-1.35 ( 60-70°C), leave it at room temperature, add ethanol to make the alcohol content 60-80%, stir well, let it stand (5-25h) to precipitate, take the supernatant and concentrate it under reduced pressure to a relative density of 1.30-1.50 (60-70°C) add 10-15% starch in the extract, dry in a drying oven for 5-12h (T=65-80°C) to obtain a dry product (dry paste rate 20-28%), add the dry matter Silica of 2%-8% by weight is packed into capsules, crushed and passed through a 24-60 mesh sieve. (Each gram of capsule is equivalent to 3.10~3.38g of crude drug)

【identification】

1. Microscopic identification

Take this product and observe it under a microscope: the parenchyma cells are spindle-shaped, the wall is slightly thick, and there are very fine oblique interlaced textures. The tubules contain yellow or brownish-yellow discharge. The diameter of the threaded conduit is 8-23 μm, and the thickened wall is interconnected, resembling a mesh-like threaded conduit, and calcium oxalate crystals exist in the parenchyma in sheets.

2. Identification of hesperidin

Take 5g of this product, add methanol 20ml, cold soak overnight, filter, and the filtrate is concentrated to 2ml, as the test solution. Take another hesperidin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the thin-layer chromatography (Appendix VIB) test, draw 2 μl of each of the above two solutions, respectively spot on the same silica gel G thin-layer plate with sodium carboxymethylcellulose as the binder, and mix with ethyl acetate-ethanol-water (8:2:1) was used as the developer, developed, taken out, dried in the air, sprayed with 5% magnesium acetate methanol solution, and inspected under ultraviolet light (365nm) after the methanol evaporated. In the chromatogram of the test product, at the position corresponding to the chromatogram of the reference product, there are fluorescent spots of the same color.

3. Identification of Radix Paeoniae Rubra

Preparation of the test solution Take 10g of finished product powder, add 50ml of 70% ethanol to dissolve, and place overnight. Filter, evaporate the ethanol to the filtrate, add water to 20ml, and extract with ethyl acetate twice, 20ml each time. The extracts were combined, dehydrated with 2 g of anhydrous sodium sulfate, filtered and concentrated to about 2 ml as the test solution.

Preparation of the reference medicinal material solution Take 2 g of Radix Paeoniae Rubra fine powder, add 30 ml of 70% ethanol to dissolve, and prepare the Radix Paeoniae Radix Radix Paeoniae Rubra reference medicinal material solution according to the method under the preparation of the test solution.

Preparation of blank control solution Remove red peony root from the prescription of Xuefu Zhuyu Capsules, prepare a blank reference substance according to the production process, and then prepare a blank reference substance solution according to the method under the preparation of the test solution.

TLC analysis Take 10 μl of each of the above three solutions and spot them on the same high-efficiency silica gel G plate, using chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) as the developing solvent, with a spread distance of about 15 cm. Remove and let dry. Spray with 5% vanillin sulfuric acid solution, and blow it with hot air until the spots appear. The chromatogram of the test product and the chromatogram of the reference medicinal material have spots of the same color at the corresponding positions, and the blank control has no interference.

4. Identification of peach kernels: Take 1g of this product, add 20ml of water to dissolve, add 20ml of ether to extract twice, discard the ether solution, extract the aqueous solution twice with 30ml of ethyl acetate, collect the ethyl acetate solution and evaporate it to dryness, add 2ml Methanol is the test solution. Take another naringin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the test of thin-layer chromatography (Appendix VIB of "Chinese Pharmacopoeia" 2000 edition), draw 5-10 μl of each of the above two solutions, respectively spot on the same silica gel G thin-layer plate, and mix with chloroform-methanol-water (45:17 : 5) The lower layer liquid left for 12 hours was developed, taken out, dried, sprayed with aluminum trichloride methanol solution and inspected under ultraviolet light (365nm). In the chromatogram of the test product, the same bright fluorescent spots appear at the positions corresponding to the reference product.

5. Identification of Citrus aurantium: Take 1g of the contents of this product, add 15ml of methanol and ultrasonically treat it for 10min to filter, evaporate the filtrate to a small volume under reduced pressure, add 0.5g of silica gel (100-160 mesh), mix well, evaporate to dryness, and add silica gel On the column (column diameter 1cm, column length 25cm, silica gel 100~160 mesh 5g, dry packed column) with chloroform:ethyl acetate:methanol:water (15:40:22:10) at 5~10°C for 12 hours in the lower layer For the eluent, elute, collect 16 ~ 21ml of liquid that is the test solution. Take another amygdalin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the test of thin-layer chromatography (Appendix VIB of "Chinese Pharmacopoeia" 2000 edition), draw 5-10 μl of each of the above two solutions, spot them on the same silica gel G thin-layer plate, develop with eluent as developing agent, and take out , dry in the air, immediately spray with phosphomolybdic acid solution (2g of phosphomolybdic acid, add 20ml of water to dissolve, then slowly add 30ml of sulfuric acid and mix well.) Heat at 105°C for about 10 minutes to develop color. On the corresponding position of the control substance, the same dark blue spots appeared.

6. Identification of licorice: take 2g of the content of this product, add 30ml of ether, sonicate for 10min, filter, discard the ether solution, add 30ml of methanol to the residue, sonicate for 10min, evaporate the filtrate to dryness under reduced pressure, add 40ml of water to dissolve, and use Extract 3 times with n-butanol, combine 20ml of n-butanol solution each time, depressurize to dryness, add 5ml of methanol to dissolve, and obtain the test solution. Take another monoammonium glycyrrhizinate reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the test of thin-layer chromatography (Appendix VIB of "Chinese Pharmacopoeia" 2000 edition), draw 2 to 4 μl of each of the above two solutions, respectively spot on the same high-efficiency silica gel GF ₂₅₄ thin-layer plate, and use n-butanol-20% ammonia water -Methanol (5:2:1.5) was developed as a developer, taken out, dried, and inspected under a UV lamp (254nm). In the chromatogram of the test product, the same blue-black spots appeared at the corresponding positions of the reference product. .

[Inspection] The difference in filling volume: take the test product, and measure it according to the method of filling volume difference under the item of capsule IL in the first appendix of "Chinese Pharmacopoeia" in 2005 edition. Compared with the standard loading volume, the loading volume of each capsule should be within ±10.0%. (legal standard should be within ±10.0%), no more than 2 capsules exceed the limit of difference in filling quantity, and no one capsule exceeds the limit by twice the limit.

Moisture: Get the content of the test product, and measure it according to "Chinese Pharmacopoeia" 2005 edition an Appendix IX H moisture determination method, it should not exceed 8.0% (legal standard should not exceed 9.0%).

Disintegration time limit: According to the "Chinese Pharmacopoeia" 2005 edition, an appendix XII A disintegration time limit inspection method, using water as the medium, the disintegration time limit should not exceed 20 minutes (legally not exceed 30 minutes).

Microbial limit inspection: according to the "Chinese Pharmacopoeia" 2005 edition of an appendix XIII C inspection, should meet the following

Column regulations: the number of bacteria shall not exceed 10,000/g (the legal standard shall not exceed 10,000/g). mold and yeast count

It should not exceed 100 pieces/g (the legal standard should not exceed 100 pieces/g). Escherichia coli and live mites shall not be detected. coliform bacteria

The group is less than 100/g (the legal standard is less than 100/g).

[Content Determination] Determination according to high performance liquid chromatography (Appendix VID)

Chromatographic conditions and system suitability test Octadecyl bonded silica gel was used as filler; acetonitrile-water (16:84) was used as mobile phase; detection wavelength was 230nm. The number of theoretical plates calculated based on the peak of paeoniflorin should not be less than 4300.

Preparation of reference substance solution Weigh an appropriate amount of paeoniflorin reference substance, weigh it accurately, add methanol to make a solution containing 0.1mg per 1ml, and get it.

Preparation of the test solution Take 0.3g of the content of this product, grind it finely, and weigh it accurately. Put 24ml of methanol into a 25ml measuring bottle, sonicate for 10min, let it cool, add methanol to the mark, shake well, filter through a microporous membrane, take the subsequent filtrate and put it in a brown bottle to obtain the product.

Determination method Precisely draw 10 μl each of the reference substance solution and the test solution, inject it into the liquid chromatograph, measure it, and obtain it.

Each capsule of this product contains paeoniflorin (C ₂₃ H ₂₈ O ₁₁ ), not less than 2.20mg.

【Functions and Indications】

Promoting blood circulation and removing blood stasis, promoting qi circulation and relieving pain. It is used for blood stasis internal resistance, chest pain or headache, internal heat and depression, insomnia and dreaminess, palpitations, irritability and anger; coronary heart disease, angina pectoris, vascular and traumatic headaches, which belong to the above symptoms.

[Usage and Dosage] Take orally, 3 capsules at a time, 2 times a day, one month as a course of treatment.

[Precautions] Do not eat spicy or cold food. Pregnant women should not take it.

【Precautions】Specification: 0.4g per capsule

【Storage】Sealed, store in a cool and dry place.

[Validity period] Three years.

Example 2

Take 1.35kg of eleven herbs such as angelica, extract with water twice, each time with 8 times the water consumption, each extraction time is 1h, filter, collect the filtrate, concentrate under reduced pressure to a relative density of 1.16 (60°C), and place it in the cold At room temperature, add ethanol to make the alcohol content 70%, stir well, let it stand for precipitation, take the supernatant and concentrate it under reduced pressure to a relative density of 1.402 (60°C), add 61.5g of starch, and dry it in a drying oven for 9h ( T=70-75° C.) to obtain 378 g of dry matter, add 22.68 g of silicon dioxide, pulverize through a 40-mesh sieve, pack into capsules, and make 1000 grains. (Each gram of capsule is equivalent to 3.375g of crude drug)

Example 3

Take 1.35 kg of eleven herbs such as angelica, extract with water 3 times, each time with 6 times the water consumption, each extraction time is 0.5h, filter, collect the filtrate, concentrate under reduced pressure to a relative density of 1.08 (60°C), and wait for cooling Leave it at room temperature, add ethanol to make the alcohol content 60%, stir well, let it stand for precipitation, take the supernatant and concentrate it under reduced pressure to a relative density of 1.43 (60°C), add 64.8g of starch, and dry in a drying oven for 9 hours (T=65-70° C.), to obtain 375 g of dry matter, add 25.53 g of silicon dioxide, pulverize through a 24-mesh sieve, pack into capsules, and make 1000 grains. (Each gram of capsule is equivalent to 3.215g of crude drug)

Example 4

Take 1.35kg of eleven herbs such as angelica, extract with water once, the water consumption is 10 times each time, and the extraction time is 1.5h each time, filter, collect the filtrate, concentrate under reduced pressure to a relative density of 1.21 (60°C), and wait for cooling Leave it at room temperature, add ethanol to make the alcohol content 80%, stir well, let it stand for precipitation, take the supernatant and concentrate it under reduced pressure to a relative density of 1.38 (60°C), add 67.9g of starch, and dry in a drying oven for 9h (T=75-80° C.), to obtain 371 g of dry matter, add 29.82 g of silicon dioxide, pulverize through a 24-mesh sieve, pack into capsules, and make 1000 grains. (Each gram of capsule is equivalent to 3.185g of crude drug)

Claims (4)

1. A preparation method of Xuefu Zhuyu Capsule, which is characterized in that the following weight ratio Chinese medicinal materials are used as raw materials: Angelica 162g Peach kernel 216g Safflower 162g Achyranthes bidentata 162g Rehmannia 162g Radix Paeoniae Rubra 108g Citrus aurantium 108g Bellflower 81g Chuanxiong 81g Bupleurum 54g Licorice 54g, The preparation method is: extract 11 medicinal herbs such as angelica with water for 1-3 times, use 6-10 times the water consumption, and filter for 0.5-1.5 hours each time, collect the filtrate, concentrate under reduced pressure to a relative density of 1.07-1.35, Measure at 60-70°C, leave it at room temperature, add ethanol to make the alcohol content 60-80%, stir well, let stand for 5-25h to precipitate, take the supernatant and concentrate it under reduced pressure to a relative density of 1.30-1.50, 60 Measured at -70°C, add 10-15% starch in the extract, dry in a drying oven for 5-12h, T=65-80°C, to obtain a dry product, the dry paste rate is 20-28%, add 2% by weight of the dry matter -8% silicon dioxide, packed into capsules, crushed through a 24-60 mesh sieve, each gram of capsule is equivalent to 3.10-3.38g of crude drug. 2. The preparation method of Xuefu Zhuyu Capsules according to claim 1, which is characterized in that the following weight ratio Chinese medicinal materials are used as raw materials: Angelica 162g Peach kernel 216g Safflower 162g Achyranthes bidentata 162g Rehmannia 162g Radix Paeoniae Rubra 108g Citrus aurantium 108g Bellflower 81g Chuanxiong 81g Bupleurum 54g Licorice 54g, The preferred preparation method is: extract 11 medicinal herbs such as angelica with water twice, each time with 8 times the water consumption, each extraction time is 1h, filter, collect the filtrate, concentrate under reduced pressure to a relative density of 1.16-1.19, 60°C Determination, put it at room temperature after cooling, add ethanol to make the alcohol content 70%, stir evenly, let it stand for precipitation, take the supernatant and concentrate it under reduced pressure to a relative density of 1.35-1.43, measure at 60°C, add 15% extract The starch was dried in a drying oven for 9h, T=70-75°C, to obtain a dry matter, the dry paste rate was 23-25%, adding silicon dioxide with a weight of 6% of the dry matter, pulverized through a 40-mesh sieve, and packed into capsules. Made into 1000 capsules, each gram of capsule is equivalent to 3.375g of crude drug. 3. A quality standard for Xuefu Zhuyu Capsules, characterized in that the identification items of peach kernel, citrus aurantium and licorice are set in the quality standard, and each capsule contains paeoniflorin C ₂₃ H ₂₈ O ₁₁ , not less than The quantitative index of 2.20mg. 4. A quality standard for Xuefu Zhuyu Capsules, characterized in that the inspection method set in the quality standard is the identification of peach kernels: take 1g of the content of this product, add 20ml of water to dissolve, add 20ml of ether to extract twice, discard the ether solution , the aqueous solution was extracted twice with 30ml of ethyl acetate, the ethyl acetate solution was collected and evaporated to dryness, and 2ml of methanol was added to obtain the test solution. Take another naringin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the thin-layer chromatography "Chinese Pharmacopoeia" 2005 edition of Appendix VIB test, draw 5-10 μl of each of the above two solutions, respectively spot on the same silica gel G thin-layer plate, and place it with chloroform-methanol-water 45:17:5 The 12-hour lower layer was developed, taken out, dried, sprayed with aluminum trichloride methanol solution and inspected under a 365nm ultraviolet lamp. In the chromatogram of the test product, the same bright fluorescent spots were displayed at the positions corresponding to the reference product; Identification of Citrus aurantium: Take 1g of the contents of this product, add 15ml of methanol and ultrasonically treat it for 10min, filter, evaporate the filtrate to a small volume under reduced pressure, add 0.5g of silica gel 100-160 mesh, mix well, evaporate to dryness, add silica gel column 1 cm in diameter, 25 cm in column length, 5 g of silica gel 100-160 mesh, dry-pack the column, use chloroform: ethyl acetate: methanol: water 15: 40: 22: 10, and place the lower layer at 5-10 ° C for 12 hours as the eluent. Elution, collect 16 ~ 21ml of liquid to obtain the test solution; take another amygdalin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference solution; according to the thin layer chromatography "Chinese Pharmacopoeia" 2005 edition One appendix VIB test, draw 5-10 μl of each of the above two solutions, spot them on the same silica gel G thin-layer plate, develop with eluent as the developer, take it out, dry it, and immediately spray with phosphomolybdic acid solution, phosphorus Add 20ml of water to dissolve 2g of molybdic acid, then slowly add 30ml of sulfuric acid and mix well; heat at 105°C to develop color for about 10 minutes, and in the chromatogram of the test sample, the same dark blue spots appear at the positions corresponding to the reference substance; Identification of licorice: Take 2g of the content of this product, add 30ml of ether, sonicate for 10min, filter, discard the ether solution, add 30ml of methanol to the residue, sonicate for 10min, evaporate the filtrate to dryness under reduced pressure, add 40ml of water to dissolve, and dissolve with n-butyl Alcohol extraction 3 times, each time 20ml combined n-butanol liquid, reduced pressure to dryness, added 5ml of methanol to dissolve, and obtained the test solution; in addition, take monoammonium glycyrrhizinate reference substance, add methanol to make each 1ml containing 1mg solution, as a reference substance solution; according to thin-layer chromatography "Chinese Pharmacopoeia" 2005 edition an appendix VIB) test, draw each 2 ~ 4 μ l of the above two solutions, respectively point on the same high-efficiency silica gel GF ₂₅₄ thin-layer plate, with normal Butanol-20% ammonia water-methanol 5:2:1.5 was developed as a developer, taken out, dried, and inspected under a UV lamp at 254nm. black spots; Determination of content: measure according to high performance liquid chromatography (appendix VI D), chromatographic conditions and system suitability test: take octadecyl bonded silica gel as filler; take acetonitrile-water 16:84 as mobile phase; detection wavelength 230nm. The number of theoretical plates calculated based on the peak of paeoniflorin should not be less than 4300. Preparation of reference substance solution: Weigh an appropriate amount of paeoniflorin reference substance, weigh it accurately, add methanol to make a solution containing 0.1mg per 1ml, and obtain it. Preparation of the test solution: Take 0.3 g of the content of this product, grind it finely, and weigh it accurately. Put 24ml of methanol into a 25ml measuring bottle, sonicate for 10min, let it cool, add methanol to the mark, shake well, filter through a microporous membrane, take the subsequent filtrate and put it in a brown bottle to obtain the product. Determination method: Precisely draw 10 μl each of the reference substance solution and the test solution, inject it into the liquid chromatograph, measure it, and obtain it.

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