Preparation process and quality control method of Zishen Shugan granules

Abstract

The application relates to a preparation process and a quality control method of Zishen Shugan granules, and belongs to the technical field of Chinese medicine preparation and quality control. The Zishen Shugan granules are mainly prepared from prepared rehmannia, Chinese yam, wine jujube, fried jujube seed, cortex moutan, gardenia, white peony root, alisma, angelica, tuckahoe, common wheat, bupleurum, and licorice. The Zishen Shugan granules provided by the application have the advantages of less loss of effective components, completed granules, high product yield, and the quality control method of the granules is provided, so that the quality of the product can be effectively ensured.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation and quality control technology, and relates to a preparation process and quality control method for Zishen Shugan granules. Background Technology

[0002] Perimenopausal syndrome refers to a series of physical and psychological symptoms caused by fluctuations or decreases in sex hormones before and after menopause in women. These symptoms often include hot flashes and sweating, dizziness, tinnitus, irritability, insomnia, and lower back and knee pain. The occurrence of this condition is closely related to the physiological characteristics of women before and after menopause.

[0003] The Zishen Shugan Decoction originates from *Shoushi Xinbian* (Qing Dynasty, Wan Qianzhai). The prescription consists of: 4-5 qian (or 7-8 qian, or more than 2 qian) of Rehmannia glutinosa (processed), 1-2 qian each of Angelica sinensis and Paeonia lactiflora, 3 qian of Ziziphus jujuba seed, 1.5-2 qian of Cornus officinalis, 3-5 qian of Poria cocos, 4-5 qian of Dioscorea opposita, a few fen or more of Bupleurum chinense, 1-2 qian of Gardenia jasminoides, 1-2 qian of Paeonia suffruticosa root bark, and 2 qian of Alisma plantago-aquatica. It is indicated for "dryness and pain in the stomach, upward flow of qi to the left hypochondrium, vomiting of sour fluid, alternating fever and chills, or fever in the heart and abdomen, or hot and dark urine." The effects of Zishen Shugan Yin (Nourishing Kidney Yin and Soothing Liver Yin) can be summarized as nourishing kidney yin and clearing liver heat. Currently, Zishen Shugan Yin is widely used in gynecology, primarily for perimenopausal syndrome, oligomenorrhea, and adolescent acne.

[0004] However, the traditional method of taking Zishen Shugan Yin (a traditional Chinese medicine formula) involves decocting the kidneys and liver, which is not easy to carry and causes inconvenience in medication. Furthermore, there is currently no quality control method for Zishen Shugan preparations, making it impossible to effectively guarantee the quality of medication. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a kidney-nourishing and liver-soothing granule and its preparation process, as well as a quality control method to ensure the quality of the granule.

[0006] The present invention employs the following technical solutions to achieve the above objectives: A kidney-nourishing and liver-soothing granule is mainly prepared from 267g of Rehmannia glutinosa (processed), 178g of Dioscorea opposita, 178g of Cornus officinalis (processed with wine), 267g of Ziziphus jujuba (fried), 178g of Paeonia suffruticosa, 178g of Gardenia jasminoides, 178g of Paeonia lactiflora, 178g of Alisma plantago-aquatica, 178g of Angelica sinensis, 178g of Poria cocos, 534g of Triticum aestivum, 178g of Bupleurum chinense, and 107g of Glycyrrhiza uralensis (processed with honey).

[0007] The preparation method of the kidney-nourishing and liver-soothing granules includes the following steps: Step 1, Extraction: Mix Rehmannia glutinosa, Dioscorea opposita, Cornus officinalis, Ziziphus jujuba var. spinosa, Paeonia suffruticosa, Gardenia jasminoides, Paeonia lactiflora, Alisma plantago-aquatica, Angelica sinensis, Poria cocos, Triticum aestivum, Bupleurum chinense, and Glycyrrhiza uralensis (processed with water) and extract. Filter the extract and keep the filtrate for later use. Step 2, Concentration and Drying: Concentrate the filtrate into a thick paste, and vacuum dry it to obtain a dry paste powder; Step 3, Mixing and Granulation: Mix the dry powder and excipients to obtain a mixture; then add ethanol solution to make a soft material, granulate, vacuum dry, granulate, sieve, mix, and package to obtain the kidney-nourishing and liver-soothing granules.

[0008] In a further preferred embodiment, the amount of water added in step 1 is 8 times the total weight of the medicinal materials.

[0009] In a further preferred embodiment, the decoction is prepared three times in step 1, each time for 1.5 hours.

[0010] More preferably, the vacuum drying conditions in step 2 are: drying at 70℃ for 8 hours and vacuum degree of -0.08~-0.1MPa.

[0011] More preferably, the weight ratio of dry powder to excipients in step 3 is 3:1.

[0012] More preferably, the excipient in step 3 is maltodextrin.

[0013] More preferably, the ethanol solution in step 3 is an ethanol solution with a mass fraction of 93%.

[0014] More preferably, the amount of ethanol solution used in step 3 is 50% of the weight of the mixture.

[0015] More preferably, the vacuum drying conditions in step 3 are: drying at 70℃ for 6 hours and vacuum degree of -0.08~-0.1MPa.

[0016] The present invention also provides a quality control method for the prepared kidney-nourishing and liver-soothing granules, which mainly includes thin-layer chromatography to detect the medicinal materials or active ingredients; and HPLC to detect the content of geniposide.

[0017] Specifically, the thin-layer chromatography detection mainly includes the following: 1. Take Zishen Shugan granules, add water to extract, extract the extract with ethyl acetate, dry the ethyl acetate solution, and redissolve it with methanol to obtain the test solution; take Rehmannia glutinosa reference material and prepare the reference material solution in the same way; spot the test solution and the reference material solution on the same thin layer plate, use chloroform-methanol-water (12:8:1) solution as the developing solvent and anisaldehyde reagent as the color developing agent, heat at 105℃ until the spots are clearly visible, and examine under sunlight. The chromatogram of the test sample shows a spot of the same color at the position corresponding to the main spot of the chromatogram of the reference material. 2. Extract the kidney-nourishing and liver-soothing granules with methanol, dry the extract, and redissolve it in methanol to obtain the test solution; prepare a 1 mg / mL solution of paeoniflorin reference standard with methanol to obtain the reference solution; spot the test solution and the reference solution separately on the same thin-layer plate, using ethyl acetate-formic acid-water (6:1:1) as the developing solvent and anisaldehyde reagent as the colorimetric reagent, heat at 105℃ until the spots are clear, and examine under sunlight; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard; 3. Extract the Zishen Shugan granules with ether, discard the ether, evaporate the residue to dryness, extract with ethyl acetate, dry the extract, and redissolve in methanol to obtain the test solution; take loganin reference standard, add methanol to prepare a 2 mg / mL solution to obtain the reference solution; spot the test solution and the reference solution separately on the same thin-layer plate, using dichloromethane-ethyl acetate-methanol-concentrated ammonia solution (8:1:4:1) as the developing solvent and 5% vanillin sulfuric acid solution as the color developing agent, heat at 105℃ until the spots are clear, and examine under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard; 4. Extract the kidney-nourishing and liver-soothing granules with methanol, dry the extract, redissolve in water, adjust the pH to 1 with hydrochloric acid, extract with water-saturated n-butanol, combine the n-butanol solutions, wash with concentrated ammonia solution, discard the washings, wash again with water-saturated n-butanol solution, discard the washings, combine the n-butanol solutions, dry, redissolve in methanol to obtain the test solution; take an appropriate amount of jujube seed saponin B reference standard, prepare a 1 mg / mL solution with methanol to obtain the reference solution; spot the test solution and the reference solution separately on the same thin-layer plate, use water-saturated n-butanol solution as the developing solvent, use 5% vanillin sulfuric acid solution as the colorimetric reagent, heat at 105℃ until clear, and examine under sunlight; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard; 5. Extract the Zishen Shugan granules with ether, discard the ether solution, extract the residue with methanol, dry the extract, redissolve in water, extract with n-butanol, wash the n-butanol solution with water, discard the aqueous solution, dry the n-butanol solution, redissolve in methanol to obtain the test solution; prepare the reference solution using the same method with licorice reference material; spot the test solution and reference solution separately on the same thin-layer plate, using ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent and 10% sulfuric acid ethanol solution as the color developing agent, heat at 105℃ until the spots are clearly visible, and examine under sunlight; in the chromatogram of the test sample, a spot of the same color appears at the position corresponding to the main spot in the chromatogram of the reference material.

[0018] Specifically, the HPLC method for detecting geniposide content mainly includes the following steps: Preparation of test solution: Add Zishen Shugan granules to the extract, filter the extract to obtain the test solution; Preparation of reference solution: Take geniposide reference standard and add methanol to prepare a 0.1 mg / mL solution, which is the reference solution; Determination: Inject the reference solution and the test solution into the liquid chromatograph separately, and determine the content of geniposide according to the following chromatographic conditions; Chromatographic conditions: Column type C 18 The column was prepared using acetonitrile-0.1% phosphoric acid aqueous solution (9:91) as the mobile phase, with isocratic elution; the flow rate was 1 ml / min; the column temperature was 30℃; and the detection wavelength was 238 nm. Results: The theoretical plate number, calculated based on the geniposide peak, should be no less than 1500; the content of gardenia is mainly geniposide (C... 17 H 24 O 10 The total amount should not be less than 86.93 mg / 15g.

[0019] The present invention has the following beneficial effects: The kidney-nourishing and liver-soothing granules prepared using the preparation method provided by this invention have a high content of effective components such as geniposide and loganin. In the extraction stage, the method described in this invention has a high yield and minimal loss of effective components. In the vacuum drying stage, the drying conditions are optimized to avoid loss of effective components. In the granulation stage, the optimal excipients and granulation process are screened to effectively increase the number of formed granules. Furthermore, the granules in this invention use fewer excipients, saving costs.

[0020] This invention provides a quality control method for Zishen Shugan granules. The identification method is highly specific and reproducible, and can effectively distinguish the characteristic components of key medicinal ingredients such as Bupleurum and Licorice in Zishen Shugan granules, ensuring the authenticity of the ingredients and the stability of the process. The chromatographic conditions have been validated by the methodology, and the Rf values ​​are reproducible, the spots are clear, and the separation is high, which meets the relevant technical requirements of the Chinese Pharmacopoeia. Attached Figure Description

[0021] Figure 1 The results of thin-layer chromatography of Rehmannia glutinosa are as follows: 1, 2, and 3 are samples from three batches of Zishen Shugan Granules; 4 is the control material of Rehmannia glutinosa; and 5 is the negative control material of Rehmannia glutinosa. Figure 2 The results of thin-layer chromatography for Paeonia lactiflora are as follows: 1, 2, and 3 are samples from three batches of Zishen Shugan Granules; 4 is a paeoniflorin reference standard; 5 is a double negative sample of Paeonia lactiflora and Paeonia suffruticosa; 6 is a negative sample of Paeonia lactiflora; and 7 is a negative sample of Paeonia suffruticosa. Figure 3 The results of thin-layer chromatography analysis of roasted jujube seeds are as follows: 1, 2, and 3 are samples from three batches of Zishen Shugan Granules; 4 is the reference standard of jujube seed saponin B; and 5 is a negative sample of roasted jujube seeds. Figure 4The results of thin-layer chromatography analysis of Cornus officinalis were as follows: 1, 2, and 3 were samples from three batches of Zishen Shugan Granules; 4 was a reference standard for strychnoside; and 5 was a negative sample of Cornus officinalis. Figure 5 Thin-layer chromatography results of prepared licorice root, where 1, 2, and 3 are samples from three batches of Zishen Shugan granules, 4 is the reference licorice root, and 5 is a negative sample of prepared licorice root; Figure 6 Example 3 shows the HPLC chromatograms, where A is the chromatogram of geniposide reference standard, B is the gardenia negative sample, and C is the whole formula sample. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.

[0023] Example 1: Preparation process of Zishen Shugan Granules prescription: Prepared Rehmannia Root 267g, Dioscorea Rhizome 178g, Cornus Fruit 178g, Fried Ziziphus Jujuba Seed 267g, Moutan Bark 178g, Gardenia Fruit 178g, White Peony Root 178g, Alisma Rhizome 178g, Angelica Root 178g, Poria 178g, Floating Wheat 534g, Bupleurum Root 178g, Prepared Licorice Root 107g; Granule preparation: Step 1, Extract Take the above 13 kinds of Chinese medicinal herbs, mix them together, add water with 8 times the total weight of the herbs, decoct 3 times, 1.5 hours each time, combine the decoctions, filter, and keep the filtrate for later use; Step 2, Concentration The filtrate was concentrated to a thick paste with a relative density of 1.20~1.25 (70℃); Step 3, Drying The thick paste was vacuum dried for 8 hours at a temperature of 70°C and a vacuum degree controlled at -0.08 to -0.1 MPa to obtain a dry paste powder. Step 4, Mixing The dry powder is pulverized into a fine powder, and maltodextrin is added at a weight ratio of 3:1. The mixture is stirred and mixed for 10-15 minutes to obtain the mixture. Step 5, Granulation Add 93% ethanol to the mixture to make a soft mass. The volume of ethanol added is 50% of the total weight of the mixture. Granulate through a 16-mesh sieve, vacuum dry at 70℃, with the vacuum degree controlled at -0.08~-0.1MPa for 6 hours, granulate through a 12-mesh sieve, sieve again, mix, and package (15g / bag, each 1g is equivalent to 2.8g of medicinal slices) to obtain granules.

[0024] Example 2: Thin-layer chromatographic identification of Zishen Shugan granules 1. Thin-layer chromatography identification of Rehmannia glutinosa: Dissolve 5g of Zishen Shugan granules in 20mL of water, heat under reflux for 30min, centrifuge the extract for 5min, extract the supernatant with ethyl acetate three times, 20mL each time, combine the ethyl acetate extracts, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to obtain the test solution; take 3g of Rehmannia glutinosa reference material and prepare the reference material solution using the same method; according to the formula of Zishen Shugan granules, take the decoction pieces excluding Rehmannia glutinosa, prepare the Rehmannia glutinosa negative sample according to the preparation process, and test according to the test... A negative control solution of Rehmannia glutinosa was prepared by the method of preparing the test solution. 8 μl each of the test solution, the reference herb solution, and the negative control solution were spotted onto the same high-performance silica gel G thin-layer plate. The plate was developed using chloroform-methanol-water (12:8:1) solution as the developing solvent. After development, the plate was removed, dried, sprayed with anisaldehyde reagent, and heated at 105℃ until the spots were clearly visible. Upon examination under sunlight, the chromatogram of the test sample showed spots of the same color at the positions corresponding to the main spots in the chromatogram of the reference herb, and there was no interference from the corresponding negative sample.

[0025] 2. Thin-layer chromatography identification of Paeonia lactiflora: Take 4g of Zishen Shugan granules, add 20mL of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to obtain the test solution; take paeoniflorin reference standard, add methanol to prepare a 1mg / mL solution to obtain the reference solution; weigh the medicinal slices according to the formula of Zishen Shugan granules, and prepare Paeonia lactiflora-Paeonia suffruticosa double negative samples, Paeonia lactiflora negative samples, and Paeonia suffruticosa negative samples according to the preparation process; prepare Paeonia lactiflora-Paeonia suffruticosa double negative control solution, Paeonia lactiflora negative control solution, and Paeonia suffruticosa negative control solution according to the test solution preparation method; test solution 1 μl each of the reference solution and negative control solution were spotted onto the same high-performance silica gel G thin-layer plate. The plate was developed using ethyl acetate-formic acid-water (6:1:1) as the developing solvent. After development, the plate was removed, dried, sprayed with anisaldehyde reagent, and heated at 105℃ until the spots were clear. The plates were then examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample. Both white peony root and peony bark contain paeoniflorin. The negative control solutions of white peony root and peony bark interfered with each other in the identification of paeoniflorin, while the double negative control solution of white peony root and peony bark showed no negative interference, indicating that this method has good identification effectiveness.

[0026] 3. Thin-layer chromatography identification of Cornus officinalis: Take 6g of Zishen Shugan granules, add 20mL of ether and shake to extract twice, discard the ether solution, evaporate the residue to dryness, add 30mL of ethyl acetate, sonicate for 20min, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to obtain the test solution; take loganin reference standard, add methanol to prepare a 2mg / mL solution to obtain the reference solution; according to the formula of Zishen Shugan granules, take the medicinal slices other than Cornus officinalis, prepare the negative sample of Cornus officinalis according to the preparation process, and dissolve the sample according to the test solution. A negative control solution of Cornus officinalis was prepared. 2 μl each of the test solution, reference solution, and negative control solution were spotted onto the same high-performance silica gel G thin-layer plate. The plate was developed using dichloromethane-ethyl acetate-methanol-concentrated ammonia solution (8:1:4:1) as the developing solvent. The plate was then removed, dried, sprayed with 5% vanillin-sulfuric acid solution, and heated at 105℃ until the spots were clear. Upon examination under sunlight, the test sample chromatogram showed spots of the same color at the corresponding positions as the loganin reference chromatogram. The negative control solution caused interference.

[0027] 4. Thin-layer chromatography identification of stir-fried jujube seed: Take 10g of Zishen Shugan granules, add 60mL of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 20mL of water to dissolve the residue, add hydrochloric acid to adjust the pH to 1, extract twice with 20mL of water-saturated n-butanol each time, combine the n-butanol solutions, wash twice with 20mL of ammonia solution each time, discard the washings, wash twice with 20mL of water-saturated n-butanol solution each time, discard the washings, combine the n-butanol solutions, concentrate to dryness, add 1mL of methanol to dissolve the residue, and prepare the test solution; take an appropriate amount of jujube seed saponin B reference standard, and prepare a 1mg / m³ solution with methanol. The solution of L is the reference solution; according to the formula of Zishen Shugan Granules, take the medicinal slices except for the fried jujube seed, and prepare the fried jujube seed negative sample according to the preparation process. Prepare the fried jujube seed negative control solution according to the preparation method of the test solution; spot 2 μl of each of the test solution, reference solution and negative control solution on the same high-performance silica gel G thin layer plate, develop with water-saturated n-butanol solution as the developing solvent, remove, air dry, spray with 5% vanillin sulfuric acid solution, heat at 105℃ until clear, and examine under sunlight; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample; the negative sample has no interference.

[0028] 5. Thin-layer chromatography identification of prepared licorice: Take 8g of Zishen Shugan granules, add 40mL of ether, heat under reflux for 1h, filter, discard the ether liquid, add 40mL of methanol to the residue, heat under reflux for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40mL of water, extract with n-butanol 3 times, 20mL each time, combine the n-butanol extracts, wash with water 3 times, discard the aqueous liquid, evaporate the n-butanol extract to dryness, dissolve the residue in 5mL of methanol to obtain the test solution; prepare the reference solution using the same method with licorice reference material; according to the formula of Zishen Shugan granules, take the medicinal slices other than prepared licorice, and prepare the prepared licorice according to the preparation process. For the licorice negative sample, a licorice negative control solution was prepared according to the test solution preparation method. 2 μl each of the test solution, control solution, and licorice negative control solution were spotted onto the same high-performance silica gel G thin-layer plate. The plate was developed using ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent. The plate was then removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plate was then examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the positions corresponding to the main spots in the chromatogram of the control medicinal material. The negative sample showed no interference.

[0029] Example 3 HPLC detection Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0030] Chromatographic conditions and system suitability test: Column: XTERRA MS C 18 4.6×250mm, 5μm, Waters; using acetonitrile-0.1% phosphoric acid aqueous solution (9:91) as the mobile phase; flow rate 1ml / min; column temperature 30℃; detection wavelength 238nm; theoretical plate number calculated based on geniposide peak should not be less than 1500.

[0031] Preparation of reference solution: Take an appropriate amount of geniposide reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per ml.

[0032] Preparation of the test solution: Take an appropriate amount of Zishen Shugan granules, grind them into a fine powder, weigh 1.4g, place them in a 100 mL brown volumetric flask, add 80% methanol to make up to about 80 mL, stopper tightly, weigh, sonicate (power 300W, frequency 50kHz) for 45 minutes, cool, add 80% methanol to make up to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0033] Determination: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the content of geniposide.

[0034] I. Factors Affecting Formulation Preparation 1. Measurement Items and Methods 1.1 Determination of geniposide and loganin content by HPLC Chromatographic conditions: Column (XTERRA MS C) 18 The column was 4.6 × 250 mm, 5 μm (Waters), with a detection wavelength of 240 nm. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (10:90), the flow rate was 1.0 mL / min, and the column temperature was 35 °C.

[0035] Preparation of reference solutions: Accurately weigh an appropriate amount of geniposide reference standard and add 50% methanol to prepare a solution containing 0.4 mg per mL. Accurately weigh an appropriate amount of loganin reference standard and add 50% methanol to prepare a solution containing 0.1 mg per mL.

[0036] Preparation of the test solution: Accurately pipette 5 mL of the herbal extract into a 25 mL volumetric flask, add 60% methanol solution to make up to volume, shake well, take an appropriate amount of solution and centrifuge (3000 r / min) for 30 min, take the supernatant and filter.

[0037] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0038] 1.2 Determination of Ointment Yield After the test solution is thoroughly mixed, accurately measure 20 mL and place it in an evaporating dish that has been dried to constant weight. Evaporate to dryness in a water bath, then dry at 105℃ for 6 hours. Cool in a desiccator for 30 minutes and quickly and accurately weigh. Calculate the paste yield using the following formula.

[0039] 1.3 Overall Evaluation The concentration of the indicator components and the yield of the ointment were used as evaluation indicators to comprehensively score the results.

[0040] 2. Effects of extraction method on extract yield and concentration of active ingredients We used a single prescription amount of medicinal materials and investigated the effects of different amounts of water, extraction times, and extraction times on the yield of extract and the concentration of active ingredients.

[0041] The results are shown in Table 1 below.

[0042] Table 1 Results of the investigation on extraction methods Based on the above experimental results, the optimal process parameters were 10 times the amount of water, 3 extractions, and 1.5 hours each time. However, considering the capacity of the production equipment and working time, the amount of water was appropriately adjusted, and the process of this invention was determined to be: 8 times the amount of water, 1.5 hours of extraction, and 3 decoctions. A single-fold prescription amount of medicinal materials was taken and extracted according to the optimal process, and the extract was concentrated to 200 mL. The contents of geniposide and loganin in the extract were determined. 20 mL of the extract was taken and the yield of the extract was determined according to the above method. A comprehensive score was then calculated.

[0043] Alternatively, a traditional extraction method was used: A single-prescription amount of medicinal materials was placed in a clay pot and decocted twice, with water added to cover the materials by approximately one inch. The first decoction lasted 35 minutes, and the second decoction lasted 25 minutes. The decoctions were combined, filtered, and concentrated to 200 mL. The concentrations of geniposide and loganin in the extract were determined according to the above method. 20 mL of the extract was taken and the yield of the extract was determined according to the above method. A comprehensive score was then calculated.

[0044] The results are shown in Table 2 below.

[0045] Table 2 Results of different extraction methods The above experimental results show that, compared with the traditional process, the optimized extraction process of this invention can significantly improve the yield of extract. 3. The Influence of Different Formulation Processes on Granules Following the optimized process described above, the medicinal materials were extracted by reflux with water. The extract was filtered, and the filtrate was concentrated. The effects of different drying methods and excipients on granulation were investigated. The properties, particle size, yield, moisture content, and solubility of the granules were evaluated, and a comprehensive score was calculated.

[0046] in, Evaluation of characteristics: The characteristics of the granules are directly observed with the naked eye. 0-4 points are uneven color and serious mottled grains; 5-7 points are relatively uniform color and slight mottled grains; 8-10 points are uniform color and no obvious mottled grains.

[0047] Particle size evaluation: The granulated particles are weighed to obtain the total weight of the particles. The resulting particles are then passed through pharmacopoeia sieves No. 1 and No. 5 in sequence. Particles that cannot pass through sieve No. 1 and those that can pass through sieve No. 5 are considered unqualified particles, weighed, and their particle size is calculated. The remaining particles are qualified particles and are bagged for later use.

[0048] Yield evaluation: Moisture content assessment: Take 3g of qualified granules and use a rapid moisture analyzer at 105℃. The measurement value when the weight loss is less than 1mg every 30s is regarded as the moisture content of the granules.

[0049] Solubility evaluation: Take 10g of each batch of granules and add 200ml of hot water, stirring for 5 minutes. Record the time required for complete dissolution and the state of the solution. After stirring for 5 minutes and standing for 5 minutes, immediately observe the state of the solution. The granules should be completely dissolved or slightly turbid. 1-10 points: sparingly soluble, with obvious turbidity or precipitation, the liquid is viscous and has poor flowability; 11-20 points: soluble, slightly turbid, the liquid is viscous but has good flowability; 21-30 points: granules dissolve rapidly, completely dissolved or slightly turbid, the liquid is clear.

[0050] 3.1 Effect of extract drying temperature on granules Weigh three times the prescribed amount of medicinal materials, add eight times the amount of water, decoct three times, 1.5 hours each time, combine the extracts, filter, concentrate to a thick paste with a relative density of 1.20 (70℃), transfer to a graduated cylinder and make up to the mark, divide into three equal parts, and vacuum dry at 60℃, 70℃ and 80℃ respectively until the moisture content is below 5%, record the drying time and determine the content.

[0051] Table 3 Effect of extract drying temperature on granules The drying times required at 60℃, 70℃, and 80℃ were 11 hours, 8 hours, and 5 hours, respectively. At all three drying temperatures, the extract was thoroughly dried without caking or uneven drying, and the content of key components remained uniform. Considering factors such as efficacy retention, drying efficiency, production cost, material stability, and equipment compatibility, vacuum drying at 70℃ for 8 hours was determined to be the most suitable process. This process effectively avoids the damage to active ingredients and equipment wear caused by drying at 80℃, while also addressing the shortcomings of low drying efficiency and high residual moisture content at 60℃.

[0052] 3.2 Effect of excipients on granules Weigh three times the prescribed amount of medicinal materials, add eight times the amount of water, decoct three times, 1.5 hours each time, combine the extracts, filter, and concentrate to a clear paste with a relative density of 1.1 (70℃). Dry the paste under vacuum at 70℃ for 8 hours to obtain a dry paste powder. Take six parts of the dry paste powder and mix them with excipients at a weight ratio of 3:1 or 4:1, using 93% ethanol as a binder to form a soft material that "can be formed into a ball by hand but crumbles easily upon light pressure." Granulate the mixture through a 16-mesh sieve, dry at 70℃ for 6 hours, and then granulate using a 12-mesh sieve. Evaluate the overall quality of the granules.

[0053] Table 4 Effect of excipients on granules The quality indicators of the granules were improved to varying degrees after the addition of excipients. In the comprehensive score, the top three ratios of dry extract powder to excipient were: maltodextrin 3:1 (97.98 points) > maltodextrin 4:1 (92.05 points) > dextrin 3:1 (91.33 points). The granules with added maltodextrin had a higher comprehensive score than those with other excipients, and the granules obtained with a 3:1 ratio were generally better. Therefore, maltodextrin was determined to be the optimal excipient for the kidney-nourishing and liver-soothing granules.

[0054] 3.3 Effect of ethanol granulation on particle size Weigh three times the prescribed amount of medicinal materials, add eight times the amount of water, decoct three times, 1.5 hours each time, combine the extracts, filter, and concentrate to a clear paste with a relative density of 1.1 (70℃). Dry the paste under vacuum at 70℃ for 8 hours to obtain a dry paste powder. Take six parts of the dry paste powder and add maltodextrin to each part at a weight ratio of 3:1, with a total mass of 50g. Granulate the mixture using ethanol solutions of different concentrations. Use the weight of the granules as the evaluation index. Perform the double sieving method according to the particle size section of the 2020 edition of the Chinese Pharmacopoeia Appendix for Granules. Granules that can pass through sieve No. 1 and those that cannot pass through sieve No. 5 are considered qualified granules.

[0055] Table 5 Effect of ethanol solution on particles As shown in the table, the highest number of shaped particles were prepared under the conditions of 93% ethanol concentration and 25 mL ethanol volume, which meets the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0056] 3.4 Effect of drying process on particles Weigh three times the prescribed amount of medicinal materials, add eight times the amount of water, decoct three times, 1.5 hours each time, combine the extracts, filter, concentrate to a clear paste with a relative density of 1.1 (70℃), and vacuum dry at 70℃ for 8 hours to obtain dry paste powder; pulverize the dry paste powder into fine powder, mix with maltodextrin, and then add 93% ethanol solution (50% of the weight of the mixture) for granulation. Investigate the effects of excipients, drying time, and drying temperature on the granules. The results are shown in Table 6.

[0057] As can be seen from the results in Table 6, for the present invention, the optimal granule forming effect is achieved when the weight ratio of dry powder to maltodextrin is 3:1, the drying temperature is 70℃, and the vacuum drying time is 6h.

[0058] Table 6 Comparison of Factors Affecting Particle Forming II. Quality Study of Zishen Shugan Granules - Thin-Layer Chromatography Identification Three batches of Zishen Shugan Granules were prepared according to the preparation process described in Example 1, and thin-layer chromatography and HPLC were performed according to the following methods.

[0059] 1. Thin-layer chromatography identification of Rehmannia glutinosa: Take 5g of Zishen Shugan granules, add 20mL of water to dissolve, heat under reflux for 30min, centrifuge the extract for 5min, extract the supernatant with ethyl acetate 3 times, 20mL each time, combine the ethyl acetate extracts, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution. Take 3g of prepared Rehmannia glutinosa reference material and prepare a reference material solution using the same method; According to the formula of Zishen Shugan Granules, except for Rehmannia glutinosa slices, Rehmannia glutinosa negative sample was prepared according to the preparation process, and Rehmannia glutinosa negative control solution was prepared according to the test solution preparation method. The thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition) was used. 8 μl each of the above-mentioned test solution (three batches), reference medicinal material solution, and negative control solution were spotted onto the same high-performance silica gel G thin-layer plate. The plate was developed using chloroform-methanol-water (12:8:1) solution as the developing solvent. After development, the plate was removed, dried, sprayed with anisaldehyde reagent, and heated at 105℃ until the spots were clearly visible. Upon examination under sunlight, the chromatogram of the test sample showed spots of the same color at the corresponding positions as the main spots in the chromatogram of the reference medicinal material, with no interference from the corresponding negative sample. Results are shown below. Figure 1 .

[0060] 2. Thin-layer chromatography identification of white peony root: Take 4g of Zishen Shugan granules, add 20mL of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to obtain the test solution; Prepare a 1 mg / mL solution of paeoniflorin reference standard by dissolving it in methanol. According to the formula of Zishen Shugan Granules, the medicinal slices were weighed and prepared according to the preparation process to obtain the double negative samples of white peony root and peony bark, the negative sample of white peony root and the negative sample of peony bark. The double negative control solution of white peony root and peony bark, the negative control solution of white peony root and the negative control solution of peony bark were prepared according to the test solution preparation method. Thin-layer chromatography (TLC) was performed according to the Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502. 1 μl each of the above-mentioned test solution (three batches), reference solution, and negative control solution were spotted separately onto the same high-performance silica gel G TLC plate. Ethyl acetate-formic acid-water (6:1:1) was used as the developing solvent. After development, the plate was removed, dried, sprayed with anisaldehyde reagent, and heated at 105℃ until the spots were clear. The plates were then examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample. Both white peony root and peony bark contain paeoniflorin. The negative control solutions of white peony root and peony bark interfered with each other in the identification of paeoniflorin, while the white peony root-peony bark double negative control solution showed no negative interference, indicating that this method has good identification effectiveness. Results are shown below. Figure 2 .

[0061] 3. Thin-layer chromatography identification of Cornus officinalis: Take 6g of Zishen Shugan granules, add 20mL of ether and shake to extract twice, discard the ether liquid, evaporate the residue to dryness, add 30mL of ethyl acetate, sonicate for 20min, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue to obtain the test solution. Prepare a 2 mg / mL solution of loganin reference standard by dissolving it in methanol. According to the formula of Zishen Shugan Granules, take the medicinal slices except for Cornus officinalis, prepare the Cornus officinalis negative sample according to the preparation process, and prepare the Cornus officinalis negative control solution according to the test solution. The thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition) was followed. 2 μl each of the above-mentioned test solution (three batches), reference solution, and negative control solution were spotted separately onto the same high-performance silica gel G thin-layer plate. The plate was developed using dichloromethane-ethyl acetate-methanol-concentrated ammonia solution (8:1:4:1) as the developing solvent. The plate was removed, dried, sprayed with 5% vanillin-sulfuric acid solution, and heated at 105℃ until the spots were clear. The plates were then examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of loganin reference standard. Interference from the negative sample was observed. Results are shown below. Figure 3 .

[0062] 4. Thin-layer chromatography identification of stir-fried jujube seeds: Take 10g of Zishen Shugan granules, add 60mL of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 20mL of water to dissolve the residue, add hydrochloric acid to adjust the pH to 1, extract twice with 20mL of water-saturated n-butanol each time, combine the n-butanol solutions, wash twice with 20mL of ammonia solution each time, discard the washings, wash twice with 20mL of water-saturated n-butanol solution each time, discard the washings, combine the n-butanol solutions, concentrate to dryness, add 1mL of methanol to dissolve the residue, and use the test solution. Take an appropriate amount of jujube seed saponin B reference standard and prepare a 1 mg / mL solution with methanol to make the reference solution; According to the formula of Zishen Shugan Granules, take the medicinal slices except for the fried jujube seed, and prepare the fried jujube seed negative sample according to the preparation process. Prepare the fried jujube seed negative control solution according to the test solution preparation method. The thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was used. 2 μl each of the above-mentioned test solution (three batches), reference solution, and negative control solution were spotted separately onto the same high-performance silica gel G thin-layer plate. Water-saturated n-butanol solution was used as the developing solvent. The plate was then removed, dried, sprayed with 5% vanillin-sulfuric acid solution, heated at 105℃ until clear, and examined under sunlight. In the chromatogram of the test sample, spots of the same color as those in the chromatogram of the reference sample appeared at the corresponding positions. No interference was observed with the negative control sample. Results are shown below. Figure 4 .

[0063] 5. Thin-layer chromatography identification of prepared licorice: Take 8g of Zishen Shugan granules, add 40mL of ether, heat under reflux for 1h, filter, discard the ether liquid, add 40mL of methanol to the residue, heat under reflux for 1h, filter, evaporate the filtrate to dryness, add 40mL of water to dissolve the residue, extract with n-butanol 3 times, 20mL each time, combine the n-butanol extracts, wash with water 3 times, discard the aqueous liquid, evaporate the n-butanol extract to dryness, add 5mL of methanol to dissolve the residue, and this is the test solution; Prepare a reference herb solution using the same method as for licorice reference material; According to the formula of Zishen Shugan Granules, take the medicinal slices except for roasted licorice root, prepare roasted licorice root negative sample according to the preparation process, and prepare roasted licorice root negative control solution according to the test solution preparation method. The thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition) was used. 2 μl each of the above-mentioned test solution (three batches), reference solution, and prepared licorice negative control solution were spotted separately onto the same high-performance silica gel G thin-layer plate. The plate was developed using ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent. The plate was then removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as the main spots in the chromatogram of the reference medicinal material. No interference was observed with the negative control sample. Results are shown below. Figure 5 .

[0064] III. Quality Study of Zishen Shugan Granules - HPLC Detection 1. Determination of indicator components Zishen Shugan Granules are composed of 13 traditional Chinese medicines, including Rehmannia glutinosa, Dioscorea opposita, and Cornus officinalis. To establish scientific quality control standards, the detection indicators (rehinoside D and verbascoside) of the principal herb, Rehmannia glutinosa, were first evaluated. The theoretical content of rehinoside D was far below the sensitivity threshold of conventional detection methods, and rehinoside D exhibits terminal absorption characteristics, requiring a detection wavelength of 205 nm. This secondary wavelength falls within the short-wavelength ultraviolet region, where solvents and impurities in the sample easily cause absorption, severely interfering with the accuracy of the detection results. The theoretical content of verbascoside was below the lower limit of quantitation of existing detection technologies, making precise quantification impossible and failing to meet the basic requirements for content determination.

[0065] Taking into account various factors, geniposide was selected as the core content determination indicator.

[0066] 2. Optimization of elution conditions Column: XTERRA MS C 18 4.6×250mm, 5μm; Flow rate 1.0 mL / min, column temperature 30℃, injection volume: 10 μl.

[0067] The mobile phases were investigated separately: acetonitrile-0.1% phosphoric acid aqueous solution (11:89), acetonitrile-0.1% phosphoric acid aqueous solution (33:67), and acetonitrile-0.1% phosphoric acid aqueous solution (9:91).

[0068] Results Analysis: When using an elution gradient of acetonitrile-0.1% phosphoric acid aqueous solution (33:67), the solvent elution intensity was too high, causing geniposide to elute rapidly with other components without effective separation, or to be missed due to insufficient retention time. Consequently, no characteristic chromatographic peak of geniposide was detected within the 30-minute detection cycle. After adjusting the elution gradient to acetonitrile-0.1% phosphoric acid aqueous solution (11:89), although a geniposide peak was detected, it overlapped with the chromatographic peaks of subsequent eluent components, and the resolution did not meet the resolution requirement (≥1.5) for component detection in the Chinese Pharmacopoeia, thus failing to achieve accurate quantification of geniposide. When the elution gradient was adjusted to acetonitrile-0.1% phosphoric acid aqueous solution (9:91), there was no interference from other impurity peaks before or after the geniposide peak, no negative interference, and the peak shape was symmetrical with a stable response value, fully meeting the sensitivity and accuracy requirements for detection.

[0069] 2. Investigation of extraction solvent This experiment investigated the use of 50% methanol, 80% methanol, and pure methanol as extraction solvents. HPLC analysis showed that with pure methanol, the characteristic peak response value of geniposide was extremely low, with almost no effective chromatographic peaks appearing, indicating that pure methanol had very poor extraction efficiency for geniposide from the particles. Simultaneously, the extract was noticeably turbid, because pure methanol has a strong dissolving ability for impurities such as polysaccharides and starch in the particles, leading to a large amount of impurities contaminating the extract, interfering with the extraction of the target component and affecting the accuracy of subsequent detection. When using 80% methanol as the extraction solvent, HPLC analysis showed that the geniposide peak was symmetrical, and the extraction area (peak area) was higher than the other two groups, indicating that this concentration resulted in the highest dissolution amount and optimal extraction rate of geniposide. Furthermore, the extract was clear, with no obvious precipitation or turbidity. Therefore, 80% methanol was ultimately determined to be the optimal extraction solvent for pretreatment of geniposide for detection.

[0070] 3. Content determination method Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0071] Chromatographic conditions and system suitability test: XTERRA MS C column 18 The column diameter was 4.6 × 250 mm and the column depth was 5 μm. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (9:91). The flow rate was 1 ml / min. The column temperature was 30 ℃. The detection wavelength was 238 nm. The theoretical plate number calculated based on the geniposide peak should not be less than 1500.

[0072] Preparation of reference solution: Take an appropriate amount of geniposide reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per ml.

[0073] Preparation of test solution: Take an appropriate amount of Zishen Shugan granules, grind them into a fine powder, weigh 1.4g, place them in a 100 mL brown volumetric flask, add 80% methanol to make up to the mark, stopper tightly, weigh, sonicate (power 300W, frequency 50kHz) for 45 minutes, cool, add 80% methanol to make up to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0074] Preparation of Gardenia negative sample solution: According to the formula of Zishen Shugan Granules, take the medicinal slices except for Gardenia and prepare Gardenia negative sample according to the preparation process. Prepare Cornus officinalis negative control solution according to the test solution. Determination: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0075] Each sachet (15g) of this product contains gardenia and geniposide (C). 17 H 24 O 10 The total amount shall not be less than 86.93mg.

[0076] 3.1 Specificity The test sample, reference standard, and negative sample solutions were injected and analyzed under the chromatographic conditions described above. The chromatograms showed the same positions as those for geniposide reference standard, and the chromatographic peaks of the test sample were consistent. Geniposide was not detected in the gardenia negative sample, and there were no other interfering peaks, indicating that this method has strong specificity.

[0077] 3.2 Accuracy Six parallel samples of Zishen Shugan Granules were prepared according to the above-described method for preparing the test solution, and analyzed under the same chromatographic conditions. The recoveries and RSDs were calculated. The results showed that the recoveries of the six samples ranged from 95.20% to 102.27%, with an average recovery of 99.56% and an RSD of 2.84%, which meets the relevant standards of the 2025 edition of the Chinese Pharmacopoeia, indicating that the method for determining geniposide has good accuracy.

[0078] 3.3 Precision Repeatability: Six parallel test solutions of Zishen Shugan Granules were prepared according to the above-described test solution preparation method, and the content and RSD of geniposide were determined according to the above-described chromatographic conditions. The results showed that the content of geniposide in the six samples was relatively consistent, with an RSD value of 0.26%, indicating that the method has good repeatability.

[0079] Intermediate precision: Six parallel test solutions of Zishen Shugan Granules were prepared using different instruments and on different dates according to the above-described test solution preparation method. An Agilent Technologies 1200 high-performance liquid chromatograph (equipped with a VWD variable wavelength ultraviolet detector) was used for determination, with the remaining chromatographic conditions the same as described above. The content of geniposide and the RSD in the test solutions were calculated. The results showed that the determination results of geniposide content in the six samples were consistent, with an RSD of 0.58%, indicating that the method has good intermediate precision. The combined RSD of repeatability and intermediate precision was 1.04%.

[0080] 3.4 Examination of Linear Relationships Preparation of reference stock solution: Take an appropriate amount of geniposide reference standard, accurately weigh it, and add methanol to dilute to volume to obtain a geniposide stock solution with a concentration of 272.718 μg / ml.

[0081] Accurately measure 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml of the above-mentioned geniposide stock solution and place them in 5 ml volumetric flasks. Dilute to the mark with methanol to prepare six different concentrations of geniposide reference solutions. Accurately inject 10 μl of each concentration solution into the high-performance liquid chromatograph (HPLC) and determine the corresponding peak area. Plot a standard curve with the standard injection concentration (μg / ml) as the abscissa (X) and peak area as the ordinate (Y). The regression equation was calculated as: Y = 14536X + 18561, with a correlation coefficient R² = 0.9992. The results show that geniposide exhibits good linearity in the concentration range of 27.272–272.718 μg / mL.

[0082] 3.5 Stability The test solution of Zishen Shugan Granules was prepared according to the above-mentioned test solution preparation method. The peak area of ​​geniposide was measured at 0, 2, 4, 8, 12, 18 and 24 h according to the above-mentioned chromatographic conditions. The peak area was recorded and the RSD value of the peak area at each time point was calculated to be 1.83%, indicating that the method has good stability within 24 h.

[0083] 3.6 Durability 3.6.1 Investigation at different column temperatures Using 30℃ as the baseline column temperature, the chromatographic behavior under conditions of 25℃ and 35℃ (i.e., 30℃±5℃) was further investigated. The results showed that when the column temperature fluctuated within the range of 30℃±5℃, there was no significant difference in the determination of geniposide in the test sample, with an RSD of 1.62%, indicating that the geniposide detection method has good tolerance to column temperature changes.

[0084] 3.6.2 Investigation of different chromatographic columns Spectroscopic columns of the same length and packing particle size (4.6 × 250 mm, 5 μm) but from different suppliers (Shimadzu, Agilent, and Waters) were selected for a robustness study. The results showed that when using columns from different suppliers, there was no significant difference in the determination of geniposide in the test sample, with an RSD of 1.57%, indicating that the geniposide detection method has good robustness.

[0085] 3.6.3 Investigation at different wavelengths Using 238 nm as the reference wavelength, the chromatographic behavior under conditions of 233 nm and 243 nm (i.e., 238 nm ± 5 nm) was further investigated, and the results are shown in Table 6. The data show that when the detection wavelength fluctuates within the range of 238 nm ± 5 nm, there is no significant difference in the determination results of geniposide in the test sample, with an RSD of 0.17%, indicating that this geniposide detection method has good tolerance to wavelength changes.

[0086] 3.6.4 Investigation of different flow velocities Using 1.0 ml / min as the baseline flow rate, the chromatographic behavior was further investigated at flow rates of 0.8 ml / min and 1.2 ml / min (i.e., 1 ml / min ± 0.2 ml / min). The results showed that when the detection flow rate fluctuated within the range of 1 ml / min ± 0.2 ml / min, there was no significant difference in the determination of geniposide in the test sample, with an RSD of 1.08%, indicating that the geniposide detection method has good tolerance to flow rate changes.

[0087] 3.6.5 Investigation of Mobile Phase Ratio Using acetonitrile-0.1% phosphoric acid aqueous solution (9:91, v / v) as the reference mobile phase ratio, the chromatographic behavior under the conditions of 8:92 and 10:90 ratios was further investigated. The results showed that under the three conditions of acetonitrile-0.1% phosphoric acid aqueous solution ratios of 8:92, 9:91, and 10:90, there was no significant difference in the determination results of geniposide in the test sample, with an RSD of 1.68%, indicating that the method for determining geniposide content has good tolerance to fluctuations in the mobile phase ratio.

[0088] 3.6.6 Investigation at different pH values Using pH 3.8 as the baseline, the chromatographic behavior was further investigated under pH 3.6 and pH 4.0 (i.e., pH 3.8 ± 0.2). These pH conditions correspond to 0.09%, 0.1%, and 0.11% phosphoric acid aqueous solutions, respectively. The results showed no significant difference in the determination of geniposide in the test sample under the three phosphoric acid aqueous solution conditions, with an RSD of 1.31%, indicating that the method for determining geniposide content has good tolerance to fluctuations in mobile phase pH.

[0089] 3.7 Content determination in three pilot-scale batches Three batches of Zishen Shugan Granules were taken and three batches of test solutions were prepared according to the above-mentioned test solution preparation method. The solutions were tested according to the above-mentioned chromatographic conditions, and the results are shown in Table 7.

[0090] Table 7. Results of Gardenin Content Detection in Three Pilot-Scale Batches The above test results show that the content of geniposide in the three pilot-scale batches ranged from 107.525 to 109.844 mg / bag, with an average content of 108.667 mg / bag. Considering actual production conditions, the content limit for geniposide is set at 80% of the average content of the three pilot-scale batches, meaning that each bag of this product contains geniposide (C...). 17 H 24 O 10 The total amount shall not be less than 86.93mg.

Claims

1. A kidney-nourishing and liver-soothing granule, characterized in that, Its preparation method includes the following steps: Step 1, Extraction: Mix Rehmannia glutinosa, Dioscorea opposita, Cornus officinalis, Ziziphus jujuba var. spinosa, Paeonia suffruticosa, Gardenia jasminoides, Paeonia lactiflora, Alisma plantago-aquatica, Angelica sinensis, Poria cocos, Triticum aestivum, Bupleurum chinense, and Glycyrrhiza uralensis (processed with water) and extract. Filter the extract and keep the filtrate for later use. Step 2, Concentration and Drying: Concentrate the filtrate into a thick paste, and vacuum dry it to obtain a dry paste powder; Step 3, Mixing and Granulation: Mix the dry powder and excipients to obtain a mixture; then add ethanol solution to make a soft material, granulate, vacuum dry, granulate, sieve, mix, and package to obtain the kidney-nourishing and liver-soothing granules.

2. The kidney-nourishing and liver-soothing granules as described in claim 1, characterized in that, In step 1, the amount of water added should be 8 times the weight of the medicinal materials; decoct 3 times, 1.5 hours each time.

3. The kidney-nourishing and liver-soothing granules as described in claim 1, characterized in that, The vacuum drying conditions in step 2 are: temperature 70℃, drying time 8h, and vacuum degree -0.08~-0.1MPa.

4. The kidney-nourishing and liver-soothing granules as described in claim 1, characterized in that, In step 3, the weight ratio of dry powder to excipients is 3:

1.

5. The kidney-nourishing and liver-soothing granules as described in claim 4, characterized in that, The excipient is maltodextrin.

6. The kidney-nourishing and liver-soothing granules as described in claim 1, characterized in that, In step 3, the ethanol solution is a 93% ethanol solution, and the amount of ethanol solution used is 50% of the weight of the mixture.

7. The kidney-nourishing and liver-soothing granules as described in claim 1, characterized in that, The vacuum drying conditions in step 3 are: temperature 70℃, drying time 6h, and vacuum degree -0.08~-0.1MPa.

8. A quality control method for the kidney-nourishing and liver-soothing granules as described in any one of claims 1-7, characterized in that, The method includes thin-layer chromatography for detecting medicinal materials or active ingredients; and high-performance liquid chromatography (HPLC) for detecting geniposide content.

9. The quality control method as described in claim 8, characterized in that, The thin-layer chromatography method includes the following detection methods: (1) Take the Zishen Shugan granules and extract them with water. Extract the extract with ethyl acetate, dry the ethyl acetate solution, and redissolve it with methanol to make the test solution. Take the Rehmannia glutinosa reference material and prepare the reference material solution in the same way. Spot the test solution and the reference material solution on the same thin layer plate. Use chloroform-methanol-water with a volume ratio of 12:8:1 as the developing solvent and anisaldehyde reagent as the color developing agent. Heat until the spots are clearly visible and examine them under sunlight. (2) Take Zishen Shugan granules and extract with methanol. Dry the extract and redissolve it with methanol to make the test solution. Take paeoniflorin reference standard and add methanol to make a reference solution. Spot the test solution and the reference solution on the same thin layer plate. Use ethyl acetate-formic acid-water with a volume ratio of 6:1:1 as the developing solvent and anisaldehyde solution as the color developing agent. Heat until the spots are clear and examine them under sunlight. (3) Take Zishen Shugan granules and extract with ether, discard the ether liquid, evaporate the residue to dryness, extract with ethyl acetate, dry the extract, redissolve with methanol to obtain the test solution; take loganin reference standard and add methanol to prepare the reference solution; spot the test solution and the reference solution on the same thin layer plate, use dichloromethane-ethyl acetate-methanol-concentrated ammonia solution with a volume ratio of 8:1:4:1 as the developing solvent, use 5% vanillin sulfuric acid solution as the color developing agent, heat until the spots are clear, and examine under sunlight; (4) Take Zishen Shugan granules and extract with methanol. Dry the extract, redissolve with water, adjust the pH to 1 with hydrochloric acid, extract with water-saturated n-butanol, combine the n-butanol solutions, wash with concentrated ammonia solution, discard the washings, wash with water-saturated n-butanol solution, discard the washings, combine the n-butanol solutions, dry, redissolve with methanol to obtain the test solution; take an appropriate amount of jujube seed saponin B reference standard, prepare a 1 mg / mL solution with methanol to obtain the reference solution; spot the test solution and the reference solution on the same thin layer plate, use water-saturated n-butanol solution as the developing solvent, use 5% vanillin sulfuric acid solution as the color developing agent, heat until clear, and examine under sunlight; (5) Take the Zishen Shugan granules and extract with ether, discard the ether liquid, extract the residue with methanol, dry the extract, redissolve with water, extract with n-butanol, wash with water, discard the water liquid, dry the n-butanol liquid, redissolve with methanol to make the test solution; take the licorice reference material to prepare the reference material solution in the same way; spot the test solution and the reference solution on the same thin layer plate, use ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 15:1:1:2 as the developing solvent, use 10% sulfuric acid ethanol solution as the color developing agent, heat until the spots are clearly visible, and examine under sunlight.

10. The quality control method as described in claim 8, characterized in that, The HPLC method for determining the content of geniposide includes the following steps: Preparation of test solution: Add Zishen Shugan granules to the extract, filter the extract to obtain the test solution; Preparation of reference solution: Take geniposide reference standard and add methanol to prepare a 0.1 mg / mL solution, which is the reference solution; Determination: Inject the reference solution and the test solution into the liquid chromatograph separately, and determine the content of geniposide according to the following chromatographic conditions; Chromatographic conditions: Column type C 18 The column was eluted isocratically using an acetonitrile-0.1% phosphoric acid aqueous solution (9:91 v / v) as the mobile phase. Results: The theoretical plate number, calculated based on the geniposide peak, should be no less than 1500; the content of gardenia, calculated as geniposide, should be no less than 86.93 mg / 15g.

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