Jinmai calculus-removing composition as well as preparation and quality detection methods thereof

Abstract

The invention discloses a radix ophiopogonis calculus-removing composition as well as a preparation method and a quality detection method thereof. A preparation method of the composition comprises the following steps: adding water to decoct the sixteen medicines (the talc and the spora lygodii are decocted in bags) twice, adding 8.5 times of water to soak for 30 minutes for the first time, adding 6 times of water to soak for 30 minutes for the second time, decocting for 30 minutes each time, and filtering to obtain a filtrate; and filtering, concentrating the filtrate into thick paste with the relative density of 1.28-1.32 (70 DEG C), drying, crushing, and adding a proper amount of auxiliary materials to prepare a corresponding dosage form, thereby obtaining the traditional Chinese medicine composition. The whole formula of the radix ophiopogonis and radix ophiopogonis calculus-removing composition has the effects of clearing away heat and toxic materials, inducing diuresis for treating stranguria, removing calculus, activating spleen and removing dampness, can remarkably improve clinical symptoms and signs of patients suffering from urolithiasis, is an effective and safe traditional Chinese medicine preparation for treating urinary calculus, and is worthy of popularization and application clinically.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a golden wheat stone-removing composition and a preparation method and a quality detection method thereof. Background Art

[0002] Urinary stones belong to the category of "stone stranguria" in traditional Chinese medicine. The name of stranguria syndrome first appeared in the "Inner Canon of Medicine" with names such as "stranguria" and "stranguria fullness". Zhang Zhongjing attributed its pathogenesis to "heat in the lower part of the body" in "Golden Chamber Synopsis"; Chao Yuanfang's "Treatise on the Causes and Symptoms of Various Diseases" clearly stated: "Stranguria is caused by kidney deficiency and bladder heat" and "stone stranguria, the kidney governs water, and water will turn into stones when it is condensed. Therefore, the kidney is invaded by sand and stone, and kidney deficiency is taken advantage of by heat, and heat will cause stranguria". Chao's pathogenesis analysis with kidney deficiency as the root and bladder heat as the target has important theoretical and practical significance. Most doctors in later generations believed that the basic pathogenesis of the disease at the beginning was damp heat in the lower part of the body. The damp heat accumulated for a long time, boiled the water, and the urine condensed, and accumulated over time into sand and stone. Once stranguria is formed, it itself becomes a pathogenic factor. If stranguria is not cured for a long time, dampness and heat will entrap the spleen, resulting in spleen qi deficiency. At the same time, dampness is a yin evil, which will damage the spleen and kidney yang qi over a long period of time; or it will accumulate and turn into heat, which will burn yin and damage the spleen and kidney qi or kidney yin and yang deficiency. In summary, the cause of stranguria with stones is mainly dampness and heat. In the early stage, it is mostly a syndrome of evil excess, and the treatment is to clear heat and dampness, relieve stranguria and expel stones, and promote qi and remove blood stasis; with the passage of time, it is mostly a syndrome of mixed deficiency and excess, and the treatment should be clearing heat and dampness, relieving stranguria and expelling stones, while either tonifying the spleen and kidney, or nourishing yin and clearing heat.

[0003] There are many treatments for urinary stones, including surgical stone removal and laser lithotripsy, but they are painful and often relapse. Taking Chinese medicine to relieve stranguria and remove stones is still an important method for urinary stones, especially for stones less than 1 cm, which have obvious advantages in Chinese medicine treatment. Based on his own Nine-ingredient Stone Removal Decoction, Professor Xiao Zhenghua optimized the Jinmai Stone Recipe after years of clinical application. In the recipe, the herb Qianqiancao clears heat and dampness, relieves stranguria and removes stones; the herb Dianthus superbus is a diuretic and urinary tract infection, and promotes blood circulation and menstruation. Sea Sand is heat-clearing and dampness-removing, and relieves pain; Chicken’s gizzard lining is stone-clearing and relieves pain; Plantago is heat-clearing and detoxifying, and is diuretic and relieves stranguria; Abutilon seeds, Imperata root, Pyrola, Rehmannia root, Gardenia cools blood and stops bleeding, and is diuretic and relieves stranguria; White Peony relieves pain and relieves urgency; Wine-scented Achyranthes bidentata nourishes the liver and kidneys, guides the downward movement of the medicine, and is combined with talcum powder to smooth the orifices, clear heat and dampness, and relieves diuresis and relieves stranguria; Stir-fried Toosendan fruit and bran-fried Citrus aurantium promote qi and blood circulation, relieves urgency and relieves pain, and is an adjuvant to increase the force of pushing; Licorice relieves urgency and relieves pain, and is a guiding medicine. The whole prescription has the effects of clearing heat and detoxifying, diuretic and relieves stranguria, stone-dispelling and stone-removing, and spleen and dampness.

[0004] Modern pharmacological studies have shown that the flavonoids rich in Lysimachia can bind to calcium ions, reduce the concentration of calcium oxalate, thereby inhibiting the growth of calcium oxalate stones, and make urine acidic, promoting the dissolution of stones in an alkaline environment. Lycium japonicum can dissolve hard stones, improve urinary tract pain, and reduce the growth of calcium oxalate stones in urine. It can also promote the increase of urinary enzyme content in patients, thereby increasing the patient's urine volume, reducing phosphorus, urea and urine calcium, and increasing the frequency of ureteral smooth muscle peristalsis, promoting the discharge of stones. Chicken gizzard contains rich trace elements, which can delay the formation of stones. Pyrrosia has an anti-urinary stone effect, which accelerates stone discharge by promoting water metabolism, increasing urine volume, and enhancing smooth muscle movement. Sake Sichuan Achyranthes bidentata can promote blood circulation and remove blood stasis, lead the medicine downward, and directly act on the ureter to enhance its peristalsis to promote the discharge of urinary stones.

[0005] The Jinmai Stone-Removing Composition provided by the invention has the effects of heat detoxification, diuresis and stranguria relief, stone removal, spleen and dampness-regulating, is an effective and safe traditional Chinese medicine preparation for treating urinary tract stones, and is worthy of promotion and application in clinical practice. Summary of the invention

[0006] The present invention aims to provide a golden wheat stone removal composition.

[0007] Another object of the present invention is to provide a method for preparing the Jinmai Stone Removal Composition.

[0008] Another object of the present invention is to provide a quality detection method of the Jinmai Stone Removal Composition.

[0009] The formula of the golden wheat stone-removing composition of the present invention is composed of the following Chinese medicinal raw materials in parts by weight: 640-680 parts of Lysimachia chinensis, 145-185 parts of Dianthus superbus, 145-185 parts of Lygodium japonicum, 90-130 parts of Gallus gallus domesticus, 200-240 parts of talc, 200-240 parts of Imperatae cyperi, 145-185 parts of Plantaine, 90-130 parts of Achyranthes bidentata, 90-130 parts of stir-fried Toosendan fruit, 120-140 parts of Pyrola, 145-185 parts of Abelomi japonica seed, 145-185 parts of white peony root, 90-130 parts of stir-fried Citrus aurantium with bran, 90-130 parts of Rehmannia root, 58-74 parts of liquorice tablets, and 90-130 parts of Gardenia jasminoides.

[0010] Preferably, the formula of the Jinmai Shi-removing composition of the present invention is composed of the following Chinese medicinal raw materials in parts by weight: 650-670 parts of Lysimachia chinensis, 155-175 parts of Dianthus superbus, 155-175 parts of Lygodium japonicum, 100-120 parts of Gallus gallus domestica, 210-230 parts of talc, 210-230 parts of Imperata cylindrica, 155-175 parts of Plantain, 100-120 parts of Achyranthes bidentata, 100-120 parts of stir-fried Toosendan fruit, 125-135 parts of Pyrrosia odoratus, 155-175 parts of Abelomi japonica seeds, 155-175 parts of white peony root, 100-120 parts of stir-fried Citrus aurantium with bran, 100-120 parts of Rehmannia glutinosa, 62-70 parts of liquorice tablets, and 100-120 parts of Gardenia jasminoides.

[0011] Further preferably, the formula of the Jinmai Shi-removing composition of the present invention is composed of the following Chinese medicinal raw materials in parts by weight: 660 parts of Lysimachia chinensis, 165 parts of Dianthus superbus, 165 parts of Lycopodiella ciliata, 110 parts of Gallus gallus domestica, 220 parts of talc, 220 parts of Imperata cylindrica, 165 parts of Plantain, 110 parts of Achyranthes bidentata, 110 parts of stir-fried Toosendan fruit, 132 parts of Pyrola, 165 parts of Amitamia flavonoids, 165 parts of Paeonia lactiflora, 110 parts of stir-fried Fructus Aurantii Immaturus with bran, 110 parts of Rehmannia glutinosa, 66 parts of Licorice tablets, and 110 parts of Gardenia jasminoides.

[0012] The preparation method of the Jinmai Shi-removing composition of the present invention is as follows: taking a prescribed amount of medicinal materials, talc and sea sand are decocted in a bag, 8-9 times of water are added for the first time to soak for 30-60 minutes, 4-8 times of water are added for the second time, decocted 1-3 times, each time for 30-60 minutes, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.18-1.40 at 70°C, dried, crushed, and added with appropriate amount of auxiliary materials to prepare a corresponding dosage form.

[0013] Preferably, the preparation method of the Jinmai Shi-removing composition of the present invention is: take the formulated medicinal materials, talc and sea sand, decoct them in a bag, add 8.5 times of water for the first time and soak them for 30 minutes, add 6 times of water for the second time, decoct them twice, each time for 30 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.28-1.32 at 70°C, dry, crush, add appropriate amount of auxiliary materials, and make corresponding dosage forms.

[0014] The quality detection method of the Jinmai Shi-removing composition of the present invention comprises thin layer chromatography identification of Lysimachia sibiricum, Rhizoma Cynanchum, stir-fried Toosendan fruit, stir-fried Fructus Aurantii with bran and Fructus Gardeniae, and content determination of Gardenia glycoside and Paeoniflorin.

[0015] The thin layer chromatography identification method of the Lysimachia sibiricum of the present invention is as follows: 3 g of the product is added with 20 ml of 70% ethanol, subjected to ultrasonic treatment for 30 minutes, filtered, and the filtrate is concentrated to 5 ml as a test solution; 3 g of a negative preparation lacking Lysimachia sibiricum is taken separately, and a negative control solution is prepared in the same manner; 1 g of a Lysimachia sibiricum control medicinal material is taken again, and a control medicinal material solution is prepared in the same manner; the thin layer chromatography method (Chinese Pharmacopoeia) is used to identify the Lysimachia sibiricum.

[0016] 2020 Edition of the General Rules for Part IV 0502) test, take 5-8 μl of each of the three solutions, spot them on the same silica gel G thin layer plate, use ethyl acetate-ethanol-water (8:2:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and immediately place under ultraviolet light (365mn) for inspection;

[0017] The thin layer chromatography identification method of the wine-flavored Cyathula is as follows: take 3g of the product, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and add 1ml of methanol to the residue to dissolve it as the test solution; take another 3g of the negative preparation lacking wine-flavored Cyathula, and prepare a negative control solution in the same way; take 2g of the control medicinal material of Cyathula, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), take 8-10μl of the test solution and the negative control solution, and 5μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, use chloroform-methanol (7:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm);

[0018] The thin layer chromatography identification method of the stir-fried Toosendan fruit is as follows: take 10g of the product, add 50ml of water to dissolve it, filter it, extract the filtrate with ethyl acetate twice, 30ml each time, combine the ethyl acetate solution, shake and extract it with ammonia test solution twice, 20ml each time, discard the ammonia test solution, take the ethyl acetate solution, evaporate it to dryness, add 1ml of ethanol to the residue, and use it as the test solution; take 10g of the negative preparation without stir-fried Toosendan fruit, and prepare the negative control solution in the same way; take 2g of Toosendan fruit control medicinal material, add water, 50ml, decoct for 30 minutes, filter, add 30ml of ethyl acetate to the filtrate, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), 10μl of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (5:2) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 2% p-dimethylaminobenzaldehyde-40% sulfuric acid solution, and heated at 105°C until the spots were clearly colored;

[0019] The thin layer chromatography identification method of the bran-fried Citrus aurantium is as follows: take 3g of the product, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation of Citrus aurantium fried without bran, and prepare a negative control solution in the same way; take 0.5g of Citrus aurantium control medicinal material, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), take 2 to 5μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, respectively, with chloroform-methanol-water (13:6:2) lower layer solution as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm);

[0020] The thin layer chromatography identification method of gardenia is as follows: take 3g of the product, add 20ml of 70% methanol, shake for 5 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation lacking gardenia, and prepare a negative control solution in the same way; then take 0.5g of gardenia control medicinal material, and prepare a gardenia control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), 2 to 5μl of each of the above three solutions are absorbed and spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-ethyl acetate-methanol-concentrated ammonia test solution (8:1:3:1) is used as the developing agent. Develop, take out, dry, and spray with 5% vanillin sulfuric acid solution.

[0021] The method for determining the content of gardenia glycoside and paeoniflorin of the present invention is as follows: chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as a filler; acetonitrile-0.1% phosphoric acid gradient elution: 0-22min (13:87), 23-38min (40:60), 39-45min (13:87), flow rate is 1ml / min, column temperature is 30°C, detection wavelength is 230nm, injection volume is 10μl, and the number of theoretical plates calculated based on the paeoniflorin peak should not be less than 2000; preparation of reference solution: take appropriate amount of gardenia glycoside reference substance and paeoniflorin reference substance , accurately weigh, add methanol to prepare a mixed solution containing 50μg and 45μg of gardenia glycoside and paeoniflorin per 1ml as the reference solution, and the result is obtained; Preparation of test solution: take 0.4g of the Jinmai Paishi composition, accurately weigh, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, seal it, weigh the weight, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the result; Sample determination: accurately aspirate 10μl of the reference solution and the test solution respectively, inject them into liquid chromatograph, and determine the result.

[0022] The Jinmai Stone-Removing Composition of the present invention or the Jinmai Stone-Removing Composition prepared by the preparation method is used in preparing medicines for treating urinary stones.

[0023] The parts described in the formulation of the present invention represent mass weight units such as g and kg.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The extraction method of the present invention is scientific and reasonable. According to the comprehensive analysis of prescription research and relevant literature, the main indicator components of the 16 drugs in the formula of the present invention are mostly flavonoid glycosides, triterpenes, and cyclopentane glycosides. The glycoside components have good water solubility and are easily soluble in hot water. Therefore, the prescription is suitable for water extraction. Among them, talc and sea sand need to be decocted as medicine. The team of the present invention optimized the water extraction method through single factor investigation and orthogonal experiment to obtain the best process combination. The content of effective ingredients in the obtained water extract is high and meets the needs of actual operation.

[0026] 2. The prescription of the present invention has significant efficacy. Clinical observation results show that Jinmai Paishi Fang can significantly improve the clinical symptoms and signs of patients with stranguria, such as lumbar and abdominal colic, hematuria, frequent urination, urgency and pain, short and yellow urine, and abnormal urination (P < 0.001). The total effective rate is 95%. This shows that Jinmai Paishi Fang can significantly improve the clinical symptoms and signs of patients with stranguria, and is an effective and safe traditional Chinese medicine preparation for treating patients with stranguria, and is worthy of promotion and application in clinical practice.

[0027] 3. The preparation of the present invention is easy to use. The extracted formula is made into granules, which overcomes the problems of poor taste and inconvenience of decoction compared with the original method of decoction, and can meet the medication needs of patients and is convenient for patients to take.

[0028] 4. The quality inspection method of the present invention is scientific and reasonable. The present invention investigates the identification method of the prepared preparation: qualitative identification is performed on nine medicinal materials in the preparation, including Herba Lysimachiae, Rhizoma Achyranthis Bidentatae, Radix Paeoniae Alba, Fructus Aurantii Immaturus Stir-fried with Glutinous Rice Bran, Fructus Gardeniae, Herba Plantaine, Rhizoma Imperatae, Fructus Toosendan and Fructus Rehmanniae. Among them, the negative control for qualitative identification of four medicinal materials, including Rhizoma Achyranthis Bidentatae, Fructus Aurantii Immaturus Stir-fried with Glutinous Rice Bran, Fructus Toosendan and Fructus Gardeniae, has no interference. After verification by three batches of pilot samples, this method has good repeatability, so it is included in the text of the draft quality standard. The conditions for determining the content of geniposide in Fructus Gardeniae and paeoniflorin in Radix Paeoniae Alba were determined through investigation experiments.

[0029] 5. The present invention inspected the particle size, moisture, filling volume difference and microbial limit of three batches of Jinmai Paishi granules. The results of various inspections showed that this product complies with the relevant provisions of the General Rules 0114 Granules of Part IV of the 2020 Edition of the Chinese Pharmacopoeia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Specific HPLC chromatogram of water extract (where A is the reference solution, B is the test solution, and C is the negative control solution);

[0031] Figure 2 Linear regression plot of paeoniflorin;

[0032] Figure 3 Moisture absorption equilibrium curve of Jinmai Paishi granules;

[0033] Figure 4 Moisture absorption curve of Jinmai Paishi granules;

[0034] Figure 5 TLC results of Lysimachia sibiricum (from left to right are the results obtained by method 1, method 2, and method 3);

[0035] Figure 6 Imperata root TLC result diagram (the left one is the result obtained by method 1, and the right one is the result obtained by method 2);

[0036] Figure 7 TLC result of Rehmannia root (the left one is the result obtained by method 1, and the right one is the result obtained by method 2);

[0037] Figure 8 Gardenia TLC result diagram (from left to right, the results obtained by method 1, the results obtained by method 2 using chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) as the developing solvent, the negative control interference diagram, and the results obtained by method 2 using chloroform-ethyl acetate-methanol-concentrated ammonia test solution (8:1:3:1) as the developing solvent).

[0038] Fig. 9 TLC result of plantain (the left one is the result obtained by method 1, and the right one is the result obtained by method 2);

[0039] Fig.10 The result of TLC of Achyranthes bidentata in Sakagawa;

[0040] Fig.11 White peony root TLC result diagram (from left to right are the results obtained by method 1, method 2, and method 3);

[0041] Fig.12 TLC result diagram of fried Citrus aurantium with bran;

[0042] Fig.13 TLC results of stir-fried Toosendan fruit (the left one is the result obtained by method 1, and the right one is the result obtained by method 2);

[0043] Fig.14 Chromatogram of TLC durability test of Lysimachia chinensis and Achyranthes bidentata in Jinmai Paishi granules;

[0044] Fig.15 Chromatogram of the durability of TLC of fried bitter orange and licorice in Jinmai Paishi granules;

[0045] Fig.16 Full wavelength scanning of Gardenia jasminoides;

[0046] Fig.17 Full wavelength scanning of paeoniflorin;

[0047] Fig.18 The mobile phase was acetonitrile-0.1% phosphoric acid (14:86) with gradient elution chromatogram;

[0048] Fig.19 The mobile phase was acetonitrile-0.1% phosphoric acid (13:87) with gradient elution chromatogram;

[0049] Fig. 20 The mobile phase was acetonitrile-0.1% phosphoric acid (12:88) with gradient elution chromatogram;

[0050] Fig.21 The mobile phase was acetonitrile-0.1% phosphoric acid (11:89) with gradient elution chromatogram;

[0051] Fig. 22 The mobile phase was acetonitrile-0.1% phosphoric acid (10:90) with gradient elution chromatogram;

[0052] Fig.23 The mobile phase was acetonitrile-0.1% phosphoric acid (10:90) with constant-flow chromatogram;

[0053] Fig.24 The mobile phase was acetonitrile-water (10:90) in a constant-flow chromatogram;

[0054] Fig.25 Chromatogram of column temperature investigation experiment (from top to bottom, column temperatures are 25℃, 30℃, and 35℃ respectively);

[0055] Fig.26 Chromatogram of flow rate investigation experiment (flow rates from top to bottom are 0.9ml / min, 1.0ml / min, 1.2ml / min respectively);

[0056] Fig. 27 Specificity test HPLC chromatogram (where a is paeoniflorin, b is the test sample, and c is the negative control);

[0057] Fig.28 Standard curve of geniposide;

[0058] Fig.29 Standard curve of paeoniflorin. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described in detail below through specific embodiments.

[0060] Example 1: Formula of Jinmai Stone Removal Composition

[0061] 660g of Centella asiatica, 165g of Dianthus superbus, 165g of Lycopodiella cernua, 110g of chicken's gizzard lining, 220g of talc, 220g of Imperata cylindrica, 165g of Plantain, 110g of Achyranthes bidentata, 110g of stir-fried Toosendan fruit, 132g of Pyrola, 165g of Amur seed, 165g of Paeonia lactiflora, 110g of stir-fried Citrus aurantium with bran, 110g of Rehmannia glutinosa, 66g of licorice tablets, and 110g of Gardenia jasminoides.

[0062] Example 2: Formula of Jinmai Stone Removal Composition

[0063] 680g of Centella asiatica, 185g of Dianthus superbus, 185g of Lycopodiella cernua, 130g of Gallus gallus domesticus, 240g of Talcum, 240g of Imperata root, 185g of Plantain, 130g of Achyranthes bidentata, 130g of stir-fried Toosendan fruit, 140g of Pyrola, 185g of Amur seed, 185g of Paeonia lactiflora, 130g of stir-fried Citrus aurantium with bran, 130g of Rehmannia root, 74g of Licorice tablets, and 130g of Gardenia jasminoides.

[0064] Example 3: Formula of Jinmai Stone Removal Composition

[0065] 640g of Centella asiatica, 145g of Dianthus superbus, 145g of Lycopodiella cernua, 90g of chicken's gizzard lining, 200g of talc, 200g of Imperata root, 145g of Plantain, 90g of Achyranthes bidentata, 90g of stir-fried Toosendan fruit, 120g of Pyrola, 145g of Amur seed, 145g of Paeonia lactiflora, 90g of stir-fried Citrus aurantium with bran, 90g of Rehmannia root, 58g of licorice tablets, and 90g of Gardenia jasminoides.

[0066] Example 4: Formula of Jinmai Stone Removal Composition

[0067] 670g of Centella asiatica, 175g of Dianthus superbus, 175g of Lycopodiella cernua, 120g of chicken's gizzard lining, 230g of talc, 230g of Imperata root, 175g of Plantain, 120g of Achyranthes bidentata, 120g of stir-fried Toosendan fruit, 135g of Pyrola, 175g of Amur seed, 175g of Paeonia lactiflora, 120g of stir-fried Citrus aurantium with bran, 120g of Rehmannia root, 70g of licorice tablets, and 120g of Gardenia jasminoides.

[0068] Example 5: Formula of Jinmai Stone Removal Composition

[0069] 650g of Centella asiatica, 155g of Dianthus superbus, 155g of Lycopodiella cernua, 100g of chicken's gizzard lining, 210g of talc, 210g of Imperata cylindrica, 155g of Plantain, 100g of Achyranthes bidentata, 100g of stir-fried Toosendan fruit, 125g of Pyrola, 155g of Amur seed, 155g of white peony root, 100g of stir-fried Citrus aurantium with bran, 100g of Rehmannia glutinosa, 62g of licorice tablets, and 100g of Gardenia jasminoides.

[0070] The formula of the Jinmai Paishi composition of Examples 1-5 is used in the following preparation method of the embodiment

[0071] Example 6 Preparation method of Jinmai Paishi composition

[0072] Take the prescribed amount of medicinal materials, talc and sea sand, wrap and decoct, add 8.5 times the water for the first time and soak for 30 minutes, add 6 times the water for the second time, decoct twice, each time for 30 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.28 at 70°C, add the auxiliary material microcrystalline cellulose according to the ratio of extract: auxiliary material of 1:0.7, add 2 g of stevioside, mix well, make into granules, boil and dry at 70°C, and make 1000 g.

[0073] Example 7 Preparation method of Jinmai Paishi composition

[0074] Take the prescribed amount of medicinal materials, talc and sea sand, wrap and decoct, add 8.5 times the water for the first time and soak for 30 minutes, add 6 times the water for the second time, decoct twice, each time for 30 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.32 at 70°C, add the auxiliary material microcrystalline cellulose according to the ratio of extract: auxiliary material of 1:0.75, add 2 g of stevioside, mix well, make into granules, boil and dry at 80°C, and make 1000 g.

[0075] Example 8 Preparation method of Jinmai Paishi composition

[0076] Take the prescribed amount of medicinal materials, talc and sea sand, wrap and decoct, add 9 times the water for the first time and soak for 60 minutes, add 8 times the water for the second time, decoct 3 times, each time for 60 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.40 at 70°C, add the auxiliary material microcrystalline cellulose at a ratio of 1:1 between the extract and the auxiliary material, add 2.5 g of stevioside, mix well, make into granules, boil and dry at 80°C, and make into 1000 g.

[0077] Example 9 Preparation method of Jinmai Paishi composition

[0078] Take the prescribed amount of medicinal materials, talc and sea sand, wrap and decoct, add 8 times the water for the first time and soak for 30 minutes, add 6 times the water for the second time, decoct once, each time for 45 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.18 at 70°C, add the auxiliary material microcrystalline cellulose at an extract: auxiliary material ratio of 1:0.6, add 1.5 g of stevioside, mix well, make into granules, boil and dry at 60°C, and make 1000 g.

[0079] The Jinmai Paishi composition prepared in Examples 6-9 was used in the following quality testing methods

[0080] Example 10 Quality testing method of Jinmai Paishi composition

[0081] (1) Thin layer chromatography identification of Lysimachia chinensis, Achyranthes bidentata, stir-fried Toosendan fruit, stir-fried Citrus aurantium, and Gardenia jasminoides

[0082] The thin layer chromatography identification method of Centella asiatica is as follows: take 3g of this product, add 20ml of 70% ethanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation lacking Centella asiatica, and prepare a negative control solution in the same way; take 1g of Centella asiatica control medicinal material, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), 5 to 8μl of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, with ethyl acetate-ethanol-water (8:2:1) as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and immediately placed under ultraviolet light (365mn) for inspection.

[0083] The thin layer chromatography identification method of wine-flavored Cyathula is as follows: take 3g of this product, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 1ml of methanol to the residue to dissolve it, and use it as the test solution; take another 3g of the negative preparation lacking wine-flavored Cyathula, and prepare the negative control solution in the same way; take 2g of the control medicinal material of Cyathula, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the "Chinese Pharmacopoeia"), take 8-10μl of the test solution and the negative control solution, and 5μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, use chloroform-methanol (7:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0084] The thin layer chromatography identification method of stir-fried Chuanlianzi is as follows: take 10g of this product, add 50ml of water to dissolve it, filter it, extract the filtrate with ethyl acetate twice, 30ml each time, combine the ethyl acetate solution, shake and extract it with ammonia test solution twice, 20ml each time, discard the ammonia test solution, take the ethyl acetate solution, evaporate it to dryness, add 1ml of ethanol to the residue as the test solution; take another 10g of the negative preparation without stir-fried Chuanlianzi, and prepare the negative control solution in the same way; take 2g of Chuanlianzi control medicinal material, add 50ml of water, and extract it with ethyl acetate twice, 30ml each time, discard the ammonia test solution, take the ethyl acetate solution, evaporate it to dryness, add 1ml of ethanol to the residue, and use it as the test solution; take another 10g of the negative preparation without stir-fried Chuanlianzi, and prepare the negative control solution in the same way; take another 2g of Chuanlianzi control medicinal material, add 50ml of water, and extract it with ethyl acetate twice, 20ml each time, discard the ammonia test solution, take the ethyl acetate solution, evaporate it to dryness, add 1ml of ethanol to the residue, and use it as the test solution; take another 10g of the negative preparation without stir-fried Chuanlianzi, and prepare the negative control solution in the same way; take another 2g of Chuanlianzi control medicinal material, add 50ml of water, and use the negative control solution in the same way; 0ml, decocted for 30 minutes, filtered, 30ml of ethyl acetate was added to the filtrate, and a control medicinal solution was prepared in the same way; according to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), 10μl of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (5:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 2% p-dimethylaminobenzaldehyde-40% sulfuric acid solution, and heated at 105°C until the spots were clearly colored.

[0085] The thin layer chromatography identification method of fried Citrus aurantium with bran is as follows: take 3g of this product, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation of fried Citrus aurantium without bran, and prepare the negative control solution in the same way; take 0.5g of Citrus aurantium control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the "Chinese Pharmacopoeia"), take 2-5μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, use chloroform-methanol-water (13:6:2) lower layer solution as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm).

[0086] The thin layer chromatography identification method of Gardenia is as follows: take 3g of this product, add 20ml of 70% methanol, shake for 5 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation lacking Gardenia, and prepare the negative control solution in the same way; then take 0.5g of Gardenia control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the "Chinese Pharmacopoeia"), take 2-5μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, and use chloroform-ethyl acetate-methanol-concentrated ammonia test solution (8:1:3:1) as the lower layer solution as the developing agent, develop, take out, dry, and spray with 5% vanillin sulfuric acid solution.

[0087] (2) Determination of the content of geniposide and paeoniflorin

[0088] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; acetonitrile-0.1% phosphoric acid gradient elution: 0-22min (13:87), 23-38min (40:60), 39-45min (13:87); flow rate 1ml / min; column temperature 30℃; detection wavelength 230nm; injection volume 10μl. The theoretical plate number calculated based on the paeoniflorin peak should not be less than 2000.

[0089] Preparation of reference solution: Take appropriate amount of gardenia glycoside reference substance and paeoniflorin reference substance, weigh accurately, add methanol to prepare a mixed solution containing 50μg gardenia glycoside and 45μg paeoniflorin per 1ml as the reference solution.

[0090] Preparation of test solution: take 0.4g of the Jinmai Paishi composite material, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper it, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0091] Determination: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0092] Each bag of this product contains Gardenia jasminoides (C 17 H 24 O 10 ) and paeoniflorin (C 23 H 28 O 11 ) shall not be less than 6.4mg and 10.4mg. In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:

[0093] 1. Screening of process conditions

[0094] (I) Water extraction process research

[0095] According to clinical medication habits, water is used for extraction. Factors that affect the water extraction process include water absorption rate of the pieces, soaking time, water addition multiple, number of decoctions and decoction time. The following will use the content of paeoniflorin as the evaluation index. Under the condition that other conditions remain unchanged, a single factor investigation will be carried out on soaking time, extraction time, water addition amount and number of extractions. Combined with the results of the single factor test, L9(3) was used to evaluate the water absorption rate of the pieces. 4 Orthogonal experiments were performed to screen out the optimal extraction process conditions.

[0096] 1. Instruments and reagents

[0097] 1.1 Instrument

[0098] Electronic balance (TP-320C, Xiangyi Balance Instrument Equipment Co., Ltd.), electronic balance (YH-A100001, Shanghai Yueheng Industrial Co., Ltd.), decoction pot, high performance liquid chromatograph (Agilent 1260, Agilent Technologies Inc., USA), electronic balance (AUW220D, Shimadzu Philippines Factory), centrifuge (H2-16K, Hunan Kecheng Instrument Equipment Co., Ltd.).

[0099] 1.2 Drug testing

[0100] Herba Lysimachiae, Herba Dianthus Superbus, Herba Lygodii, Gallinae Gallifoliae, Talc, Rhizoma Imperatae, Herba Plantaine, Herba Cynoglossi, Fructus Toosendan, Fructus Abutilii, Fructus Alba, Fructus Aurantii Immaturus, Radix Rehmanniae, Glycyrrhizae Sinensis Tablets, and Fructus Gardeniae were purchased from Guizhou Tongjitang Chinese Medicine Pieces Co., Ltd. with batch numbers of 230105, 230524, 211202, 230428, 211122, 221124, 221207, 221120, 20221101, 230129, 230517, 230413, 221217, 230115, 230317, and 230113) all comply with the standards of Part I of the 2020 edition of the Chinese Pharmacopoeia; peony inoside reference substance (batch number: 110736-202145, content 94.6%) was purchased from the China Food and Drug Inspection Institute; acetonitrile (chromatographic grade, TEDIA, USA); water (pure water, manufactured by Guiyang Wahaha Food Co., Ltd.); phosphoric acid (HPLC grade, Tianjin Comeo Chemical Reagent Co., Ltd.); Jinmai Paishi water extract was homemade by the laboratory of the Second Affiliated Hospital of Guizhou University of Chinese Medicine.

[0101] 2 Methods and Results

[0102] 2.1 Screening of inspection indicators

[0103] According to the positioning of the "king, minister, assistant and messenger" of drugs in traditional Chinese medicine compound preparations, the index components of the king drug Lysimachia chinensis and Dianthus superbus in Jinmai Paishi granules were examined. The index components of Lysimachia chinensis are mainly quercetin and kaempferol. Quercetin and kaempferol are widely present in dicotyledonous plants as flavonoids. Therefore, they are present in the herbs of Lysimachia chinensis, Dianthus superbus, fried Toosendan fruit, and Pyrrosia odoratus in this prescription. Therefore, it is impossible to conduct a specific investigation on the king drug Lysimachia chinensis and Dianthus superbus. The main component of the minister drug white peony is paeoniflorin, and the terpenoid components are easily soluble in hot water. The test shows that paeoniflorin can be used as a content determination indicator to evaluate the extraction process.

[0104] 2.2 Determination of paeoniflorin content in Jinmai Paishi water extract

[0105] 2.2.1 Chromatographic conditions and system suitability test: Chromatographic column Welch Ultimate XB-C18 (250mm×4.6mm, 5μm); acetonitrile-0.1% phosphoric acid gradient: 0~14min(16:84), 15~30min(30:70), 31~35min(16:84); column temperature 30℃; detection wavelength 230nm; injection volume 10μl. The theoretical plate number calculated based on the paeoniflorin peak should not be less than 2000.

[0106] 2.2.2 Solution preparation

[0107] 2.2.2.1 Preparation of reference solution

[0108] Accurately weigh an appropriate amount of paeoniflorin reference substance and add methanol to prepare a reference substance solution of about 60 μg / ml.

[0109] 2.2.2.2 Preparation of test solution

[0110] Accurately measure 1 ml of the water extract, place it in a 10 ml volumetric flask, add dilute ethanol to the scale, shake well, filter, and filter the filtrate through a 0.45 μm microporous filter membrane to obtain the product.

[0111] 2.2.2.3 Preparation of negative control solution

[0112] Accurately measure 1 ml of the Jinmai Shi-pai Yin aqueous extract lacking white peony root, and prepare it according to the method under "2.2.2.2".

[0113] 2.2.3 Exclusivity

[0114] Accurately measure the above-mentioned reference solution, test solution, and negative control solution respectively, and measure them according to the chromatographic conditions. The results are shown in Figure 1 .

[0115] 2.2.4 Investigation of linear relationship

[0116] Weigh 12.11 mg of paeoniflorin standard using a 1 / 10,000 electronic balance, dissolve in methanol and dilute to volume in a 20 ml volumetric flask to obtain a paeoniflorin reference solution with a mass concentration of 0.5728 mg / ml. Take 4 10 ml volumetric flasks, add 1 ml, 2 ml, 3 ml, and 5 ml of the paeoniflorin reference solution, respectively, and dilute to volume with methanol to obtain a series of concentration reference solutions of 57.28, 114.56, 171.84, and 286.40 μg / ml. Pipette 2 ml and 5 ml of the paeoniflorin reference solution with a mass concentration of 57.28 μg / ml into 2 10 ml volumetric flasks, dilute to volume with methanol, and obtain a series of concentration reference solutions of 11.46 and 28.64 μg / ml. Accurately pipette 10 μl of the above series of concentration reference solutions into the liquid chromatograph for determination. The results are shown in Table 1, and the linear relationship is shown in Table 1. Figure 2 .

[0117] Table 1 Linear data of paeoniflorin

[0118]

[0119] Linear regression equation of paeoniflorin: Y=1299.1x-29.005, R 2 =0.9993.

[0120] The results showed that the linear relationship of paeoniflorin was good in the range of 0.115μg~2.864μg.

[0121] 2.2.5 Precision test

[0122] The paeoniflorin reference substance with a concentration of 57.28 μg / ml was accurately aspirated and injected six times continuously. The results are shown in Table 2.

[0123] Table 2 Results of precision study of paeoniflorin

[0124]

[0125] Results: The average peak area of ​​paeoniflorin was 719.1, and the RSD value was 0.24%, indicating that the instrument had good precision.

[0126] 2.2.6 Repeatability test

[0127] Accurately pipette 6 portions of Jinmai Paishi Fang water extract of the same concentration, 1 ml each, and prepare according to the preparation method of "2.2.2.2", and inject and measure. See Table 3.

[0128] Table 3 Extraction solution repeatability test results

[0129]

[0130] Results: The average content of paeoniflorin in the extract of Jinmai Paishi prescription was 0.71 mg / ml, and the RSD value was 0.30%, indicating that this preparation method had good repeatability.

[0131] 2.2.7 Stability study

[0132] Take the same sample solution and inject it at different times at room temperature for determination. See Table 4.

[0133] Table 4 Extract stability test results

[0134]

[0135] Results: The average peak area of ​​paeoniflorin within 14 hours was 890.6, and the RSD value was 0.33%, indicating that the Jinmai Paishi Fang extract was stable at room temperature within 14 hours.

[0136] 2.2.8 Sample recovery test

[0137] Preparation of reference solution: Accurately weigh 14.68 mg of paeoniflorin reference substance, place it in a 20 ml volumetric flask, add methanol to dissolve and dilute to the mark to prepare a 0.6944 mg / ml reference substance stock solution. Accurately measure 5 ml of the stock solution and place it in a 10 ml volumetric flask, add methanol to prepare a 0.3472 mg / ml reference substance solution.

[0138] Accurately pipette 0.5 ml of Jinmai Paishi Fang extract with known content, a total of 6 portions, accurately add 1 ml of paeoniflorin reference substance (C = 0.3472 mg / ml) to each, add dilute ethanol to make up to 10 ml, shake well to prepare 6 sample solutions, and inject for determination. The results are shown in Table 5.

[0139] Table 5 Extraction solution recovery

[0140]

[0141] Results: The recovery rates of paeoniflorin were between 99.32% and 101.17%, and the average recovery rate was 100.33%, indicating that the method had good accuracy.

[0142] 2.3 Investigation of water absorption rate of medicinal materials

[0143] Weigh 253g of the slices according to the prescription ratio, place in a 6000ml stainless steel pot, add 2500ml of water to soak, and after the slices are completely soaked, pour out all the unabsorbed solvents, and calculate the water absorption rate according to water absorption rate (%) = [water addition amount (ml) - filtrate amount (ml)] / medicinal material amount (g) × 100%. Repeat 3 times. The results are shown in Table 6.

[0144] Table 6 Water absorption test results of decoction pieces

[0145]

[0146] From Table 6, we know that the average water absorption rate of the slices is 246.9%. For the sake of convenience, the water absorption rate is calculated as 2.5 times. Therefore, 2.5 times the weight of the slices should be added during the first water extraction.

[0147] 2.4 Investigation of immersion time

[0148] Take 126.5g of 1 / 2 times the clinical prescription amount of medicinal materials, a total of 12 portions, add 6 times the amount of water, soak for 0h, 0.5h, 1h, 1.5h, 2h and 2.5h, decoct twice, 30 minutes each time, filter, concentrate the liquid, and make up to 250ml to obtain the extract. Prepare the test solution according to the method under "2.1.2.3", inject it into the liquid chromatograph for detection, and investigate the effect of different soaking times on the content of paeoniflorin in the water extract of Jinmai Paishi. The results are shown in Table 7.

[0149] Table 7 The results of the investigation on the content of paeoniflorin in the water extract of Jinmai Paishi at different soaking times (n=2)

[0150]

[0151] The results showed that there was no significant difference in the content of paeoniflorin after soaking for 30 minutes. According to the need of saving time and cost, the best soaking time was determined to be 30 minutes.

[0152] 2.5 Decoction time study

[0153] Weigh the medicinal material slices according to the 1 / 2 prescription ratio, first add 6 times the amount of water to soak for 30 minutes, then decoct for 30, 45, 60, 90, and 120 minutes respectively, decoct twice, filter, concentrate the filtrate and make up to 250ml. Prepare the test solution according to the method under "2.1.2.3", inject it into the liquid chromatography instrument for determination, and investigate the effect of different decoction time on the content of paeoniflorin. See Table 8.

[0154] Table 8 The results of the investigation on the content of paeoniflorin in the water extract of Jinmai Paishi at different decoction times (n=2)

[0155]

[0156] The results showed that after 30 minutes of decoction, the content of paeoniflorin gradually decreased. Considering pharmacoeconomics, the extraction time was selected as 30 minutes, 45 minutes, and 60 minutes.

[0157] 2.6 Investigation of water addition

[0158] Weigh the medicinal pieces according to the 1 / 2 prescription ratio, add 4, 6, 8, and 10 times of water in accordance with the water absorption rate of the medicinal materials, soak for 30 minutes, decoct twice, each time for 30 minutes, filter the filtrate, concentrate and adjust the volume to 250 ml, prepare the test solution, inject and measure, and investigate the effect of the amount of water added on the content of paeoniflorin in the Jinmai Paishi water extract. See Table 9.

[0159] Table 9 Effect of water addition on the content of paeoniflorin in Jinmai Paishi water extract (n=2)

[0160]

[0161] The results showed that the content of peony glycosides was the highest when the amount of water added was 6 and 8 times. However, the more times the water was added, the longer the concentration time was. After comprehensive consideration, the final material-liquid ratio was selected to be 6, 8, and 10 times.

[0162] 2.7 Investigation of decoction frequency

[0163] Weigh the medicinal material slices according to the 1 / 2 prescription ratio, add 6 times the amount of water when the water absorption rate of the medicinal material is met, soak for 30 minutes, decoct for 1, 2, 3, and 4 times, 30 minutes each time, filter, and concentrate the filtrate to 250ml. Prepare the test solution according to the method under "2.1.2.3", inject it into the liquid chromatograph for detection, and investigate the effect of different decoction times on the content of paeoniflorin. See Table 10.

[0164] Table 10 Results of investigation on the number of decoctions (n=2)

[0165]

[0166] The results showed that there was no difference in the content of paeoniflorin after decoction for two times. Therefore, after comprehensive consideration, the extraction times were selected as 1, 2, and 3 times.

[0167] 2.8 Orthogonal experiment and result analysis

[0168] Taking the content of paeoniflorin as the evaluation index, based on the results of the single factor test, about 126.5 g of medicinal materials were weighed according to the 1 / 2 prescription ratio, a total of 18 portions, and the extraction time, water addition amount, and extraction times were used as the investigation factors. L9(3) 4 Orthogonal experiment (factor levels see Table 11) was used to screen the optimal extraction process parameters. The results are shown in Tables 12 and 13.

[0169] Table 11 Orthogonal test factor level table

[0170]

[0171] Table 12 Orthogonal test results (n = 2)

[0172]

[0173] Note: K is the mean value of paeoniflorin; R is the range.

[0174] Table 13 Results of variance analysis of paeoniflorin extract

[0175]

[0176] Note: F0.05(1,2)=19.00.

[0177] Through the orthogonal test results (Table 12), it can be intuitively concluded that the influence of the three factors on the content of paeoniflorin is C, A, and B, and the best combination is A3B2C3. Through variance analysis (Table 13), it can be known that the number of extractions has a significant difference (P < 0.05). Based on the actual clinical application, it is clear that the optimal combination of water extraction process is A1B1C2, that is, adding 6 times of water for decocting twice, each time for 30 minutes.

[0178] 2.9 Water extraction process verification experiment

[0179] The optimal extraction process was prepared in parallel for 3 times, and the verification results can be seen in Table 14. The results showed that the RSD of the paeoniflorin content of the 3 batches of verification samples was below 3.0%, which indicated that the extraction process was stable, could be repeated, and was suitable for large-scale production.

[0180] Table 14 Extraction process validation test results

[0181]

[0182]

[0183] In summary, through single factor and orthogonal test investigations, the water extraction process conditions of Jinmai Paishi granules were determined as follows: prescription 16 (talc, sea sand wrapped in decoction), add 8.5 times the water for the first time to soak for 30 minutes, add 6 times the water for the second time, decocted twice, each time for 30 minutes.

[0184] (II) Research on formulation molding process

[0185] 1. Instruments and Materials

[0186] Electronic balance (TP-320C, Xiangyi Balance Instrument Equipment Co., Ltd.), electronic balance (YH-A100001, Shanghai Yueheng Industrial Co., Ltd.), electronic balance (Shimadzu Philippines Factory, model AUW220D), electronic balance (Shanghai Jinghai Instrument Co., Ltd., model FA2204N), electric blast drying oven (Tianjin Test Instrument Co., Ltd., model 101-2AB), moisture meter (Ohaus Instrument (Changzhou) Co., Ltd., model MB27ZH), temperature-attached specific gravity bottle 25ml (Shanghai Xinyi Instrument Factory Co., Ltd.), pharmacopoeia sieve (Zhejiang Shangyu Jinding Standard Sieve Factory). Soluble starch (Anhui Shanhe Pharmaceutical Excipients Co., Ltd., 210401), dextrin (Guangrao Lifeng Biotechnology Co., Ltd., 2022062301), corn starch (Qufu Tianli Pharmaceutical Excipients Co., Ltd., 20230227), microcrystalline cellulose (Anhui Shanhe Pharmaceutical Excipients Co., Ltd., B220925), stevioside (Qufu Shengren Pharmaceutical Co., Ltd., 20230201), medicinal materials (Guizhou Tongjitang Chinese Medicine Co., Ltd.).

[0187] 2 Methods and Results

[0188] This experiment adopts mechanical wet granulation, first prepares Jinmai Paishi extract, then adds a certain proportion of auxiliary materials to mix, and makes soft material, with the degree of "holding into a ball, kneading and loosening", the soft material is passed through a 14-mesh screen to make wet granules, dried at 70-80℃, the moisture content of the granules is controlled at 3-5%, and passed through a 10-mesh screen for whole granules. The following is an investigation from five aspects: the selection of auxiliary materials, the ratio of extract to auxiliary materials, the relative density of the extract, the drying temperature and method, and the amount of flavoring agents. The quality of Jinmai Paishi granules is evaluated in terms of soft materials and granulation conditions, formability, moisture absorption rate and fluidity, and the best forming process of Jinmai Paishi granules is selected.

[0189] 2.1 Evaluation indicators

[0190] 2.1.1 Determination of Forming Rate: According to the requirements for particle size under the granules (General Rule 0104), qualified granules are those that can pass through the No. 1 sieve and cannot pass through the No. 5 sieve. Granule Forming Rate (%) = Mass of qualified granules / Total mass of granules before sieve × 100%.

[0191] 2.1.2 Determination of moisture absorption rate: Place about 1g of particles at the bottom of a weighing bottle that has been dried to constant weight, shake gently to make it evenly distributed, weigh accurately, remove the weighing bottle cap, place it in a desiccator (with saturated sodium chloride solution at the bottom), place it for 72 hours, take it out, weigh it immediately and calculate the moisture absorption rate. The calculation formula is as follows:

[0192]

[0193] 2.1.3 Determination of the angle of repose: The fixed funnel method is used to determine the angle of repose. Take qualified particles and let them fall naturally from a funnel at a certain height onto a watch glass. When all the particles have been drained, the height formed with the plane is the cone height (h), and the diameter of the cone is the diameter of the vertebra (d). According to the trigonometric function α = degrees (atan (2h / d)), the angle of repose α is calculated. The smaller α is, the better the fluidity.

[0194] 2.2 Preparation of extract

[0195] Weigh 3 times the amount of the prescription medicinal material slices, about 1265g, add 6 times the amount of water, decoct twice, 30 minutes each time, filter, and concentrate the filtrate to an extract with a relative density of 1.28 (70℃). Seal and store in an environment below 10℃.

[0196] 2.3 Screening of different types of excipients

[0197] The investigation was mainly based on single-flavor excipients to screen the most suitable excipients. Weigh 20g of Jinmai Paishi extract under "2.2" for a total of 4 portions, mix them evenly with corn starch, dextrin, soluble starch, and microcrystalline cellulose respectively, and wet granulate them. The soft material and granulation conditions, particle formation rate, moisture absorption rate, repose angle, and excipient dosage were evaluated to screen out the best excipients. The results are shown in Table 15.

[0198] Table 15 Granulation screening results of different excipient types

[0199]

[0200] The results showed that the granulation rate of the four excipients was good, all greater than 95%, and the angle of repose was less than 40°. However, the granules made from dextrin were slightly harder, and the granules made from soluble starch had a slightly higher moisture absorption rate than the granules made from other excipients. Considering the amount of excipients, the amount of corn starch, dextrin, and soluble starch was too large compared to microcrystalline cellulose, which was 1.6 to 2 times that of microcrystalline cellulose, which would cause patients to take too much and have poor compliance. Therefore, microcrystalline cellulose was selected as an excipient.

[0201] 2.4 Screening of extract and excipient ratio

[0202] According to the screening results of single-flavor excipients, the extract and microcrystalline cellulose were mixed for granulation. The extract and excipient ratios were set at 1:1, 1:0.9, 1:0.85, 1:0.8, 1:0.75, 1:0.7, 1:0.65, and 1:0.6, respectively. The effects of different ratios of extract and excipient on the soft material and granulation, molding rate, and particle properties were observed, and the optimal ratio of extract and excipient was selected. The results are shown in Table 16.

[0203] Table 16 Granulation screening results of different extract and microcrystalline cellulose ratios

[0204]

[0205] As shown in Table 16, the granule forming rates of different drug-excipient ratios are good. However, when the drug-excipient ratio is 1:0.8-1, the prepared granules have more fine powder or fine powder; when the drug-excipient ratio is 1:0.6-0.65, the prepared soft material becomes sticky and sticks to the sieve, and the prepared granules have hard particles; when the drug-excipient ratio is 1:0.7-0.75, the prepared soft material is moderate and the particles are uniform. Therefore, the extract:excipient ratio is 1:0.7-0.75.

[0206] 2.5 Determination of relative density of extract

[0207] Take 15 times the amount of clinical prescription medicinal materials, extract according to the best water extraction process, concentrate to 2000ml, and set aside. Accurately measure equal amounts of extracts and concentrate to different relative densities, namely 1.18, 1.24, 1.28, 1.32, 1.35, 1.38, and 1.40, add appropriate amounts of microcrystalline cellulose, mix well, and wet granulate. Use soft materials and granulation conditions, molding rate, and particle properties as indicators to screen out the appropriate relative density range of the extract. The results are shown in Table 17.

[0208] Table 17 Effect of different relative density extracts on granule preparation

[0209]

[0210]

[0211] It can be seen from Table 17 that when the relative density of Jinmai Paishi extract is 1.18-1.24 (70°C), the obtained granules contain more fine powder and the amount of auxiliary materials is too much; when the relative density is 1.28-1.32 (70°C), the obtained soft material is moderate and the granules are uniform; when the relative density is

[0212] When the relative density is 1.35-1.40 (70℃), the soft material is slightly sticky and sticks to the sieve, and the particles are coarse. Therefore, the relative density of the extract is 1.28-1.32 (70℃).

[0213] 2.6 Selection of flavoring agents and dosage

[0214] The Jinmai Shi Paishi prescription has a bitter taste, and the granules obtained from it have a strong bitter taste, and the patient compliance is poor. Therefore, a small amount of flavoring agent needs to be added to improve patient compliance. Commonly used sweeteners for preparing granules include sucrose, stevioside, etc. Sucrose is generally used as a flavoring agent in an amount of 2% to 10%, and the sweetness of stevioside is about 150-300 times that of sucrose. Considering that the Jinmai Shi Paishi prescription has many medicinal flavors, the total solids of the water extract will also be relatively large. In order not to increase the patient's dosage, a very small amount of stevioside is selected for flavoring. This study selected the amount of stevioside at 1‰, 1.5‰, 2‰, and 2.5‰ of the granule ratio. The results are shown in Table 18.

[0215] Table 18 Effect of different flavor corrective ratios on the taste of Jinmai Paishi granules

[0216]

[0217] According to the above test results, the granules prepared by adding 2‰ stevioside have a moderate taste and are easy to accept. Therefore, the stevioside concentration is selected to be 2‰ of the total mass.

[0218] 2.7 Investigation of Granule Drying Process

[0219] 2.7.1 Investigation of particle drying temperature

[0220] Different drying temperatures have different drying times. When the temperature is too low, the drying time is long; when the temperature is too high, the drying time is short, but false drying is likely to occur. When the wet granules are dried, the general drying temperature is 60-80°C. The test set the drying temperature at 60°C, 70°C, and 80°C. The paeoniflorin content and water content in the granules were used as evaluation indicators to select the optimal drying temperature. The results are shown in Table 19.

[0221] Table 19 Effect of different drying temperatures on paeoniflorin content and water content

[0222]

[0223] Results: Drying temperature had no significant effect on the content of paeoniflorin. Considering that the drying time would be longer if the temperature was too low, on the basis of ensuring the stability of the drug, this experiment controlled the drying temperature between 70℃ and 80℃ and dried until the moisture content was less than 5.0%.

[0224] 2.7.2 Investigation of granule drying methods

[0225] Commonly used drying methods include hot air circulation drying, boiling drying, vacuum drying, spray drying, etc. According to the existing production conditions of the preparation center, boiling drying and hot air circulation drying are compared to select the appropriate drying method. Take an appropriate amount of the prescribed medicinal materials, extract them according to the optimal water extraction process, concentrate them to an extract with a relative density of 1.30 (70°C), and add an appropriate amount of auxiliary materials for mixing and granulation. The same batch of prepared wet granules are placed in a boiling drying oven and a hot air circulation drying oven, respectively, and the temperature is set to 70-80°C. Dry until the moisture content of the granules is less than 3.0%, and determine its effect on the content of gardenia glycosides and paeoniflorin. At the same time, the moisture content of the granules is determined to determine the final drying method. The results are shown in Table 20.

[0226] Table 20 Effect of different drying temperatures on paeoniflorin content and moisture

[0227]

[0228] The results showed that there was no difference in the content of peonygin between hot air circulation drying and boiling drying. With boiling drying, the wet particles are constantly in a boiling state in the hot air, with rapid heat exchange and fluidized drying, fast drying speed and short time. The operation process is simple. As long as the inlet air temperature is set, the process from wet to dry is carried out under negative pressure in a closed container. In addition, the sealing of the boiling dryer is also very good. There is no need to turn the material during the drying process, no dust is flying, and turning the material is convenient and quick. If hot air circulation drying is used, it is inconvenient to turn the material, and the oven must be opened from time to time, which will increase the drying time and the risk of microbial contamination. Taking all factors into consideration, boiling drying is used as the final drying method.

[0229] 2.8 Mixing process research

[0230] 2.8.1 Experimental instruments

[0231] HCT-800 single column lifting hopper mixer.

[0232] 2.8.2 Experimental methods and results

[0233] Mixing uniformity is an important indicator of granule quality. In order to ensure the uniformity of component mixing, from the perspective of actual production, this experiment used a mixer to mix for 15 minutes, 30 minutes, and 45 minutes, observed the color of the finished product, and took samples at 6 different positions. The samples were prepared according to the preparation method of the test sample mentioned above, and the content of paeoniflorin was determined. The results are shown in Table 21. The results showed that the color of the samples mixed for 15 minutes, 30 minutes, and 45 minutes was uniform, but the paeoniflorin content measured in the samples mixed for 15 minutes was uneven (RSD = 20.93% > 3%), and the paeoniflorin content measured in the samples mixed for 30 minutes and 45 minutes was uniform, with RSDs of 0.38% and 0.39%, respectively. Considering production costs, the mixing process of Jinmai Paishi Granules was set to mix for 30 minutes.

[0234] Table 21 Results of investigation on the content of paeoniflorin (mg / g) in granules obtained at different mixing times

[0235]

[0236] 2.9 Forming process verification test

[0237] About 50 g of the same batch of Jinmai Paishi extract (relative density of 1.30, 70°C) was taken and pelletized three times according to the preferred molding process conditions. The results are shown in Table 22, indicating that the process has good reproducibility and operability.

[0238] Table 22 Forming process verification results

[0239]

[0240]

[0241] 3. Small test to confirm

[0242] According to the optimized water extraction process and molding process conditions, three batches of 5-fold small-scale production were carried out to investigate the feasibility and rationality of the preparation process of this product. The paeoniflorin content and water content of three batches of Jinmai Paishi Granules were determined. The results are shown in Table 23. The results show that the process preparation conditions are highly operable and can be used for large-scale production.

[0243] Table 23 Small test confirmation batch test results

[0244]

[0245] (IV) Determination of particle flowability and critical relative humidity

[0246] 1 Determination of the angle of repose

[0247] The fluidity of the particles was measured according to the method under "(II) 2.1.3", and each batch of samples was measured three times. The results are shown in Table 24. The average repose angles of the three batches of particles were all less than 40°, indicating that the particles had good fluidity and could ensure that the particle loading met the requirements.

[0248] Table 24 Repose angle measurement results

[0249]

[0250] 2 Determination of critical relative humidity of particles

[0251] The humidity of the packaging environment has a great influence on the packaging of particles, so it is necessary to determine the critical relative humidity of the particles.

[0252] 2.1 Determination of moisture absorption equilibrium time

[0253] Take a glass desiccator with a supersaturated NaCl solution (relative humidity 75%) at the bottom, and a weighing bottle (5×3cm) built into the desiccator to reach constant weight in a constant temperature drying oven at 25℃. Take about 1.2g of sample, accurately weigh it, put it in the weighing bottle with constant weight, shake it gently to make it evenly distributed, accurately weigh it, open the weighing bottle cap, put it in the upper part of the desiccator, and store it in a constant temperature drying oven at 25℃. Weigh it once at 1, 3, 5, 7, 24, 48, 72, 96, and 120h, and calculate the moisture absorption rate at different times. Calculation formula: Moisture absorption rate = (total weight after moisture absorption - total weight before moisture absorption) / total amount before moisture absorption × 100%. The test results show that the samples of three batches (20230703, 20230704, and 20230705) were tested separately, and the experimental results were similar. The experimental results are now listed for analysis. The moisture absorption equilibrium time of the particles is shown in Table 25, and the moisture absorption equilibrium curve is shown in Figure 3 .

[0254] Table 25 Moisture absorption equilibrium time (n=3)

[0255]

[0256] From Table 25, Figure 3 It can be seen that the moisture absorption of this product basically reaches equilibrium after 72 hours, and the moisture absorption rate no longer increases significantly. The moisture absorption equilibrium time of the particles is 72 hours.

[0257] 2.2 Determination of critical relative humidity of particles

[0258] Weigh 14 portions of granules accurately, each about 1.2g, spread them flat in a weighing bottle (5×3cm) that has been dried and weighed accurately, and put them into a constant temperature and humidity chamber (see Table 26 for setting humidity). Open the weighing bottle cap, keep it at 25℃ for 72h, then weigh it, measure the weight change of the sample, calculate the moisture absorption rate, and draw a curve with the moisture absorption rate as the ordinate and the relative humidity as the abscissa. Draw tangents from both ends of the curve, and the abscissa corresponding to the intersection of the two tangents is the critical relative humidity. The results show that the critical relative humidity of the granules is about 72% (see Table 26). Figure 4 ). Therefore, during granulation, packaging and storage, the ambient humidity must be controlled below 72% to reduce the impact of moisture on the properties and stability of the drug.

[0259] Table 26 Determination of critical relative humidity of particles

[0260]

[0261] In summary, the preparation process of this product is determined as follows: control the humidity of the mixing and packaging environment to <72%, add 8.5 times the water for the above 16 flavors (talc and sea sand for decoction) for 30 minutes for the first time, add 6 times the water for the second time, decoct twice, each time for 30 minutes, filter, and concentrate the filtrate to a thick paste with a relative density of 1.28-1.32 (70°C), add an appropriate amount of microcrystalline cellulose and 2g of stevioside, mix well, make into granules, dry, and make into 1000g.

[0262] (V) Determination of daily dosage of prescription

[0263] The two-day prescription of Jinmai Paishi Fang is 253g, which is taken in 6 doses. According to experimental research, the two-day Jinmai Paishi Fang is extracted and concentrated by water to obtain a thick paste of about 70g, and then an appropriate amount of microcrystalline cellulose and stevioside are added and mixed to make granules of about 75g, that is, each gram of granules is equivalent to 3.4g of crude drug. According to a single prescription, it is taken for 2 days, 3 times / day, and the dosage for each dose is about 10g. Therefore, the specification of Jinmai Paishi granules is set at 10g per bag.

[0264] 2. Research on Preparation Quality Standards

[0265] 1. Properties: All three batches of pilot samples are brown to tan granules and powders; slight odor, slightly sweet and slightly bitter taste.

[0266] 2. Thin layer identification

[0267] 2.1 Instruments and reagents

[0268] ZF-20D dark box UV analyzer (Gongyi Yuhua Instrument Co., Ltd.); TP-320C electronic balance (Xiangyi Balance Instrument Equipment Co., Ltd.); DZKW-4 electronic constant temperature water bath (Shanghai Jinqiao Kexi Instrument Factory); 98-1-B electronic temperature control heating jacket (Tianjin Taisite Instrument Co., Ltd.); SK5010HP ultrasonic cleaning machine (power 200W, frequency 53kHz; Shanghai Kedao Ultrasonic Instrument Co., Ltd.); medical centrifuge (H2-16k, Hunan Kecheng Instrument Equipment Co., Ltd.); silica gel G thin layer plate (purchased from Qingdao Ocean Chemical Plant); methanol, chloroform, ethyl acetate, formic acid, ammonia water, 2-butanone, dichloromethane, acetone, toluene, ethyl formate, n-butanol, petroleum ether Glacial acetic acid, ether, cyclohexane, dilute hydrochloric acid, 5% vanillin sulfuric acid solution, 10% sulfuric acid ethanol solution, 3% aluminum chloride ethanol solution, etc. were of analytical grade.

[0269] 2.2 Methods and Results

[0270] The preparation consists of 16 Chinese medicinal materials including Herba Lysimachiae, Herba Lysimachiae, Herba Imperatae, and Herba Cynoglossi. With reference to the methods in the Chinese Pharmacopoeia and related literature, qualitative identification was performed on 9 medicinal materials in the preparation, including Herba Lysimachiae, Herba Cynoglossi, Herba Paeoniae Alba, Fructus Aurantii Immaturus, Fructus Gardeniae, Herba Plantaine, Herba Imperatae, Fructus Toosendan and Fructus Rehmanniae. The negative control for qualitative identification of Fructus Chuanxiong, Fructus Aurantii Immaturus, Fructus Toosendan and Fructus Gardeniae had no interference. After verification by 3 batches of pilot samples, this method had good repeatability, so it was included in the draft text of the quality standard. Herba Lysimachiae, Herba Paeoniae Alba, Herba Plantaine, Fructus Imperatae and Fructus Rehmanniae were all extracted by two extraction methods and systematic experiments were carried out, but they were not included in the text of the quality standard due to negative interference. The test results are as follows.

[0271] 2.2.1 Thin layer chromatography identification of Lysimachia chinensis

[0272] The 2020 edition of the "Chinese Pharmacopoeia" records that the thin-layer chromatography identification method of Lycopodiella is to use quercetin and kaempferol as controls for thin-layer chromatography identification. However, the Chinese medicinal materials in the prescription, including Dianthus superbus, fried Toosendan fruit, Plantago, and Pyrola, also contain quercetin and kaempferol ingredients. Therefore, the thin-layer chromatography identification of Lycopodiella was not carried out using the pharmacopoeia method for thin-layer chromatography identification.

[0273] Reference to the literature "Qualitative identification of Lysimachia and Gallus gallus domesticus in Kidney Stone Granules by TLC by Liao Liying et al.", the qualitative identification method of Lysimachia was studied: Take 5g of this product, add 30ml of ether, heat and reflux for 30 minutes, filter, evaporate the filtrate, and dissolve the residue in 1ml of methanol as the test solution. Take another 5g of the negative preparation lacking Lysimachia, and prepare the negative control solution in the same way. Take 1g of Lysimachia control medicinal material, add 50ml of water, decoct for 0.5h, filter, take the filtrate, add ether and shake to extract twice, 20ml each time, combine the ether solution, evaporate, and dissolve the residue in 1ml of methanol as the control medicinal material solution. According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (9.5:0.5) was used as the developing agent. After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and dried at 105°C until the spots were clearly colored, and then examined under ultraviolet light (365mn). The test solution and the negative control solution interfered with each other (see Figure 5 Left).

[0274] Method 2: Optimize the preparation method and development conditions of the test solution of the Chinese Pharmacopoeia in the 2020 edition of the Chinese Pharmacopoeia: Take 3g of this product, add 20ml of 70% ethanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution. Take another 3g of the negative preparation lacking the Chinese Pharmacopoeia, and prepare the negative control solution in the same way. Take 1g of the Chinese Pharmacopoeia control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia), take 5 to 8μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, ethyl acetate-2-butanone-formic acid-water (5:3:3:1) as the developing agent, saturate for 10min, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365mn). Results The test solution and negative control solution interfered with each other (see Figure 5 middle).

[0275] Method three: Take 3g of this product, add 20ml of 70% ethanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution. Take another 3g of the negative preparation lacking Centella asiatica, and prepare a negative control solution in the same way. Take another 1g of Centella asiatica control medicinal material, and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), 5-8μl of each of the three solutions were taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-ethanol-water (8:2:1) was used as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and immediately place under ultraviolet light (365mn) for inspection. As a result, in the chromatogram of the test product, fluorescent spots of the same color appeared at the corresponding positions of the control medicinal materials, and there was no interference with the negative control. (See Figure 5 right).

[0276] 2.2.2 TLC Identification of Imperata cylindrica Rhizome

[0277] Method 1: Refer to the thin-layer identification method of Imperata root on page 111 of the 2020 edition of the Chinese Pharmacopoeia for experimental research: Take 5g of this product, add 25ml of ether, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, and add 1ml of ether to the residue to dissolve it as the test solution. Take another 5g of the negative preparation lacking Imperata root, and prepare a negative control solution in the same way. Take 1g of Imperata root control medicinal material, and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia), 10μl of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, and dichloromethane was used as the developing agent. Develop, take out, dry, spray with 10% ethanolic sulfuric acid solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365mn). In the chromatogram of the test product, there are no spots of the same color at the corresponding position of the chromatogram of the control medicinal material (see Figure 6 Left).

[0278] Method 2: Refer to the thin layer identification method of Imperata root in "Thin layer identification study of Jiannao Yizhi capsule" for processing: Take 3g of this product, put it in a 100ml stoppered triangular flask, add 30ml of methanol, ultrasonically treat for 30min, filter, evaporate the filtrate, and dissolve the residue in 1ml of methanol to obtain it. Take another 3g of negative preparation lacking Imperata root, and prepare negative control solution in the same way. Take 1g of Imperata root control medicinal material, and prepare control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia), take 10μl of each of the above three solutions and spot them on the same silica gel G thin layer plate, use chloroform-methanol-water-formic acid (7.5:2.5:1:3) as the developing agent, pre-saturate for 10min, develop, take out, dry, spray with 10% sulfuric acid ethanol solution and heat until the spots are clearly colored, and inspect under ultraviolet light (365mn). The test solution and the negative control solution interfere with each other (see Figure 6 right).

[0279] 2.2.3 Thin layer chromatography identification of Rehmannia root

[0280] Method 1: Refer to the thin-layer identification method of Rehmannia glutinosa on pages 129-130 of the 2020 edition of the Chinese Pharmacopoeia for experimental research: take 5g of this product, add 25ml of methanol, ultrasonicate for 30min, filter, and take the filtrate as the test solution. Take another 5g of the negative preparation lacking Rehmannia glutinosa, and prepare the negative control solution in the same way. Take 1g of Rehmannia glutinosa control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia), 5μl of each of the three solutions were taken and spotted on the same silica gel G thin layer plate, and chloroform-methanol-water (14:6:1) was used as the developing agent. Develop, take out, spray with anisaldehyde test solution, and heat at 105°C until the spots are clearly colored. The test solution and the negative control solution interfere with each other (see Figure 7 Left).

[0281] Method 2: Take 3g of this product, add 25ml of 70% methanol, ultrasonicate for 30min, filter, and concentrate the filtrate to 3ml as the test solution. Take another 3g of the negative preparation lacking Rehmannia glutinosa, and prepare a negative control solution in the same way. Take another 1g of Rehmannia glutinosa control medicinal material, and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the "Chinese Pharmacopoeia"), 5μl of each of the three solutions mentioned above were taken and spotted on the same silica gel G thin layer plate, and chloroform-ethyl acetate-methanol-concentrated ammonia test solution (8:1:4:1) was used as the developing agent. Develop, take out, dry, spray vanillin test solution (prepare now for use), and bake at 105°C until the spots are clearly colored. Results In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material, and there was no interference with the negative control (see Figure 7 Right), but its sensitivity is low and the existing photographic equipment cannot meet the photographic requirements, so the thin layer identification of plantain was not included in the draft quality standard.

[0282] 2.2.4 Thin layer chromatography identification of Gardenia

[0283] Method 1: Refer to the thin layer identification method of gardenia on pages 259-260 of the 2020 edition of the "Chinese Pharmacopoeia" for experimental research: take 3g of this product, add 20ml of 50% methanol, ultrasonically treat for 40 minutes, filter, and take the filtrate as the test solution. Also take 3g of the negative preparation lacking gardenia and 0.5g of gardenia control medicinal materials, and prepare negative control solution and control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part 4 of the 2020 edition of the "Chinese Pharmacopoeia"), 3-5μl of each of the three solutions were taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-acetone-formic acid-water (5:5:1:1) was used as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. The test solution and the negative control solution interfere with each other (see Figure 8 Left 1).

[0284] Method 2: Take 3g of this product, add 20ml of 70% methanol, shake for 5 minutes, filter, and concentrate the filtrate to 5ml as the test solution. Also take 3g of the negative preparation lacking Gardenia and 0.5g of Gardenia control medicinal materials, and prepare negative control solution and control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the "Chinese Pharmacopoeia"), 4μl of the control medicinal material solution, 8-10μl of the test solution and the negative control solution are respectively spotted on the same silica gel G thin layer plate, and chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) is used as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and there is no interference with the negative control (see Figure 8 Left 2). However, after many experiments, it was found that negative controls sometimes have certain interference (see Figure 8 2 on the right). Then, the lower layer solution of chloroform-ethyl acetate-methanol-concentrated ammonia solution (8:1:3:1) was used as the developing agent for development, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and inspected under a fluorescent lamp. As a result, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the control medicinal material, and the negative control solution had no interference. After verification by 3 batches of pilot samples, this method has good reproducibility, so it is included in the text of the draft quality standard (see Figure 8 Right 1).

[0285] 2.2.5 Thin layer chromatography identification of plantain

[0286] Method 1: Conduct experimental research with reference to the thin-layer identification method for plantain on pages 70-71 of the 2020 edition of the Chinese Pharmacopoeia: Take 3 g of this product, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, and take the filtrate as the test solution. Also take 3 g of the negative preparation lacking plantain and 1 g of plantain control medicinal material, and prepare the negative control solution and control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-methanol-formic acid-water (18:3:1.5:1) was used as the developing agent. Develop, take out, dry, and inspect under ultraviolet light (365mn). The test solution and the negative control solution interfere with each other (see Fig. 9 Left).

[0287] Method 2: Conduct experimental research with reference to the method of plantain formula granules: Take 5g of this product, add 30ml of ethyl acetate, ultrasonicate for 40 minutes, filter, evaporate the filtrate, dissolve the residue in 1ml of methanol, and use it as the test solution. Take another 5g of the negative preparation without plantain, and prepare the negative control solution in the same way. Take 2g of plantain control medicinal material, add 50ml of water, boil for 30 minutes, filter, take the filtrate and extract it with ethyl acetate twice, 20ml each time, combine the ethyl acetate solution, evaporate, and dissolve the residue in 1ml of methanol, and use it as the control medicinal material solution. According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and petroleum ether (30-60°C)-chloroform-ethyl acetate-acetone (5:5:3:2) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and dried at 105°C until the spots were clearly colored, and then examined under ultraviolet light (365nm). The test solution and the negative control solution interfere with each other (see Fig. 9 right).

[0288] 2.2.6 Thin layer chromatography identification of Achyranthes bidentata

[0289] The experimental study was conducted with reference to the thin layer identification method of Cyathula officinalis on pages 39-40 of the 2020 edition of the Chinese Pharmacopoeia: 3 g of this product was added with 20 ml of methanol, ultrasonically treated for 30 minutes, filtered, the filtrate was evaporated to dryness, and the residue was dissolved with 1 ml of methanol as the test solution. Another 3 g of the negative preparation of Cyathula officinalis without alcohol was taken, and the negative control medicinal material solution was prepared in the same way. Another 2 g of Cyathula officinalis control medicinal material was taken, and the control medicinal material solution was prepared in the same way. According to the thin layer chromatography method (General Rules 0502 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia), 8 to 10 μl of the test solution and the negative control solution and 5 μl of the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, respectively, with chloroform-methanol (7:1) as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light (365nm). In the chromatogram of the test sample, the same color fluorescent spot appears at the corresponding position of the chromatogram of the control medicinal material. Fig.10 ), this method has good reproducibility, so it is included in the text of the draft quality standard.

[0290] 2.2.7 Thin layer chromatography identification of white peony root

[0291] Method 1: Refer to the white peony thin layer identification method on pages 108-109 of the 2020 edition of the "Chinese Pharmacopoeia" for experimental research: take 3g of this product, add 25ml of ethanol, shake for 5 minutes, filter, evaporate the filtrate to dryness, and add 1ml of ethanol to the residue to dissolve it as the test solution. Take another 3g of the missing negative preparation and prepare the negative control solution in the same way. Take another 1g of white peony control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part 4 of the 2020 Edition of the "Chinese Pharmacopoeia"), 10μl of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, and chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) was used as the developing agent. Develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored. The test solution and the negative control solution interfere with each other (see Fig.11 Left).

[0292] Method 2: Take 3g of this product, add 20ml of 70% methanol, shake for 5 minutes, filter, and concentrate the filtrate to 5ml as the test solution. Take another 3g of the negative preparation lacking white peony root, and prepare the negative control solution in the same way. Take another 0.5g of white peony control medicinal material, add 10ml of 70% ethanol, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), 8-10μl of each of the three solutions were taken and spotted on the same silica gel G thin layer plate, and chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) was used as the developing agent. Develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored. The test solution and the negative control solution interfere with each other (see Fig.11 middle).

[0293] Method three: Take 3g of this product, add 20ml of 70% methanol, shake for 5 minutes, filter, and concentrate the filtrate to 5ml as the test solution. Take another 3g of the negative preparation lacking white peony root, and prepare the negative control solution in the same way. Take 0.5g of white peony root control medicinal material, add 10ml of 70% methanol, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 8-10μl of each of the above three solutions, spot them on the same silica gel G thin layer plate, and develop them with chloroform-ethyl acetate-methanol-concentrated ammonia test solution (8:1:3:1) as the developing agent. Take it out, dry it, spray it with 5% vanillin sulfuric acid solution, and bake it at 105°C until the spots are clearly colored. In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and there is no such spot in the negative control solution (see Fig.11 right), but its durability is poor and therefore it was not included in the draft quality standard.

[0294] 2.2.8 TLC Identification of Fructus Aurantii Immaturus Stir-fried with Glutinous Rice Bran

[0295] Refer to the thin layer identification method of Citrus aurantium on pages 257-258 of the 2020 edition of the Chinese Pharmacopoeia for experimental research: Take 3g of this product, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution. Take another 3g of the negative preparation lacking Citrus aurantium, and prepare the negative control solution in the same way. Take another 0.2g of Citrus aurantium control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia), take 2-5μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, with chloroform-methanol-water (13:6:2) as the lower layer solution as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position in the chromatogram of the control medicinal material, there is a fluorescent spot of the same color, and there is no interference with the negative control. Fig.12 ), this method has good reproducibility, so it is included in the text of the draft quality standard.

[0296] 2.2.9 TLC Identification of Stir-fried Toosendan Fruit

[0297] Method 1: Refer to the TLC identification method of Toosendan fruit on page 44 of the 2020 edition of the Chinese Pharmacopoeia for experimental research: Take 5g of this product, add 40ml of water to dissolve, filter, extract the filtrate with dichloromethane 3 times, 25ml each time, combine the dichloromethane solution, evaporate to dryness, and add 2ml of methanol to the residue to dissolve it as the test solution. Take another 5g of the negative preparation without stir-fried Toosendan fruit, and prepare the negative control solution in the same way. Take another 2g of Toosendan fruit control medicinal material, add 80ml of water, decoct for 30 minutes, cool, centrifuge, take the supernatant, and prepare the control medicinal material solution in the same way as the test solution preparation method. According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and dichloromethane-methanol (16:1) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with p-dimethylaminobenzaldehyde test solution, and heated at 105°C until the spots were clearly colored. The test solution and the negative control solution interfered with each other (see Fig.13 Left).

[0298] Method 2: Refer to the thin layer chromatography identification method of Toosendan in Toosendan Fructus Toosendan in "Thin layer chromatography identification of Toosendan in Toosendan Fructus Toosendan" for treatment: Take 10g of this product, add 50ml of water to dissolve, filter, extract the filtrate with ethyl acetate twice, 30ml each time, combine the ethyl acetate solution, shake and extract with ammonia test solution twice, 20ml each time. Discard the ammonia test solution, take the ethyl acetate solution, evaporate to dryness, add 1ml of ethanol solution to the residue as the test solution. Take another 10g of the negative preparation without stir-frying Toosendan Fructus Toosendan, and prepare the negative control solution in the same way. Take another 2g of Toosendan Fructus Toosendan control medicinal material, add 50ml of water, decoct for 30 minutes, filter, add 30ml of ethyl acetate to the filtrate, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of each of the three solutions mentioned above were taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (5:2) was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 2% dimethylaminobenzaldehyde-40% sulfuric acid solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding position in the chromatogram of the control medicinal material, and there was no interference with the negative control. This method was verified by 3 batches of pilot samples and had good reproducibility, so it was included in the text of the draft quality standard (see Fig.13 right).

[0299] 2.3 Study on the durability of TLC of Herba Lysimachiae, Rhizoma Cynanchum, Fructus Toosendan, Fructus Aurantii Immaturus and Fructus Gardeniae

[0300] Three batches of pilot samples (20240301, 20240302, 20240303) were taken. According to the thin layer chromatography conditions used in the experiment, the effects of different batches (20220418, 20221107) of silica gel G thin layer plates, different temperatures (4°C, 25°C, 35°C), and different humidity (18%, 58%, 88%) on the TLC results of Lysimachia scabra, Achyranthes bidentata, stir-fried Toosendan fruit, stir-fried Fructus Aurantii Immaturus, and Gardenia jasminoides were investigated. The results are shown in Table 1. Figure 14-15 The chromatographic behaviors of the test samples on thin-layer plates of different batches, at different temperatures and under different humidity conditions were basically consistent, and the qualitative identification methods used in the description were all of good durability.

[0301] 3. Inspection: Inspection shall be carried out in accordance with the relevant provisions under Granules (General Rules 0114 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), and the particle size, moisture, filling volume difference, and microbial limit shall be checked respectively.

[0302] Particle size: According to the particle size and particle size distribution determination method (General Rule 0982 Method 2 Double Screening Method), the sum of the particles that cannot pass through the No. 1 sieve and the particles that can pass through the No. 5 sieve shall not exceed 15%. The results are shown in Table 27.

[0303] Table 27 Particle size determination results

[0304]

[0305] Moisture content: Determined according to the moisture determination method (General Rules 0832 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), it shall not exceed 8.0%. See Table 28 for the moisture determination results.

[0306] Table 28 Moisture determination results

[0307]

[0308] Filling volume difference: Take 5 bags of this product and check the filling volume difference under the granules (General Rule 0104). The filling volume difference limit should be within ±5% of the labeled filling volume. No more than 2 bags should exceed the filling volume difference limit, and no bag should exceed the limit by 1 times. The results are shown in Table 29.

[0309] Table 29 Results of filling volume difference determination

[0310]

[0311] Microbial limit: According to the microbial limit inspection for non-sterile products: microbial counting method (General Rule 1105) and control bacteria inspection method (General Rule 1106) and microbial limit standard for non-sterile drugs (General Rule 1107), the total aerobic bacteria count of this product shall not exceed 103 cfu / g, the total mold and yeast count shall not exceed 102 cfu / g, and Escherichia coli shall not be detected. The results are shown in Table 30, all of which meet the requirements.

[0312] Table 30 Microbial limit test results

[0313]

[0314] The results of various inspections show that this product complies with the relevant provisions under General Chapter 0114 Granules of Part IV of the 2020 Edition of the Chinese Pharmacopoeia.

[0315] 4. Extractables determination

[0316] 4.1 Selection of extract solvent

[0317] Referring to the solvent selection of the extract of the medicinal materials of Jinmai Pai Shi Granules in the "Chinese Pharmacopoeia" 2020 edition, combined with the fact that the chemical components of the medicinal materials that make up Jinmai Pai Shi Granules are mainly flavonoids, triterpenes, steroids, etc., water, 50% ethanol, 75% ethanol, and 95% ethanol were used as leaching solvents, respectively, and the hot leaching method was used to determine the contents of water-soluble and alcohol-soluble extracts in Jinmai Pai Shi Granules, see Table 31.

[0318] Table 31 Extract solvent selection results

[0319]

[0320] As shown in Table 31, the extract content of Jinmai Paishi granules extracted with water and 50% ethanol is the highest, which are 45.5% and 44.11% respectively, and there is no difference between the two. When considering the extract content with water as the leaching solvent, it is difficult to filter and centrifuge is required; when 50% ethanol is used as the leaching solvent, there is no centrifuge step. In order to simplify the operation method, 50% ethanol is selected as the leaching solvent.

[0321] 4.2 Determination of alcohol-soluble extracts from Jinmai Paishi granules

[0322] Take 2g of the test sample, weigh it accurately, put it in a 150ml conical flask, add 50ml of 50% ethanol accurately, plug it tightly, weigh it, let it stand for 1h, connect the reflux condenser, heat it to boiling, and keep it boiling for 1h. After cooling, remove the conical flask, plug it tightly, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it with a drying filter, accurately measure 25ml of the filtrate, put it in an evaporating dish that has been dried to constant weight, evaporate it on a water bath, dry it at 105℃ for 3h, move it to a desiccator, cool it for 30min, and quickly weigh it accurately. The calculation formula is as follows:

[0323]

[0324] 4.3 Determination results of alcohol-soluble extracts from Jinmai Paishi granules

[0325] The alcohol-soluble extracts of 9 batches of Jinmai Paishi Granules were measured, and the results showed that the alcohol-soluble extracts were about 38.6-41.9%. According to the difficulty of mass production, the extracts of this product were calculated by reducing the content of the 9 batches of samples by 20%, that is, 38.6%-38.6%×20%=30.9%. The extract content of Jinmai Paishi Granules was tentatively not less than 30.9%. The results are shown in Table 32.

[0326] Table 32 Test results of alcohol-soluble extracts from different batches of Jinmai Paishi granules

[0327]

[0328] 5 Experimental study on the determination method of paeoniflorin content

[0329] 5.1 Instruments and reagents

[0330] Instruments: Agilent 1260 high performance liquid chromatograph; G4212B1260DAD diode array detector; AUW220D electronic balance; electronic temperature control heating mantle (98-1-B, Tianjin Test Instrument Co., Ltd.); ultrasonic cleaner (SK8210HP, Shanghai Kedao Ultrasonic Instrument Co., Ltd., power 250W, frequency 40kHz).

[0331] Test drugs: phosphoric acid (HPLC grade, Tianjin Komiou Chemical Reagent Co., Ltd.); acetonitrile (chromatographic grade, TEDIA, USA), water (purified water, manufactured by Chengdu Wahaha Food Co., Ltd.); paeoniflorin reference substance (batch number: 110736-202246, content of 94.6%, purchased from China Food and Drug Inspection Institute); Jinmai Paishi Granules and White Peony Negative Preparation.

[0332] 5.2 Solution preparation

[0333] With reference to the determination method of geniposide in gardenia and paeoniflorin in white peony in Volume 1 of Chinese Pharmacopoeia, the contents of paeoniflorin in white peony and geniposide in gardenia were determined by HPLC.

[0334] 5.2.1 Preparation of reference solution

[0335] Preparation of mixed reference substance 1 of gardenia glycoside and paeoniflorin: accurately weigh 12.89 mg of gardenia glycoside reference substance and 11.86 mg of paeoniflorin reference substance, place them in a 50 ml volumetric flask, add methanol to the mark, shake well, and obtain the mixed reference substance stock solution (gardenia glycoside 0.2529 mg / ml, paeoniflorin 0.2244 mg / ml). Take 2 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute with methanol, and obtain the reference substance solution (gardenia glycoside 50.58 μg / ml, paeoniflorin 44.88 μg / ml).

[0336] Preparation of mixed reference substance 2 of gardenia glycoside and paeoniflorin: accurately weigh 12.85 mg of gardenia glycoside reference substance and 11.81 mg of paeoniflorin reference substance, place them in a 50 ml volumetric flask, add methanol to the mark, shake well, and obtain the mixed reference substance stock solution (gardenia glycoside 0.2521 mg / ml, paeoniflorin 0.2234 mg / ml). Take 2 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute with methanol, and obtain the reference substance solution (gardenia glycoside 50.42 μg / ml, paeoniflorin 44.69 μg / ml).

[0337] 5.2.2 Preparation of test solution

[0338] Take 0.4 g of Jinmai Paishi granules, weigh accurately, put it in a stoppered conical flask, add 25 ml of 50% ethanol accurately, stopper it, weigh it, ultrasonicate it for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it, take the filtrate, filter it with a 0.45 μm microporous filter membrane, and use it as the test solution.

[0339] 5.2.3 Preparation of negative control solution

[0340] Take the negative samples lacking Gardenia jasminoides and Paeonia lactiflora and prepare them according to the method of “5.2.2 Preparation of test solution” to obtain the negative control solutions lacking Gardenia jasminoides and Paeonia lactiflora.

[0341] 5.3 Investigation of chromatographic conditions

[0342] 5.3.1 Detection wavelength

[0343] The full wavelength scanning of 190-400 nm for gardenia glycoside and paeoniflorin revealed that the maximum absorption wavelength of gardenia glycoside was around 240 nm (see Fig.16 ), the maximum absorption wavelength of paeoniflorin is around 230nm (see Fig.17 ), since the elution times of the two are close, switching wavelengths is not conducive to detection, and the chromatographic peak area of ​​gardenia glycoside is much larger than that of paeoniflorin, so 230nm was selected as the detection wavelength.

[0344] 5.3.2 Investigation of mobile phase

[0345] When selecting the chromatographic conditions for gardenia glycoside and paeoniflorin, Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) was used as the chromatographic column, the detection wavelength was 230 nm, the flow rate was 1 ml / min, the column temperature was 30°C, and different proportions of the mobile phase acetonitrile-0.1% phosphoric acid were mainly screened. See Table 33.

[0346] Table 33 Selection of chromatographic conditions for gardenia glycoside and paeoniflorin

[0347]

[0348]

[0349] By comparing the chromatographic peaks under the above several different chromatographic conditions, the results show that when the mobile phase ratio is acetonitrile-0.1% phosphoric acid gradient elution: 0-22min (13:87), 23-38min (40:60), 39-45min (13:87), the chromatographic peaks of gardenia glycoside and paeoniflorin are completely separated from other components, and the separation effect is good. However, under other chromatographic conditions, either the separation effect of gardenia glycoside or the separation effect of paeoniflorin is poor. Therefore, the mobile phase is determined to be: acetonitrile-0.1% phosphoric acid gradient elution: 0-22min (13:87), 23-38min (40:60), 39-45min (13:87).

[0350] 5.3.3 Investigation of column temperature

[0351] Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) was used as the chromatographic column to investigate the chromatographic separation effects at 25 ° C, 30 ° C, and 35 ° C. The results are shown in Table 34 and Fig.25 .

[0352] Table 34 Determination results of paeoniflorin content in Jinmai Paishi granules at different column temperatures

[0353]

[0354] The results show that the column temperature of 25℃, 30℃ and 35℃ has no difference in the separation effect of the chromatographic peaks. Considering the actual use, the column temperature of 30℃ was selected.

[0355] 5.3.4 Investigation of flow rate

[0356] Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) was used as the chromatographic column to investigate the chromatographic separation effects at different flow rates (0.9 ml / min, 1.0 ml / min, 1.2 ml / min). The results are shown in Table 35 and Fig.26 .

[0357] Table 35 Determination results of gardenia glycoside and paeoniflorin content in Jinmai Paishi granules at different flow rates

[0358]

[0359] The results show that the flow rates of 0.9 ml / min, 1.0 ml / min and 1.2 ml / min have little effect on the separation effect of the chromatographic peaks. According to the actual laboratory use, the flow rate of 1.0 ml / min was selected.

[0360] In summary, the chromatographic conditions of gardenia jasminoides and paeoniflorin were determined as follows: octadecylsilane bonded silica gel as filler; acetonitrile-0.1% phosphoric acid gradient elution: 0-22 min (13:87), 23-38 min (40:60), 39-45 min (13:87); flow rate: 1 ml / min; column temperature: 30 °C; detection wavelength: 230 nm; injection volume: 10 μl.

[0361] 5.4 System suitability test

[0362] 5.4.1 Test method for sample solution treatment

[0363] 5.4.1.1 Investigation of extraction solvent

[0364] Take about 0.4g of this product, weigh it accurately, put it in a stoppered conical flask, number 1 to 12; add solvent accurately, No. 1 and No. 2 solvents are 30% methanol; No. 3 and No. 4 solvents are 50% methanol; No. 5 and No. 6 solvents are 70% methanol; No. 7 and No. 8 solvents are 30% ethanol; No. 9 and No. 10 solvents are 50% ethanol; No. 11 and No. 12 solvents are 70% ethanol; process according to the preparation method of the test sample, calculate the content of gardenia glycoside and paeoniflorin, and use SPSS23 software for statistical analysis. The results are shown in Tables 36-39.

[0365] Table 36 Effect of extraction solvent on the content of gardenia glycoside and paeoniflorin

[0366]

[0367] Table 37 Single factor analysis of variance of extraction solvents for gardenia glycoside and paeoniflorin

[0368]

[0369] Table 38 Waller-Duncan test results of paeoniflorin content under different extraction solvents

[0370]

[0371] Note: The average values ​​of each group in the homogeneous subset will be displayed;

[0372] a: Using harmonic mean sample size = 2.000; b: Type I / Type II error severity ratio = 100.

[0373] Table 39 Waller-Duncan test results of gardenia glycoside content under different extraction solvents

[0374]

[0375] Note: The average values ​​of each group in the homogeneous subset will be displayed;

[0376] a: Using harmonic mean sample size = 2.000; b: Type I / Type II error severity ratio = 100.

[0377] Results: One-way ANOVA showed that the p values ​​of gardenia glycoside and paeoniflorin were both less than 0.05, indicating that different extraction solvents had significant differences in the contents of gardenia glycoside and paeoniflorin; and the homogeneous subset results of the Waller-Duncan method showed that the average values ​​of gardenia glycoside and paeoniflorin extracted by 70% methanol were the largest, therefore, this method used 70% methanol solvent as the solvent for extracting samples.

[0378] 5.4.1.2 Investigation of extraction methods

[0379] Take about 0.4g of this product, weigh accurately, a total of 6 portions, put in a stoppered conical bottle, accurately add 25ml of 70% methanol to each, weigh the weight, 2 of which are heated to reflux for 30min; 2 portions are ultrasonically treated for 30min; 2 portions are cold soaked for 30min, let cool, weigh again, make up the reduced weight with 70% methanol, shake well, filter, and filter with a 0.45μm microporous filter membrane to obtain. Accurately take 10μl of each of the above 6 solutions and the paeoniflorin reference substance, inject into the high performance liquid chromatograph, record the chromatogram, and determine the content of gardenia glycoside and paeoniflorin in the sample. The results are shown in Table 40.

[0380] Table 40 Investigation of extraction methods

[0381]

[0382] The results showed that the contents of gardenia glycoside and paeoniflorin in Jinmai Pai Shi granules extracted by ultrasound for 30 minutes and heating reflux for 30 minutes were equivalent. However, considering that the experimental steps of ultrasound treatment for 30 minutes were simple, ultrasound treatment for 30 minutes was selected as the extraction method for the test solution in this experiment.

[0383] 5.4.2 Specificity test

[0384] Accurately pipette 10 μl of reference solution, test solution, negative sample solution lacking Gardenia jasminoides and Paeonia lactiflora into the chromatograph and record the chromatogram. Fig. 27 As can be seen from the chromatogram, the negative control solution has no corresponding chromatographic peak at the same retention time position as the reference substance, indicating that other medicinal materials except paeoniflorin have no interference with the determination.

[0385] 5.5 Linearity and Range

[0386] Weigh 12.89 mg of Gardenia glycoside reference substance and 11.86 mg of Paeoniflorin reference substance with a 1 / 10,000 electronic balance, place them in a 50 ml volumetric flask, add methanol to the mark, shake well, and obtain a mixed reference substance stock solution (Gardenia glycoside 0.2529 mg / ml, Paeoniflorin 0.2244 mg / ml). Take 3 10 ml volumetric flasks, add 1, 2, and 5 ml of the mixed reference substance stock solution, respectively, and dilute with methanol to obtain a series of mixed reference substance solutions (Gardenia glycoside: 25.29, 50.58, 126.45 μg / ml; Paeoniflorin: 22.44, 44.88, 112.20 μg / ml). Separately, pipette 1 ml, 2 ml, and 5 ml of mixed reference solution of 25.29 μg / ml gardenia glycoside and 22.44 μg / ml paeoniflorin into three 10 ml volumetric flasks, and dilute to volume with methanol to obtain a series of mixed reference solutions of 11.46 and 28.64 μg / ml (gardenia glycoside: 2.53, 5.06, 12.65 μg / ml; paeoniflorin: 2.24, 4.49, 11.22 μg / ml). Accurately pipette 10 μl of the above series of mixed reference solutions into the liquid chromatograph and record the peak area. Perform regression processing on the injection volume (X) and the corresponding peak area (Y) to obtain two standard curves (see Tables 41-42, Figure 28-29 ). The regression equation of gardenia glycoside is: Y = 1460.1X-12.83, R2 = 0.9992 (n = 6); the regression equation of paeoniflorin is: Y = 1260.1X-12.77, R2 = 0.9996 (n = 6). The results showed that the injection amount and peak area showed a good linear relationship in the range of 0.0506-2.5290μg for gardenia glycoside and in the range of 0.0449-2.2440μg for paeoniflorin.

[0387] Table 41 Gardenia glycoside linear data

[0388]

[0389] Table 42 Paeoniflorin linear data

[0390]

[0391] 5.6 Precision test

[0392] Accurately pipette 10 μl of the mixed reference solution (Gardenia glycoside: 50.58 μg / ml; Paeoniflorin: 40.88 μg / ml), repeat the injection 6 times, measure the peak area, calculate the average peak area value and relative standard deviation (RSD), see Table 43.

[0393] Table 43 Precision results of reference substances

[0394]

[0395] Results: The RSD values ​​of gardenoside and paeoniflorin were 1.89% and 1.43%, respectively, indicating that the instrument had good precision.

[0396] 5.7 Stability test

[0397] Accurately pipette the test solution for the repeatability test, and inject 10 μl at 0, 3, 6, 9, and 12 h respectively. Record the peak areas of gardeniain and paeoniflorin, and calculate the average peak areas and RSD (%) of gardeniain and paeoniflorin, see Table 44.

[0398] Table 44 Stability test results

[0399]

[0400] Results: The RSD values ​​of the peak areas of gardenia glycoside and paeoniflorin were 0.3% and 1.3%, respectively, indicating that the test solution had good stability within 12 hours.

[0401] 5.8 Repeatability test

[0402] Take about 0.4 g of the sample (batch number 20230901), accurately weigh it, and prepare 6 test solutions in parallel according to the preparation method of the test solution. Take 10 μl of each solution, inject it into the liquid chromatograph, measure the peak area, and calculate the content, average content and RSD%. The results are shown in Table 45.

[0403] Table 45 Repeatability test

[0404]

[0405] Results: The average content of gardenoside in the sample was 3.66 mg / g, and the RSD was 0.61% (n=6); the average content of paeoniflorin was 2.58 mg / g, and the RSD was 1.62% (n=6), indicating that this method has good repeatability.

[0406] 5.9 Sample recovery rate

[0407] Preparation of mixed reference stock solution: Accurately weigh 15.24 mg of gardenia glycoside reference substance and 10.85 mg of paeoniflorin reference substance, place in a 20 ml volumetric flask, add methanol to the scale, shake well, and obtain the mixed reference stock solution (gardenia glycoside 0.7475 mg / ml, paeoniflorin 0.5132 mg / ml).

[0408] Using the sample addition recovery method, 6 portions of Jinmai Pai Shi granules with known content (gardenoside content 3.66 mg / g, paeoniflorin content 2.58 mg / g) of the same batch were accurately weighed, each portion was about 0.2 g, and 1 ml of mixed reference solution (peoniflorin 0.5132 mg / ml, gardeniaflorin 0.7475 mg / ml) was accurately added to each portion. According to the preparation method of the test solution, 10 μl of the test solution was respectively aspirated, and the contents of gardeniaflorin and paeoniflorin were determined according to the above-mentioned chromatographic conditions. The sample addition recoveries were calculated respectively. The results are shown in Table 46.

[0409] The recovery rate calculation formula is: recovery rate = (measured amount - content in preparation) / added amount * 100%.

[0410] Table 46 Sample recovery test results (n=6)

[0411]

[0412] The results showed that the average recoveries of gardenoside and paeoniflorin measured by this method were 99.11% and 101.04%, respectively, and the RSD values ​​of both were less than 3%, indicating that the recoveries of gardenoside and paeoniflorin were good.

[0413] 5.10 Sample determination

[0414] Accurately weigh 0.4 g of 6 batches of Jinmai Paishi granules, prepare according to the preparation method of the test solution, inject 10 μl of each, record the chromatogram, measure the peak area, and calculate. The results are shown in Table 47.

[0415] Table 47 Determination results of gardenoside and paeoniflorin in 6 batches of Jinmai Paishi granules samples

[0416]

[0417] The results showed that the contents of gardenia glycoside and paeoniflorin in the test solution were approximately 0.8-1.7 mg / g and 1.3-2.3 mg / g, respectively. Considering the actual situation of large-scale production, the content limits of gardenia glycoside and paeoniflorin were temporarily set at no less than 0.64 mg / g and 1.04 mg / g, respectively. Based on the actual 10 g per bag, each bag of this product contains gardenia glycoside (C 17 H 24 O 10 ) and paeoniflorin (C 23 H 28 O 11 ) shall not be less than 6.4mg and 10.4mg.

[0418] 3. Clinical efficacy verification

[0419] 1. Experimental Design

[0420] 1.1 Source of cases: All cases were from outpatients and inpatients diagnosed with gonorrhea in the Department of Gastroenterology of the Second Affiliated Hospital of Guizhou University of Chinese Medicine, and they were also voluntary clinical researchers who joined this project.

[0421] 1.2 Collection time: January 2020 - March 2024.

[0422] 1.3 Diagnostic criteria: refer to the guidelines for clinical research of new Chinese medicines.

[0423] 1.3.1 Western medicine diagnostic criteria

[0424] Refer to the diagnostic criteria in the relevant chapters of the "Guidelines for Diagnosis and Treatment of Urological Diseases in China" (2019 edition):

[0425] ① Medical history and symptoms: Typical symptoms include sudden onset of lumbar and abdominal colic, accompanied by microscopic or macroscopic hematuria, etc.; or a history of stone excretion; or only dull pain, aching and discomfort in the lower back and abdomen.

[0426] ②Physical signs: During acute attacks, there is often percussion pain or tenderness in the kidney area and ureteral area;

[0427] ③ Color Doppler ultrasound examination: can detect X-ray positive or negative stones larger than 2 mm;

[0428] ④ Urinary tract X-ray: Urinary tract X-ray can detect about 90% of X-ray positive stones and can roughly determine the location, size, number and shape of the stones.

[0429] ⑤ Urine examination: After an attack of colic, an increase in red blood cells is often seen, and more white blood cells can also be seen. When a urinary tract infection occurs, the white blood cell count increases significantly, and urine bacterial culture can be performed.

[0430] ⑥CT examination: CT examination can detect stones as small as 1 mm and can clearly show the shape and size of stones, including negative stones. Therefore, CT plain scan is the first choice for patients with renal colic.

[0431] ⑦Other examinations: intravenous urography, enhanced CT, etc.

[0432] The diagnosis can be made if any of the above imaging tests reveal kidney stones.

[0433] 1.3.2 TCM diagnostic criteria

[0434] Refer to the diagnostic criteria in the relevant chapters of the "Standards for Diagnosis and Efficacy of TCM Diseases and Syndromes" (2017 edition):

[0435] Damp-heat syndrome in the lower part of the body:

[0436] Main symptoms: colic in waist and abdomen, painful urination, blood in urine, or interrupted urination, unbearable stabbing pain during urination. Secondary symptoms: constipation. Tongue: yellow and greasy. Pulse: stringy or rapid. The quantitative table of symptoms is shown in Table 48.

[0437] Table 48 Grading and quantification table of stranguria symptoms

[0438]

[0439]

[0440] 1.4 Inclusion criteria

[0441] ①Age 18-65 years old;

[0442] ② Patients diagnosed with unilateral renal stones by preoperative CT (stone diameter > 2 cm);

[0443] ③ Patients who underwent percutaneous nephrolithotomy (in accordance with the indications for percutaneous nephrolithotomy in the Chinese Guidelines for the Diagnosis and Treatment of Urological and Andrological Diseases (2019 Edition));

[0444] ④ The operation went smoothly, with no major bleeding, infection, or damage to surrounding organs during and after the operation;

[0445] ⑤ Two weeks after surgery, a CT scan was performed to confirm the diagnosis of residual stones, and the diameter of the residual stones was <0.6 cm;

[0446] ⑥ Patients who voluntarily participate in this study, cooperate throughout the whole process, and actively respond to follow-up.

[0447] 1.5 Exclusion criteria

[0448] ① Those who do not meet the above inclusion criteria;

[0449] ② Those suffering from severe cardiovascular, cerebrovascular, liver and kidney diseases;

[0450] ③Women who are preparing for pregnancy, pregnant or breastfeeding;

[0451] ④Those who are taking other stone-removing drugs;

[0452] ⑤ Patients with ureteral stenosis or those who have recently undergone ureteral stenosis dilation or other related surgeries;

[0453] ⑥ Those who refuse to take Chinese medicine or have a history of allergy to the Chinese medicine used in this study.

[0454] 3.6 Criteria for exclusion or dropout of cases

[0455] ① Subjects who develop other diseases during medication that affect the efficacy of the trial;

[0456] ② Those who cannot tolerate the medication;

[0457] ③ Those who fail to take the medicine according to the doctor's instructions or take other medicines on their own, which affects the efficacy evaluation;

[0458] ④ The subject voluntarily withdraws from the study;

[0459] 2. Observation indicators

[0460] 2.1 Safety Observation

[0461] For general physical examination items, if adverse reactions occur, the clinical manifestations, time of occurrence, severity, symptomatic treatment methods and results should be recorded to objectively evaluate its safety.

[0462] 2.2 Efficacy observation (evaluated once before treatment and within 24 hours after treatment)

[0463] (1) Relevant symptoms and tongue and pulse required by this test, and pay attention to the onset time and recovery time.

[0464] (2) Related symptoms: lumbar and abdominal cramps, hematuria, frequent urination, urgency and pain, short and yellow urine, abnormal urination, etc.

[0465] (3) Related signs: tongue, pulse, etc.

[0466] (4) Imaging examination: Perform abdominal CT / B-ultrasound examination once before and after taking the medicine, and record the location, long diameter, and number of stones;

[0467] 3. Efficacy evaluation

[0468] 3.1 Criteria for evaluating efficacy

[0469] (1) Total effective rate of clinical efficacy after medication (calculation formula: total effective rate = (number of cured cases + number of effective cases) ÷ total number of cases × 100%);

[0470] (2) TCM symptom score: The symptoms are scored from mild to severe, from no to present, with scores of 0, 1, 2, and 3. Tongue and pulse are not included in the score. The score is recorded in detail based on the patient's description. The total score of TCM symptoms before and after medication is compared to evaluate the improvement of the condition.

[0471] 4. Statistical analysis

[0472] SPSS 26.0 software was used for statistical analysis of the data. The Kolmogorov-Smirnov test was used to test the normal distribution of continuous variables. Normally distributed quantitative data were expressed in the form of x±s. The paired sample t test was used to compare the differences between the two groups before and after treatment. P<0.05 was considered to be statistically significant.

[0473] 5. Test results

[0474] 5.1 Comprehensive efficacy

[0475] The total effective rate after treatment was 95%. See Table 49.

[0476] Table 49 Comparison of the efficacy of TCM symptoms before and after treatment (x 2 , n=30)

[0477]

[0478] 5.2 Comparison of clinical symptom and sign scores between groups

[0479] As shown in Table 50, compared with before treatment, the clinical symptom and sign scores after treatment were significantly higher than those before treatment, and the difference was extremely statistically significant (P < 0.001).

[0480] Table 50 Comparison of clinical symptom and sign scores between groups before and after treatment

[0481]

[0482] Note: Intra-group comparison: compared with before treatment, # P<0.001.

[0483] 7.3 Comparison of symptom scores between groups

[0484] As shown in Table 51, compared with before treatment, the scores of clinical symptoms and signs between the groups after treatment were significantly higher than those before treatment, and the difference was extremely statistically significant (P < 0.001).

[0485] Table 51 Comparison of symptoms and signs between groups before and after treatment

[0486]

[0487] Note: Comparison of symptoms and signs within the same group: compared with before treatment, #P<0.001.

[0488] 6. Adverse reactions: No adverse reactions occurred during the observation of this subject.

[0489] 7. Comprehensive efficacy analysis

[0490] The results of this study showed that Jinmai Paishi Fang can improve the symptoms and signs of patients with stranguria, with a total effective rate of 95%. This shows that Jinmai Paishi Fang can significantly improve the clinical symptoms and signs of patients with stranguria, and is an effective and safe traditional Chinese medicine preparation for treating stranguria, and is worthy of promotion and application in clinical practice.

[0491] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A golden wheat stone removal composition, characterized in that: The invention is composed of the following Chinese medicinal raw materials in parts by weight: 640-680 parts of Lysimachia chinensis, 145-185 parts of Dianthus superbus, 145-185 parts of Lygodium japonicum, 90-130 parts of Gallus gallus domesticus, 200-240 parts of talc, 200-240 parts of Imperata cyperus rhizome, 145-185 parts of Plantain, 90-130 parts of Cyathula officinalis, 90-130 parts of stir-fried Toosendan fruit, 120-140 parts of Pyrola, 145-185 parts of Abelmoschus flavonoids, 145-185 parts of Paeonia lactiflora, 90-130 parts of stir-fried Citrus aurantium with bran, 90-130 parts of Rehmannia root, 58-74 parts of liquorice tablets and 90-130 parts of Gardenia jasminoides.

2. A Jinmai stone removal composition according to claim 1, characterized in that: The invention is composed of the following Chinese medicinal raw materials in parts by weight: 650-670 parts of Lysimachia chinensis, 155-175 parts of Dianthus superbus, 155-175 parts of Lygodium japonicum, 100-120 parts of Gallus gallus domesticus, 210-230 parts of talc, 210-230 parts of Imperata cylindrica, 155-175 parts of Plantain, 100-120 parts of Cyathula officinalis, 100-120 parts of stir-fried Toosendan fruit, 125-135 parts of Pyrola, 155-175 parts of Abutilon seeds, 155-175 parts of white peony root, 100-120 parts of stir-fried Citrus aurantium with bran, 100-120 parts of Rehmannia root, 62-70 parts of liquorice tablets and 100-120 parts of Gardenia jasminoides.

3. A golden wheat stone removal composition according to claim 2, characterized in that: The invention is composed of the following Chinese medicinal raw materials in parts by weight: 660 parts of Herba Lysimachiae, 165 parts of Dianthus Superbus, 165 parts of Lycopodiella, 110 parts of Gallus gallus domesticus, 220 parts of Talcum, 220 parts of Imperatae Rhizoma, 165 parts of Plantago, 110 parts of Cyathula officinalis, 110 parts of stir-fried Toosendan Fructus, 132 parts of Pyrrosiae, 165 parts of Abelmoschus seeds, 165 parts of Paeonia lactiflora, 110 parts of stir-fried Fructus Aurantii Immaturus with bran, 110 parts of Rehmannia glutinosa, 66 parts of Licorice tablets and 110 parts of Gardenia jasminoides.

4. A method for preparing the Jinmai stone-removing composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: taking a prescribed amount of medicinal materials, talc and sea sand, decocting the mixture in a bag, adding 8-9 times of water for the first time and soaking for 30-60 minutes, adding 4-8 times of water for the second time, decocting for 1-3 times, each time for 30-60 minutes, filtering, concentrating the filtrate to a thick paste with a relative density of 1.18-1.40 at 70° C., drying, crushing, adding appropriate amount of auxiliary materials, and preparing the corresponding dosage form.

5. The method for preparing the Jinmai stone-removing composition according to claim 4, characterized in that: The preparation method comprises the following steps: taking a prescribed amount of medicinal materials, talcum powder and sea sand, decocting the mixture in a bag, adding 8.5 times of water for soaking for 30 minutes for the first time, adding 6 times of water for the second time, decocting twice, each time for 30 minutes, filtering, concentrating the filtrate to a thick paste with a relative density of 1.28-1.32 at 70° C., drying, crushing, adding appropriate amount of auxiliary materials, and preparing the corresponding dosage form.

6. A method for detecting the quality of the Jinmai Stone Removal Composition according to any one of claims 1 to 3 or the Jinmai Stone Removal Composition prepared by the preparation method according to any one of claims 4 to 5, characterized in that: It includes thin layer chromatography identification of Herba Lysimachiae, Rhizoma Achyranthis Bidentatae, stir-fried Fructus Toosendan, stir-fried Fructus Aurantii Immaturus with bran, and Fructus Gardeniae, as well as the content determination of geniposide and paeoniflorin.

7. The quality inspection method according to claim 6, characterized in that: The thin layer chromatography identification is specifically as follows: The thin layer chromatography identification method of Lysimachia chinensis is as follows: take 3g of the product, add 20ml of 70% ethanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation lacking Lysimachia chinensis, and prepare the negative control solution in the same way; take 1g of Lysimachia chinensis control medicinal material, and prepare the control medicinal material solution in the same way; according to the general rule 0502 thin layer chromatography test, take 5-8μl of each of the above three solutions, spot them on the same silica gel G thin layer plate, use ethyl acetate-ethanol-water = 8:2:1 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and immediately place under ultraviolet light for 365min for inspection; The thin layer chromatography identification method of wine Sichuan Achyranthes bidentata is as follows: take 3g of this product, add 20ml of methanol, and ultrasonically treat for 30 minutes, evaporate the filtrate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution; take another 3g of the negative preparation lacking wine Sichuan Achyranthes bidentata, and prepare the negative control solution in the same way; Take 2g of Sichuan Cyathulae Radix as control medicinal material, and prepare control medicinal material solution in the same way; according to the thin layer chromatography test in General Rule 0502, take 8-10μl of the test solution and negative control solution, and 5μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, respectively, use chloroform-methanol = 7:1 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light at 365nm; The thin layer chromatography identification method of stir-fried Toosendan fruit is as follows: take 10g of this product, add 50ml of water to dissolve, filter, extract the filtrate with ethyl acetate twice, 30ml each time, combine the ethyl acetate solution, shake and extract twice with ammonia test solution, 20ml each time, discard the ammonia test solution, take the ethyl acetate solution, evaporate to dryness, add 1ml of ethanol to the residue as the test solution; take another 10g of the negative preparation without stir-fried Toosendan fruit, and prepare the negative control solution in the same way; take 2g of Toosendan fruit control medicinal material, add 50ml of water, decoct for 30 minutes, filter, add 30ml of ethyl acetate to the filtrate, and prepare the control medicinal material solution in the same way; according to the general rule 0502 thin layer chromatography test, take 10μl of each of the above three solutions, spot them on the same silica gel G thin layer plate, use chloroform-acetone = 5:2 as the developing solvent, develop, take out, dry, spray with 2% p-dimethylaminobenzaldehyde-40% sulfuric acid solution, and heat at 105°C until the spots are clearly colored; The thin layer chromatography identification method of bran-fried Citrus aurantium is as follows: take 3g of the product, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation of Citrus aurantium fried without bran, and prepare the negative control solution in the same way; take 0.5g of Citrus aurantium control medicinal material, and prepare the control medicinal material solution in the same way; according to the general rule 0502 thin layer chromatography test, take 2-5μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, use chloroform-methanol-water=13:6:2 lower layer solution as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light at 365nm; The thin layer chromatography identification method of gardenia is as follows: take 3g of this product, add 20ml of 70% methanol, shake for 5 minutes, filter, and concentrate the filtrate to 5ml as the test solution; take another 3g of the negative preparation lacking gardenia, and prepare the negative control solution in the same way; take 0.5g of gardenia control medicinal material, and prepare the control medicinal material solution in the same way; according to the general rule 0502 thin layer chromatography test, take 2-5μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-concentrated ammonia test solution = 8:1:3:1 lower layer solution as the developing agent, develop, take out, dry, and spray with 5% vanillin sulfuric acid solution.

8. The quality inspection method according to claim 6, characterized in that: The content determination method of gardeniside and paeoniflorin is as follows: chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.1% phosphoric acid gradient elution: 0-22min, acetonitrile-0.1% phosphoric acid is 13:87; 23~38min, acetonitrile-0.1% phosphoric acid is 40:60, 39~45min, acetonitrile-0.1% phosphoric acid is 13:87, flow rate is 1ml / min, column temperature is 30℃, detection wavelength is 230nm, injection volume is 10μl, theoretical plate number calculated based on paeoniflorin peak should be not less than 2000; Preparation of reference solution: Take appropriate amount of gardenia glycoside reference substance and paeoniflorin reference substance, accurately weigh, add methanol to make 1ml containing 50μg gardenia glycoside and paeoniflorin, 45 μg of mixed solution is used as the reference solution; preparation of the test solution: take 0.4g of the Jinmai Paishi composite material, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, seal it, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it; sample determination: accurately aspirate 10μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine it.

9. Use of the Jinmai stone-removing composition as claimed in any one of claims 1 to 3 or the Jinmai stone-removing composition prepared by the preparation method as claimed in any one of claims 4 to 5 in preparing a medicine for treating urinary stones.

Images