A quality detection method of a kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition

Abstract

The present application belongs to the field of Chinese medicine preparation, and relates to a quality detection method of a Chinese medicine compound composition for benefiting kidney and dispelling wind. The Chinese medicine compound composition for benefiting kidney and dispelling wind comprises the following components in weight fractions: 9-30 parts of raw astragalus, 6-12 parts of wine holly fruit, 3-6 parts of scorpion, 5-10 parts of fried cantharis, 9-30 parts of prince salvia, 6-12 parts of fried atractylodes, 6-30 parts of pyrrosia, 3-10 parts of safflower, 3-10 parts of chuanxiong, 15-30 parts of white flower snake tongue, 15-30 parts of poria bark, and 9-30 parts of plantago seed. The quality detection method of the Chinese medicine compound composition adopts thin layer chromatography to identify astragalus and wine holly fruit, and further improves the comprehensiveness and reliability of the quality monitoring of the Chinese medicine compound composition by determining the content of calycosin-7-glucoside, and more perfectly characterizes the effective active components and quality of the medicine, which is beneficial to the comprehensive monitoring of the active components and further guarantees the stability, consistency, controllability, safety and effectiveness of the Chinese medicine compound composition.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine preparations, and relates to a quality detection method of a kidney-yi and wind-qi traditional Chinese medicine compound composition. BACKGROUND

[0002] The prevalence rate of adult chronic nephritis in China is about 10%, most of chronic nephritis has insidious onset, prolonged and repeated illness, and can be accompanied by different degrees of renal function decline, and even gradually progresses to chronic renal failure. Early patients can have no obvious symptoms, which is easy to be ignored. The basic clinical manifestations are proteinuria, hematuria, hypertension and edema, among which proteinuria can not only damage the glomerular filtration membrane and renal tubular cells, but also promote glomerular sclerosis and tubulointerstitial fibrosis, which is an independent risk factor for renal function deterioration and disease progression to end-stage renal disease. Therefore, the treatment of reducing proteinuria is very important, which is related to the prognosis of patients with chronic nephritis.

[0003] At present, there is no specific treatment for proteinuria in chronic nephritis and nephrotic syndrome in western medicine. The research mainly focuses on reducing urinary protein and blood pressure control, and the measures to control proteinuria mainly include ACEI and ARB drugs. In clinical practice, glucocorticoids, cytotoxic drugs and immunosuppressive agents also have significant effects, but some patients are not sensitive, long-term use brings side effects, easy to repeat after reducing or stopping the drug, and expensive. In view of the fact that traditional Chinese medicine has exact curative effect in the treatment of chronic nephritis, and plays a role in synergistic effect and reducing toxicity when combined with hormones or immunosuppressive agents, it is highly praised and trusted by the majority of patients. At present, the traditional Chinese medicines for reducing proteinuria on the market include Bailin capsules, Ziling mycosporine (main component is artificially cultured Cordyceps sinensis), Huangkui capsules (main component is Abelmoschus manihot flower), etc. These traditional Chinese medicines have relatively single components, and cannot take into account the complex and refractory proteinuria. Therefore, there is an urgent need in clinical practice to develop a kind of traditional Chinese medicine preparation which is efficient and convenient for patients with chronic nephritis, and to bring greater benefits to the majority of patients with kidney disease.

[0004] In view of traditional Chinese medicine is a complex system composed of multiple components, and its components are affected by multiple factors, such as variety, origin, harvesting, processing, etc., but the diversity and complexity of the components in traditional Chinese medicine are the material basis for its good curative effect, wide application and small side effects. This material basis has been in a state of ambiguity and is difficult to evaluate comprehensively for a long time. After the compatibility and decoction of multiple flavors, the quality of the compound cannot be evaluated with clear, effective and reasonable indicators. However, the treatment effect of traditional Chinese medicine compound is directly related to its quality, and poor quality of traditional Chinese medicine compound will directly lead to poor treatment effect, which requires quality control of traditional Chinese medicine compound to ensure the stability and controllability of curative effect. The quality control of traditional Chinese medicine needs to monitor all the effective components, and the quality control system established can ensure the effectiveness, controllability and safety of the drug. Therefore, establishing a modern quality control system with the characteristics of basic theory of traditional Chinese medicine, solving the problems of traditional Chinese medicine analysis and improving the existing quality control method are the research hotspots today. SUMMARY

[0005] The technical problem solved by the present application is to provide a quality detection method for a kidney-yielding and wind-dispelling traditional Chinese medicine compound composition, which has high accuracy and sensitivity, realizes effective control of the quality of the medicine, and has important significance for controlling the quality of the traditional Chinese medicine compound composition and ensuring the clinical curative effect.

[0006] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] The present application discloses a quality detection method for a kidney-yielding and wind-dispelling traditional Chinese medicine compound composition, which is composed of characteristics, identification, inspection, content determination and microbial limit test, wherein the identification is the identification of Astragalus and wine plum, and the content determination is the determination of the content of calycosin-7-glucoside in the kidney-yielding and wind-dispelling traditional Chinese medicine compound composition granules by high performance liquid chromatography.

[0008] Specifically, the characteristics are that the product is yellow to yellow-brown granules, and the taste is sweet and slightly sour.

[0009] Specifically, the identification includes the identification of Astragalus and the identification of wine plum.

[0010] Further, the identification of Astragalus includes the following steps:

[0011] Preparation of test sample solution: take the kidney-yielding and wind-dispelling traditional Chinese medicine compound composition granules, grind, dissolve in water, extract with saturated n-butanol, and obtain n-butanol extract; wash the n-butanol extract with ammonia test solution, water bath dry the n-butanol, and add methanol to dissolve the obtained solid, to obtain the test sample solution.

[0012] Preparation of negative sample solution: take astragalus negative sample particles, grind, dissolve with water, extract with saturated n-butanol, obtain n-butanol extract; wash the n-butanol extract with ammonia test solution, evaporate the n-butanol in water bath, add methanol to the obtained solid to dissolve, and obtain;

[0013] Preparation of control solution: take astragaloside, add methanol to dissolve, and obtain;

[0014] Identification of astragalus: adopt thin layer chromatography to identify, respectively take test solution, negative sample solution and control solution, place them on a thin layer plate, develop with chloroform-methanol-10wt% sodium hydroxide solution as developing agent, take out, air dry, spray with sulfuric acid ethanol solution, heat until the spots on the thin layer plate develop clear color, and place under ultraviolet light to observe.

[0015] Specifically, the preparation method of the ammonia test solution is as follows: add water to 400mL 25wt%-28wt% concentrated ammonia water to make the volume 1000mL.

[0016] Further, the identification of wine jumumei includes the following steps:

[0017] Preparation of test solution: take Yishen Qufeng traditional Chinese medicine compound composition particles, grind, dissolve with water, extract with ethyl acetate, obtain ethyl acetate extract; evaporate the solvent of the ethyl acetate extract, add methanol to the obtained solid to dissolve, and obtain;

[0018] Preparation of negative sample solution: take wine jumumei negative sample particles, grind, dissolve with water, extract with ethyl acetate, obtain ethyl acetate extract; evaporate the solvent of the ethyl acetate extract, add methanol to the obtained solid to dissolve, and obtain;

[0019] Preparation of control solution: take wine jumumei control medicinal materials, add water to boil, filter, extract the filtrate with ethyl acetate, obtain ethyl acetate extract; evaporate the solvent of the ethyl acetate extract, add methanol to the obtained solid to dissolve, and obtain;

[0020] Identification of wine jumumei: adopt thin layer chromatography to identify, respectively take test solution, negative sample solution and control solution, place them on a thin layer plate, develop with toluene-ethyl acetate-formic acid solvent as developing agent, take out, air dry, spray with sulfuric acid ethanol solution, heat until the spots on the thin layer plate develop clear color, and place under sunlight to observe.

[0021] Specifically, the inspection includes particle size, moisture, solubility and weight difference.

[0022] Specifically, the particle size is inspected according to the double sieve method in the fourth part of the Chinese Pharmacopoeia 2020 edition general rules 0982; the double sieve method stipulates that the sum of the particles that cannot pass through the first sieve and the particles that can pass through the fifth sieve shall not exceed 15%.

[0023] Among them, the moisture is checked according to the drying method in the fourth part of the People's Republic of China Pharmacopoeia 2020 edition general rule 0832; the moisture in the drying method is not more than 8.0%.

[0024] Among them, the solubility is checked according to the soluble granules inspection method under the solubility item in the People's Republic of China Pharmacopoeia 2020 edition general rule 0104 granules; the soluble granules inspection method is provided that all solubility or slight turbidity.

[0025] Among them, the weight variation is checked according to the method under the weight variation item in the fourth part of the People's Republic of China Pharmacopoeia 2020 edition general rule 0104 granules; the weight variation method is provided that the weight variation of each bag compared with the labeled weight is not more than 2 bags of granules exceeding the weight variation limit (± 5%), and not more than 1 bag exceeding the weight variation limit (± 5%) 1 times.

[0026] Specifically, the content determination of calycosin-7-glucoside in the traditional Chinese medicine compound composition granules for benefiting kidney and dispelling wind by high performance liquid chromatography comprises the following steps:

[0027] Preparation of test sample solution: take the traditional Chinese medicine compound composition granules for benefiting kidney and dispelling wind, add methanol, ultrasonic, constant volume, shake well, filter membrane, and get it;

[0028] Preparation of negative sample solution: take the astragalus negative sample granules, add methanol, ultrasonic, constant volume, shake well, filter membrane, and get it;

[0029] Preparation of control solution: take calycosin-7-glucoside, add methanol, ultrasonic, shake well, filter membrane, and get it;

[0030] High performance liquid chromatography conditions: chromatographic column: Agilent Porshell 120EC-C18, specification 4.6mm x 100mm, 2.7μm; mobile phase A is 0.2% phosphoric acid aqueous solution, mobile phase B is acetonitrile, the volume ratio of mobile phase A and mobile phase B is 90:10, isocratic gradient elution; column temperature: 30℃; flow rate: 1.0mL / min; detection wavelength: 260nm; injection volume: 10μL; collection time 40min;

[0031] The determination method of high performance liquid chromatography is: respectively take the test sample solution, the negative sample solution and the control solution, inject into the high performance liquid chromatograph, determine and calculate.

[0032] Specifically, the microbial limit test is checked according to the People's Republic of China Pharmacopoeia 2020 edition 1105 microbial count method and 1106 control bacteria inspection method.

[0033] In some embodiments, the kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition comprises the following components in the weight ratio: 9-30 parts of raw Huangqi, 6-12 parts of Jiumeur, 3-6 parts of Quanxie, 5-10 parts of fried Jiegeng'e, 9-30 parts of Taizi'gen, 6-12 parts of fried Baishu, 6-30 parts of Shivei, 3-10 parts of Honghua, 3-10 parts of Chuanmou, 15-30 parts of Baihuashexie, 15-30 parts of Fulingpi, and 9-30 parts of Cheqianzi. The preparation method of the kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition comprises the following steps: decocting raw Huangqi, Jiumeur, Quanxie, fried Jiegeng'e, Taizi'gen, fried Baishu, Shivei, Honghua, Chuanmou, Baihuashexie, Fulingpi, and Cheqianzi with water, filtering, concentrating the filtrate under reduced pressure to obtain a concentrated solution, placing conventional excipients in a fluidized bed, setting the inlet air temperature, feeding the concentrated solution into the fluidized bed for atomization and drying to obtain the kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition granules.

[0034] In some embodiments, the concentrating under reduced pressure comprises first and second concentrating under reduced pressure. The filtrate is first concentrated under reduced pressure to a relative density of 1.05-1.14 at 60°C, and then centrifuged. The filtrate is further second concentrated under reduced pressure to a relative density of 1.15-1.20 at 60°C, and then conventional excipients are added to obtain the concentrated solution.

[0035] In some embodiments, the inlet air temperature is 100°C. The feeding rate of the concentrated solution is 100-150 r / min. The atomization pressure is 0.2 MPa outside and 0.3 MPa inside. The drying is performed at 60°C for 1-1.5 h.

[0036] Specifically, the kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition comprises the following components in the weight ratio: 9-30 parts of raw Huangqi, 6-12 parts of Jiumeur, 3-6 parts of Quanxie, 5-10 parts of fried Jiegeng'e, 9-30 parts of Taizi'gen, 6-12 parts of fried Baishu, 6-30 parts of Shivei, 3-10 parts of Honghua, 3-10 parts of Chuanmou, 15-30 parts of Baihuashexie, 15-30 parts of Fulingpi, and 9-30 parts of Cheqianzi. The preparation method of the kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition comprises the following steps: decocting raw Huangqi, Jiumeur, Quanxie, fried Jiegeng'e, Taizi'gen, fried Baishu, Shivei, Honghua, Chuanmou, Baihuashexie, Fulingpi, and Cheqianzi with water, filtering, first concentrating the filtrate under reduced pressure to a relative density of 1.05-1.14 at 60°C, centrifuging, further second concentrating the filtrate under reduced pressure to a relative density of 1.15-1.20 at 60°C to obtain a concentrated solution, placing conventional excipients in a fluidized bed, setting the inlet air temperature to 100°C, feeding the concentrated solution into the fluidized bed at a rate of 100-150 r / min, atomizing at an atomization pressure of 0.2 MPa outside and 0.3 MPa inside, and drying at 60°C for 1-1.5 h to obtain the kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition granules.

[0037] The aforementioned herbal compound composition for tonifying the kidneys and dispelling wind uses "tonifying the kidneys and dispelling wind" as its main treatment principle. The prescription is precise and the medicinal effect is specific, closely addressing the core pathogenesis and main contradictions of proteinuria in nephritis: insufficient kidney qi and wind-evil invasion. The selection of prescriptions and medications is based on years of clinical experience, avoiding the drawbacks of trying to treat everything at once and causing mutual restraint. It uses fewer but more effective herbs, achieving significant therapeutic effects. The herbal compound composition for tonifying the kidneys and dispelling wind is more targeted in treating various pathological types of nephritis and mild to severe proteinuria, with a clear overall effective rate and low adverse reactions, making it easier to promote and treat. The dosage of whole scorpion in the herbal compound composition of this invention is slightly higher than in general prescriptions. Whole scorpion is an insect-based medicine, often used to treat proteinuria in nephritis. The conventional daily dose is 1-3g. The inventor inherited the medication experience of the national master of traditional Chinese medicine, Zou Yanqin, and combined it with many years of clinical experience, breaking through the dosage limits of insect-based medicines such as whole scorpion for proteinuria in nephritis. Through repeated clinical reflection and summarization, it has been verified that a higher dose of whole scorpion can be used in clinical treatment, with more significant efficacy at lower doses and no obvious toxic side effects.

[0038] The compatibility principle of the herbal compound composition for tonifying the kidney and dispelling wind is as follows:

[0039] In this traditional Chinese medicine compound, raw astragalus and wine-processed cornus are the principal herbs. Raw astragalus replenishes vital energy and kidney qi, invigorates qi, strengthens essence, and tonifies the lungs and strengthens the exterior, making it an essential medicine for treating kidney disease. Wine-processed cornus strengthens yin, benefits essence, and tonifies the liver and kidneys, which are its special functions. Combining this with Zhang Xichun's statement that "it astringes the righteous qi without astringing the evil qi, unlike other astringent medicines," it astringes essence and stops leakage without astringing the evil. The two herbs together tonify and strengthen the kidneys, treating the root cause. Scorpion and stir-fried silkworm are the assistant herbs, with "dispelling wind" as their key function. Wind evil is difficult to eliminate when it lingers, and cannot be dispersed by ordinary wind-dispelling medicines. Over time, it is accompanied by stagnation of kidney meridians. These two insects are used to dispel wind and unblock meridians to search out the lurking evil. Silkworm is light and dispersing, dispersing wind-heat evil qi externally, and benefiting the throat to dispel wind. It addresses the evil qi from the throat as well, thus resolving both internal and external wind.

[0040] Codonopsis pilosula, a smaller form of ginseng, enters the lung and spleen meridians. It nourishes while clearing heat, and its effects are flexible, addressing deficiencies in the five internal organs according to its function. The yang of the five internal organs generates yin and blood; yang arises naturally, and yin grows accordingly. It clears deficiency fire, stops sweating, generates fluids, and replenishes the original qi of the lungs and spleen.

[0041] Atractylodes macrocephala: Li Zongzi believed that "Atractylodes macrocephala is sweet and warm in nature, and it has the harmonious Qi of the middle Jiao. Therefore, it is the best medicine for tonifying the spleen and stomach." When the middle Jiao is weak, Atractylodes macrocephala can help to regulate the middle Qi, strengthen the spleen and dry dampness. When the earth is strong, it can restore the original position of clearing and ascending and descending of turbidity. It can also strengthen the body, remove dampness and fill the interior. When the interior Qi is full, the exterior Qi will be solidified. When combined with raw Astragalus membranaceus, it can fill the interior and strengthen the exterior at the same time.

[0042] Safflower and Ligusticum chuanxiong: These herbs promote blood circulation and harmonize the meridians throughout the treatment. The combination of safflower and Ligusticum chuanxiong gently disperses blood stasis. They penetrate deep into the blood to remove stubborn blood stasis and stagnant blood, gradually relieving lower back pain and kidney stagnation, and restoring harmony to the blood vessels.

[0043] Pyrrosia lingua, Poria cocos peel, Plantago asiatica seed, and Hedyotis diffusa: The developers inherited the principles of traditional Chinese medicine master Zou Yanqin, who favored a mild diuretic approach and a heavy dose of light medicine. In clinical practice, they frequently combine mild diuretics such as Poria cocos peel and Plantago asiatica seed in a heavy dose to promote diuresis without excessively damaging Qi and Yin. For patients with chronic nephritis accompanied by edema, diuresis is the primary focus, combined with herbs like Hedyotis diffusa and Pyrrosia lingua that clear damp-heat, achieving a synergistic effect of mild diuresis and clearing heat. Furthermore, these herbs guide the medicine to the kidney meridian, serving as adjuvant herbs.

[0044] Invention Concept: The inventors believe that whether proteinuria is caused by primary or secondary kidney disease, the primary location of the disease is in the kidneys, with the spleen and kidneys as key factors, involving the lungs, liver, triple burner, and bladder. The etiology is inseparable from both congenital deficiency and acquired malnutrition. External and internal factors such as the six pathogenic factors, drug toxicity, the seven emotions, diet, and overwork are both the cause and the cause of the disease's progression, often persisting throughout its development and requiring careful clinical treatment. This disease is characterized by deficiency in the root and excess in the branch, with spleen and kidney deficiency as the fundamental cause, especially kidney qi insufficiency. During the course of the disease, pathological factors such as wind, dampness, damp heat, and blood stasis often coexist, leading to persistent and difficult-to-cure proteinuria.

[0045] ① Spleen and Kidney Deficiency as the Root Cause: Proteinuria in chronic nephritis is a Western medical term, and there is no direct related content in ancient Chinese medical texts. Traditional Chinese medicine research can explore its origins in diseases such as edema, lower back pain, urinary retention, general weakness, and dizziness. Combining ancient Chinese medical texts, the Zou family's nephrology tradition, and many years of clinical experience, the inventor believes that spleen and kidney deficiency is the root cause of this disease, with insufficient kidney qi being the primary culprit. The spleen and kidneys represent the innate and acquired constitution of the human body. The kidneys are the foundation of innate constitution, inherited from parents and replenished by acquired constitution. The essence and yang qi in the kidneys can nourish and warm the five internal organs. If kidney qi is weak, it will inevitably affect the nourishment and functional state of the five internal organs. At the same time, insufficient kidney qi weakens the body's vital energy and reduces its ability to transform qi, allowing pathogenic factors to directly damage the kidneys. The kidneys are the organ of water and the foundation of water storage. If their function is disordered, problems will arise in water metabolism, the storage of essential substances, and the excretion of waste products, leading to a series of manifestations such as edema and proteinuria. The spleen is the foundation of postnatal health, responsible for transporting and transforming the essence of food and fluids. It nourishes the innate constitution with the acquired constitution. If the innate constitution is deficient, it can be restored with postnatal nourishment. However, if the innate constitution is deficient and the postnatal constitution is also insufficient, there is little chance of survival. Therefore, regulating the spleen and kidneys is the key to treating the kidneys. At the same time, the spleen and stomach are the hub of the ascending and descending of Qi. They are responsible for ascending and transporting the essence. When the clear Yang ascends, the essence is consolidated, and when the turbid Yin descends, the Qi is harmonized, and urination and defecation remain unchanged.

[0046] ② Wind evil runs through the whole process: In clinic, wind evil is particularly emphasized in the treatment of pathological factors of proteinuria in kidney disease. It is believed that wind evil damages kidney throughout the whole process of the occurrence and development of proteinuria, and is also the main cause of the intermittent and prolonged proteinuria. As Professor Yan Dexin said: 'Water is calm and clear without wind, but it becomes turbulent and turbid when it encounters wind. Chronic nephritis with proteinuria is lingering and intractable, and the root cause is often wind evil.' The treatment of wind evil needs to consider external wind, internal wind and mixed wind. External wind is mainly exogenous wind evil, which is caused by wind-cold and wind-heat attacking the body surface, and is often accompanied by surface symptoms. Throat symptoms are common in clinic, such as sore throat, even tonsillitis and redness, which are caused by wind-heat evil. Skin itching and even skin rash are also manifestations of external wind. As for internal wind, on the one hand, external wind disturbs the kidney and wind evil hides in the kidney, becoming latent evil, which is difficult to eliminate and often accompanied by other evil. On the other hand, some patients with chronic nephritis are accompanied by high blood pressure, dizziness and even headache, which is caused by internal liver wind, and is caused by deficiency of liver and kidney and lack of kidney yin. Mixed wind is also common in clinic, and proteinuria often increases significantly when it attacks. It is caused by external wind invasion and internal wind activation, and is caused by mixed wind evil. Internal wind is not eliminated, and the healthy qi is not sufficient. External wind will also be invaded.

[0047] ③ Water wet, damp, blood clots mixed: kidney disease more spleen and kidney weakness, or with lung qi deficiency, water metabolism disorder, water and wet evil is one of the main pathological factors, wet heavy turbidity, clinical patients more lower extremity edema, with water and wet evil corresponding to the low high swelling; a part of patients eyelid face swelling mainly, then is wind evil mixed with water and wet evil; stool not into shape or loose sticky, tired and weak also more, more by spleen and kidney deficiency, water wet spleen caused by. Damp heat is often caused by water and wet heat and long-term retention, wet and hot and mutual resistance, combined and difficult to distinguish, sticky and difficult to solve, also difficult to eliminate in symptoms; two evil who more who less, who master who auxiliary, pathogenesis complex difficult to identify, also for clinical prescription drug added a great difficulty; three wet diffuse nature, often diffuse Sanjiao, proteinuria formation and lower jiao most, but can not ignore the upper and middle two jiao; five wet belong to evil, injury and injury, heat belong to evil, consume Yin liquid, the course of the disease, gas Yin dark consumption, deficiency, more obvious. Zou kidney disease from Zou Yunxiang old Mr. treatment of kidney edema considering blood water disease, that is, considering the pathological factors of blood stasis, on this basis, the inventor inherits Zou's experience and combines years of clinical experience, that chronic nephritis proteinuria blood stasis and wind evil throughout the disease, when the blood stasis or visible or difficult to capture, there are light, often with other pathological factors, so that wind, heat and blood stasis combined with the disease, exacerbating the complexity of pathogenesis, the intractability of the disease.

[0048] According to its etiology and pathogenesis and pathological factors, the inventor established the basic method of treating nephrotic proteinuria "benefiting qi, dispelling wind, removing blood stasis and clearing and draining", and self-made Yishen Qufeng Decoction, which mainly includes Huangqi, Taizishen, Jiurusu, Chuaibai-shu, Quanxie, Chaojiangcan, Shiwu, Honghua, Chuanmiao, Baihuashexie, Fulingpi, Cheqianzi. Yishen Qufeng Decoction is mainly for lung, spleen and kidney, and can dispel water and dampness, damp heat, blood stasis and wind evil, and treat deficiency and excess simultaneously. It has achieved good results in clinical application. There is an urgent need for efficient and convenient Chinese patent medicine preparations for patients with chronic nephritis to bring greater benefits to kidney patients.

[0049] The development of the project has wide clinical and market development prospects. According to the inventor's single outpatient volume, about 40% to 60% of kidney disease patients have proteinuria, and the patient demand of the inventor has reached 2000 person-times / year. The annual outpatient volume of the Nephrology Department of Jiangsu Provincial Hospital of Traditional Chinese Medicine is about 150,000 times, and it is estimated that at least 50,000 person-times / year have the demand of reducing urinary protein, which has great economic benefits, and can help patients delay the progression of kidney function, reduce the use of expensive immunosuppressive agents, and reduce the economic burden on individuals, families and society. In the future, on the basis of clinical use of hospital preparations, new proteinuria-lowering drugs with clear mechanism of action, clear effective components, stable and controllable quality, advanced production process, definite curative effect and safe and reliable can be further developed, which is expected to create huge economic and social benefits, and is also an important part of promoting Chinese medicine culture and promoting the modernization of traditional Chinese medicine.

[0050] In some embodiments, in the identification of Astragalus, the volume ratio of the developing agent trichloromethane-methanol-10wt% sodium hydroxide solution is 13:7:2; the ultraviolet light has a wavelength of 366nm.

[0051] In some embodiments, in the identification of Astragalus, the sulfuric acid ethanol solution has a solvent of ethanol and a solute of sulfuric acid, and the concentration of sulfuric acid is 10wt%.

[0052] In some embodiments, in the identification of Wine Junmei, the volume ratio of the developing agent toluene-ethyl acetate-formic acid solvent is 5:8:1.

[0053] In some embodiments, in the identification of Wine Junmei, the sulfuric acid ethanol solution has a solvent of ethanol and a solute of sulfuric acid, and the concentration of sulfuric acid is 10wt%.

[0054] In some embodiments, the Astragalus negative sample particles are prepared by the preparation method of the kidney-yi and wind-removing traditional Chinese medicine compound composition except for Astragalus.

[0055] In some embodiments, the Wine Junmei negative sample particles are prepared by the preparation method of the kidney-yi and wind-removing traditional Chinese medicine compound composition except for Wine Junmei.

[0056] In some embodiments, in the content determination of calycosin-7-glucoside, the calculation method is the external standard method.

[0057] The idea of identifying only Astragalus and Wine Junmei in the traditional Chinese medicine compound composition in the present application is as follows: Astragalus and Wine Junmei are the monarch drugs in the kidney-yi and wind-removing traditional Chinese medicine compound composition of the present application, so thin layer chromatography is used to identify Astragalus and Wine Junmei in the quality detection method of the present application.

[0058] The idea of determining the content of calycosin-7-glucoside in the traditional Chinese medicine compound composition granules for benefiting kidney and dispelling wind in the present application is as follows: the traditional water decoction method is used to extract the traditional Chinese medicine compound composition to prepare granules, and the index component of Jiu Gushu is not found in the granules after detection, and the index component of Jiu Gushu cannot be extracted by the water extraction method, but the index component of calycosin-7-glucoside of Astragalus in the pharmacopoeia is found in the detection, so the content of calycosin-7-glucoside in the traditional Chinese medicine compound composition granules for benefiting kidney and dispelling wind is determined by the high performance liquid chromatography method.

[0059] Advantages:

[0060] (1) The quality detection method of the traditional Chinese medicine compound composition disclosed by the present application further improves the comprehensiveness and reliability of the quality monitoring of the traditional Chinese medicine compound composition by determining the content of calycosin-7-glucoside; the test solution, negative sample solution and control solution are extracted and treated, and the appropriate mobile phase and gradient elution program are selected, so that the detection method is accurate and reliable, has strong specificity, and meets the requirements of linear relationship, precision and recovery rate, and is simple and convenient to operate.

[0061] (2) The quality detection method of the traditional Chinese medicine compound composition disclosed by the present application realizes the identification of Astragalus in the traditional Chinese medicine compound composition by dissolving the traditional Chinese medicine compound composition granules with water, extracting with n-butanol, washing with ammonia test solution, dissolving with methanol to prepare a test solution, and developing the test solution with chloroform-methanol-10wt% sodium hydroxide solution as a developing agent; the identification of Jiu Gushu in the traditional Chinese medicine compound composition is realized by dissolving the traditional Chinese medicine compound composition granules with water, extracting with ethyl acetate, dissolving with methanol to prepare a test solution, and developing the test solution with toluene-ethyl acetate-formic acid as a developing agent, so that the quality detection of the traditional Chinese medicine compound composition is more comprehensive, and the effective active components and the quality of the medicine are more perfectly characterized, which is beneficial to the comprehensive monitoring of the active components, further guarantees the stability, consistency and controllability of the traditional Chinese medicine compound composition, and ensures the safety and effectiveness of the traditional Chinese medicine compound composition. BRIEF DESCRIPTION OF DRAWINGS

[0062] The above and / or other aspects of the present application will become apparent by the following description of the application with reference to the accompanying drawings, in which:

[0063] Figure 1 The thin layer plate coloration chart for identifying Astragalus with chloroform-methanol-10wt% sodium hydroxide solution as a developing agent; wherein, 1 is a control solution, 2 is a test solution, and 3 is a negative sample solution.

[0064] Figure 2This study investigated the identification of Astragalus membranaceus by thin-layer chromatography using silica gel G-plate spotting at Qingdao Ocean Chemical Co., Ltd. In the sample, 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0065] Figure 3 This study investigated the identification of Astragalus membranaceus by thin-layer chromatography using silica gel G-plate spotting at Anhui Liangchen Silicon Source Materials Co., Ltd. 1 is a reference solution, 2 is the test solution, and 3 is a negative sample solution.

[0066] Figure 4 Thin-layer chromatograms were prepared to investigate the identification of Astragalus membranaceus by thin-layer chromatography at 10℃; in the figure, 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0067] Figure 5 Thin-layer chromatograms were prepared to investigate the identification of Astragalus membranaceus by thin-layer chromatography at 25℃; in the figure, 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0068] Figure 6 Thin-layer chromatograms were prepared to investigate the identification of Astragalus membranaceus by thin-layer chromatography at 32% humidity. In the figure, 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0069] Figure 7 Thin-layer chromatograms were prepared to investigate the identification of Astragalus membranaceus by thin-layer chromatography at 72% humidity. In the figure, 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0070] Figure 8 The thin-layer chromatography (TLC) image is shown for identifying Cornus officinalis using toluene-ethyl acetate-formic acid as the developing solvent; where 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0071] Figure 9 This study investigated the identification of Cornus officinalis by thin-layer chromatography using silica gel G-plate spotting for Qingdao Ocean Chemical Co., Ltd. In the sample, 1 was the reference solution, 2 was the test solution, and 3 was the negative sample solution.

[0072] Figure 10 This study investigated the identification of Cornus officinalis by thin-layer chromatography using silica gel G-plate spotting at Anhui Liangchen Silicon Source Materials Co., Ltd. 1 is a reference solution, 2 is the test solution, and 3 is a negative sample solution.

[0073] Figure 11 Thin-layer chromatograms were prepared to investigate the identification of Cornus officinalis by thin-layer chromatography at 10℃; in the figure, 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0074] Figure 12 Thin-layer chromatograms were prepared to investigate the identification of Cornus officinalis by thin-layer chromatography at 25℃; in the figure, 1 is the reference solution, 2 is the test solution, and 3 is the negative sample solution.

[0075] Figure 13 The thin layer chromatography plate for identifying Jiu Gushu under 32% humidity is shown in the following figure, wherein 1 is a control solution, 2 is a test solution, and 3 is a negative sample solution.

[0076] Figure 14 The thin layer chromatography plate for identifying Jiu Gushu under 72% humidity is shown in the following figure, wherein 1 is a control solution, 2 is a test solution, and 3 is a negative sample solution.

[0077] Figure 15 The special composition figure of traditional Chinese medicine compound is shown in the following figure, wherein Figure 15 A is the chromatogram of calycosin-7-glucoside control sample, Figure 15 B is the chromatogram of test sample, Figure 15 C is the negative sample of Astragalus membranaceus.

[0078] Figure 16 The linear graph of the standard curve for determining the content of calycosin-7-glucoside is shown in the following figure.

[0079] Figure 17 The statistical chart of the effect of Yishen Qufeng Decoction on the last 24h-UTP (mg / d) of rats at the 8th week is shown in the following figure, wherein Con is a blank group, Hem is a model group, YSQF-L is a low-dose Yishen Qufeng Decoction group, YSQF-M is a medium-dose Yishen Qufeng Decoction group, and YSQF-H is a high-dose Yishen Qufeng Decoction group.

[0080] Figure 18 The Masson staining chart of the kidney tissue of the model group of rats is shown in the following figure.

[0081] Figure 19 The Masson staining chart of the kidney tissue of the high-dose Yishen Qufeng Decoction treatment group of rats is shown in the following figure.

[0082] Figure 20 The PASM staining chart of the kidney tissue of the model group of rats is shown in the following figure.

[0083] Figure 21 The PASM staining chart of the kidney tissue of the high-dose Yishen Qufeng Decoction treatment group of rats is shown in the following figure. DETAILED DESCRIPTION

[0084] The embodiments of the present application will be described in detail below with examples, and the specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be purchased in the market.

[0085] 1 The materials, reagents and medicinal materials used in the following examples are as follows:

[0086] 1.1 Experimental materials

[0087] Silica gel plate (batch number: 20210329, silica gel G plate of Qingdao Haoyang Chemical Industry) / (batch number: 20210320, silica gel G plate of Anhui Liangchen Silicon Source Material).

[0088] 1.2 Experimental reagents and medicinal materials

[0089] Ammonia test solution preparation: 400 mL of 25wt%-28wt% concentrated ammonia was added to 1000 mL with water, and then the volume was adjusted to 1000 mL.

[0090] In the study of the quality standard of the traditional Chinese medicine compound composition of the application, the control samples are all collected in the fourth edition of the Pharmacopoeia of the People's Republic of China 2020, and are purchased from the China Institute for Drug Control. According to the “Technical Requirements for Research on Control Samples for Quality Standards of Traditional Chinese Medicine New Drugs” issued by the State Drug Administration, they can be directly used according to the category, and the following is explained:

[0091] Astragaloside reference substance, batch number: 110781-201717; calycosin-7-glucoside, batch number: 111920-201304; wine jumume medicinal material, batch number 121495-200702; all purchased from China Institute for Drug Control.

[0092] Example 1: Preparation of kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition granules

[0093] A kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition comprises the following components in parts by weight: 30 parts of raw astragalus, 10 parts of wine jumume, 4 parts of scorpion, 10 parts of fried silkworm, 15 parts of prince ginseng, 10 parts of fried atractylodes, 10 parts of pyrrosia herb, 10 parts of safflower, 10 parts of chuanxiong, 15 parts of white flower snake tongue, 15 parts of poria cocos skin, and 15 parts of plantago seed.

[0094] Preparation method: take the above medicines and decoct twice with water (conventional decoction, add 6 times the mass of water to the total mass of medicinal materials each time), filter separately, combine the two filtrates, and perform first vacuum concentration (-0.06 MPa, 60℃) on the filtrate to 60℃ relative density of 1.05-1.14. After standing, centrifuge with a high-speed centrifuge (16000 r / min, 6 L / min). Perform second vacuum concentration (-0.06 MPa, 60℃) on the filtrate to 60℃ relative density of 1.15-1.20 to obtain a concentrated solution. Weigh an appropriate amount of dextrin and place it in a fluidized bed. Set the inlet air temperature to 100℃, and feed the concentrated solution at a rate of 120 r / min. The atomization pressure is 0.2 MPa outside and 0.3 MPa inside. After spraying, dry at 60℃ for 1.2 h to obtain kidney-nourishing and wind-dispelling traditional Chinese medicine compound composition granules.

[0095] Example 2: Preparation of astragalus negative sample granules

[0096] Huangqi Yin sample, comprising the following components in parts by weight: Jiumuru 10 parts, Quanxie 4 parts, Chaojiangcan 10 parts, Taizipen 15 parts, Chao-baizhu 10 parts, Shiwu 10 parts, Honghua 10 parts, Chuanmiao 10 parts, Baihuasheshecail 15 parts, Fulingpi 15 parts, Cheqianzi 15 parts.

[0097] Preparation method: take the above drugs and decoct twice with water (conventional decoction, add 6 times of water to the total mass of medicinal materials each time), filter separately, combine the two filtrates, and perform first vacuum concentration (-0.06 MPa, 60°C) on the filtrate to 60°C relative density of 1.05-1.14, centrifuge after standing with a high-speed centrifuge (16000 r / min, 6L / min), perform second vacuum concentration (-0.06 MPa, 60°C) on the filtrate to 60°C relative density of 1.15-1.20, and obtain concentrated liquid; take an appropriate amount of dextrin and place it in a fluidized bed, set the inlet air temperature to 100°C, and input the concentrated liquid at a rate of 120 r / min, with atomization pressure outside 0.2 MPa and atomization pressure inside 0.3 MPa, spray until the end, and dry at 60°C for 1.2 h to obtain Huangqi Yin sample granules.

[0098] Example 3: Preparation of Jiumuru Yin sample granules

[0099] Jiumuru Yin sample, comprising the following components in parts by weight: Shenghuangqi 30 parts, Quanxie 4 parts, Chaojiangcan 10 parts, Taizipen 15 parts, Chao-baizhu 10 parts, Shiwu 10 parts, Honghua 10 parts, Chuanmiao 10 parts, Baihuasheshecail 15 parts, Fulingpi 15 parts, Cheqianzi 15 parts.

[0100] Preparation method: take the above drugs and decoct twice with water (conventional decoction, add 6 times of water to the total mass of medicinal materials each time), filter separately, combine the two filtrates, and perform first vacuum concentration (-0.06 MPa, 60°C) on the filtrate to 60°C relative density of 1.05-1.14, centrifuge after standing with a high-speed centrifuge (16000 r / min, 6L / min), perform second vacuum concentration (-0.06 MPa, 60°C) on the filtrate to 60°C relative density of 1.15-1.20, and obtain concentrated liquid; take an appropriate amount of dextrin and place it in a fluidized bed, set the inlet air temperature to 100°C, and input the concentrated liquid at a rate of 120 r / min, with atomization pressure outside 0.2 MPa and atomization pressure inside 0.3 MPa, spray until the end, and dry at 60°C for 1.2 h to obtain Jiumuru Yin sample granules.

[0101] Example 4: Quality detection of traditional Chinese medicine compound composition for benefiting kidney and dispelling wind

[0102] 1. Study of properties

[0103] According to the results of the pilot sample, three batches of pilot samples (batch numbers 20221001, 20221002 and 20221003, each batch of sample has the same weight fraction ratio of each traditional Chinese medicine raw material and the preparation method as in Example 1, and the total mass of the used traditional Chinese medicine raw materials is about 84.7 kg) are consistent with the description of the results, and are included in the quality control method text. The product is yellow to yellow-brown granules, with a sweet and slightly sour taste.

[0104] 2. Identification of Astragalus

[0105] 2.1. Identification of Astragalus in the Traditional Chinese Medicine Compound Composition for Benefiting Kidney and Dispelling Wind by Thin Layer Chromatography

[0106] Preparation of test sample solution: Astragalus membranaceus was used as a negative sample. The preparation method was the same as that of the traditional Chinese medicine compound composition for benefiting kidney and dispelling wind in Example 2. The total mass of the used traditional Chinese medicine raw materials was about 2728 g. The powder was obtained by grinding. 10 g of the powder was weighed and dissolved in water. Saturated n-butanol was used for extraction twice, 20 mL each time. The organic phase (i.e. n-butanol extract) was separated and combined. The organic phase was washed with ammonia solution twice, and the water phase was discarded. The organic phase was dried by water bath. The obtained solid residue was dissolved in 1 mL of methanol to obtain the test sample solution.

[0107] Preparation of test sample solution: Astragalus membranaceus was used as a negative sample. The preparation method was the same as that of the traditional Chinese medicine compound composition for benefiting kidney and dispelling wind in Example 2. The total mass of the used traditional Chinese medicine raw materials was about 2728 g. The powder was obtained by grinding. 10 g of the powder was weighed and dissolved in water. Saturated n-butanol was used for extraction twice, 20 mL each time. The organic phase (i.e. n-butanol extract) was separated and combined. The organic phase was washed with ammonia solution twice, and the water phase was discarded. The organic phase was dried by water bath. The obtained solid residue was dissolved in 1 mL of methanol to obtain the test sample solution.

[0108] Preparation of control sample solution: 11.87 mg of astragaloside A was weighed and dissolved in methanol to prepare a control sample solution with a concentration of 0.4748 mg / mL.

[0109] Sample application: 10 μL of each of the above test sample solution, negative sample solution and control sample solution was applied to the same silica gel G thin layer plate (silica gel G plate from Qingdao Haoyang Chemical Industry).

[0110] Developing agent: (1) The developing agent was trichloromethane-methanol-water (volume ratio of 13:7:2), the separation degree was poor, and the spots were not clear.

[0111] (2) with chloroform-methanol-10wt% sodium hydroxide solution (volume ratio of the three is 13:7:2) as the developing agent, take out, air dry, spray with 10wt% sulfuric acid ethanol solution, heat to the spots on the thin layer plate to develop clear color, place under ultraviolet light (366nm) for inspection, the results are shown in Figure 1 As shown in Figure 1 , this method has good separation and clear spots, so it is used as the thin layer chromatography identification of Huangqi, and the durability test is carried out.

[0112] 2.2, the thin layer chromatography identification method of Huangqi in the kidney and wind dispelling traditional Chinese medicine compound composition

[0113] (1) Different thin layer plates

[0114] Take 10μL of the test solution, negative sample solution and control solution in "Example 4, 2.1" respectively, and point them on the silica gel G plate of Qingdao Haoyang Chemical Industry (batch number: 20210329); then take 10μL of the above three solutions respectively and point them on the silica gel G plate of Anhui Liangchen Silicon Source Material (batch number: 20210320); under the same temperature and humidity conditions, develop with chloroform-methanol-10wt% sodium hydroxide solution (volume ratio of the three is 13:7:2) as the developing agent, take out, air dry, spray with 10wt% sulfuric acid ethanol solution, heat to the spots on the thin layer plate to develop clear color, place under ultraviolet light (366nm) for inspection, the results are shown in Figure 2 and Figure 3 , clear separation, proving that the method has good adaptability to different thin layer plates.

[0115] (2) Different temperatures

[0116] Under the conditions of 10℃ and 25℃, respectively, develop the thin layer chromatography identification of Huangqi by the method of "Example 4, 2.1" above (using the silica gel G plate of Qingdao Haoyang Chemical Industry), the results are shown in Figure 4 and Figure 5 , there is no obvious difference between the chromatograms under the conditions of 10℃ and 25℃, so the method has good adaptability to temperature.

[0117] (3) Different humidity

[0118] Under the conditions of 32% humidity and 72% humidity, respectively, develop the thin layer chromatography identification of Huangqi by the method of "Example 4, 2.1" above (using the silica gel G plate of Qingdao Haoyang Chemical Industry), the results are shown in Figure 6 and Figure 7 , there is no obvious difference between the chromatograms under the conditions of 32% humidity and 72% humidity, so the method has good adaptability to humidity.

[0119] 3, identification of Jiuju meat

[0120] 3.1, Identification of Jiu Gushan in the Chinese medicine compound composition of Yishen Qufeng by thin layer chromatography

[0121] Preparation of test sample solution: the Chinese medicine compound composition granules prepared by the method of Example 1 (the weight ratio of each raw material and the preparation method are the same as Example 1, and the total mass of the raw materials used is about 84.7 kg) were weighed, ground, and powder was obtained; 8 g of the powder was weighed, dissolved in water, and extracted twice with 20 mL of ethyl acetate each time, and the organic phase (i.e. ethyl acetate extract) was combined. The ethyl acetate in the ethyl acetate extract was evaporated, and the obtained solid was dissolved in 1 mL of methanol to obtain the test sample solution.

[0122] Preparation of negative sample solution: the Jiu Gushan negative sample granules prepared by the method of Example 3 (the weight ratio of each raw material and the preparation method are the same as Example 3, and the total mass of the raw materials used is about 3168 g) were weighed, ground, and powder was obtained; 8 g of the powder was weighed, dissolved in water, and extracted twice with 20 mL of ethyl acetate each time, and the organic phase (i.e. ethyl acetate extract) was combined. The ethyl acetate in the ethyl acetate extract was evaporated, and the obtained solid was dissolved in 1 mL of methanol to obtain the test sample solution.

[0123] Preparation of control sample solution: 5 g of Jiu Gushan control drug was added to 50 mL of water, boiled for 30 min, filtered, and the filtrate was concentrated to about 20 mL. The solution was extracted twice with 20 mL of ethyl acetate each time, and the organic phase (i.e. ethyl acetate extract) was combined. The ethyl acetate in the ethyl acetate extract was evaporated, and the obtained solid was dissolved in 1 mL of methanol to obtain the control sample solution.

[0124] Sample application: 10 μL of each of the test sample solution, negative sample solution, and control sample solution was applied to the same silica gel G thin layer plate (silica gel G plate from Qingdao Haoyang Chemical Industry) respectively.

[0125] Developing agent: the developing agent was toluene-ethyl acetate-formic acid (volume ratio of 5:8:1), and the plate was taken out, dried, and sprayed with 10 wt% sulfuric acid ethanol solution. The plate was heated until the spots on the plate were clearly colored, and observed under daylight. The results are shown in Table 1, and the separation degree was good and the spots were clear, so this method was used as the thin layer identification of Jiu Gushan, and a durability test was conducted. Figure 8

[0126] 3.2, Durability test of the thin layer chromatography identification method of Jiu Gushan in the Chinese medicine compound composition

[0127] (1) Different thin layer plates

[0128] ​Take 10 μL of the test solution, negative sample solution and control solution in "Example 4, 3.1" respectively, and spot them on the silica gel G plate (batch number: 20210329) of Qingdao Haoyang Chemical Industry; then take 10 μL of the above three solutions and spot them on the silica gel G plate (batch number: 20210320) of Anhui Liangchen Silicon Source Material; under the same temperature and humidity conditions, develop them with toluene-ethyl acetate-formic acid (volume ratio of the three is 5:8:1) as the developing agent, take them out, air dry, spray with 10 wt% sulfuric acid ethanol solution, heat until the spots on the thin layer plate develop clear color, and observe under daylight, and the results are shown in Figure 9 and Figure 10 The separation is clear, and this method is well adaptable to different thin layer plates.

[0129] (2) Investigation at different temperatures

[0130] Without changing other conditions, develop the thin layer chromatogram for identification at 10 ℃ and 25 ℃ respectively using the method in "Example 4, 3.1" above (use the silica gel G plate of Qingdao Haoyang Chemical Industry), and the results are shown in Figure 11 and Figure 12 The chromatograms at the two temperatures have no obvious difference, so this method is well adaptable to temperature.

[0131] (3) Investigation at different humidities

[0132] Without changing other conditions, develop the thin layer chromatogram for identification at 32% humidity and 72% humidity respectively using the method in "Example 4, 3.1" above (use the silica gel G plate of Qingdao Haoyang Chemical Industry), and the results are shown in Figure 13 and Figure 14 The chromatograms at the two humidities have no obvious difference, so this method is well adaptable to humidity.

[0133] 4. Inspection

[0134] 4.1. Particle size inspection

[0135] According to the particle size of "People's Republic of China Pharmacopoeia" 2020 edition volume four (general rule 0982 second method—double sieve method), the total of the particles that can pass through No. 1 sieve and No. 5 sieve shall not exceed 15%, and the particle size determination of the three batches of trial products (i.e. 20221001, 20221002 and 20221003, the three batches of drug particles in "1. Study on the properties" in Example 4) is shown in Table 1.

[0136] Table 1: Particle size determination results of three batches of trial samples

[0137]

[0138] The determination results in Table 1 show that the particle sizes of the three batches of trial samples meet the requirements.

[0139] 4.2, Moisture test

[0140] According to the moisture determination method (Chinese Pharmacopoeia 2020 edition volume IV (general rule 0832 second method - drying method), the moisture should not exceed 8.0%, and the moisture determination results of three batches of pilot samples (i.e. 20221001, 20221002 and 20221003, three batches of drug particles in the item of "1, study on the properties" in example 4) are shown in table 2.

[0141] Table 2 moisture determination results of three batches of pilot samples

[0142]

[0143] The determination results in table 2 show that the moisture of the three batches of pilot samples meets the requirements.

[0144] 4.3, Solubility test

[0145] According to the granules preparation general rule (Chinese Pharmacopoeia 2020 edition general rule) "0104 granules"

solubility

[0146] Table 3 solubility test results of three batches of pilot samples

[0147]

[0148] The determination results in table 3 show that the solubility of the three batches of pilot samples meets the requirements.

[0149] 4.4, Weight variation test

[0150] According to the granules preparation general rule (Chinese Pharmacopoeia 2020 edition volume IV general rule) "0104 granules"

weight variation

[0151] Table 4 weight variation test results of three batches of pilot samples

[0152]

[0153] The results in Table 4 show that the difference in loading of the three batches of pilot samples meets the requirements.

[0154] 5. Determination of Calycosin-7-Glucoside in Traditional Chinese Medicine Compound Composition Granules by High Performance Liquid Chromatography

[0155] 5.1. Methodological Investigation

[0156] (1) Chromatographic Conditions

[0157] Chromatographic column: Agilent Porshell 120EC-C18 (4.6 mm x 100 mm, 2.7 μm); mobile phase: mobile phase A is 0.2% phosphoric acid aqueous solution, mobile phase B is acetonitrile, the volume ratio of mobile phase A to mobile phase B is 90:10, isocratic gradient elution; column temperature: 30°C; flow rate: 1.0 mL / min; detection wavelength: 260 nm; injection volume: 10 μL; collection time: 40 min.

[0158] (2) Preparation of Test Solution

[0159] Take 1 g of Yishen Qufeng Traditional Chinese Medicine Compound Composition Granules prepared by the method of Example 1 (the weight fraction ratio of each traditional Chinese medicine raw material and the preparation method are the same as in Example 1, and the total mass of the traditional Chinese medicine raw materials used is about 84.7 kg), accurately weigh, place in a 10 mL volumetric flask, add an appropriate amount of methanol, ultrasonic for 15 min (ultrasonic power 280 W, frequency 50 Hz, ultrasonic temperature 40°C), after standing to room temperature, add methanol to the mark, shake well, pass through a 0.22 μm filter membrane, and obtain the test solution.

[0160] (3) Preparation of Negative Sample Solution

[0161] Take 1 g of Huangqi negative sample granules prepared by the method of Example 2 (the weight fraction ratio of each traditional Chinese medicine raw material and the preparation method are the same as in Example 2, and the total mass of the traditional Chinese medicine raw materials used is about 84.7 kg), accurately weigh, place in a 10 mL volumetric flask, add an appropriate amount of methanol, ultrasonic (ultrasonic power 280 W, frequency 50 Hz, ultrasonic temperature 40°C) for 15 min, after standing to room temperature, add methanol to the mark, shake well, pass through a 0.22 μm filter membrane, and obtain the test solution.

[0162] (4) Preparation of Reference Solution

[0163] Take 10 mg of calycosin-7-glucoside reference substance, accurately weigh, add an appropriate amount of methanol, ultrasonic (ultrasonic power 280 W, frequency 50 Hz, ultrasonic temperature 40°C) for 15 min, shake well, pass through a 0.22 μm filter membrane, and prepare a reference solution containing about 1.187 mg of calycosin-7-glucoside per 1 mL.

[0164] (5) Specificity Test

[0165] The test solution, Astragalus membranaceus negative sample solution, and Verrucous isoflavone glucoside reference solution prepared under “(2) Preparation of test solution, (3) Preparation of negative sample solution, and (4) Preparation of reference solution” were respectively injected into the high performance liquid chromatograph for analysis according to “5.1, Methodological Investigation (1) Chromatographic conditions”. The results are as follows. Figure 15 As shown, the results indicate that verbascoside isoflavone glucoside was well separated in the sample and had no negative interference.

[0166] (6) Plotting the standard curve

[0167] Take the verbascoside glucoside reference solution (obtained in the preparation of reference solution under methodological investigation (4)) and dilute it to prepare a solution containing 47.48 μg·mL of verbascoside glucoside. -1 The reference solution was diluted 2, 4, 8, 16, and 32 times sequentially (numbered 1, 2, 3, 4, 5, and 6 respectively). Appropriate amounts were taken and measured according to the chromatographic conditions under "5.1, Methodological Investigation (1)". The results are shown in Table 5. A standard curve was then plotted with peak area (Y) as the ordinate and reference concentration (X) as the abscissa. The standard curve is shown in Table 5. Figure 16 As shown.

[0168] The results showed that the regression equation for verbascoside isoflavone glucoside was y = 35578x + 12124 (R0). 2 =0.9997), in the range of 1.4838–47.48 μg·mL -1 The linear relationship was good within the concentration range. The linearity graph of the standard curve for the determination of verbascoside glucoside content is shown below. Figure 16 .

[0169] Table 5. Linearity Results Test Table

[0170]

[0171] (7) Precision test

[0172] Take 23.74 μg / mL of verbascoside isoflavone glucoside. -1 Reference solution (prepared in the same way as in 5.1, under methodological investigation (4)), diluted to 23.74 μg·mL. -1 The chromatographic conditions were determined according to “5.1, Methodological Investigation (1) Chromatographic Conditions”. Six consecutive injections were performed, and the peak area values ​​were recorded. The results are shown in Table 6. The experimental results show that RSD = 0.75%, which is less than 5%, and the instrument precision is good.

[0173] Table 6 Precision Test Results

[0174]

[0175]

[0176] (8) Reproducibility test

[0177] The same batch number (batch number 20221001, the weight ratio of each traditional Chinese medicine raw material and the preparation method are the same as in Example 1, and the total mass of the used traditional Chinese medicine raw materials is about 84.7 kg) traditional Chinese medicine compound composition granules, 0.3 g, 6 portions, were accurately weighed, the test sample solution was prepared according to the method of “5.1, Methodology Investigation (2) Preparation of Test Sample Solution”, and was injected into the high performance liquid chromatograph, and was determined according to “5.1, Methodology Investigation (1) Chromatographic Conditions”, the peak area value was recorded, and the determination data is shown in Table 7, the experimental results show that: the reproducibility RSD = 3.42%, less than 5%, and the sample reproducibility is good.

[0178] Table 7 Reproducibility test results

[0179]

[0180] (9) Stability test

[0181] The same test sample solution in “(8) Reproducibility test” was taken, and was injected into the high performance liquid chromatograph at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively, and was determined according to “5.1, Methodology Investigation (1) Chromatographic Conditions”, and the peak area value was calculated, and the results are shown in Table 8, the experimental results show that: the stability RSD = 4.92%, less than 5%, and the sample is stable within 24 h.

[0182] Table 8 Stability test results

[0183]

[0184] (10) Spiked recovery test

[0185] 0.2 g of traditional Chinese medicine compound composition granules (batch number 20221001, the weight ratio of each traditional Chinese medicine raw material and the preparation method are the same as in Example 1, and the total mass of the used traditional Chinese medicine raw materials is about 84.7 kg) with a known content were accurately weighed, 6 portions, and 1 mL of the control sample solution containing calycosin-7-glucoside (the concentration of calycosin-7-glucoside in the solution was 23.74 μg·mL -1, the preparation method is the same as that in "(7) Precision test") were respectively placed in 10 mL volumetric flasks, an appropriate amount of methanol was added, and ultrasonic treatment (ultrasonic power 280 W, frequency 50 Hz, ultrasonic temperature 40℃) was performed for 15 min. After being left to room temperature, methanol was added to the mark, shaken well, and filtered through a 0.22 μm filter membrane. The obtained solution was injected into a high performance liquid chromatograph, and determined according to "5.1, Methodology investigation (1) Chromatographic conditions". The peak area value was recorded, and the recovery rate was calculated. The results are shown in Table 9. The recovery rate and RSD meet the requirements.

[0186] Table 9: Recovery test table

[0187]

[0188]

[0189] 5.2, Determination of the content of calycosin-7-glucoside in three batches of pilot samples

[0190] Three batches of pilot samples (batch numbers 20221001, 20221002 and 20221003, three batches of drug granules in "1, Study of properties" in Example 4) were prepared according to "5.1, Methodology investigation (2) Preparation of test solution". Then, they were determined according to "5.1, Methodology investigation (1) Chromatographic conditions". The peak area value was recorded, and the content of calycosin-7-glucoside was calculated. The results are shown in Table 10. The results show that the content of calycosin-7-glucoside in each batch has little difference.

[0191] Table 10: Data table for determination of the content of calycosin-7-glucoside in pilot samples

[0192]

[0193] 6, Microbial limit test

[0194] Culture medium: tryptone soy peptone liquid medium (batch number: 20200302), tryptone soy peptone agar medium (batch number: 20200528), sabouraud glucose liquid medium (batch number: 20200731), sabouraud glucose agar medium (batch number: 20200520), McConkey liquid medium (batch number: 20200824), and McConkey agar medium (batch number: 20200526) were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0195] Preparation of test solution: 10 g of traditional Chinese medicine compound composition granules prepared in Example 1 was weighed, added with tryptone soy peptone liquid medium to 100 mL, and shaken uniformly to serve as 0.1 g / mL test solution for standby.

[0196] 6.1, Aerobic bacteria, mold and yeast count

[0197] 6.1.1, Aerobic bacterial count

[0198] 6.1.1.1, Bacterial count determination:

[0199] Take 1 mL of the above-prepared 0.1 g / mL test solution in a sterile flat dish, and prepare two flat dishes in parallel. Immediately pour 15-20 mL of tryptone soy broth agar medium with a temperature not exceeding 45°C, mix well, solidify, and cultivate at 30-35°C for 3 days. Count the colonies.

[0200] 6.1.1.2, Negative control:

[0201] Take 1 mL of the same batch of prepared tryptone soy broth liquid medium in a sterile flat dish, and prepare two flat plates in parallel. Immediately pour 15-20 mL of tryptone soy broth agar medium with a temperature not exceeding 45°C, mix well, solidify, and cultivate at 30-35°C for 3 days. There should be no bacterial growth.

[0202] 6.1.2, Mold and yeast count

[0203] 6.1.2.1, Mold and yeast count determination:

[0204] Take 1 mL of the above-prepared 0.1 g / mL test solution in a sterile flat dish, and prepare two flat dishes in parallel. Immediately pour 15-20 mL of tryptone soy broth agar medium with a temperature not exceeding 45°C, mix well, solidify, and cultivate at 30-35°C for 3 days. Count the colonies.

[0205] 6.1.2.2, Negative control:

[0206] Take 1 mL of the same batch of prepared tryptone soy broth liquid medium in a sterile flat dish, and prepare two flat plates in parallel. Immediately pour 15-20 mL of tryptone soy broth agar medium with a temperature not exceeding 45°C, mix well, solidify, and cultivate at 30-35°C for 3 days. There should be no bacterial growth.

[0207] 6.2, Control bacteria inspection

[0208] Escherichia coli inspection: Take 10 mL of the above-prepared 0.1 g / mL test solution and add it to 100 mL of tryptone soy broth liquid medium. After 24 hours of cultivation at 30-35°C, if there is bacterial growth, take 1 mL of this culture solution and cultivate it in 100 mL of MacConkey liquid medium at 42-44°C for 48 hours. Then, streak inoculate it on MacConkey agar medium plates to determine whether it is contaminated.

[0209] Negative control: Take 10 mL of the same batch of prepared tryptone soy broth liquid medium and follow the above sample inspection method. After cultivation, there should be no bacterial growth. The specific experimental results are shown in Table 11.

[0210] Microbial limit test results of the traditional Chinese medicine compound composition in Table 11

[0211]

[0212] The microbial limit test results in Table 11 show that the microbial limits of the three batches of pilot samples meet the requirements.

[0213] Example 5: Preparation of a Yishen Qufeng traditional Chinese medicine compound composition decoction

[0214] 30 parts by mass of raw Astragalus membranaceus, 10 parts by mass of wine Fructus Corni, 4 parts by mass of scorpion, 10 parts by mass of fried Philodina, 15 parts by mass of Radix Pseudostellariae, 10 parts by mass of fried Atractylodes, 10 parts by mass of Pyrrosia calcicola, 10 parts by mass of safflower, 10 parts by mass of Ligusticum chuanxiong, 15 parts by mass of Belamcanda chinensis, 30 parts by mass of Poria cocos skin, and 15 parts by mass of psyllium were soaked and decocted in water (the amount of water added was 8 times the total weight of the traditional Chinese medicinal materials) for 30 min; after the soaking was completed, the decoction was first decocted, and after boiling with a strong fire, it was decocted with a weak fire for 30 min; after the decoction was completed, it was filtered, and the filtrate was retained; the filter residue was added with water (the amount of water added was 6 times the total weight of the traditional Chinese medicinal materials) for second decoction, and after boiling with a strong fire, it was decocted with a weak fire for 30 min; after the decoction was completed, it was filtered, and the two filtrates were combined; the filtrate was concentrated under reduced pressure to obtain a concentrated liquid.

[0215] Example 6: Animal experiment

[0216] All the researches of the present application comply with the animal research ethics of the Affiliated Hospital of Nanjing University of Chinese Medicine.

[0217] 1. Selection of animals

[0218] SPF level 5-week-old SD male rats, weighing 180±20g.

[0219] 2. Establishment of passive Heyman nephritis membranous nephropathy model in SD male rats

[0220] (1) Modeling method

[0221] Forty SD male rats were selected, and were bred in a specific pathogen free (Specific pathogen free, SPF) level experimental animal room. The rats in the modeling group were injected with goat anti-Fx1A antiserum through the tail vein once, and the dose was 0.5mL / 100g. The rats in the normal control group were injected with the same amount of normal saline through the tail vein once. The 24-hour urine protein quantification of the rats was determined at the end of the first week, the 24h urine was collected in a metal metabolism cage, the 24h urine protein was determined by a urine protein kit, and the modeling was considered to be successful when the 24h urine protein quantification was >10mg / d.

[0222] (2) Grouping and administration

[0223] 40 SD rats, after adaptive feeding for 1 week, were randomly divided into 5 groups, 8 in each group:

[0224] ① blank group (healthy rats, standard rat feed diet);

[0225] ② PHN model group (modeling + physiological saline gavage);

[0226] ③ low-dose Yishen Qufeng Decoction group (modeling + 9.27 g / kg / d gavage);

[0227] ④ medium-dose Yishen Qufeng Decoction group (modeling + 18.54 g / kg / d gavage);

[0228] ⑤ high-dose Yishen Qufeng Decoction group (modeling + 37.08 g / kg / d gavage);

[0229] The above dosing was performed after successful modeling.

[0230] The above Yishen Qufeng Decoction was prepared by Example 5, and the dosage was based on the mass of the total medicinal materials.

[0231] 3. Specimen processing

[0232] (1) On the last day of adaptive feeding, 24h urine protein quantification was uniformly measured, and rats with 24h-UTP > 10 mg / d were excluded (rats with spontaneous kidney disease were excluded), urine specimens were taken every week after modeling began, 24h urine protein quantification was measured, serum specimens were taken every 2 weeks, and kidney tissue specimens were taken after fasting for 12h at the 8th week.

[0233] (2) Urine collection: 24-hour urine was collected in a rat metabolic cage.

[0234] (3) Anesthesia: rats were anesthetized by intraperitoneal injection of 4wt% sodium pentobarbital, 0.15 mL / 100g.

[0235] (4) Kidney tissue sampling: experimental rats were anesthetized by intraperitoneal injection (4wt% sodium pentobarbital, 0.15 mL / 100g), after successful anesthesia, blood samples were collected by heart puncture, and heart perfusion was immediately performed when the rat still had a weak heartbeat. First, fix the blood needle and connect it to physiological saline, slowly push it forward, and observe the organ fullness. When the organ gradually swells, cut open the right auricle, about 2-3 minutes later, the kidney starts to turn white, and immediately stop perfusion after the kidney is completely white. Remove the capsule and surrounding fat, and divide the kidney tissue into 3 parts for pathology, immunohistochemistry, and western blot, respectively. The samples were stored in a -80℃ refrigerator.

[0236] 4. Index detection

[0237] (1) Urine: urine protein quantification was detected by the biuret method;

[0238] (2) Kidney tissue staining: The kidney tissue was prepared into 3μm paraffin sections and stained with Masson and PASM according to the standard method.

[0239] 5. Experimental Results

[0240] (1) The effect of Yishen Qufeng Formula on 24h-UTP (mg / d) of rats (3 rats in each group were randomly tested) at week 8. The results are shown in Table 12.

[0241] Table 12 Results of 24h-UTP (mg / d) in rats at week 8

[0242]

[0243] Note: Con represents the blank group, Hem represents the model group, YSQF-L represents the low-dose Yishen Qufeng decoction group, YSQF-M represents the medium-dose Yishen Qufeng decoction group, and YSQF-H represents the high-dose Yishen Qufeng decoction group.

[0244] Figure 17 This is a statistical chart showing the average 24-hour UTP (mg / d) results at the end of week 8 in rats. Figure 17 The results showed that after 8 weeks of treatment, the high-dose Yishen Qufeng decoction group (modeling + 37.08g / kg / d gavage) was significantly different from the model group (*P<0.05).

[0245] (2) Masson staining of kidney tissue

[0246] like Figure 18 As shown, according to the MASSON (200X field of view) slices of the randomly selected model group, the GBM of the rats in the model group was thickened, with epithelial eosinophilic glomerulonephrine (immune complex) deposition, obvious protein casts, and significant collagen deposition in the glomeruli.

[0247] like Figure 19 As shown, according to the MASSON (200X field of view) sections of the randomly selected treatment group, the thickness of the GBM region in rats treated with high-dose Yishen Qufeng decoction was reduced compared with that in the model group, the deposition of epithelial eosinophilic glomerulone (immune complex) was reduced, no obvious protein casts were observed, the glomerular collagen deposition was less, the glomerular structure was clear, the tubular cells were arranged in an orderly manner, and no obvious mesangial cells, endothelial cells and matrix proliferation were observed.

[0248] (3) PASM staining of kidney tissue

[0249] like Figure 20 As shown, PASM (200X field of view) slices from a randomly selected model group showed partial proliferation in the glomerular mesangial area, diffuse thickening of the basement membrane with partial band-like vacuolar degeneration, and formation of nail-like structures.

[0250] As Figure 21 shown in the slice of PASM (200X field) of the treatment group treated with randomly selected high-dose Yishen Qufeng Decoction, no obvious proliferation was observed in the mesangial area of the glomerulus, the basement membrane returned to normal, and clear glomerular structure was observed. The tubular cells were arranged in order, and there was no endothelial cell and matrix proliferation.

[0251] Example 7: Clinical trial

[0252] 1. Preparation of decoction

[0253] Soak raw Huangqi 30g, Jiumu 10g, Quanxie 5g, Chaojiangcan 10g, Taizishen 15g, Chao Baishu 10g, Shiwu 15g, Honghua 10g, Chuanqiong 10g, Baihuasheshecao 30g, Fulingpi 30g, and Cheqianzi 30g in water (the weight of water added is 8 times the total weight of the medicinal materials) for 30 minutes. After soaking, the decoction is boiled with a strong fire and then simmered for 30 minutes with a weak fire. After the decoction is filtered, the filtrate is reserved. The residue is added with water (the weight of water added is 6 times the total weight of the medicinal materials) for the second decoction. After boiling with a strong fire, the decoction is simmered for 30 minutes with a weak fire. After the decoction is filtered, the two filtrates are combined. The combined filtrate is evenly divided into two portions (one dose is taken in the morning and one in the evening) and stored at room temperature for later use.

[0254] 2. Time of taking medicine and dosage

[0255] Method of taking medicine: oral decoction, one dose per day.

[0256] Dosage of medicine for patients: 2.93g / kg, i.e. 205g of the compound of traditional Chinese medicine corresponds to a patient weighing 70kg.

[0257] The dosage of Quanxie in the compound of traditional Chinese medicine in the treatment group is 5g.

[0258] 3. Criteria for determining the efficacy of traditional Chinese medicine and western medicine

[0259] 3.1. Criteria for determining the efficacy of traditional Chinese medicine

[0260] Refer to the criteria for determining the efficacy of chronic glomerulonephritis in the 2002 edition of "Guiding Principles for Clinical Research of New Drugs of Traditional Chinese Medicine":

[0261] The efficacy index is calculated by the method of Nimodipine, and the efficacy index of syndrome = (pre-treatment score - post-treatment score) / pre-treatment score x 100%;

[0262] (1) Clinical recovery: clinical symptoms and signs are reduced or completely disappeared, and the reduction of syndrome score is ≥95%;

[0263] (2) Significant effect: clinical symptoms and signs are obviously improved, and the reduction of syndrome score is ≥70%;

[0264] (3) Effective: significant improvement in clinical symptoms and signs, and reduction of syndrome score ≥ 30%;

[0265] (4) Ineffective: no significant improvement or even worsening of clinical symptoms and signs, and reduction of syndrome score < 30%.

[0266] Table 13 Classification and quantification of TCM symptoms

[0267]

[0268]

[0269] 3.2 Western medical efficacy criteria

[0270] Refer to the 2002 edition of "Guidelines for Clinical Research of New Drugs of Traditional Chinese Medicine" for the evaluation of chronic glomerulonephritis urinary protein efficacy:

[0271] (1) Clinical control: 24-hour urinary protein quantification is normal;

[0272] (2) Marked effect: 24-hour urinary protein quantification is reduced by ≥ 40%;

[0273] (3) Effective: 24-hour urinary protein quantification is reduced by < 40%;

[0274] (4) Ineffective: 24-hour urinary protein quantification is not reduced or increased.

[0275] 4. Grouping and experimental results

[0276] From September 2018 to January 2021, 55 patients with chronic nephritis proteinuria were collected from the outpatient and inpatient departments of Jiangsu Provincial Hospital of Traditional Chinese Medicine. The treatment group had 28 cases, and the control group had 27 cases, all of which met the inclusion criteria of this study: (1) age between 20 and 75 years old, gender not limited; (2) met the western medical diagnostic criteria for chronic glomerulonephritis, with proteinuria as the main clinical manifestation; (3) TCM syndrome differentiation was spleen and kidney qi deficiency, combined with wind damage to the kidney; (4) 24-hour urinary protein quantification was 0.5-3.5 g / d; (5) serum creatinine < 110 μmol / L; (6) infection, hypertension, and hyperlipidemia could be effectively controlled.

[0277] The treatment group was treated with the compound composition of traditional Chinese medicine on the basis of treatment (control of blood pressure, blood sugar, blood lipid, correction of electrolyte acid-base balance disorder, diuresis, tripterygium glycoside 1 mg / kg / d, etc.), while the control group was only given basic treatment. The observation time was 3 months, and the changes of 24-hour urinary protein quantification, urea nitrogen, serum creatinine, serum albumin, and TCM syndrome score of the two groups before and after treatment were observed, and the occurrence of adverse reactions was recorded to evaluate the clinical efficacy and safety of the compound composition of traditional Chinese medicine.

[0278] 4.1 Comparison of Western medical effects of two groups of patients after treatment

[0279] The comparison of Western medical effects between the two groups was made by rank sum test, Z = -2.183, P = 0.033 (P < 0.05), and the difference was statistically significant. The total effective rate of the treatment group was 92.86%, and the total effective rate of the control group was 81.48%, as shown in Table 14. The experimental results show that the Western medical effect of the treatment group is higher than that of the control group.

[0280] Table 14 Comparison of Western medical effects of two groups of patients after treatment

[0281]

[0282] 4.2 Comparison of TCM effects of two groups of patients after treatment

[0283] The comparison of TCM effects between the two groups was made by rank sum test, Z = -4.124, P = 0 (P < 0.01), and the difference was statistically significant. The total effective rate of the treatment group was 89.29%, and the total effective rate of the control group was 37.04%, as shown in Table 15. The experimental data show that the TCM effect of the treatment group is significantly higher than that of the control group.

[0284] Table 15 Comparison of TCM effects of two groups of patients after treatment

[0285]

[0286] 4.3 Comparison of 24-hour urinary protein quantification (UTP (g / d)) of two groups of patients after 3 months of treatment before and after treatment

[0287] Compared with before treatment, the 24-hour urinary protein quantification of the two groups of patients after 3 months of treatment was significantly improved, with a statistically significant difference (P < 0.01). The comparison between the two groups after treatment showed that P = 0.021 < 0.05, and the difference was statistically significant. The experimental data show that the urinary protein quantification of the treatment group decreased more significantly than that of the control group, as shown in Table 16.

[0288] Table 16 Comparison of 24-hour urinary protein quantification (g / d) of two groups of patients after 3 months of treatment before and after treatment

[0289]

[0290] 4.4 Comparison of 24-hour urinary protein quantification (UTP (g / d)) of two groups of patients after 1 month, 2 months, and 3 months of treatment before and after treatment

[0291] By t-test, the 24-hour urinary protein quantification of the two groups of patients after 1 month, 2 months, and 3 months of treatment was significantly lower than that before treatment (P < 0.01), with a significant difference, as shown in Table 17.

[0292] Table 17 Comparison of 24-hour UTP (g / d) of two groups of patients before treatment and after 1 month, 2 months and 3 months of treatment

[0293]

[0294] 4.5 Comparison of albumin, urea nitrogen and blood creatinine of two groups of patients before and after treatment

[0295] By t test, there was no statistically significant difference in albumin and urea nitrogen of the treatment group before and after treatment (P>0.05), and the blood creatinine of the treatment group decreased after treatment, which had a statistically significant difference (P=0.006<0.01); there was no statistically significant difference in albumin, urea nitrogen and blood creatinine of the control group before and after treatment (P>0.05); by paired sample t test, it was found that there was no statistically significant difference in albumin, urea nitrogen and blood creatinine of two groups of patients after treatment (P>0.05), as shown in Table 18.

[0296] Table 18 Comparison of albumin (g / L), blood urea nitrogen (mmol / L) and blood creatinine (μmol / L) of two groups of patients before and after treatment

[0297]

[0298] The inclusion criteria of the chronic nephritis proteinuria patients in the clinical study of the application is that the blood creatinine is less than 110 μmol / L, and the collected clinical cases of blood creatinine, albumin and urea nitrogen are in the normal range, so there is almost no statistically significant difference in albumin, urea nitrogen and blood creatinine of two groups of patients before and after treatment (except for the comparison of blood creatinine in the treatment group).

[0299] The chronic glomerulonephritis in the clinical study of the application is mainly manifested by proteinuria, and the treatment is also focused on reducing proteinuria, while albumin, blood creatinine and urea nitrogen reflect liver and kidney function, and these three indexes are not the core of the treatment of chronic glomerulonephritis, but are more used as indexes for long-term efficacy observation and safety evaluation.

[0300] The application provides a quality detection method for a kidney-yang and wind-eliminating traditional Chinese medicine compound composition, and there are many methods and approaches for specifically implementing the technical scheme, and the above description is only a preferred embodiment of the application, and it should be pointed out that, for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principle of the application, and these improvements and refinements should also be regarded as the protection scope of the application. The components not explicitly described in the embodiment can be realized by the prior art.

Claims

1. A method for quality testing of a traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind, characterized in that, The quality testing method consists of appearance, identification, inspection, content determination and microbial limit test. Identification is the identification of Astragalus membranaceus and Cornus officinalis. Content determination is the determination of the content of isoflavone glucoside in the compound Chinese medicine composition of kidney tonifying and wind-dispelling medicine by high performance liquid chromatography. The product is described as having the following characteristics: it is a yellow to yellowish-brown granule with a sweet and slightly sour taste; The identification methods include the identification of Astragalus membranaceus and Cornus officinalis. The identification of Astragalus membranaceus includes the following steps: Preparation of the test solution: Take the granules of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind, grind them, dissolve them in water, and extract them with saturated n-butanol to obtain n-butanol extract; wash the n-butanol extract with ammonia test solution, evaporate the n-butanol to dryness in a water bath, and dissolve the obtained solid in methanol to obtain the test solution; Preparation of negative sample solution: Take Astragalus negative sample particles, grind them, dissolve them in water, and extract them with saturated n-butanol to obtain n-butanol extract; wash the n-butanol extract with ammonia test solution, evaporate the n-butanol to dryness in a water bath, and dissolve the obtained solid in methanol to obtain the solution; Preparation of reference solution: Dissolve astragaloside A in methanol to obtain the solution; Identification of Astragalus membranaceus: Thin-layer chromatography was used for identification. The test solution, negative sample solution and reference solution were respectively placed on a thin-layer plate and developed with chloroform-methanol-10 wt% sodium hydroxide solution as the developing solvent. The plates were then removed, dried, sprayed with sulfuric acid ethanol solution, and heated until the spots on the thin-layer plate were clearly visible. The plates were then examined under ultraviolet light. The identification of the mentioned Cornus officinalis fruit includes the following steps: Preparation of the test solution: Take the granules of the compound Chinese medicine composition for tonifying the kidney and dispelling wind, grind them, dissolve them in water, and extract them with ethyl acetate to obtain an ethyl acetate extract; evaporate the solvent from the ethyl acetate extract, and dissolve the resulting solid in methanol to obtain the test solution. Preparation of negative sample solution: Take the negative sample particles of Cornus officinalis, grind them, dissolve them in water, and extract them with ethyl acetate to obtain ethyl acetate extract; evaporate the solvent from the ethyl acetate extract, dissolve the resulting solid in methanol to obtain the solution; Preparation of the reference solution: Take the Cornus officinalis reference material, decoct it with water, filter it, extract the filtrate with ethyl acetate to obtain the ethyl acetate extract; evaporate the solvent from the ethyl acetate extract, dissolve the solid obtained in methanol to obtain the final product; Identification of Cornus officinalis: Thin-layer chromatography was used for identification. The test solution, negative sample solution and reference solution were respectively placed on a thin-layer plate and developed with toluene-ethyl acetate-formic acid solvent. The plates were then removed, dried, sprayed with sulfuric acid ethanol solution, and heated until the spots on the thin-layer plate were clearly visible. The plates were then examined under sunlight. The inspections include particle size, moisture content, solubility, and fill weight variation; The particle size was examined according to the double sieve method in General Chapter 0982 of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The moisture content was tested according to the drying method in General Chapter 0832 of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The solubility was tested according to the Solubility of Granules under General Chapter 0104 of the Pharmacopoeia of the People's Republic of China 2020 edition; The aforementioned content difference was checked according to the method under the "Content Difference of Granules" section of General Chapter 0104 in Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China (2020 edition). The determination of the content of verbascoside isoflavone glucoside in the granules of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind by high performance liquid chromatography includes the following steps: Preparation of test solution: Take the granules of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind, add methanol, sonicate, make up to volume, shake well, filter through a membrane, and the solution is obtained. Preparation of negative sample solution: Take Astragalus negative sample particles, add methanol, sonicate, make up to volume, shake well, filter through membrane, and the solution is obtained; Preparation of reference solution: Take verbenafil isoflavone glucoside, add methanol, sonicate, shake well, filter through membrane, and the solution is obtained; High-performance liquid chromatography (HPLC) conditions: Column: Agilent Porshell 120 EC-C18, 4.6 mm × 100 mm, 2.7 μm; Mobile phase A: 0.2% phosphoric acid aqueous solution; Mobile phase B: acetonitrile; Volume ratio of mobile phase A to mobile phase B: 90:10; Isogradient elution; Column temperature: 30℃; Flow rate: 1.0 mL / min; Detection wavelength: 260 nm; Injection volume: 10 µL; Acquisition time: 40 min. The high-performance liquid chromatography (HPLC) method is as follows: inject the test solution, negative sample solution, and reference solution into the HPLC instrument, measure, and calculate. The aforementioned microbial limit tests were conducted according to the Pharmacopoeia of the People's Republic of China 2020 edition, specifically the 1105 Microbial Counting Method and the 1106 Control Bacteria Test Method. The aforementioned herbal compound composition for tonifying the kidney and dispelling wind includes the following components in parts by weight: 30 parts raw Astragalus membranaceus, 10 parts Cornus officinalis, 4 parts scorpion, 10 parts stir-fried Bombyx mori, 15 parts Codonopsis pilosula, 10 parts stir-fried Atractylodes macrocephala, 10 parts Pyrrosia lingua, 10 parts Carthamus tinctorius, 10 parts Ligusticum chuanxiong, 15 parts Hedyotis diffusa, 15 parts Poria cocos peel, and 15 parts Plantago asiatica. In the identification of Astragalus membranaceus, the volume ratio of the developing solvent chloroform-methanol-10 wt% sodium hydroxide solution is 13:7:2; In the identification of Cornus officinalis, the volume ratio of the developing solvent toluene-ethyl acetate-formic acid is 5:8:

1.

2. The method for quality testing of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind according to claim 1, characterized in that, The preparation method of the kidney-tonifying and wind-dispelling traditional Chinese medicine compound composition involves decocting raw astragalus, cornus officinalis, scorpion, stir-fried silkworm pupae, codonopsis pilosula, stir-fried atractylodes macrocephala, pyrrosia lingua, safflower, chuanxiong rhizome, oldenlandia diffusa, poria cocos peel, and plantain seed in water, filtering, and concentrating the filtrate under reduced pressure to obtain a concentrated solution; weighing conventional excipients and placing them in a fluidized bed, setting the inlet air temperature, atomizing the concentrated solution, and drying to obtain kidney-tonifying and wind-dispelling traditional Chinese medicine compound composition granules.

3. The method for quality testing of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind according to claim 2, characterized in that, The vacuum concentration includes a first vacuum concentration and a second vacuum concentration, wherein the filtrate is concentrated to 60% by the first vacuum concentration. o The relative density of C is 1.05–1.

14. After standing and centrifugation, the filtrate is further concentrated under reduced pressure to 60. o After the relative density of C is 1.15 to 1.20, conventional excipients are added to obtain a concentrated solution.

4. The method for quality testing of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind according to claim 2, characterized in that, The air inlet temperature is 100°C. o C; The feed rate of the concentrate is 100~150 r / min; The atomization pressure is 0.2 MPa externally and 0.3 MPa internally; The drying is carried out at 60°C. o Dry at C for 1~1.5 h.

5. The method for quality testing of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind according to claim 1, characterized in that, In the identification of Astragalus membranaceus, the ultraviolet light used is 366 nm in wavelength.

6. The method for quality testing of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind according to claim 1, characterized in that, The Astragalus negative sample particles are the components of the kidney-tonifying and wind-dispelling traditional Chinese medicine compound composition, excluding raw Astragalus, prepared according to the preparation method of the kidney-tonifying and wind-dispelling traditional Chinese medicine compound composition.

7. The method for quality testing of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind according to claim 1, characterized in that, The negative sample particles of Cornus officinalis were prepared by the method described above for preparing the compound Chinese medicine composition for tonifying the kidney and dispelling wind, excluding Cornus officinalis.

8. The method for quality testing of the traditional Chinese medicine compound composition for tonifying the kidney and dispelling wind according to claim 1, characterized in that, In the determination of the content of verbascoside isoflavone glucoside, the external standard method was used for calculation.

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