Traditional chinese medicine composition, preparation method therefor, and use thereof

Abstract

Disclosed are a traditional Chinese medicine composition, a preparation method therefor, and use thereof. The present invention belongs to the technical field of traditional Chinese medicine compositions. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 61-119 parts of Radix Astragali, 61-119 parts of Fructus Ligustri Lucidi, 37-71 parts of Radix Paeoniae Alba, 37-71 parts of Rhizoma Atractylodis Macrocephalae, 80-105 parts of Flos Lonicerae, 49-95 parts of Rhizoma Curcumae, and 49-95 parts of Rhizoma Sparganii. The preparation method comprises steps such as extracting volatile oils from Rhizoma Atractylodis Macrocephalae and Rhizoma Curcumae. The traditional Chinese medicine composition prepared by the present invention can effectively treat kidney diseases, and compared with the prior art, the present invention has a better therapeutic effect on kidney diseases.

Description

A traditional Chinese medicine composition and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine compositions, and relates to a therapeutic traditional Chinese medicine composition, a preparation method and an application thereof. Background Art

[0002] Chronic glomerulonephritis (CGN), also known as chronic nephritis, is a primary glomerular disease caused by multiple etiologies and pathological types, with chronic glomerular lesions being the predominant manifestation. Common clinical symptoms include hematuria, proteinuria, hypertension, and edema, most of which are insidious and develop slowly. Prolonged disease can progress to varying degrees of renal function impairment, renal fibrosis, and ultimately end-stage renal failure, threatening the lives of millions of patients. The disease is difficult to cure and prone to relapse, and modern medicine currently lacks a specific treatment.

[0003] The current treatment status of chronic glomerulonephritis is divided into the following categories: (1) General treatment: Patients with chronic nephritis who have edema, hypertension, large amounts of proteinuria, and renal function impairment should all get adequate rest and be given a high-quality low-protein diet according to their renal function status, while controlling the intake of phosphorus in food; (2) Control of hypertension: Effective control of hypertension plays an important role in protecting target organs and reducing target organ damage, especially controlling intrarenal capillary hypertension is an important measure to delay the progression of chronic renal failure; (3) Reduce proteinuria: Currently, the following types of drugs are mainly used to reduce urine protein in clinical practice: angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor antagonists (ARB), adrenal glucocorticoids, and immunosuppressants; (4) Improve renal microcirculation: The use of alprostadil can improve the patient's renal tissue blood rheology indicators by supplementing exogenous prostaglandin E1.

[0004] Modern medical treatments primarily use hormones and immunosuppressants, which have significant side effects. Traditional Chinese Medicine (TCM), believing that the kidneys are the foundation of one's constitution, adheres to the principle of "treating the root of the disease and treating the three causes appropriately," and uses TCM syndrome differentiation and treatment to achieve significant results in reducing proteinuria, improving hematuria, and slowing the progression of renal function.

[0005] CN1565594A discloses a Chinese patent medicine for treating nephropathy and its preparation method. The Chinese patent medicine (Shenhua Tablets) is made from Astragalus, Fructus Ligustri Lucidi, White Peony Root, Flos Lonicerae, Rhizoma Sparganii, Atractylodes Macrocephalae and Rhizoma Curcumae. Atractylodes Macrocephalae, Rhizoma Curcumae, Rhizoma Sparganii, White Peony Root and Rhizoma Curcumae are distilled with water to obtain volatile oil. Astragalus and Fructus Ligustri Lucidi are subjected to alcohol extraction to obtain an extractum. The residues after volatile oil extraction and the residues after alcohol extraction are subjected to water extraction to obtain a clear paste. Finally, the extractum, clear paste and volatile oil are combined to prepare the Chinese patent medicine. The Chinese patent medicine can reduce urine protein, serum creatinine and urinary nitrogen in patients with chronic nephritis and chronic renal insufficiency, increase plasma albumin, alleviate pathological changes in the kidneys, improve hemorheological indicators, and has a good therapeutic effect on kidney diseases such as chronic nephritis and chronic renal insufficiency.

[0006] How to further improve the efficacy of Chinese patent medicines in order to better treat kidney disease is an urgent problem that needs to be solved. Summary of the Invention

[0007] One of the purposes of the present invention is to provide a traditional Chinese medicine composition, a preparation method and application thereof. By adjusting the amount of raw materials and increasing the proportion of honeysuckle in the prescription, the prepared traditional Chinese medicine composition can effectively treat kidney disease.

[0008] The second object of the present invention is to provide a method for establishing a characteristic spectrum of a Chinese medicine composition or its preparation and its application. The similarity between the characteristic spectrum of the Chinese medicine composition or its preparation obtained by this construction method and the control characteristic spectrum is greater than 0.9, which can comprehensively and effectively characterize the quality of the Chinese medicine composition and its preparation, improve the product quality evaluation system, and provide the prerequisite for comprehensive quality detection and overall quality control of the Chinese medicine composition.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a traditional Chinese medicine composition, which is prepared from the following raw materials in parts by weight: 61-119 parts of Astragalus, 61-119 parts of Ligustrum lucidum, 37-71 parts of White Peony, 37-71 parts of Atractylodes, 80-105 parts of Honeysuckle, 49-95 parts of Curcuma and 49-95 parts of Trillium.

[0011] In some embodiments, the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 72-108 parts of Astragalus, 72-108 parts of Ligustrum lucidum, 42-66 parts of White Peony, 42-66 parts of Atractylodes, 80-105 parts of Honeysuckle, 54-90 parts of Curcuma and 54-90 parts of Trigonosciadium japonicum.

[0012] In some embodiments, the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 88-92 parts of Astragalus, 88-92 parts of Ligustrum lucidum, 52-55 parts of White Peony, 52-55 parts of Atractylodes, 86-89 parts of Honeysuckle, 69-72 parts of Curcuma and 69-72 parts of Trigonosciadium japonicum.

[0013] In some embodiments, the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 88-92 parts of Astragalus, 88-92 parts of Ligustrum lucidum, 52-55 parts of White Peony, 52-55 parts of Atractylodes, 87.8 parts of Honeysuckle, 69-72 parts of Curcuma and 69-72 parts of Trigonosciadium japonicum.

[0014] In some embodiments, the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 90.1 parts of Astragalus, 90.1 parts of Ligustrum lucidum, 53.5 parts of White Peony Root, 53.5 parts of Atractylodes, 87.8 parts of Honeysuckle, 70.9 parts of Curcuma and 70.9 parts of Trigonosciadium japonicum.

[0015] The astragalus described in the present invention includes astragalus medicinal materials and decoction pieces thereof. In some embodiments, the decoction pieces are processed astragalus.

[0016] The glossy privet fruit described in the present invention includes glossy privet fruit medicinal materials and decoction pieces thereof. In some embodiments, the decoction pieces are processed glossy privet fruit.

[0017] The white peony root described in the present invention includes white peony root medicinal materials and decoction pieces thereof. In some embodiments, the decoction pieces are processed white peony root.

[0018] The Atractylodes macrocephala described in the present invention includes Atractylodes macrocephala medicinal material and its decoction pieces. In some embodiments, the decoction pieces are processed Atractylodes macrocephala. In some embodiments, the decoction pieces are fried Atractylodes macrocephala with bran.

[0019] The honeysuckle described in the present invention includes honeysuckle medicinal materials and decoction pieces thereof.

[0020] The zedoary turmeric described in the present invention includes zedoary turmeric medicinal materials and decoction pieces thereof. In some embodiments, the decoction pieces are processed zedoary turmeric.

[0021] The Trigonum gracile described in the present invention includes Trigonum gracile medicinal materials and their decoction pieces. In some embodiments, the decoction pieces are processed Trigonum gracile.

[0022] The astragalus, glossy privet fruit, white peony root, atractylodes macrocephala, honeysuckle, zedoaria and trillium described in the present invention are preferably prepared according to the processing method for the above-mentioned medicinal materials under the 2020 edition of the "Chinese Pharmacopoeia".

[0023] Through research, the inventors discovered that Astragalus membranaceus is sweet and slightly warm in nature, making it an excellent herb for tonifying the spleen and replenishing qi. The spleen is located in the middle of the body, like a dam in the middle, and therefore has the function of restraining the overflow of kidney water and consolidating the loss of kidney essence. Astragalus membranaceus is used to tonify the spleen by promoting earth and overcoming dampness, raising the clear and consolidating essence, thus achieving the effect of collecting earth and sealing the kidney. Ligustrum lucidum fruit is sweet, bitter, and cooling in nature, making it a key herb for nourishing the kidney and clearing away heat. When these two herbs are used together, Astragalus membranaceus replenishes qi and generates yin, while Ligustrum lucidum fruit nourishes yin and transforms qi, thus achieving a wonderful mutual transformation of qi and yin. Atractylodes macrocephala is bitter and sweet in nature, and can not only replenish qi and strengthen the spleen, but also dry dampness and invigorate the spleen, thus supporting Astragalus membranaceus's ability to promote earth and overcome dampness, consolidate the kidney and seal the meridians. White peony root is bitter, sour, and slightly cold in nature, and is good at nourishing blood and nourishing yin. When used with Ligustrum lucidum fruit, it can not only enhance the function of nourishing the liver and kidneys, but also have the effect of promoting blood circulation and promoting urination. Both Trigonosciadium japonicum and Curcuma zedoaria are pungent and warm in nature, and are effective in promoting blood circulation and removing blood stasis. Their combined efficacy is even stronger. Honeysuckle, on the other hand, is sweet and cold in nature, and is specialized in clearing heat and detoxifying. Based on the principles of this formula, the dosage of each component is adjusted to achieve optimal therapeutic efficacy in treating kidney disease.

[0024] In a second aspect, the present invention provides a method for preparing the above-mentioned Chinese medicine composition, comprising the following steps:

[0025] (1) Extracting volatile oil: extracting volatile oil from Atractylodes macrocephala and Curcuma zedoaria, and obtaining volatile oil, medicinal residue and extract I after solid-liquid separation;

[0026] (2) Alcohol extraction: Astragalus and Ligustrum lucidum are extracted with alcohol, and solid-liquid separation is performed to obtain alcohol extract and alcohol-extracted medicinal residue;

[0027] (3) Water extraction: extracting the medicinal residue obtained in step (1), the alcohol-extracted medicinal residue obtained in step (2), and the remaining raw materials by water extraction, and separating the solid and liquid to obtain aqueous extract II;

[0028] (4) Concentration: The extract I obtained in step (1) and the aqueous extract II obtained in step (3) are combined and concentrated to obtain aqueous extract concentrate III;

[0029] (5) alcohol precipitation: subjecting the aqueous extract concentrate obtained in step (4) to alcohol precipitation to obtain an alcohol precipitation supernatant;

[0030] (6) combining the alcohol extract obtained in step (2) and the alcohol precipitation supernatant obtained in step (5), and concentrating to obtain a thick paste;

[0031] (7) The thick paste obtained in step (6) is dried and mixed with volatile oil to obtain the Chinese medicine composition.

[0032] The extraction in step (1) of the present invention is a conventional volatile oil extraction process, such as distillation.

[0033] In some embodiments, the extraction in step (1) is a steam distillation method, including a heating distillation method after the raw material is immersed in water and a distillation method after the raw material is contacted with hot steam.

[0034] In one embodiment, the amount of water used in the extraction in step (1) is 1-8 times the total weight of Atractylodes macrocephala and Curcuma zedoaria, preferably 2-4 times, and most preferably 3 times.

[0035] In one embodiment, soaking is further included before the extraction; preferably, the soaking time is 0.2-8 hours, more preferably 0.2-6 hours, and most preferably 0.5 hours.

[0036] In one embodiment, the extraction time of the extraction in step (1) is 3-10 hours, preferably 4-6 hours, and most preferably 5 hours.

[0037] The alcohol extraction in step (2) of the present invention is a conventional process in the art, which utilizes the selectivity of alcohol solvents to dissolve and separate the active ingredients in traditional Chinese medicine.

[0038] In one embodiment, the alcohol extraction solvent used in the alcohol extraction in step (2) has an alcohol content of 50-80%, preferably 50-69%, and most preferably 60%.

[0039] In one embodiment, the amount of the alcohol extraction solvent used in the alcohol extraction in step (2) is 6-18 times the total weight of Astragalus and Ligustrum lucidum, preferably 6-12 times, and most preferably 10 times.

[0040] In one embodiment, the alcohol extraction in step (2) is performed 1-5 times, preferably 2-4 times, and most preferably 3 times.

[0041] In one embodiment, the extraction time of the alcohol extraction in step (2) is 1-5 hours, preferably 1-3 hours, and most preferably 1.5 hours.

[0042] In one embodiment, the alcohol extract in step (2) can be used directly without concentration or after concentration. In one embodiment, the concentration temperature is 50-75°C, preferably 50-70°C, and more preferably 60-70°C. In one embodiment, the relative density of the alcohol extract after concentration is 1.00-1.25 at 50-75°C, preferably 1.03-1.15, and more preferably 1.05-1.10.

[0043] The water extraction in step (3) of the present invention can be a conventional water extraction process in the art, such as room temperature extraction, hot water extraction, microwave-assisted extraction, steam distillation, ultrasonic-assisted extraction, aqueous phase extraction or countercurrent extraction.

[0044] In one embodiment, the amount of water used in the water extraction in step (3) is 6-18 times, preferably 6-14 times, and most preferably 10 times the total weight of the traditional Chinese medicine composition.

[0045] In one embodiment, the water extraction in step (3) is performed 1-4 times, preferably 1-3 times, and optimally 2 times.

[0046] In one embodiment, the water extraction time in step (3) is 1-5 hours, preferably 1-3 hours, and optimally 2 hours.

[0047] The concentration in step (4) of the present invention is performed by conventional methods in the art, such as freeze concentration, reduced pressure concentration or membrane concentration.

[0048] In one embodiment, the concentration in step (4) is concentration under reduced pressure.

[0049] In one embodiment, the concentration temperature in step (4) is 50-75°C, preferably 50-70°C, further 60-70°C.

[0050] In one embodiment, the relative density of the water-extracted concentrated solution III in step (4) is 1.10-1.40 at 50-75°C, preferably 1.10-1.30, and more preferably 1.15-1.20.

[0051] In one embodiment, the alcohol solvent used in the alcohol precipitation in step (5) is ethanol.

[0052] In one embodiment, after the alcohol solvent is added to the alcohol precipitation in step (5), the alcohol content is 50-80%, preferably 50-70%, and most preferably 60%.

[0053] In one embodiment, the temperature of the alcohol precipitation in step (5) is 0-10°C, preferably 2-8°C, more preferably 4-6°C, and most preferably 4°C.

[0054] In one embodiment, the standing time of the alcohol precipitation in step (5) is 12-84 hours, preferably 12-36 hours, and most preferably 24 hours.

[0055] In one embodiment, the alcohol precipitation supernatant in step (5) can be used directly without concentration or after concentration. In one embodiment, the concentration is concentrated under reduced pressure; in one embodiment, the concentration temperature is 50-75°C, preferably 50-70°C, and more preferably 60-70°C; in one embodiment, the relative density of the concentrated solution obtained by the concentration at 50-75°C is 1.00-1.25, preferably 1.05-1.15, and most preferably 1.05-1.10.

[0056] The concentration in step (6) of the present invention is concentration under reduced pressure.

[0057] In one embodiment, the concentration temperature in step (6) is higher than 45°C, preferably 45-90°C, more preferably 50-80°C, and even more preferably 55-75°C.

[0058] In one embodiment, the relative density of the thick paste in step (6) is 1.00-1.25 at 55-75°C, preferably 1.03-1.20, and more preferably 1.05-1.10.

[0059] In one embodiment, the relative density of the thick paste in step (7) is 1.15-1.45 at 55-75°C, preferably 1.20-1.40, and more preferably 1.25-1.30.

[0060] The drying in step (7) of the present invention is a conventional drying method in the art, including freeze drying, vacuum drying, oven drying or spray drying.

[0061] In one embodiment, the drying temperature in step (7) is 40-80°C, preferably 50-75°C, more preferably 50-70°C, and even more preferably 60-70°C.

[0062] The solid-liquid separation mentioned in the present invention is a conventional method in the art, such as centrifugation and / or filtration, which is not limited here.

[0063] In one embodiment, the preparation method comprises the following steps:

[0064] (1) Extracting volatile oil: Take Atractylodes macrocephala and Curcuma zedoaria, add 2-8 times water, soak for 0.2-8 hours, extract by steam distillation for 3-10 hours, collect volatile oil, filter the aqueous extract, and obtain aqueous extract I and medicinal residue for later use;

[0065] (2) Alcohol extraction: Take Astragalus and Ligustrum lucidum, add 6-18 times the amount of 50-80% ethanol, reflux and extract 1-5 times, each extraction time is 1-5 hours, filter, collect the alcohol solution, and concentrate under reduced pressure to obtain an alcohol extraction solution with a relative density of 1.00-1.25 (50-75°C) and alcohol extraction residue for use;

[0066] (3) Water extraction: Combine the medicinal residues and alcohol-extracted medicinal residues with the medicinal materials of Trigonella ternata, White Peony Root, and Honeysuckle, add 6-18 times the amount of water, reflux and extract 1-4 times, each extraction time is 1-5 hours, filter, and obtain aqueous extract II;

[0067] (4) Concentration: The aqueous extract I and the aqueous extract II are combined and concentrated to obtain an aqueous extract concentrate having a relative density of 1.10-1.40 (50-75° C.);

[0068] (5) Alcohol precipitation: Add ethanol to the aqueous extract concentrate to make the alcohol content reach 50-80%, let it stand at 0-10°C for 12-84 hours, filter to obtain the alcohol precipitate, and concentrate under reduced pressure to obtain the alcohol precipitation supernatant with a relative density of 1.00-1.25 (50-75°C);

[0069] (6) combining the alcohol extraction solution and the alcohol precipitation supernatant, and concentrating under reduced pressure to obtain a thick paste with a relative density of 1.15-1.45 (50-75°C);

[0070] (7) Drying the thick paste at 40-80° C. and mixing it with volatile oil to obtain the Chinese medicine composition.

[0071] In some embodiments, the Chinese medicine composition can also be prepared by subjecting the volatile oil obtained in step (1) of the above preparation method to inclusion treatment, and the inclusion compound used in the inclusion treatment is a conventional choice in the art, such as β-cyclodextrin.

[0072] In one embodiment, the preparation method comprises the following steps:

[0073] (1) Soaking Atractylodes macrocephala and Curcuma zedoaria in water, extracting volatile oil, encapsulating, and drying to obtain an inclusion compound, and collecting the aqueous solution and the medicinal residue after extraction for later use;

[0074] (2) extracting Astragalus and Ligustrum lucidum with ethanol, concentrating to obtain an alcohol extract, and collecting the alcohol-extracted medicinal residue for later use;

[0075] (3) combining the alcohol-extracted medicinal residue obtained in step (2) and the medicinal residue obtained in step (1) with Trigonella ternate, white peony root, and honeysuckle, extracting with water, concentrating to obtain a water extract, precipitating with alcohol, and concentrating the supernatant to obtain an alcohol precipitate;

[0076] (4) combining the alcohol extract obtained in step (2) and the alcohol precipitate obtained in step (3), drying them, and mixing them with the inclusion compound obtained in step (1) to obtain;

[0077] In one embodiment, the preparation method of the traditional Chinese medicine composition is:

[0078] (1) Grind Atractylodes macrocephala and Curcuma zedoaria into coarse powder, add 3 times the amount of water, soak for 0.5 hours, extract for 5 hours, extract volatile oil, collect the aqueous solution and medicinal residue after extraction separately for later use; enclose the volatile oil in β-cyclodextrin, dry, and grind;

[0079] (2) Extracting Astragalus and Ligustrum lucidum by reflux extraction with 10 times the amount of 60% ethanol for 3 times, each time for 1.5 hours, and concentrating to obtain an alcohol extract with a relative density of 1.05-1.10. The alcohol-extracted residue was collected for later use;

[0080] (3) combining the alcohol-extracted medicinal residue obtained in step (2) and the medicinal residue obtained in step (1) with Trigonella ternate, white peony root, and honeysuckle, adding 10 times the amount of water and reflux extracting twice, each time for 2 hours, concentrating to obtain a water extract with a relative density of 1.18-1.20, precipitating with 60% ethanol, and letting the precipitate stand at 0-4°C for 24-48 hours. Taking the supernatant and concentrating it to obtain an alcohol precipitate with a relative density of 1.25-1.30;

[0081] (4) combining the alcohol extract obtained in step (2) and the alcohol precipitate obtained in step (3), drying them under vacuum or vacuum belt drying, pulverizing them, and mixing them with the inclusion compound obtained in step (1) to obtain the traditional Chinese medicine composition.

[0082] Preferably, the inclusion process in step (1) is as follows: grinding β-cyclodextrin with water, adding volatile oil ethanol solution dropwise after grinding evenly and continuing grinding, refrigerating, taking out and filtering, drying, and crushing to obtain volatile oil β-cyclodextrin inclusion complex.

[0083] Further preferably, the amount of β-cyclodextrin added is calculated by volume mass ratio (ml:g): volatile oil:β-cyclodextrin = 1:7-12; more preferably 10.

[0084] Further preferably, the amount of water added is calculated in terms of mass-to-volume ratio (g:ml): β-cyclodextrin:water=1:2-6; further preferably, it is 4.

[0085] Further preferably, the volatile oil ethanol solution is prepared by dissolving the volatile oil in anhydrous ethanol.

[0086] More preferably, the grinding time is 20-60 minutes, and more preferably 30 minutes.

[0087] More preferably, the refrigeration is: placing at -4-4°C for 12-24 hours; more preferably, placing at 4°C for 24 hours.

[0088] More preferably, the drying is forced air drying, and the drying temperature is 35-45°C, and more preferably 40°C.

[0089] In a third aspect, the present invention provides the use of the above-mentioned traditional Chinese medicine composition and the traditional Chinese medicine composition prepared by the above-mentioned preparation method in the preparation of drugs for treating kidney disease.

[0090] The kidney disease described in the present invention includes chronic nephritis, proteinuria, or early or mid-stage renal insufficiency.

[0091] In one embodiment, the renal disease is chronic glomerulonephritis.

[0092] In one embodiment, the kidney disease is chronic nephritis with deficiency of both Qi and Yin, and blood stasis and toxins, or symptoms include fatigue, dry mouth and throat; dull complexion, hot palms and soles, soreness of waist and knees, fixed or stabbing pain in the waist, sore throat, pale red, reddish or dark red tongue, thin white or thin yellow or little coating, and thin, weak or thready pulse.

[0093] In some embodiments, the drug comprises the Chinese medicine composition (hereinafter referred to as the active ingredient) and a pharmaceutically acceptable excipient. The drug is also called a Chinese medicine preparation, and the preparation is prepared by extracting the raw materials using a solvent, or the preparation is a decoction, and further, the preparation is prepared by dry granulation.

[0094] The medicaments of the present invention may be prepared by any method known in pharmacy. Generally speaking, these preparation methods include associating the active ingredient with a carrier or excipient and / or one or more other auxiliary ingredients, and if necessary and / or desired, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0095] The drug of the present invention can be prepared according to known methods, such as those described in the general rules for preparation of the Japanese Pharmacopoeia, 16th edition, the United States Pharmacopoeia, and the European Pharmacopoeia, 9th edition. The specific method depends on the dosage form.

[0096] The active ingredient and excipients in the medicaments of the present invention will vary depending on the identity, size and / or condition of the subject being treated and further on the route of administration of the medicament. The medicament may contain between 0.1% and 100% (w / w) active ingredient.

[0097] As used herein, "treating" unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, or preventing the condition or disorder, or one or more symptoms of such condition or disorder, to which the term applies.

[0098] In a fourth aspect, the present invention provides a method for establishing a characteristic spectrum of the above-mentioned Chinese medicine composition or its preparation, which adopts high performance liquid chromatography, and the chromatographic conditions are:

[0099] Chromatographic column: reversed phase silica gel bonded phase chromatographic column;

[0100] Mobile phase A: organic solvent;

[0101] Mobile phase B: acid-containing aqueous solution;

[0102] Elution was performed using a gradient elution program;

[0103] The traditional Chinese medicine composition is prepared from astragalus, glossy privet fruit, white peony root, atractylodes macrocephala, honeysuckle, zedoaria and trigonosciadium japonicum.

[0104] In some embodiments, the establishment method further comprises the following steps:

[0105] (1) preparing a test solution, wherein the test solution comprises the Chinese medicine composition or a preparation thereof;

[0106] (2) Detection: Perform liquid chromatography on the test solution and collect the chromatogram;

[0107] (3) Analyze the chromatogram obtained in step (2) using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software, and use the chromatographic peaks present in the chromatograms of different batches of the Chinese medicine composition or its preparation as common peaks; generate a reference characteristic spectrum of the Chinese medicine composition or its preparation using the average value calculation method; and calculate the relative retention time and relative peak area of ​​each common peak.

[0108] In some embodiments, the organic solvent is any one or a combination of acetonitrile, methanol, ethanol and isopropanol, preferably acetonitrile; the acid in the acid-containing aqueous solution is any one or a combination of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid, preferably phosphoric acid; the mass percentage of the acid-containing aqueous solution is 0.05-0.3%, preferably 0.08-0.2%; more preferably 0.08-0.15%; further preferably 0.1%.

[0109] In some embodiments, in the gradient elution, the initial mobile phase is: mobile phase A 5-9%, mobile phase B 95-91%; the final mobile phase is: mobile phase A 28-65%, mobile phase B 72-35%.

[0110] In some embodiments, the gradient elution program is:

[0111] In some embodiments, the high performance liquid chromatography detection further includes the following conditions: the reverse phase silica gel bonded phase chromatographic column is a C18 or C8 chromatographic column, preferably a C18 chromatographic column; the flow rate of the mobile phase is 0.5-1.5 mL / min, preferably 0.8-1.2 mL / min, and more preferably 1.0 mL / min; the column temperature is 20-50°C, preferably 25-40°C, and more preferably 30°C; the injection volume is 5-20 μL, preferably 10 μL; and the detection wavelength is 230 nm.

[0112] In some embodiments, the establishment method further comprises detecting the test solution by high performance liquid chromatography; preferably, the preparation method of the test solution comprises taking a test sample of a traditional Chinese medicine composition or a preparation thereof, extracting it with a solvent, and performing solid-liquid separation, and the resulting liquid is the test solution; preferably, the preparation method of the test solution further satisfies any one or more of the following conditions:

[0113] a. The solvent for the solvent extraction process is an alcohol aqueous solution or an alcohol solvent; preferably, the alcohol is a C1-C6 alcohol, preferably any one or a combination of methanol, ethanol, propanol and butanol, more preferably ethanol; preferably, the volume ratio of the alcohol solvent in the alcohol aqueous solution is 10-80%, preferably 10-70%; more preferably 10-50%; most preferably 30%;

[0114] b. In g / mL, the ratio of the test sample to the solvent is 0.3-1.2:40-100; preferably 0.3-1.2:40-80; more preferably 0.8-1.2:50;

[0115] c. The extraction method includes ultrasonic extraction or heating reflux extraction; preferably, when ultrasonic extraction is used, the extraction temperature is 20-40 ° C, the power is 100-500W, and the frequency is 10-50Kz;

[0116] d. The extraction time is more than 15 min, preferably 15 min-2 h, more preferably 15-60 min;

[0117] e. The solid-liquid separation method is centrifugation and / or filtration.

[0118] In some embodiments, the establishment method further includes detecting the reference solution by high performance liquid chromatography; preferably, the reference solution is a mixed solution prepared by mixing any one or more combinations of chlorogenic acid, paeoniflorin, calycosin glucoside, luteolin and teruginol with a solvent; preferably, the solvent is an alcohol, preferably a C1-C6 alcohol, further preferably any one or more combinations of methanol, ethanol, propanol and butanol, and further preferably methanol; preferably, the volume fraction of the methanol is 30-80%, preferably 30-70%, and further preferably 50%; preferably, the concentrations of the components in the reference solution are as follows: chlorogenic acid is 40 μg / mL, paeoniflorin is 50 μg / mL, calycosin glucoside is 40 μg / mL, luteolin is 40 μg / mL, and teruginol is 80 μg / mL.

[0119] The control characteristic spectrum obtained by the construction method of the present invention includes 14 common characteristic peaks, and the chromatographic peak of the privet glycoside is used as a reference peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±5% of the specified value. The specified value is: peak 1 0.17, peak 2 0.27, peak 3 0.32, peak 4 0.59, peak 5 0.67, peak 6 0.82, peak 7 0.90, peak 8 0.95, peak 9 0.99, peak 10 1.00, peak 11 1.02, peak 12 1.09, peak 13 1.23, and peak 14 1.27.

[0120] Among them, the chromatographic peak of privet glycoside is used as the reference peak, and the peak No. 1 in the reference characteristic spectrum is chlorogenic acid, the peak No. 4 is paeoniflorin, the peak No. 6 is caerulein glucoside, and the peak No. 7 is luteolin.

[0121] The present invention also provides the application of the establishment method in constructing a characteristic spectrum or fingerprint spectrum of a traditional Chinese medicine composition or its preparation.

[0122] The traditional Chinese medicine composition is prepared from astragalus, glossy privet fruit, white peony root, atractylodes macrocephala, honeysuckle, zedoaria and trigonosciadium japonicum.

[0123] The beneficial effects of the present invention are:

[0124] (1) Based on the composition described in CN1565594A, the present invention adjusts the amount of raw materials and increases the proportion of honeysuckle in the formula to prepare a traditional Chinese medicine composition that can effectively treat kidney disease. Compared with the existing technology, the present invention has a better therapeutic effect on kidney disease.

[0125] (2) The present invention provides a method for constructing a characteristic spectrum of the above-mentioned Chinese medicine composition or its preparation, which can effectively detect more characteristic peaks by optimizing the chromatographic conditions. The construction method has the advantages of simplicity, good stability, high precision, and good reproducibility. The similarity between the characteristic spectrum obtained by the construction method and the control characteristic spectrum is greater than 0.9, which can comprehensively and effectively characterize the quality of the Chinese medicine composition and its preparation, improve the quality evaluation system of the product, and further provide the prerequisite for the comprehensive quality detection and overall quality control of the Chinese medicine composition (Shenhua Tablet). BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 is a chromatogram of the test solution (a) and the mixed reference solution (b) in Example 9.

[0127] FIG2 is a characteristic spectrum of the Chinese medicine composition of Example 9.

[0128] FIG3 is a comparative characteristic spectrum of Example 9.

[0129] FIG4 is a chromatogram showing the attribution of the common peaks in the characteristic spectrum of the Chinese medicine composition of Example 10.

[0130] FIG5 is a chromatogram of Example 12 using acetonitrile-formic acid aqueous solution as the mobile phase.

[0131] FIG6 is a chromatogram of Example 12 using acetonitrile-glacial acetic acid aqueous solution as the mobile phase.

[0132] Figure 7 is a chromatogram of Example 12 using acetonitrile-phosphoric acid aqueous solution as the mobile phase. DETAILED DESCRIPTION

[0133] The embodiments of the present invention will be described in detail below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0134] The medicinal materials used in the following examples are as follows:

[0135] Astragalus is the dried root of Astragalus membranaceus (Fisch.) Bge.var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of the leguminous family.

[0136] Ligustrum lucidum fruit is the dried mature fruit of Ligustrum lucidumAit., a plant of the Oleaceae family.

[0137] White peony root is the dried root of Paeonia lactiflora Pall., a plant of the Ranunculaceae family.

[0138] Atractylodes is the dried rhizome of Atractylodes macrocephala Koidz., a plant of the Asteraceae family.

[0139] Honeysuckle is the dried buds or initially opened flowers of Lonicera japonica Thunb. of the Caprifoliaceae family.

[0140] Curcuma zedoaria is the dried rhizome of Curcumaphaeocaulis Val., a plant of the ginger family.

[0141] Sparganium is the dried tuber of Sparganium stoloniferum Buch.-Ham., a plant of the Sparganaceae family.

[0142] Astragalus, Ligustrum lucidum fruit, white peony root, white atractylodes, honeysuckle, zedoaria, and trillium are prepared by drying fresh medicinal materials. According to the processing methods and preparation regulations of the above-mentioned medicinal materials in the 2020 edition of the "Chinese Pharmacopoeia", the various regulations were checked and the results showed that all the medicinal materials used in the test were in compliance with the pharmacopoeia regulations.

[0143] Example 1 Chinese medicine composition

[0144] Weigh 428 g of Astragalus, 428 g of Ligustrum lucidum fruit, 254 g of White Peony Root, 254 g of Atractylodes Macrocephala, 417 g of Honeysuckle, 337 g of Curcuma and 337 g of Trigonosciadium japonicum as raw materials.

[0145] Extract volatile oil: Take Atractylodes macrocephala and Curcuma zedoariae and place them in a distiller, add 3 times the amount of water and soak for 0.5 hours, distill and extract for 5 hours, collect the volatile oil, filter the water extract, collect it in another container, and set the residue aside.

[0146] Alcohol extraction: Take Astragalus and Ligustrum lucidum, add 10 times the amount of 60% ethanol, reflux and extract 3 times, each extraction time 1.5 hours, filter, combine the filtrate, recover the ethanol, and concentrate under reduced pressure to an alcohol-extracted clear paste with a relative density of 1.07 (60°C). The residue is set aside.

[0147] Water extraction and concentration: the residues after alcohol extraction and volatile oil extraction are combined with the medicinal materials of Trigonum multiflorum, white peony root and honeysuckle, and reflux extraction is performed twice with 10 times the amount of water, each extraction is 2 hours, filtered, and the filtrate is combined with the distilled aqueous solution, and concentrated under reduced pressure to a water extract concentrate with a relative density of 1.15 (60°C).

[0148] Alcohol precipitation of the water extract concentrate: add ethanol to the water extract concentrate to make the alcohol content reach 65%, refrigerate at 4℃ and let it stand for 24 hours. Take the supernatant and concentrate it under reduced pressure to an alcohol-precipitated paste with a relative density of 1.05 (60℃).

[0149] Concentration: Combine the alcohol precipitation paste and the alcohol extraction paste, and concentrate under reduced pressure to a thick paste with a relative density of 1.25 (60°C).

[0150] Combining: The thick paste is dried (60° C.), crushed into fine powder, and volatile oil is added for further combining and mixing to obtain a medicine for treating kidney disease.

[0151] Example 2 Chinese medicine composition

[0152] Weigh 445 g of Astragalus, 445 g of Ligustrum lucidum fruit, 277.5 g of White Peony Root, 277.5 g of Atractylodes macrocephala, 462.5 g of Honeysuckle, 360 g of Curcuma, and 360 g of Trigonosciadium japonicum as raw materials.

[0153] Extract volatile oil: Take Atractylodes macrocephala and Curcuma zedoariae and place them in a distiller, add 4 times the amount of water and soak for 0.2 hours, distill and extract for 6 hours, collect the volatile oil, filter the water extract, collect it in another container, and set aside the residue.

[0154] Alcohol extraction: Take the medicinal materials of Astragalus and Ligustrum lucidum, add 12 times the amount of 50% ethanol, reflux and extract 4 times, each extraction for 1 hour, filter, combine the filtrate, recover the ethanol, and concentrate under reduced pressure to an alcohol-extracted clear paste with a relative density of 1.05 (70°C), and the residue is set aside.

[0155] Water extraction and concentration: the residues after alcohol extraction and volatile oil extraction are combined with the medicinal materials of Trigonosciadium japonicum, White Peony Root, and Honeysuckle, and reflux extracted 3 times with 6 times the amount of water, each extraction time is 3 hours, filtered, and the filtrate is combined with the distilled aqueous solution, and concentrated under reduced pressure to a water extract concentrate with a relative density of 1.10 (70°C).

[0156] Alcohol precipitation of the water extract concentrate: add ethanol to the water extract concentrate to make the alcohol content reach 70%, refrigerate at 0℃ and let it stand for 48 hours. Take the supernatant and concentrate it under reduced pressure to an alcohol-precipitated paste with a relative density of 1.03 (70℃).

[0157] Concentration: Combine the alcohol precipitation paste and the alcohol extraction paste, and concentrate under reduced pressure to a thick paste with a relative density of 1.30 (70°C).

[0158] Combining: The thick paste is dried (40° C.), crushed into fine powder, and volatile oil is added for further combining and mixing to obtain a medicine for treating kidney disease.

[0159] Example 3 Chinese medicine composition

[0160] Weigh 595 g of Astragalus, 595 g of Ligustrum lucidum, 355 g of White Peony Root, 355 g of Atractylodes Macrocephala, 525 g of Honeysuckle, 475 g of Curcuma, and 475 g of Trigonosciadium japonicum as raw materials.

[0161] Extract volatile oil: Take Atractylodes macrocephala and Curcuma zedoariae and place them in a distiller, add 6 times the amount of water and soak for 2.5 hours, distill and extract for 8 hours, collect the volatile oil, filter the water extract, collect it in another container, and set aside the residue.

[0162] Alcohol extraction: Take the medicinal materials of Astragalus and Ligustrum lucidum, add 18 times the amount of 70% ethanol, reflux and extract once, each extraction time is 4 hours, filter, combine the filtrate, recover the ethanol, and concentrate under reduced pressure to an alcohol-extracted clear paste with a relative density of 1.10 (60°C), and the residue is set aside.

[0163] Water extraction and concentration: the residues after alcohol extraction and volatile oil extraction are combined with the medicinal materials of Trigonosciadium japonicum, White Peony Root, and Honeysuckle, and reflux extraction is performed twice with 10 times the amount of water, each extraction is 2 hours, filtered, and the filtrate is combined with the distilled aqueous solution, and concentrated under reduced pressure to a water extract concentrate with a relative density of 1.20 (60°C).

[0164] Alcohol precipitation of the water extract concentrate: add ethanol to the water extract concentrate to make the alcohol content reach 70%, refrigerate at 6℃ and let it stand for 12 hours. Take the supernatant and concentrate it under reduced pressure to an alcohol-precipitated paste with a relative density of 1.10 (60℃).

[0165] Concentration: Combine the alcohol precipitation paste and the alcohol extraction paste, and concentrate under reduced pressure to a thick paste with a relative density of 1.35 (55°C).

[0166] Combining: The thick paste is dried (50° C.), crushed into fine powder, and volatile oil is added for further combining and mixing to obtain a medicine for treating kidney disease.

[0167] Example 4 Chinese medicine composition

[0168] Weigh 540 g of Astragalus, 540 g of Ligustrum lucidum fruit, 360 g of White Peony Root, 360 g of Atractylodes Macrocephala, 900 g of Honeysuckle, 450 g of Curcuma, and 450 g of Trigonosciadium japonicum as raw materials.

[0169] Extract volatile oil: Take Atractylodes macrocephala and Curcuma zedoariae and place them in a distiller, add twice the amount of water and soak for 8 hours, distill and extract for 3 hours, collect the volatile oil, filter the water extract, collect it in another container, and set aside the residue.

[0170] Alcohol extraction: Take the medicinal materials of Astragalus and Ligustrum lucidum, add 18 times the amount of 80% ethanol, reflux and extract once, each extraction time is 2.5 hours, filter, combine the filtrate, recover the ethanol, and concentrate under reduced pressure to an alcohol-extracted clear paste with a relative density of 1.16 (75°C), and the residue is set aside.

[0171] Water extraction and concentration: Combine the residues after alcohol extraction and volatile oil extraction with the medicinal materials of Trigonum multiflorum, White Peony Root, and Honeysuckle, add 18 times the amount of water and reflux and extract once, each extraction time is 3.5 hours, filter, combine the filtrate with the distilled aqueous solution, and concentrate under reduced pressure to a water extract concentrate with a relative density of 1.25 (75°C).

[0172] Alcohol precipitation of the water extract concentrate: add ethanol to the water extract concentrate to make the alcohol content reach 75%, refrigerate at 8℃ and let it stand for 60 hours. Take the supernatant and concentrate it under reduced pressure to an alcohol-precipitated paste with a relative density of 1.15 (75℃).

[0173] Concentration: Combine the alcohol precipitation paste and the alcohol extraction paste, and concentrate under reduced pressure to a thick paste with a relative density of 1.40 (75°C).

[0174] Combining: The thick paste is dried (80° C.), crushed into fine powder, and volatile oil is added for further combining and mixing to obtain a medicine for treating kidney disease.

[0175] Example 5 Chinese medicine composition

[0176] Weigh 295 g of Astragalus, 295 g of Ligustrum lucidum fruit, 200 g of White Peony Root, 200 g of Atractylodes Macrocephala, 400 g of Honeysuckle, 245 g of Curcuma, and 245 g of Trigonosciadium japonicum as raw materials.

[0177] Extract volatile oil: Take Atractylodes macrocephala and Curcuma zedoariae and place them in a distiller, add twice the amount of water and soak for 1.5 hours, distill and extract for 4 hours, collect the volatile oil, filter the water extract, collect it in another container, and set aside the residue.

[0178] Alcohol extraction: Take the medicinal materials of Astragalus and Ligustrum lucidum, add 15 times the amount of 60% ethanol, reflux and extract 5 times, each extraction time is 3.5 hours, filter, combine the filtrate, recover the ethanol, and concentrate under reduced pressure to an alcohol-extracted clear paste with a relative density of 1.03 (65°C), and the residue is set aside.

[0179] Water extraction and concentration: the residues after alcohol extraction and volatile oil extraction are combined with the medicinal materials of Trigonum multiflorum, white peony root and honeysuckle, and reflux extraction is performed 3 times with 16 times the amount of water, each extraction is 5 hours, filtered, and the filtrate is combined with the distilled aqueous solution, and concentrated under reduced pressure to a water extract concentrate with a relative density of 1.30 (65°C).

[0180] Alcohol precipitation of water extract concentrate: add ethanol to the water extract concentrate to make the alcohol content reach 55%, refrigerate at 10℃ and let it stand for 36 hours. Take the supernatant and concentrate it under reduced pressure to an alcohol-precipitated paste with a relative density of 1.00 (65℃).

[0181] Concentration: Combine the alcohol precipitation paste and the alcohol extraction paste, and concentrate under reduced pressure to a thick paste with a relative density of 1.15 (80°C).

[0182] Combining: The thick paste is dried (70° C.), crushed into fine powder, and volatile oil is added for further combining and mixing to obtain a medicine for treating kidney disease.

[0183] Example 6 Chinese medicine composition

[0184] Weigh 360 g of Astragalus, 360 g of Ligustrum lucidum fruit, 180 g of White Peony Root, 180 g of Atractylodes Macrocephala, 360 g of Honeysuckle, 270 g of Curcuma, and 270 g of Trigonosciadium japonicum as raw materials.

[0185] Extract volatile oil: Take Atractylodes macrocephala and Curcuma zedoariae and place them in a distiller, add 8 times the amount of water and soak for 6 hours, distill and extract for 10 hours, collect the volatile oil, filter the water extract, collect it in another container, and set aside the residue.

[0186] Alcohol extraction: Take Astragalus and Ligustrum lucidum medicinal materials, add 6 times the amount of 80% ethanol, reflux extraction twice, each extraction time is 5 hours, filter, combine the filtrate, recover the ethanol, and concentrate under reduced pressure to an alcohol-extracted clear paste with a relative density of 1.20 (50°C), and the residue is set aside.

[0187] Water extraction and concentration: the residues after alcohol extraction and volatile oil extraction are combined with the medicinal materials of Trigonum multiflorum, white peony root and honeysuckle, and reflux extraction is performed 4 times with 14 times the amount of water, each extraction is 1 hour, filtered, and the filtrate is combined with the distilled aqueous solution, and concentrated under reduced pressure to a water extract concentrate with a relative density of 1.35 (50°C).

[0188] Alcohol precipitation of the water extract concentrate: add ethanol to the water extract concentrate to make the alcohol content reach 50%, refrigerate at 4°C and let it stand for 84 hours. Take the supernatant and concentrate it under reduced pressure to an alcohol-precipitated paste with a relative density of 1.20 (50°C).

[0189] Concentration: Combine the alcohol precipitation paste and the alcohol extraction paste, and concentrate under reduced pressure to a thick paste with a relative density of 1.20 (60°C).

[0190] Combining: The thick paste is dried (75° C.), crushed into fine powder, and further combined and mixed with volatile oil to obtain a medicine for treating kidney disease.

[0191] Example 7 Chinese medicine composition

[0192] Weigh 445 g of Astragalus, 445 g of Ligustrum lucidum fruit, 277.5 g of White Peony Root, 277.5 g of Atractylodes macrocephala, 462.5 g of Honeysuckle, 360 g of Curcuma, and 360 g of Trigonosciadium japonicum as raw materials.

[0193] Extract volatile oil: Take Atractylodes macrocephala and Curcuma zedoariae and place them in a distiller, add 4 times the amount of water and soak for 1 hour, distill and extract for 6 hours, collect the volatile oil, filter the water extract, collect it in another container, and set the residue aside.

[0194] Alcohol extraction: Take the medicinal materials of Astragalus and Ligustrum lucidum, add 12 times the amount of 50% ethanol, reflux and extract 4 times, each extraction for 1 hour, filter, combine the filtrate, recover the ethanol, and set aside the alcohol extract and the residue.

[0195] Water extraction and concentration: the residues after alcohol extraction and volatile oil extraction are combined with the medicinal materials of Trigonosciadium japonicum, White Peony Root, and Honeysuckle, and reflux extracted 3 times with 6 times the amount of water, each extraction time is 3 hours, filtered, and the filtrate is combined with the distilled aqueous solution, and concentrated under reduced pressure to a water extract concentrate with a relative density of 1.10 (70°C).

[0196] Alcohol precipitation of the aqueous extract concentrate: Add ethanol to the aqueous extract to make the alcohol content reach 70%, and refrigerate at 4°C for 48 hours. Take the supernatant for later use to obtain the alcohol precipitation supernatant.

[0197] Concentration: Combine the alcohol precipitation supernatant with the alcohol extract and concentrate under reduced pressure to a thick paste with a relative density of 1.25 (45°C).

[0198] Combining: The thick paste is dried (40° C.), crushed into fine powder, and volatile oil is added for further combining and mixing to obtain a medicine for treating kidney disease.

[0199] Example 8 Study on the efficacy of Chinese herbal medicine compositions prepared by different extraction processes on the SD rat adriamycin nephropathy model

[0200] The Chinese medicine composition of Example 1 was used as raw material to investigate the effects of extract powder A, extract powder B, and extract powder C prepared according to processes A, B, and C on the adriamycin nephropathy model of SD rats. Among them, (1) process A is water decoction, specifically: the Chinese medicine composition is decocted with water for 3 times, each time with 10 times the amount of water for 6 hours, the filtrate is combined, concentrated under reduced pressure, and dried to obtain Chinese medicine composition A; (2) process B adopts the extraction process of Example 1 in CN1565594A, specifically: Atractylodes macrocephala, Curcuma zedoaria, Trigonosciadium japonicum, Paeonia lactiflora, and Lonicera japonica are added with 10 times the amount of water, distilled for 7 hours to obtain volatile oil, and the medicinal liquid is stored in a separate container for standby use; Astragalus membranaceus and Ligustrum lucidum are refluxed and extracted twice with 80% ethanol, the first time with 5 times the amount and 2 hours, the second time with 5 times the amount and 4 hours, the ethanol solution is combined, the ethanol is recovered, and the extract is concentrated under reduced pressure to a relative density of 1.6. The residue after volatile oil extraction is combined with the residue after alcohol extraction, decocted with 6 times water for 4 hours, filtered, the filtered liquid and the liquid after volatile oil extraction are combined, and concentrated under reduced pressure to a clear paste with a relative density of 1.2, ethanol is added to make the alcohol content reach 60%, stirred, and allowed to stand for 30 hours. The supernatant is taken, ethanol is recovered under reduced pressure, concentrated to a clear paste with a relative density of 1.6, combined with the alcohol extract, and dried to obtain a dry powder; (3) Process C is the extraction process of Example 1 of the present invention.

[0201] 1. Experimental Animals

[0202] 180 male Sprague-Dawley rats, SPF grade. Weight at model establishment: 180-350 g. Animal use license number: SYXK (Su) 2016-0032. Room temperature and humidity control range: 20-26°C; 40-70%. Air changes: ≥15 / hour. Working lighting: ≥200 Lux; animal lighting: 15-20 Lux (lighting hours: 12 hours / day (lights on: 06:00 AM - off: 6:00 PM).

[0203] 2. Experimental animal grouping and dosage design

[0204] The proposed clinical dosage of extract powder C is 3.48g / day (calculated as extract powder). The average adult weight is 70kg, and the average SD rat weight is 300g. The equivalent dose for SD rats is 0.26g / kg. In this study, the doses of extract powder A, extract powder B, and extract powder C were all equivalent to 0.26g / kg. A normal control group, a model group, and a positive control group were also set up. The normal control group and the model group were gavaged with an equal volume of drinking water for the test animals, and the positive control group was given losartan potassium at a dose of 10mg / kg. A total of 6 groups were divided. Among them, the ratio of crude drug dosage corresponding to the same dose of extract powder B and extract powder C was 1.27:1.

[0205] The specific groups and numbers of animals are shown in Table 1 .

[0206] Table 1 Dosage groups for process screening pharmacodynamic studies Note: All groups were given the test substance, control substance or drinking water of the experimental animals at 1 mL / 100 g body weight.

[0207] 3. Test methods

[0208] Prepare doxorubicin 0.4 mg / mL and inject 1 mL / 100 g body weight of SD rats through the tail vein as a single injection. The modeling dose is 4 mg / kg. Detect 24-hour proteinuria 21 days after modeling. Screen rats with 24-hour urine protein greater than 44 mg and randomly group them according to the amount of 24-hour urine protein. Administer the drug on the day of grouping. Administer the drug for 8 weeks, and detect 24-hour urine protein at 4, 6, and 8 weeks of administration. Detect urine creatinine at the same time in the 8th week. The rats were killed the day after the last dose, and blood was collected from the abdominal aorta. The serum was centrifuged and the blood biochemical related indicators were measured. At the same time, the kidneys were taken and weighed, and the left kidney was taken for pathological examination.

[0209] 4. Administer medication

[0210] Administration: Oral administration.

[0211] Dosage volume: 1 mL / 100 g body weight.

[0212] Dosing cycle: 8 weeks.

[0213] 5. Detection indicators

[0214] 5.1 Weight

[0215] The experiment started with weekly weighing.

[0216] 5.2 General status observation

[0217] Observation time and frequency: After modeling or drug administration, observe the general condition of the animals every day;

[0218] Observation content: including but not limited to the animal's appearance, general behavior, posture, movement status, coat and skin, feces and urine, mental state, glandular secretion, respiratory status, fecal characteristics and survival period observation, etc.

[0219] 5.3 Blood collection and hematological indicators

[0220] Detection time: Detect after the end of drug administration.

[0221] Test animals: All living animals

[0222] Sampling method: Blood was collected from the abdominal aorta after the test.

[0223] Blood sample processing method: centrifuge at 3000g for 10 min and collect serum.

[0224] Test items and test methods: see Table 2 below.

[0225] Table 2 Blood biochemical test items

[0226] 5.4 Urine testing

[0227] Testing time: once about two weeks after model establishment, and once about 4 weeks, 6 weeks, and 8 weeks after administration.

[0228] Test animals: All surviving animals.

[0229] Sampling method: 24-hour urine collection was performed using the metabolic cage method.

[0230] The test items and methods are shown in Table 3 below.

[0231] Table 3 Urine test items

[0232] 5.5 Gross anatomy and histopathological examination

[0233] 5.5.1 Gross anatomy

[0234] Dissection time: After the experiment is completed.

[0235] Animals for autopsy: All living animals.

[0236] 5.5.2 Histopathological examination

[0237] Organ examination: Focus on observing changes in the kidney's texture, color, and volume. The left kidney of all surviving animals was sampled.

[0238] Animals examined: All animals.

[0239] Fixation method: Formalin fixation.

[0240] Preparation and staining.

[0241] 6. Statistical analysis

[0242] The results are presented in the form of charts, etc. SPSS23.0 software was used for statistical analysis, and the data were expressed as mean ± standard deviation ( ) indicated that the groups were compared using one-way analysis of variance, and post hoc comparisons showed that P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0243] 7. Test results

[0244] 7.1 Animal screening and grouping

[0245] A total of 170 animals were modeled in the experiment, and 24-hour urine protein was detected on the 21st day after modeling. The average urine protein in the normal control group was 29.2 mg. 55 rats with urine protein between 44.0-156.0 mg were screened and randomly divided into 5 groups according to the 24-hour urine protein range, namely model group, extract powder A, extract powder B, extract powder C and positive control group.

[0246] 7.2 Animal Death and General Condition Observation

[0247] All animals in the experiment had smooth fur, normal behavior, and normal feces and urine. No animals died during the experiment.

[0248] 7.3 weight

[0249] The body weight of each group during the experiment is shown in Table 4.

[0250] Table 4 Body weight results of each group Note: Compared with the normal control group, *p<0.05, **p<0.01.

[0251] The results showed that compared with the normal control group, the body weights of the animals in the other groups showed extremely significant differences (p < 0.01) after four weeks of administration, and extract powder C had a better effect on body weight in all treatment groups.

[0252] 7.424hr urine protein results

[0253] The 24-hour urine protein test results of each group of animals during the experiment are shown in Table 5.

[0254] Table 5 24hr urine protein results Note: Compared with the normal control group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01.

[0255] The results showed that before administration, compared with the normal control group, the 24-hour urine protein of rats in other groups was extremely significantly increased (p < 0.01), indicating that the model was successful. Compared with the model group, there was no significant difference in the 24-hour urine protein of rats in the extract powder A, B, C and positive control groups (p > 0.05), and the groups were evenly divided.

[0256] After six weeks of administration, the extract powder C group showed a significant decrease in 24-hour urine protein compared to the model group (p < 0.05). This experiment demonstrates that the Chinese herbal composition prepared using the extraction process of the present invention is fast-acting and superior to the Chinese herbal compositions prepared using processes A and B in reducing 24-hour urine protein, and also superior to positive-acting drugs.

[0257] 7.5 Kidney weight and renal index

[0258] The results of the left and right kidneys and renal indexes of the animals in each group after 8 weeks of drug administration are shown in Table 6.

[0259] Table 6 Kidney weight and renal index results Note: Compared with the normal control group, *p<0.05, **p<0.01.

[0260] The results showed that after 8 weeks of administration, compared with the normal control group, except for the extremely significant increase in the renal index of rats in the model group (p < 0.01), there was no significant difference in the renal index of rats in other groups (p > 0.05).

[0261] 7.6 Blood biochemistry test results

[0262] The blood biochemical results of animals in each group after 8 weeks of drug administration are shown in Table 7.

[0263] Table 7 Animal blood biochemical results Note: Compared with the normal control group, *p<0.05, **p<0.01.

[0264] The results showed that compared with the normal control group, the serum ALB level of rats in the model group was extremely significantly reduced (p < 0.01). However, compared with the model group, the ALB levels of extract powder B, extract powder C, and the positive control group increased, but there was no significant difference compared with the normal group. The results showed that, compared with the model group, extract powders B, C, and the positive control group, with the exception of the traditional Chinese medicine composition prepared by process A, all had the effect of increasing ALB levels in rat serum.

[0265] 7.7 Pathological test results

[0266] The left kidney was routinely stained with HE and examined under a light microscope. Scoring was based on the severity of basophilic tubules, proteinaceous casts, glomerular degeneration, and interstitial inflammatory cell infiltration. No lesions were scored as 0, and 1, 2, 3, and 4 indicated mild, moderate, and severe lesions, respectively.

[0267] Pathological results showed that compared with the normal control group, the model group showed pathological changes such as basophilic renal tubules, tubular protein casts, swelling and degeneration of glomerular visceral epithelial cells, and interstitial inflammatory cell infiltration in the kidneys. Compared with the model group, each treatment group showed similar pathological changes. The degree of lesions in the extract powder group A was not significantly reduced compared with the model group, while the degree of lesions in the extract powder group B was slightly reduced compared with the model group. The degree of renal lesions in the extract powder group C and the positive control group was significantly reduced compared with the model group. This shows that compared with conventional processes, the Chinese medicine composition prepared by the extraction process of the present invention is more effective in treating kidney disease.

[0268] Example 9 Study on the efficacy of the Chinese medicine composition prepared in Example 1 on the SD rat adriamycin nephropathy model

[0269] The specific test method was the same as in Example 8, except that the Chinese medicine composition prepared in Example 1 was used as the test group of the present invention. Specifically, male SD rats were selected as the test animals, and a total of 84 animals were modeled. After the 24-hour urine protein in the modeled rats was significantly increased, they were randomly divided into 5 groups based on the 24-hour urine protein range, namely the model group, the low-, medium-, and high-dose groups of the present invention, and a positive control group. In addition, 11 unmodeled animals were taken as the normal control group. Drug administration began immediately after grouping. The dosages of the low-, medium-, and high-dose groups of the present invention were 0.09, 0.26, and 0.78 g / kg, respectively. The administration volume was 0.5 ml / 100 g, with administration once in the morning and once in the afternoon. The normal control group and the model group were given equal volumes of drinking water for the test animals. The positive control group was given losartan potassium tablets at a dosage of 15 mg / kg. The drugs were administered for a total of 12 weeks.

[0270] During the administration process, the animals' survival status and general symptoms were observed every day, and their body weights were measured weekly. 24-hour urine protein was detected at 3, 5, 8, and 12 weeks after administration, respectively. After 12 weeks of administration, the animals were anesthetized, blood was collected from the abdominal aorta, and serum was centrifuged to measure TP (total protein), ALB (albumin), BUN (urea nitrogen), and CRE (creatinine) levels in the serum. The left and right kidneys were taken, weighed, and the kidney coefficient was calculated. The left kidney was formalin-fixed and further pathological examination was performed.

[0271] The results of animal screening and grouping showed that 24-hour urine protein was detected on the 18th day after modeling. The average urine protein in the normal control group was 14.1 mg (8.5-19.8 mg). Rats with urine protein between 40.0-170.6 mg were randomly divided into 5 groups, with 11 rats in each group.

[0272] Observations of animal mortality and general condition showed that all groups of animals had smooth fur and no abnormalities in behavior, feces, or urine. No animals died during the experiment. 24-hour urine protein testing revealed a highly significant difference in 24-hour urine protein between the positive control group and the model group after 5 and 8 weeks of administration (p < 0.01). Compared with the model group, the low-, medium-, and high-dose groups of the present invention were able to inhibit the rapid increase in urine protein after 5 and 8 weeks of administration, with an average inhibition rate of approximately 20%, reaching a maximum of 26.3%.

[0273] Kidney weight and kidney coefficient results showed that after 12 weeks of administration, the left and right kidneys in the model group were significantly enlarged compared with the normal control group (p < 0.05), and the kidney coefficient was extremely significantly increased (p < 0.01). Compared with the model group, the left and right kidney weights and kidney coefficients of the drug group and the positive control group were reduced. Blood biochemistry test results showed that after 12 weeks of administration, the ALB levels of the normal control group, model group, low-, medium-, and high-dose groups of the present invention, and the positive control group were 37.3, 29.7, 30.0, 31.2, 30.8, and 31.6 g / L, respectively. Compared with the normal control group, the ALB level of the model group was significantly reduced (p < 0.05). Compared with the model group, there were no significant differences in TP, ALB, BUN, and CRE in the other groups (p > 0.05). Pathological results showed that the degree of renal lesions in the positive control group was significantly reduced compared with the model group. Compared with the model group, the low-, medium-, and high-dose groups of the present invention demonstrated good pharmacodynamic effects in terms of the number of lesions and the lesion scores, and were dose-related.

[0274] Example 10 Toxicity test of Chinese medicine composition in rats after repeated oral administration for 26 weeks

[0275] The Chinese medicine combination provided in the embodiment of the present invention is a brown to tan powder, and is mainly used clinically for chronic nephritis, proteinuria or early and middle stage renal insufficiency. This experiment detects the potential toxicity of the test product of the present invention by orally administering it to SD rats for 26 consecutive weeks, and evaluates the reversibility, persistence or delayed effect of the toxic effects of the test product through a 4-week recovery period to achieve the following purposes: ① determine the toxic target organs or target tissues of repeated administration of the test product; ② if possible, determine the dose level at which no clinical adverse reactions are observed; ③ infer the starting dose of the first clinical trial to provide a safe dose range for subsequent clinical trials; ④ provide a reference for monitoring and prevention of clinical adverse reactions; ⑤ predict clinical adverse reactions that may be caused by the test product.

[0276] Experimental design

[0277] The present invention provides 30g crude drug / kg and 15g crude drug / kg as the high and low doses of the test substance, respectively. A solvent control group is also set up, and the experimental animals are given drinking water. The animals are randomly divided into groups according to body weight, with 30 animals in each group, half male and half female. The dosing volume for all animals is 1mL / 100g body weight, wherein the test substance is the Chinese medicine composition prepared in Example 1.

[0278] During the experiment, the behavior and physical signs of the animals were observed and recorded. The body weight and food intake were measured once a week during the experiment. At the end of the administration period (week 26) and the recovery period (four weeks of recovery), 80 and 40 animals were sacrificed by bleeding from the abdominal aorta after anesthesia, respectively. Blood, blood biochemical indices, urine, and developmental indicators were measured. The animals were systematically dissected and their organs were weighed. The organ coefficients were calculated, and organ tissues were taken for pathological examination.

[0279] The results showed that during the 26-week repeated oral administration of the Chinese medicine composition provided in the embodiment of the present invention to rats, the rats in each administration group were in good general condition and had no abnormal behavior or activities.

[0280] During the dosing period and the recovery period, no test substance-related effects were found in body weight and food intake compared with the vehicle control group. Clinical test results showed that there were no toxicological changes in all test substance groups.

[0281] The histopathological results showed that after 26 weeks of administration, there was no obvious change in the liver cells and other organs after administration of the test substance.

[0282] Therefore, by orally administering the test substance to SD animals for 26 consecutive weeks at doses of 15 and 30 crude drugs / kg, the following conclusions can be drawn: Combined with the clinical examination and pathological results, observation of daily symptoms and the prognosis after drug withdrawal, it is believed that under the conditions of this experiment, after the animals were repeatedly orally administered with the Chinese medicine composition provided by the present invention for 26 weeks, no toxic reaction was observed at 30g crude drug / kg.

[0283] Example 11

[0284] The method for establishing a characteristic spectrum of a Chinese medicine composition or its preparation is described in detail below with reference to the examples.

[0285] [Instruments and reagents]

[0286] Instruments: Ultimate 3000 high-performance liquid chromatograph (Thermo Fisher Scientific, USA); Agilent 1260 high-performance liquid chromatograph (Agilent Scientific, USA); DAD detector (Thermo Fisher Scientific, USA); ELSD detector (Agilent Scientific, USA); XPE105 analytical balance (Mettler-Toledo Instrument Co., Ltd.); JM-A300 analytical balance (Yuyao Jiming Weighing Equipment Co., Ltd.); S60H CNC ultrasonic cleaner (ELMA, Germany).

[0287] Test drugs: Astragaloside IV reference substance (batch number: 110781-201717), terunculoside reference substance (batch number: 111926-201605), paeoniflorin reference substance (batch number: 110736-201842), and chlorogenic acid reference substance (batch number: 110753-201817) were all purchased from the China Food and Drug Inspection Institute; calycoside isoflavone reference substance (batch number: PS011743) and luteolin reference substance (batch number: PS021031) were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; acetonitrile was chromatographic grade, Merck; water was ultrapure water; and the other reagents were of analytical grade.

[0288] The batch numbers of the Chinese medicine compositions are: 2020122201, 2020122202, 2020122203, 2020122301, 2020122302, 2020122303, 2020122401, 2020122402, 2020122403, 2020122501, 2020122502, and 2020122503, all of which are produced by Suzhong Pharmaceutical Group Co., Ltd.

[0289] Example 9 Method for Establishing Characteristic Spectra of Traditional Chinese Medicine Compositions

[0290] (1) Preparation of test solution

[0291] Take the Chinese medicine compositions with batch numbers 2020122201, 2020122202, 2020122203, 2020122301, 2020122302, 2020122303, 2020122401, 2020122402, 2020122403, 2020122501, 2020122502, and 2020122503 prepared according to the method of Example 1, and number them S1-S12 respectively. Accurately weigh 1.0 g of each, place it in a stoppered conical flask, add 50 mL of 30% ethanol, ultrasonically treat it at 250w / 40kHz for 15 min, cool it, filter it, and take the filtrate to obtain 12 batches of test solution.

[0292] (2) Preparation of mixed reference solution

[0293] Take appropriate amounts of chlorogenic acid, paeoniflorin, calycosin isoflavone glucoside, luteolin, and terglucosidoside reference substances, accurately weigh them, dissolve them in 50% methanol, dilute to the mark, and shake well to obtain a mixed reference solution. The mass concentrations of chlorogenic acid, paeoniflorin, calycosin isoflavone glucoside, luteolin, and terglucosidoside in the mixed reference solution are 40 μg / ml, 50 μg / ml, 40 μg / ml, 40 μg / ml, and 80 μg / ml, respectively.

[0294] (3) HPLC conditions

[0295] Chromatographic column: Agilent XDB-C18 reverse phase column (specifications: 250 nm × 4.6 mm, 5 μm);

[0296] Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution;

[0297] The elution method is gradient elution, and the gradient elution method is shown in Table 8;

[0298] Flow rate: 1.0 mL / min;

[0299] Column temperature: 30°C;

[0300] Injection volume: 10 μL;

[0301] The detection wavelength is: 230nm.

[0302] Table 8 Gradient elution table

[0303] (4) Accurately pipette 10 μL of the test solution and the mixed reference solution, respectively, and perform the test using the above-mentioned HPLC conditions to obtain the liquid chromatograms of the test solution and the chromatograms of the mixed reference solution, respectively.

[0304] The chromatogram of the mixed reference solution is shown in FIG1 , wherein peak 2 is chlorogenic acid, peak 4 is paeoniflorin, peak 6 is calycosin glucoside, peak 7 is luteolin, and peak 10 is privetside.

[0305] The chromatogram of the test solution was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Edition" for analysis, and the evaluation method was used for calculation, multi-point correction, and data matching to generate an overlaid chromatogram, which is the characteristic spectrum of the Chinese medicine composition, as shown in Figure 2.

[0306] Figure 3 shows the reference spectrum generated based on the chromatograms of the 12 batches of test products. Similarity analysis was performed between the characteristic spectra of the 12 batches of preparations and the generated reference spectrum. The relative retention times of the common peaks and the reference peaks were calculated, and the RSD values ​​were calculated. The results are shown in Tables 9 and 10.

[0307] Table 9 Similarity of characteristic spectra of 12 batches of Chinese medicine compositions

[0308] Table 10 Determination results of relative retention time of common peaks in characteristic spectra of 12 batches of traditional Chinese medicine compositions

[0309] As can be seen from Tables 9 and 10, the similarity between the characteristic spectra of different batches of Chinese medicine compositions and the control spectra is above 0.9, indicating that the similarity between different batches is high; the relative retention time RSD% of each common peak of different batches of Chinese medicine compositions is less than 5%, indicating that the test method provided by the present invention has good stability.

[0310] Example 10: Attribution Analysis of Common Peaks in the Characteristic Spectrum of Traditional Chinese Medicine Compositions in Medicinal Materials

[0311] The UV absorption of the chromatographic peaks in the positive control chromatograms and negative control chromatograms of the Chinese herbal medicine composition were measured according to the test sample assay method. The retention times of the chromatographic peaks were compared to determine the attribution of the common peaks in the characteristic chromatogram of the Chinese herbal medicine composition. The results are shown in Table 11.

[0312] Table 11. Common chromatographic peaks on the medicinal material spectrum

[0313] Comparing the negative characteristic spectra, the 14 chromatographic peaks attributed to the characteristic spectrum of the traditional Chinese medicine composition accounted for 88% of the total peak area. Among them, 4 chromatographic peaks originated from Ligustrum lucidum fruit, accounting for 53% of the attributed peak area, 8 chromatographic peaks originated from honeysuckle flower, accounting for 35% of the attributed peak area, 1 originated from white peony slices, accounting for 8% of the attributed peak area, and 1 originated from Astragalus slices, accounting for 4% of the attributed peak area. Using single-standard solution and reference solution, it can be confirmed that peak 2 is chlorogenic acid, peak 4 is paeoniflorin, peak 6 is calycosin isoflavone glucoside, peak 7 is luteolin, and peak 10 is terebutrin. Among them, the chromatographic peaks of confirmed chemical components accounted for 65% of the attributed peak area, as shown in Figure 4.

[0314] Example 11 Methodological Verification

[0315] 1. Precision test

[0316] The Chinese medicine composition with batch number 2020122201 prepared according to the method of Example 1 was tested according to the basic steps of chromatographic testing of the test sample in Example 9. 10 μl of the test sample was accurately aspirated and injected six times, and the chromatogram was recorded. The relative retention time and relative peak area of ​​the main chromatographic peak (accounting for more than 1% of the total peak area) were calculated. The results are shown in Tables 12 and 13.

[0317] The results showed that the instrument had good precision.

[0318] Table 12 Results of precision investigation of characteristic spectra of Chinese medicine compositions (relative retention time)

[0319] Table 13 Precision investigation results of characteristic spectrum of Chinese medicine composition (relative peak area)

[0320] 2. Stability test

[0321] The Chinese medicine composition with batch number 2020122201 prepared according to the method of Example 1 was tested according to the basic steps of chromatographic detection of the test sample in Example 9. The same test solution was injected at 0 h, 7 h, 14 h, 25 h, 32 h, and 39 h, and the chromatogram was recorded. The relative retention time and relative peak area of ​​the main chromatographic peak (accounting for more than 1% of the total peak area) were calculated. The results are shown in Tables 14 and 15.

[0322] The results showed that the test solution had good stability within 39 hours at room temperature.

[0323] Table 14 Results of stability study of characteristic patterns of Chinese medicine compositions (relative retention time)

[0324] Table 15 Results of stability study of characteristic spectrum of Chinese medicine composition (relative peak area)

[0325] 3. Repeatability test

[0326] The Chinese medicine composition with batch number 2020122201 prepared according to the method of Example 1 was tested according to the basic steps of chromatographic testing of the test sample in Example 9. Six test sample solutions were prepared in the same manner, and the samples were injected and measured separately, and the chromatograms were recorded. The relative retention time and relative peak area of ​​the main chromatographic peaks (accounting for more than 1% of the total peak area) were calculated. The results are shown in Tables 16 and 17.

[0327] The results showed that the method had good reproducibility.

[0328] Table 16 Repeatability test results of characteristic spectrum of Chinese medicine composition (relative retention time)

[0329] Table 17 Repeatability test results of characteristic spectrum of Chinese medicine composition (relative peak area)

[0330] Example 12 Method Screening

[0331] 1. Selection of detection wavelength

[0332] Except for the detection wavelength conditions, the basic steps for chromatographic detection of the sample in Example 9 were the same. The sample was scanned at a full wavelength of 190-400 nm using a DAD detector, and the isoabsorption diagram was analyzed. The wavelengths with more absorption peaks were extracted for comparison. It was shown that the 230 nm detection wavelength could more comprehensively reflect the chemical components in the aforementioned traditional Chinese medicine composition, and the chromatographic peaks were well separated and the baseline was stable. Therefore, 230 nm was selected as the acquisition wavelength for the characteristic spectrum of the traditional Chinese medicine composition.

[0333] 2. Optimization of mobile phase

[0334] Except for the mobile phase, the basic steps for chromatographic detection of the sample were the same as those in Example 9. Comparisons were made with acetonitrile-formic acid aqueous solution, acetonitrile-glacial acetic acid aqueous solution, and acetonitrile-phosphoric acid aqueous solution, and the results are shown in Figures 5-7.

[0335] The results showed that the separation degree of each component in the acetonitrile-phosphoric acid aqueous solution system was good and the baseline was relatively stable, so the acetonitrile-phosphoric acid aqueous solution system was selected as the mobile phase.

[0336] 3. Selection of extraction method

[0337] Aside from the extraction method, the basic steps for chromatographic testing of the sample in Example 9 were the same as those for the sample in Example 9. 1.0 g of the Chinese medicinal composition of batch number 2020122201, prepared according to the method of Example 1, was placed in a stoppered conical flask. 50 mL of 30% ethanol was added, and ultrasonic treatment and reflux extraction were performed at 250W / 40kHz for 15 minutes, respectively. The mixture was cooled, filtered, and each filtrate was collected for analysis. The number of chromatographic peaks and the main peak area divided by the sample volume were used as evaluation indicators. The test results showed that there was no significant difference in the effects of ultrasonic treatment and reflux extraction. Considering the simplicity of ultrasonic treatment, ultrasonic treatment was selected.

[0338] 4. Selection of extraction solvent

[0339] Except for the extraction solvent, the other basic steps for chromatographic detection of the test sample in Example 9 are the same. Take 1.0g of the Chinese medicine composition of batch number 2020122201 prepared according to the method of Example 1, weigh 5 portions, place in a stoppered conical flask, accurately add water, methanol, 30% ethanol, 60% ethanol, and 50mL of ethanol, respectively, and ultrasonically treat at 250w / 40kHz for 15min, cool, filter, and take each filtrate for determination. The chromatographic peak behavior, the number of chromatographic peaks, and the main peak area divided by the sample volume are used as evaluation indicators. The test results show that the sample information extracted with different concentrations of ethanol is more comprehensive, and there is no significant difference in the number of chromatographic peaks and the main peak area divided by the sample volume ratio. 30% ethanol is selected as the extraction solvent.

[0340] 5. Selection of extraction time

[0341] Except for the extraction time, the other basic steps for chromatographic detection of the test sample in Example 9 were the same. 1.0 g of the Chinese medicine composition of batch number 2020122201 prepared according to the method of Example 1 was taken, and three portions were weighed and placed in stoppered conical flasks. 50 mL of 30% ethanol was accurately added to each portion. The mixture was ultrasonically treated at 250W / 40kHz for 15 minutes, 30 minutes, and 45 minutes. The mixture was cooled, filtered, and the filtrates were collected for analysis. The number of chromatographic peaks and the peak area of ​​the main peak divided by the sample size were used as evaluation indicators. The test results showed that the components in the sample were fully extracted after 15 minutes of ultrasonication, so the extraction time was selected as 20 minutes.

[0342] Based on the above test results, the characteristic spectrum of the traditional Chinese medicine composition was determined. The preparation method of the test solution was as follows: 1.0 g of the powder of the product was placed in a stoppered conical flask, 50 mL of 30% ethanol was accurately added, and ultrasonic treatment was performed at 250w / 40kHz for 15 minutes. The product was cooled, filtered, and the filtrate was obtained.

[0343] Example 13 Characteristic Spectrum of Chinese Medicine Composition Preparation

[0344] Take 100 g of the traditional Chinese medicine composition, add 25 g of starch, mix well, dry granulate, sieve the whole granules with a 65-mesh sieve, add 0.1% magnesium stearate, mix well, press into tablets, use Opadry 290W665025-CN as a coating excipient, and film-coat with a coating liquid solid content of 17%. Package to obtain the traditional Chinese medicine composition preparation.

[0345] Wherein, the preparation of the Chinese medicine composition is as follows:

[0346] Weigh 254g of Atractylodes macrocephala and 337g of Curcuma zedoaria respectively, grind them into coarse powder, add 3 times the amount of water, soak for 0.5 hours, steam distill and extract for 5 hours, collect the volatile oil, and collect the aqueous solution and the medicinal residue after extraction for use; the volatile oil is included in the β-cyclodextrin (take 10 times the amount of β-cyclodextrin of the volatile oil, add 4 times the amount of purified water of β-cyclodextrin, grind it thoroughly, grind it evenly, slowly add the volatile oil ethanol solution dropwise, grind it for 30 minutes, refrigerate it at 4°C for 24h, take it out, filter it, and dry it with air at 40°C), grind it to obtain the volatile oil β-cyclodextrin inclusion complex; weigh 428g of Astragalus membranaceus and 428g of Ligustrum lucidum respectively, add 10 times the amount of 60v / v% ethanol Reflux extraction is performed for 3 times, each time for 1.5 hours, and the extract is concentrated to obtain an alcohol extract with a relative density of 1.05-1.10; the residues after alcohol extraction and volatile oil extraction are combined with 337g of Trigonum multiflorum, 254g of white peony root, and 417g of honeysuckle, and 10 times the amount of water is added for reflux extraction for 2 times, each time for 2 hours, and the extract is concentrated to obtain a water extract with a relative density of 1.18-1.20, and 60v / v% ethanol is precipitated. The precipitate is allowed to stand at 0-4°C for 24-48 hours, and the supernatant is concentrated to obtain an alcohol precipitate with a relative density of 1.25-1.30. The precipitate is combined with the alcohol extract, vacuum dried, crushed, and mixed with the volatile oil β-cyclodextrin inclusion compound to obtain an extract of the traditional Chinese medicine composition.

[0347] The moisture content of the extract of this product is controlled within 8%.

[0348] The method of Example 9 was used to establish a characteristic spectrum for a Chinese medicine composition preparation, and a spectrum substantially identical to that of the Chinese medicine composition was obtained, indicating that the characteristic spectrum establishment method of the present invention can also be used for quality control of Chinese medicine composition preparations.

[0349] The above further describes the present invention in conjunction with specific embodiments. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition, characterized in that The invention is prepared from the following raw materials in parts by weight: 61-119 parts of astragalus, 61-119 parts of glossy privet fruit, 37-71 parts of white peony root, 37-71 parts of atractylodes macrocephala, 80-105 parts of honeysuckle, 49-95 parts of zedoary zedoary and 49-95 parts of trigonosciadium japonicum.

2. The Chinese medicine composition according to claim 1, characterized in that The invention is prepared from the following raw materials in parts by weight: 72-108 parts of astragalus, 72-108 parts of glossy privet fruit, 42-66 parts of white peony root, 42-66 parts of atractylodes macrocephala, 80-105 parts of honeysuckle, 54-90 parts of zedoary zedoary and 54-90 parts of trigonosciadium japonicum.

3. The Chinese medicine composition according to claim 1, characterized in that The invention is prepared from the following raw materials in parts by weight: 88-92 parts of astragalus, 88-92 parts of glossy privet, 52-55 parts of white peony, 52-55 parts of atractylodes, 86-89 parts of honeysuckle, 69-72 parts of zedoary and 69-72 parts of trigonosciadium, and is further prepared from the following raw materials in parts by weight: 88-92 parts of astragalus, 88-92 parts of glossy privet, 52-55 parts of white peony, 52-55 parts of atractylodes, 87.8 parts of honeysuckle, 69-72 parts of zedoary and 69-72 parts of trigonosciadium.

4. The Chinese medicine composition according to claim 1, characterized in that The invention is prepared from the following raw materials in parts by weight: 90.1 parts of astragalus, 90.1 parts of glossy privet fruit, 53.5 parts of white peony root, 53.5 parts of atractylodes macrocephala, 87.8 parts of honeysuckle, 70.9 parts of zedoary zedoaria and 70.9 parts of trigonosciadium japonicum.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The steps include: (1) Extracting volatile oil: extracting volatile oil from Atractylodes macrocephala and Curcuma zedoaria, and obtaining volatile oil, medicinal residue and extract I after solid-liquid separation; (2) Alcohol extraction: Astragalus and Ligustrum lucidum are extracted with alcohol, and solid-liquid separation is performed to obtain alcohol extract and alcohol-extracted medicinal residue; (3) Water extraction: extracting the medicinal residue obtained in step (1), the alcohol-extracted medicinal residue obtained in step (2), and the remaining raw materials by water extraction, and separating the solid and liquid to obtain aqueous extract II; (4) Concentration: The extract I obtained in step (1) and the aqueous extract II obtained in step (3) are combined and concentrated to obtain aqueous extract concentrate III; (5) alcohol precipitation: subjecting the aqueous extract concentrate III obtained in step (4) to alcohol precipitation to obtain an alcohol precipitation supernatant; (6) combining the alcohol extract obtained in step (2) and the alcohol precipitation supernatant obtained in step (5), and concentrating to obtain a thick paste; (7) The thick paste obtained in step (6) is dried and mixed with volatile oil to obtain the Chinese medicine composition.

6. The preparation method according to claim 5, characterized in that The amount of water used in the extraction in step (1) is 1-8 times the total weight of Atractylodes macrocephala and Curcuma zedoaria, preferably 2-4 times, and most preferably 3 times; Preferably, before the extraction in step (1), soaking is further included; further preferably, the soaking time is 0.2-8 hours, more preferably 0.2-6 hours, and most preferably 0.5 hours; The extraction time of the extraction in step (1) is 3-10 hours, preferably 4-6 hours, and most preferably 5 hours.

7. The preparation method according to claim 5, characterized in that The alcohol extraction solvent used in the alcohol extraction in step (2) has an alcohol content of 50-80%, preferably 50-69%, and most preferably 60%; The amount of the alcohol extraction solvent in the alcohol extraction in step (2) is 6-18 times the total weight of the Astragalus and Ligustrum lucidum fruit, preferably 6-12 times, and most preferably 10 times; The number of extractions of the alcohol extraction in step (2) is 1-5 times, preferably 2-4 times, and most preferably 3 times; The extraction time of the alcohol extraction in step (2) is 1-5 hours, preferably 1-3 hours, and most preferably 1.5 hours; The alcohol extract in step (2) can be used directly without being concentrated or after being concentrated.

8. The preparation method according to claim 5, characterized in that The amount of water used in the water extraction in step (3) is 6-18 times the total weight of the Chinese medicine composition, preferably 6-14 times, and most preferably 10 times; The water extraction in step (3) is performed 1-4 times, preferably 1-3 times, and most preferably 2 times; The extraction time of the water extraction in step (3) is 1-5 hours, preferably 1-3 hours, and optimally 2 hours.

9. The preparation method according to claim 5, characterized in that The concentration in step (4) is concentration under reduced pressure; the concentration temperature in step (4) is 50-75°C, preferably 50-70°C, further 60-70°C; The relative density of the water-extracted concentrated solution III in step (4) is 1.10-1.40 at 50-75°C, preferably 1.10-1.30, and more preferably 1.15-1.

20.

10. The preparation method according to claim 5, characterized in that The alcohol solvent used in the alcohol precipitation in step (5) is ethanol; After adding the alcohol solvent to the alcohol precipitation in step (5), the alcohol content is 50-80%, preferably 50-70%, and most preferably 60%; The temperature of the alcohol precipitation in step (5) is 0-10°C, preferably 2-8°C, more preferably 4-6°C, and most preferably 4°C; The standing time of the alcohol precipitation in step (5) is 12-84 hours, preferably 12-36 hours, and most preferably 24 hours; The alcohol precipitation supernatant in step (5) can be used directly without concentration or after concentration.

11. The preparation method according to claim 5, characterized in that The relative density of the thick paste in step (6) is 1.00-1.25 at 55-75°C, preferably 1.03-1.20, and more preferably 1.05-1.

10.

12. The preparation method according to claim 5, characterized in that The relative density of the thick paste in step (7) is 1.15-1.45 at 55-75°C, preferably 1.20-1.40, and more preferably 1.25-1.30; The drying temperature in step (7) is 40-80°C, preferably 50-75°C, more preferably 50-70°C, and even more preferably 60-70°C.

13. Use of the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine composition prepared by the method according to any one of claims 5 to 12 in preparing a drug for treating kidney disease.

14. The use according to claim 13, characterized in that The kidney disease is chronic nephritis, proteinuria, or accompanied by early or mid-stage renal insufficiency; preferably, the kidney disease is chronic glomerulonephritis; further preferably, the chronic glomerulonephritis is chronic nephritis with deficiency of both qi and yin, blood stasis and toxins, or symptoms include fatigue, dry mouth and throat; dull complexion, hot palms and soles, soreness of waist and knees, fixed or stabbing pain in waist, sore throat, pale red or reddish or dark red tongue, thin white or thin yellow or sparse coating, and thin or weak or thready pulse.

15. The use according to claim 13, characterized in that The medicine comprises the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 12 and pharmaceutically acceptable excipients.

16. A traditional Chinese medicine preparation comprising the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 12 and pharmaceutically acceptable excipients; further, the preparation is prepared by extracting the raw materials using a solvent, or the preparation is a decoction and is further prepared into a non-liquid preparation, and further, the preparation is prepared by dry granulation.

Images