Traditional Chinese medicine composition for treating kidney diseases and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating kidney diseases, which is prepared from the following eleven medicinal materials: radix rehmanniae recen, artemisia capillaris, radix scutellariae, fructus aurantii, folium eriobotryae, dendrobe, radix ophiopogonis, perilla stem, fructus amomi, cortex magnoliae officinalis and radix salviae miltiorrhizae, the radix rehmanniae recen and the artemisia capillaris are monarch drugs, the radix scutellariae, the fructus aurantii, the folium eriobotryae, the dendrobe, the radix ophiopogonis, the perilla stem and the fructus amomi are ministerial drugs, the cortex magnoliae officinalis and the radix salviae miltiorrhizae are adjuvant drugs, and the radix rehmanniae recen and the fructus amomi are adjuvant drugs. The traditional Chinese medicine composition has the effects of nourishing yin, clearing stomach, resolving dampness and enlivening spleen, and also discloses a medicine for treating kidney diseases, which is prepared from the traditional Chinese medicine composition and pharmaceutically acceptable auxiliary materials, and application of the traditional Chinese medicine composition in preparation of the medicine for treating kidney diseases, especially kidney diseases caused by damp-heat impairment of yin. The traditional Chinese medicine composition is used for clearing damp-heat and nourishing yin, has a good clinical curative effect, can improve the renal function and relieve renal injury, and is high in safety and free of toxic and side effects.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and specifically relates to a Chinese medicine composition for treating kidney diseases and application thereof. Background Art

[0002] Affected by social development, population aging, and changes in lifestyle and dietary structure, the prevalence of chronic kidney disease (CKD) is on the rise. About 10% of the world's population is affected by CKD (World Health Organization survey data). In China, the overall prevalence of CKD in adults aged 18 years or older is 10.8% (An update on the global disparities in kidney disease burden and care across world countries and regions. Lancet Glob Health. 2024, 12(3): e382-e395.). Its high morbidity, high mortality, low awareness, and low prevention and treatment rates have brought a huge burden to the global health care system.

[0003] Modern medicine mainly treats it by changing lifestyle and treating primary diseases and complications. If the patient has entered the end-stage renal disease stage when it is discovered, the kidney function is severely lost, and a variety of complications that threaten the patient's life occur, renal replacement therapy (dialysis, kidney transplantation) is required. Kidney transplantation is limited by time, economy, and insufficient donors and cannot be popularized, so dialysis treatment is often the preferred treatment option. However, whether it is dialysis or kidney transplantation, long-term treatment will put pressure on the patient's physiology, psychology, and economy, and reduce the patient's quality of life.

[0004] Therefore, in order to effectively treat chronic kidney disease, protect kidney function, delay the entry into end-stage renal disease, protect patients' lives, and reduce the medical burden on patients' families and society, it is very important to find effective methods to treat chronic kidney disease.

[0005] Traditional Chinese medicine has rich experience in treating kidney diseases. It has progressed with the development of the times and combined with modern medical technology. It diagnoses and examines the causes, adjusts and treats diseases holistically, and shows the unique advantages of traditional Chinese medicine in the treatment of chronic kidney disease.

[0006] Chronic renal failure is caused by spleen and kidney deficiency, dampness, turbidity and toxic evils that turn into heat. However, dampness and heat will damage stomach yin over time, and the strength of spleen and stomach function is related to the prognosis of the disease. The Chinese medicine compositions in the prior art include Chinese medicine compositions for protecting kidneys and promoting urination, delaying the progression of chronic kidney disease, etc., but there is no research on Chinese medicine compositions for removing dampness, heat and nourishing yin to improve kidney function, reduce kidney damage or treat kidney diseases. Summary of the invention

[0007] The present invention aims to provide a Chinese medicine composition for treating kidney diseases.

[0008] The present invention also aims to provide a medicine for treating kidney disease made from the above-mentioned Chinese medicine composition and pharmaceutically acceptable excipients and a preparation method thereof.

[0009] The last object of the present invention is to provide the use of the above-mentioned Chinese medicine composition in the preparation of medicines for treating kidney diseases.

[0010] The above-mentioned first purpose of the present invention can be achieved by the following technical scheme: a traditional Chinese medicine composition for treating kidney disease, consisting of the following raw materials in parts by weight: 15-20 parts of raw rehmannia root, 10-20 parts of artemisia capillaris, 10-15 parts of scutellaria baicalensis, 10-15 parts of fructus aurantii, 15-20 parts of loquat leaf, 15-20 parts of dendrobium, 15-20 parts of ophiopogon, 10-15 parts of perilla stem, 7-15 parts of amomum, 15-20 parts of magnolia bark, and 15-20 parts of salvia miltiorrhiza, wherein raw rehmannia root and artemisia capillaris are monarch drugs, baicalensis, fructus aurantii, loquat leaf, dendrobium, ophiopogon, perilla stem and amomum are ministerial drugs, and magnolia bark and salvia miltiorrhiza are adjuvant drugs.

[0011] Preferably, the traditional Chinese medicine composition for treating kidney disease of the present invention is composed of the following raw materials in parts by weight: 15-20 parts of Rehmannia root, 10-15 parts of Artemisia capillaris, 10-15 parts of Scutellaria baicalensis, 10-15 parts of Fructus Aurantii Immaturus, 15-20 parts of Loquat leaf, 15-20 parts of Dendrobium officinale, 15-20 parts of Ophiopogon japonicus, 10-15 parts of Perilla stem, 7-10 parts of Amomum villosum, 15-20 parts of Magnolia officinalis, and 15-20 parts of Salvia miltiorrhiza.

[0012] More preferably, the Chinese medicine composition for treating kidney disease of the present invention is composed of the following raw materials in parts by mass: 15 parts of Rehmannia root, 15 parts of Artemisia capillaris, 10 parts of Scutellaria baicalensis, 15 parts of Fructus Aurantii Immaturus, 15 parts of Eriobotrya japonica, 15 parts of Dendrobium officinale, 15 parts of Ophiopogon japonicus, 15 parts of Perilla stem, 7 parts of Amomum villosum, 15 parts of Magnolia officinalis, and 15 parts of Salvia miltiorrhiza.

[0013] Among the raw materials of the present invention:

[0014] Rehmannia glutinosa is the fresh or dried root of Rehmannia glutinosa Libosch., a plant of the Scrophulariaceae family. Functions and indications: clearing away heat and promoting the production of body fluid, cooling blood and dispersing blood stasis, detoxifying and relieving pain; used for heat entering the blood, fever caused by warm toxins, vomiting blood and epistaxis, damage to the yin due to febrile diseases, red tongue and thirst, constipation caused by damage to body fluid, fever caused by yin deficiency, fever caused by bone steaming and fatigue, and thirst caused by internal heat. The main components of Rehmannia glutinosa are iridoids, ionones and phenylethanol compounds. Rehmannia glutinosa has the effects of increasing myocardial blood flow, lowering blood pressure and blood sugar in mice; Rehmannia glutinosa extract can counteract the inhibitory effect of dexamethasone on the pituitary-adrenal cortex system, and prevent adrenal cortex atrophy and decreased corticosterone levels caused by glucocorticoids; Rehmannia glutinosa can increase cellular immune function, promote the phagocytic function of the reticuloendothelial system and increase peripheral blood T lymphocytes; it also has anti-tumor, sedative and promotion of protein synthesis in rat liver and kidney tissues; it also has hemostatic, cardiotonic, diuretic and anti-inflammatory effects. Studies have found that Shengdi can reduce the levels of creatinine and urea nitrogen in the serum of UUO rats, and improve glomerular sclerosis, tubular damage and interstitial fibrosis.

[0015] Artemisia capillaris is the dried aerial part of Artemisia scoparia Waldst.et Kit. or Artemisia capillaris Thunb. of the Asteraceae family. Functions and indications: clearing away dampness and heat, promoting bile and relieving jaundice; used for jaundice, oliguria, damp-heat, summer-heat, wet sores, itching and other symptoms. Artemisia capillaris contains a variety of chemical components with medicinal functions, mainly including artemisia coumarins, volatile oils, chromones, flavonoids and chlorogenic acid. In addition to the traditional effects of clearing away heat and dampness, promoting bile and relieving jaundice, Artemisia capillaris also has antipyretic, analgesic, anti-inflammatory, antiviral, anti-tumor, blood pressure lowering, blood lipid regulating, anti-osteoporosis, neuroprotection, metabolism regulation, T cell immune activity enhancement, and immune regulation. Modern pharmacological studies have shown that flavonoids in Artemisia capillaris can reduce atherosclerosis in rabbits with hypercholesterolemia and reduce visceral fat deposition; Artemisia capillaris water extract can inhibit the proliferation of Hs68 cells by acting on HaCaT cells, thereby playing an immunosuppressive role.

[0016] Scutellaria baicalensis is the dried root of Scutellaria baicalensis Georgi. of the Labiatae family. Functions and indications: clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus. It is used for damp-heat, summer-heat, chest tightness and nausea, damp-heat fullness, diarrhea, jaundice, lung heat cough, high fever and thirst, blood heat vomiting and bleeding, carbuncle, sore, and fetal movement disorder. The main active ingredients in Scutellaria baicalensis are flavonoids, polysaccharides, and volatile oils. It has multiple pharmacological effects. Baicalin can improve mucin-induced renal toxicity by activating the antioxidant defense mechanism in the kidney and downregulating the inflammatory response. It also has the effects of reducing the production of reactive oxygen species, inhibiting cell apoptosis, and reducing the expression level of inflammatory factors. Studies have shown that β-sitosterol in Scutellaria baicalensis has anti-inflammatory, blood sugar, blood lipid regulation, etc., and can protect renal tissue from damage caused by renal ischemia / reperfusion.

[0017] Citrus aurantium is the dried immature fruit of Citrus aurantium L. and its cultivated varieties. Functions and indications: Regulates qi and relieves fullness, relieves stagnation and relieves bloating. It is used for chest and flank qi stagnation, fullness and pain, indigestion, phlegm and fluid retention, and ptosis of organs. Studies have shown that the main active ingredients in Citrus aurantium are Citrus aurantium flavonoids, volatile oils, alkaloids, etc. Modern studies have shown that Citrus aurantium can increase blood pressure, promote diuresis in a short period of time, and reduce kidney volume.

[0018] Loquat leaf is the dried leaf of Eriobotrya japonica (Thunb) Lindl., an evergreen small tree of the Rosaceae family. Functions and indications: moistening the lungs and relieving cough, lowering adverse reactions and relieving vomiting. It is used to treat lung heat cough, vomiting blood, stomach heat vomiting, irritability and thirst, and other diseases. Pharmacochemical studies have shown that loquat mainly contains chemical components such as volatile oils, triterpenes, sesquiterpenes, flavonoids, and amino acids. Loquat leaf has pharmacological effects such as anti-inflammatory, antioxidant, hypoglycemic, antitussive and expectorant, anti-tumor, antibacterial and antiviral, liver protection, scavenging oxygen free radicals, and enhancing the body's immune function. Loquat leaf can significantly reduce the content of 5-hydroxytryptamine (5-HT) and dopamine (DA) in the brain tissue of the mouse vomiting model induced by cisplatin, and has a good antiemetic effect.

[0019] Dendrobium is a cultivated product of Dendrobium nobile Lindl., Dendrobium chrysotoxum Lindl. or Dendrobium fimbriatum Hook. of the orchid family and the fresh or dried stems of similar species of plants of the same genus. Functions and indications: nourishing the stomach and promoting the production of body fluid, nourishing yin and clearing away heat. It is used for damage to body fluid caused by febrile diseases, dry mouth and thirst, insufficient stomach yin, poor appetite and retching, persistent deficiency heat after illness, excessive yin and fire, bone steaming and fatigue fever, blurred vision, and weakness of tendons and bones. Dendrobium mainly contains alkaloids such as dendrobine, dendrobamine, dendrobine alkaloids, and mucus. Studies have shown that Huoshan Dendrobium can reduce the serum interleukin-6 content of mice with kidney yin deficiency, and has the effect of regulating cytokine disorders and enhancing the body's antioxidant capacity.

[0020] Ophiopogon japonicus is the dried root of Ophiopogon japonicus (Thunb.) Ker-Gawl., a plant of the Liliaceae family. Functions and indications: nourishing yin and promoting body fluid, moistening the lungs and clearing the heart. It is used to treat dry cough due to lung dryness, vomiting blood, lung abscess, fatigue and fever, thirst, damage to body fluid due to fever, dry throat and mouth, constipation, etc. Ophiopogon japonicus contains a variety of steroidal saponins, carotene, mucus, sugars, stigmasterol and other ingredients. Its pharmacological effects have been gradually developed and improved, and it has a variety of pharmacological effects such as protecting the cardiovascular system, regulating immunity, anti-tumor, lowering blood sugar, and anti-inflammatory. Ophiopogon furostanal saponin A and Ophiopogon furostanal saponin B extracted from Ophiopogon japonicus have a certain inhibitory effect on the expression of eosinophil-activated chemokine induced by the combination of interleukin (IL)-4 and tumor necrosis factor α.

[0021] Perilla frutescens (L.) Britt. var. arguta (Benth) Hand.-Mazz. is a plant of the genus Perilla in the family Lamiaceae. Functions and indications: regulating qi and relieving fullness, calming the fetus, and harmonizing blood. Traditionally, it is commonly used to treat wind-cold exterior syndrome, spleen and stomach qi stagnation, vomiting, fish and crab poisoning and other diseases. Perilla contains a variety of chemical components, and the above-ground part mainly contains volatile oils, flavonoids and their glycosides, terpenes, lipids and other components. Modern pharmacological studies have shown that perilla extract perilla aldehyde can exert anti-inflammatory function by reducing the expression levels of inflammatory factors IL-6 and TNF-α. Perilla extract can also reduce renal damage induced by trimethylamine n-oxide (TMAO). The specific mechanism is to reduce renal tubular apoptosis induced by trimethylamine n-oxide by inhibiting the phosphorylation of ASK1-JNK.

[0022] Amomum villosum Lour., the dried mature fruit of the ginger family plants Amomum viUosum Lour., Amomum villosum Lour. var. xanthioides TLWu et Senjen or Amomum longiligulare TLWu. Functions and indications: removing dampness and stimulating appetite, warming the spleen and stopping diarrhea, regulating qi and calming the fetus. It is used for dampness and turbidity blocking the middle part of the body, abdominal distension without hunger, spleen and stomach deficiency and cold, vomiting and diarrhea, nausea during pregnancy, and restless fetus. Amomum villosum mainly contains volatile oils, flavonoids, polysaccharides and other bioactive ingredients. The pharmacological effects of Amomum villosum mainly include gastrointestinal protection, anti-inflammatory, analgesic, antidiarrheal, and hypoglycemic effects. Studies have shown that the water extract of Amomum villosum has a significant restorative effect on intestinal flora imbalance caused by antibiotics.

[0023] Magnolia officinalis is the dried bark, root bark and branch bark of Magnolia officinalis Rehd.et Wils. or Magnolia offinalis Rehd.et Wils.var.biloba Rehd.et Wils. of the Magnoliaceae family. Functions and indications: Drying dampness and eliminating phlegm, lowering qi and eliminating fullness. It is used for dampness stagnation, abdominal distension, vomiting and diarrhea, food stagnation and qi stagnation, abdominal distension and constipation, phlegm and cough. Magnolia officinalis mainly contains phenolic substances (magnolia officinalis, honokiol, isomagnokiol, etc.), volatile oil (β-eudesmol), etc. Studies have shown that magnolia officinalis can inhibit the upregulation of inflammatory cytokines in human glomerular mesangial cells induced by LPS and high glucose, and improve renal fibrosis by inhibiting extracellular matrix and proinflammatory factors in vivo and in vitro.

[0024] Danshen is the dried root and rhizome of SalviamiltiorrhizaBge., a plant of the Labiatae family. Its functions and indications include promoting blood circulation and removing blood stasis, relieving pain through menstruation, clearing the heart and eliminating vexation, cooling blood and eliminating carbuncle. It is used for chest and abdominal pain, rheumatic pain, palpitations and insomnia, menstrual disorders, amenorrhea and dysmenorrhea, sores, carbuncle, swelling and pain, etc. Danshen contains chemical components such as tanshinones, salvianolic acids, and volatile oils. Clinical studies have found that tanshinone IIA may reduce the expression of inflammatory factors tumor necrosis factor-α and interleukin-6 by reducing TGF-β1, thereby alleviating kidney inflammation damage. Salvianolic acid, an important component of Danshen, can significantly improve indicators such as plasma viscosity, fibrinogen and whole blood viscosity, and has a positive effect on blood rheology.

[0025] The present invention is carefully formulated under the guidance of traditional Chinese medicine theory. The motherland's medicine believes that although chronic renal failure is caused by spleen and kidney deficiency, dampness, turbidity and toxic evil are internally contained and transformed into heat, but dampness and heat will damage stomach yin over time, and the strength of spleen and stomach function is related to the prognosis of the disease. Weak spleen and stomach, difficult to digest food, poor appetite, affect the absorption of drugs and the performance of drug efficacy. Lack of essence and micro, no source of qi and blood biochemistry, and then anemia, fatigue and other symptoms appear. Spleen and stomach ascending and descending disorder, clear and turbid are not distinguished, and symptoms such as poor appetite, nausea and vomiting often occur. Weak spleen and stomach, water metabolism disorder, gather in the body and become damp, damp evil condenses, and the spleen and earth are trapped, so that the middle jiao qi machine is blocked, and the ascending and descending power is weak. Damp heat in the stomach affects its function of descending turbidity and receiving, and damp heat and toxic evil are stored, and the syndromes are multiplied.

[0026] The raw rehmannia root in the present invention is sweet, bitter, cold, enters the heart, liver, and kidney meridians, enters the kidney meridian and nourishes yin, is bitter and cold, clears away heat, and nourishes yin and produces body fluid; Artemisia capillaris is bitter, pungent, slightly cold, enters the spleen, stomach, liver, and gallbladder meridians, controls wind, cold, dampness, and heat evil spirits, and can relieve the dampness and heat of spleen and earth; Scutellaria baicalensis is bitter and cold, enters the lung, gallbladder, spleen, stomach, large intestine, and small intestine meridians, is good at treating dampness and heat blocking the qi mechanism and causing chest tightness, and stops vomiting; Citrus aurantium is bitter, pungent, sour, warm, enters the spleen, stomach, and large intestine meridians, is pungent and bitter, is good at promoting qi and harmonizing the stomach; Loquat leaf is bitter and slightly cold, enters the lung and stomach meridians, harmonizes the stomach and descends qi, descends adverse qi and stops vomiting; Dendrobium officinale is sweet, slightly cold, enters the stomach and kidney meridians, is good at nourishing stomach yin, and clears qi Stomach heat can produce fluid, and can nourish kidney yin. Together with Ophiopogon japonicus, it nourishes the yin of the spleen and stomach and clears away heat. Ophiopogon japonicus is sweet, slightly bitter, and slightly cold, and it enters the stomach, lung, and heart meridians. It is good at nourishing stomach yin, producing fluid and quenching thirst, and clearing away stomach heat. Perilla stem is pungent and warm, and it enters the lung and spleen meridians. This product is pungent and can move qi, promote qi circulation to harmonize the stomach and stop vomiting. Amomum villosum is pungent and warm, and it enters the spleen, stomach, and kidney meridians. It is pungent and dispersing, warms and unblocks, warms and invigorates the spleen, eliminates dampness and promotes qi circulation. Magnolia officinalis is bitter, pungent, and warm, and it enters the spleen, stomach, lung, and large intestine meridians. This product is bitter, dry, pungent and dispersing, can dry dampness, and can eliminate bloating. Salvia miltiorrhiza is bitter, slightly cold, and it enters the heart, pericardium, and liver meridians. It is mild in nature and can move blood, promote blood circulation, nourish blood, and remove stasis and heat.

[0027] In the Chinese medicine composition, Radix Rehmanniae and Artemisiae Scopariae are the main drugs, nourishing the yin of the spleen and stomach, clearing dampness and heat of the spleen and stomach; Radix Scutellariae is bitter and cold, clearing heat and drying dampness, and purging heat to preserve yin; Radix Ophiopogonis and Herba Dendrobii nourish the yin of the spleen and stomach, clearing deficiency heat; Folium Eriobotryae and Fructus Aurantii Immaturus clear stomach heat; Radix Scutellariae increases the function of clearing dampness and heat; Perilla stem is pungent and warm, harmonizes the stomach and stops vomiting; Fructus Amomum is aromatic and invigorates the spleen and eliminates dampness, and the above are all ministerial drugs; Radix Salviae Miltiorrhizae promotes blood circulation and nourishes blood, removes stasis and heat; and Cortex Magnoliae Officinalis promotes qi and harmonizes the stomach are all adjuvant drugs. When all the drugs are used together, dampness and heat are eliminated, yin fluid is nourished, and there are the effects of nourishing yin and clearing the stomach, eliminating dampness and invigorating the spleen, which hits the pathogenesis of the disease. It is confirmed by clinical application and pharmacological experiments that the Chinese medicine composition of the present invention has obvious effect and definite curative effect.

[0028] The invention is composed of 11 medicinal materials including Radix Rehmanniae, Artemisia Capillaris, Radix Scutellariae, Fructus Aurantii Immaturus, Fructus Loquatae, Herba Dendrobii, Radix Ophiopogonis, Perilla Stem, Fructus Amomi, Fructus Magnoliae, and Radix Salviae Miltiorrhizae, and has the functions of nourishing yin and clearing the stomach, removing dampness and invigorating the spleen. It is used for poor appetite, dry mouth, fatigue, abdominal distension, nausea, and sticky mouth caused by insufficient stomach yin and damp-heat damaging yin. Clinicians will adjust the dosage ratio of the prescription according to the different stages of the patient's condition, so as to determine the optimal ratio more scientifically and reasonably.

[0029] The above second object of the present invention can be achieved by the following technical solution: a medicine for treating kidney disease, which is made from the above traditional Chinese medicine composition and pharmaceutically acceptable excipients.

[0030] Preferably, the drug is in the form of an aqueous solution, granules or concentrated pills.

[0031] Preferably, the auxiliary material is one or more of dextrin, starch, edible alcohol and flavoring agent, and the flavoring agent is aspartame or sucrose.

[0032] The preparation method of the medicine for treating kidney disease comprises the following steps: taking various raw materials of the traditional Chinese medicine composition, soaking them in water, decocting and extracting them once, filtering them to obtain medicine residues and a primary filtrate; adding water to the medicine residues, decocting and extracting them twice, filtering them to obtain a secondary filtrate; combining the primary filtrate and the secondary filtrate, concentrating them, and adding auxiliary materials as needed to prepare the medicine.

[0033] Preferably, during the first decoction extraction and the second decoction extraction, 5-10 times the total mass of water of the traditional Chinese medicine composition is added, and the decoction extraction time for each time is 60-90 minutes.

[0034] Preferably, the soaking time in water is 60-120 minutes.

[0035] The medicine for treating kidney diseases of the present invention has a reasonable prescription, a simple preparation method and a simple process, and is easy to operate. The obtained medicine has a significant therapeutic effect on kidney diseases.

[0036] The last object of the present invention can be achieved by the following technical solution: use of the above-mentioned traditional Chinese medicine composition in the preparation of medicines for treating kidney diseases.

[0037] Furthermore, the present invention also provides the use of the above-mentioned Chinese medicine composition in the preparation of a medicine for treating kidney diseases caused by damp-heat damaging yin.

[0038] The present invention has the following advantages: the Chinese medicine composition of the present invention can be used together with the medicine for clearing away dampness and heat and the medicine for nourishing yin, and the composition has good clinical efficacy, can improve renal function, can alleviate the effect of renal damage, and is highly safe and has no toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1The results of HE staining of renal tissues of mice in the normal group, model group, Ganluyin low-dose group and Ganluyin high-dose group after pathological injury of cisplatin-induced AKI mice in Example 4;

[0040] Figure 2 The creatinine and urea nitrogen levels of renal tissues of mice in the normal group, model group, Ganluyin low-dose group and Ganluyin high-dose group after cisplatin-induced AKI mice pathological injury in Example 4;

[0041] Figure 3 is the protein expression of ACSL4, LPCAT3, Bax, and CHOP in the normal group, model group, and Ganluyin high-dose group in Example 4, and AKI and GLY represent the model group and Ganluyin high-dose group, respectively;

[0042] Figure 4 The effect of Ganluyin on the renal GPR43 protein in cisplatin-induced AKI mice in Example 4;

[0043] Figure 5 The effect of Ganluyin in Example 4 on the expression and activation of mTORC1, a key protein controlling autophagy activation in the kidneys of cisplatin-induced AKI mice;

[0044] Figure 6 is the change in the protein level of the lipophage marker protein PLIN2 in the kidneys of each group of mice in Example 4;

[0045] Figure 7 The results of Oil Red O staining of kidney tissues of AKI mice in each group in Example 4;

[0046] Figure 8 The effect of Ganluyin on the levels of blood creatinine, urea nitrogen and eGFR in patients with AKI in Example 4;

[0047] Fig. 9 The changes in intestinal flora of AKI patients before and after treatment with Ganluyin in Example 4;

[0048] Fig.10 The figure shows the changes in intestinal flora of AKI mice before and after treatment with Ganluyin in Example 4. Sham, AKI and GLY represent the normal group, model group and Ganluyin high-dose group, respectively. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with specific examples. The following examples are only for illustrative purposes and are not to be construed as limiting the present invention. Unless otherwise specified, the raw materials used in the following examples are conventional commercially available or commercially available raw materials, and unless otherwise specified, the methods and equipment used in the following examples are conventional methods and equipment used in the art.

[0050] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0051] Example 1

[0052] The Chinese medicine composition for treating kidney disease provided in this embodiment is composed of the following raw materials of different qualities: 15g of raw rehmannia root, 15g of artemisia capillaris, 10g of scutellaria baicalensis, 15g of fructus aurantii, 15g of loquat leaf, 15g of dendrobium, 15g of ophiopogon, 15g of perilla stem, 7g of amomum villosum, 15g of magnolia bark, and 15g of salvia miltiorrhiza, wherein raw rehmannia root and artemisia capillaris are monarch drugs, baicalensis, fructus aurantii, loquat leaf, dendrobium, ophiopogon, perilla stem and amomum villosum are ministerial drugs, and magnolia bark and salvia miltiorrhiza are adjuvant drugs.

[0053] The drug for treating kidney disease provided in this embodiment is an aqueous solution, and its preparation method is as follows:

[0054] Add all the above medicinal materials into the multifunctional Chinese medicine decoction pot at one time, add 8 times the mass of pure water to fully soak the medicinal materials for 30 minutes, select "automatic decoction" for 45 minutes, filter with sterile gauze, add 8 times the mass of pure water to decoct for 2 times for 45 minutes, and then filter the liquid again. Combine the filtered liquid, heat and condense the filtered liquid, and then place it in the refrigerator for natural cooling and standby use. When using, it can be diluted according to the concentration required for the experiment, and flavoring agents such as aspartame or sucrose can be added as needed.

[0055] Example 2

[0056] The Chinese medicine composition for treating kidney disease provided in this embodiment is composed of the following raw materials of different qualities: 15g of raw rehmannia root, 20g of artemisia capillaris, 10g of scutellaria baicalensis, 15g of fructus aurantii, 15g of loquat leaf, 20g of dendrobium, 15g of ophiopogon japonicus, 15g of perilla stem, 7g of amomum villosum, 20g of magnolia bark, and 15g of salvia miltiorrhiza, wherein raw rehmannia root and artemisia capillaris are monarch drugs, baicalensis, fructus aurantii, loquat leaf, dendrobium, ophiopogon japonicus, perilla stem and amomum villosum are ministerial drugs, and magnolia bark and salvia miltiorrhiza are adjuvant drugs.

[0057] The drug for treating kidney disease provided in this embodiment is a granule, which is prepared by adding auxiliary material starch to the concentrated solution prepared in Example 1.

[0058] Example 3

[0059] The Chinese medicine composition for treating kidney disease provided in this embodiment is composed of the following raw materials of different qualities: 20g of raw rehmannia root, 10g of artemisia capillaris, 15g of scutellaria baicalensis, 10g of fructus aurantii, 20g of loquat leaf, 15g of dendrobium, 20g of ophiopogon, 10g of perilla stem, 15g of amomum, 15g of magnolia bark, and 20g of salvia miltiorrhiza, wherein raw rehmannia root and artemisia capillaris are monarch drugs, baicalensis, fructus aurantii, loquat leaf, dendrobium, ophiopogon, perilla stem and amomum are ministerial drugs, and magnolia bark and salvia miltiorrhiza are adjuvant drugs.

[0060] The dosage form of the drug for treating kidney disease provided in this embodiment is concentrated pills, which are prepared by adding auxiliary material starch to the concentrated solution prepared in Example 1.

[0061] Example 4

[0062] This example confirms through clinical application and pharmacological experiments that the Chinese medicine composition of the present invention has obvious effects and definite therapeutic effects, as shown in the following.

[0063] 1. Experimental Materials

[0064] 1. Preparation of medicinal materials

[0065] The drug in Example 1.

[0066] 2. Experimental animals and housing conditions

[0067] 2.1 Experimental animals

[0068] SPF grade C57BL / 6 male mice, 8 weeks old, weighing 20-25 g, were purchased from Guangdong Sijiajingda Biotechnology Co., Ltd. and were raised in strict accordance with the Regulations on the Administration of Experimental Animals of the People's Republic of China and approved by the Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine.

[0069] 2.2 Rearing conditions

[0070] Feeding and management: All experimental animals were kept in the Experimental Animal Center of Guangdong Provincial Academy of Traditional Chinese Medicine and were managed by dedicated personnel.

[0071] Facility conditions: temperature (22±2)℃, humidity 40%~60%, 12h day and night alternation. During the experiment, the 3R principle was strictly followed and humanitarian care was given.

[0072] Feeding groups: After the mice were adaptively fed, they were randomly divided into 4 groups: a normal group, an AKI model group, a low-dose Ganluyin group (the Chinese medicine composition prepared in Example 1), and a high-dose Ganluyin group (the Chinese medicine composition prepared in Example 1), with 10 mice in each group.

[0073] The low-dose group corresponds to the clinical equivalent dose, and the high-dose group corresponds to 1.5 times the clinical equivalent dose; the normal group and the model group were gavaged with the same amount of pure water at the same time, and the drug was administered continuously for 7 days. Samples were collected 72 hours after modeling, and after anesthetizing the mice, blood and kidney tissue specimens were collected and stored in liquid nitrogen or -80℃ for future use.

[0074] 3. Experimental testing

[0075] 3.1 Renal function test in each group

[0076] Blood sample 3 000r·min -1Centrifuge for 15 minutes and take the upper serum; detect the serum creatinine and urea nitrogen levels in each sample according to the kit instructions.

[0077] 3.2 Renal histopathological observation and tubular injury score

[0078] Mouse kidney tissue was fixed in 4% paraformaldehyde solution for 24 hours, then embedded in paraffin and sliced ​​to a thickness of about 3 μm. HE and PAS staining were performed, and the pathological changes of renal tissue were observed under an optical microscope. Under a light microscope, 5 renal tubular fields at the corticomedullary junction were randomly selected from each HE slice for injury scoring, and the injury scores of mice in each group were averaged.

[0079] 3.3 Oil Red O staining

[0080] Mouse kidney tissue was used to make frozen sections. After fixation with 10% formalin for 10 minutes, the sections were washed three times with water and then immersed in 60% ethanol for 20-30 seconds. The sections were placed in Oil Red O staining solution, sealed and stained in the dark for 10-15 minutes. The sections were briefly washed in 60% ethanol to remove the staining solution. After washing with distilled water for 3 times, the nuclei were counterstained with hematoxylin staining solution for 1 minute. The sections were then differentiated with 1% hydrochloric acid solution. The sections were rinsed with tap water for 10 minutes and dried in a 60°C oven. The sections were sealed with glycerol gelatin.

[0081] 3.4 Western blot analysis of the expression levels of ferroptosis, apoptosis, and lipophagy-related proteins in renal tissue

[0082] Mouse kidney tissue was extracted with RIPA buffer, and the total protein content of the sample was determined by BCA protein quantification kit. The sample was leveled according to the minimum protein concentration, and SDS-PAGE gel electrophoresis was performed after high temperature denaturation. The sample was transferred to a PVDF membrane, blocked with 5% skim milk powder at room temperature for 2 h, and the strips were washed with TBST. The diluted primary antibodies were added and incubated at 4°C overnight. The membrane was washed with TBST 5 times, each time for 5 min, and incubated with HRP-labeled goat secondary antibody (1:3 000) at room temperature for 1 h. The membrane was washed with TBST 5 times, each time for 5 min, and then chemiluminescence development was performed. The gray value of the target protein and the internal reference protein was analyzed using ImageJ software. The relative expression of the target protein = gray value of the target protein / gray value of the internal reference protein.

[0083] 2. Test results

[0084] 1. Ganluyin alleviates pathological damage and renal function damage in mice with cisplatin-induced AKI

[0085] Ganluyin alleviated the pathological damage of mice with AKI induced by cisplatin. As HE staining showed, the renal tubular epithelial cells of mice in the AKI model group underwent significant atrophy, necrosis and shedding, the brush border of the renal tubules disappeared, a large number of protein casts filled the tubules, the tubules vacuolar degeneration, the renal interstitium widened and edema was visible, and the renal structure was severely damaged; Ganluyin significantly improved the renal structural damage. By testing the serum creatinine and urea nitrogen levels of mice in each group, it was found that the renal tubular epithelial cell necrosis, protein casts, vacuolar degeneration and interstitial edema of the mice in the high-dose Ganluyin group of AKI mice were significantly reduced ( Figure 1 ), urea nitrogen (BUN) and serum creatinine (Scr) levels were significantly higher than those in the normal group. Compared with the model group, the urea nitrogen and serum creatinine levels in the high-dose Ganluyin group were significantly decreased ( Figure 2 ).

[0086] 2. Ganluyin can effectively inhibit ferroptosis and apoptosis of renal cells in AKI

[0087] ACSL4 is one of the main enzymes in fatty acid metabolism, responsible for catalyzing long-chain polyunsaturated fatty acids into the corresponding acyl-CoA, and then LPCAT3 acts as an intermediate medium to insert CoA into the plasma membrane to form a ferroptosis-specific lipid peroxidation reaction substrate, triggering ferroptosis. Bax and CHOP are classic pro-apoptotic proteins and can be used as marker proteins for apoptosis. By detecting the renal tissue proteins of each group, it was found that compared with the normal group, the protein expression levels of ACSL4, LPCAT3, Bax, and CHOP in the AKI model group were significantly increased; and the Ganluyin intervention group could effectively reduce their expression, indicating that Ganluyin can effectively inhibit ferroptosis and apoptosis in acute kidney injury ( Figure 3 ).

[0088] 3. Ganluyin inhibits the decrease of GPR43 expression in the kidneys of cisplatin-induced AKI mice

[0089] In order to observe the effect of Ganluyin on the renal GPR43 of mice with cisplatin-induced AKI, the protein level of GPR43 in the renal kidneys of mice in each group was detected. It was found that compared with the normal group, the expression of GPR43 protein in the renal kidneys of mice with cisplatin-induced AKI was significantly decreased, while Ganluyin drug intervention could significantly promote the expression of GPR43 protein ( Figure 4 ).

[0090] 4. Ganluyin inhibits the decrease in the expression and activity of mTORC1 in the kidneys of AKI mice

[0091] In order to observe the effect of Ganluyin on the expression and activation of mTORC1, a key protein controlling autophagy activation in the kidneys of cisplatin-induced AKI mice, the protein levels of mTORC1 and p-mTORC1 in the kidneys of mice in each group were detected. The results showed that compared with the normal group, the expression of mTORC1 and p-mTORC1 proteins in the kidneys of cisplatin-induced AKI mice was significantly decreased, while Ganluyin drug intervention could significantly increase the expression of mTORC1 and p-mTORC1 proteins ( Figure 5 ).

[0092] 5. Ganluyin significantly inhibits renal lipophagy in AKI mice

[0093] In order to observe the effect of Ganluyin on renal lipophagy events in mice with cisplatin-induced AKI, the protein level changes of PLIN2, a marker of renal lipophagy, in each group of mice were detected and analyzed. The results showed that compared with the normal group, PLIN2 in the kidneys of mice with cisplatin-induced AKI was significantly increased, indicating that obvious lipophagy events occurred in the kidneys of mice with cisplatin-induced AKI; Ganluyin administration intervention can significantly increase the expression of PLIN2 protein, indicating that Ganluyin can significantly inhibit the renal lipophagy events of mice with cisplatin-induced AKI ( Figure 6 ).

[0094] 6. Ganluyin can restore the number of renal lipid droplets in cisplatin-induced AKI mice to homeostasis

[0095] In order to observe the effect of Ganluyin on the number of renal fat droplets in cisplatin-induced AKI mice, the renal tissues of mice in each group were stained with Oil Red O. The results showed that compared with the normal group, the number of renal fat droplets in cisplatin-induced AKI mice increased significantly, and Ganluyin intervention could restore the number of renal fat droplets in cisplatin-induced AKI mice to a steady state ( Figure 7 ).

[0096] 3. Clinical sample testing of Ganluyin's efficacy in improving acute kidney injury and intestinal flora research

[0097] 1. Ganluyin can significantly improve renal function damage in patients with AKI

[0098] Figure 8 The results showed that Ganluyin (high-dose group) can significantly reduce blood creatinine and urea nitrogen in AKI patients and increase eGFR levels.

[0099] 2. Ganluyin can significantly restore the abundance of bacteria producing acetic acid and other SCFAs in the intestine of AKI patients

[0100] Changes in intestinal flora in AKI patients before and after treatment with Ganluyin (high-dose group): Fecal 16SrRNA results showed that the abundance of SCFAs-producing bacteria Faecalibacterium, Anaerostipes, Dialister, Megasphaera, Roseburia, Kineothrix, Blautia, Lachnospiraceae, Streptococcaceae, and Enterobacteriaceae in the intestine of patients treated with Ganluyin increased significantly, among which Lachnospiraceae, Streptococcaceae, and Enterobacteriaceae had stronger acetic acid production effects ( Fig. 9 ).

[0101] Therefore, Ganluyin (high-dose group) can significantly restore the abundance of intestinal SCFAs-producing bacteria in AKI patients.

[0102] 3. Ganluyin can significantly restore the abundance of bacteria producing acetic acid and other SCFAs in AKI mice

[0103] Changes in intestinal flora of AKI mice before and after treatment with Ganluyin (high-dose group): Ganluyin pretreatment can significantly increase the abundance of SCFAs-producing bacteria Faecalibaculum, Bacteroides, Bifidobacterium, AnaerostipesLactobacillaceae, and Lachnospiracea in the AKI intestine, among which Bacteroides, Bifidobacterium, Lactobacillaceae, and Lachnospiraceae have stronger acetic acid production effects ( Fig.10 ).

[0104] IV. Clinical Application

[0105] Zhang, female, 49 years old, has a history of chronic renal failure for 10 years. One week before the visit, the patient had a blood creatinine of 696 μmol / L in the local hospital due to nausea and fatigue. The patient refused the recommended renal replacement therapy. Diagnosis: dry mouth, ammonia taste in the mouth, nausea, poor appetite, fatigue, abdominal distension, constipation, red tongue, yellow and greasy fur, and slippery pulse. Ultrasound showed: left kidney 8.0cm×3.8cm×3.5cm, parenchyma thickness 1.0cm, right kidney 7.7cm×3.2cm×4.0cm, parenchyma thickness 0.9cm. Diffuse lesions in both kidneys. Western medicine diagnosis: chronic renal failure; Chinese medicine syndrome differentiation belongs to damp-heat injury to yin. Given the present invention (the medicine in Example 1, twice a day), the patient was still weak, dry mouth, nausea, shortness of breath, and poor appetite after one week, and the blood creatinine was 551.8 μmol / L. Continue to give the present invention (the medicine in Example 1, 2 times a day), fatigue and shortness of breath were relieved after 2 weeks, nausea, dry mouth, pale complexion, no abdominal distension, stool once a day, blood creatinine was checked 442.9μmol / L. After 3 months, the above symptoms were improved, and the blood creatinine was checked 388.9μmol / L. The patient was treated with modified Ganluyin (20g of Rehmannia glutinosa, 15g of Artemisia capillaris, 15g of Scutellaria baicalensis, 15g of Citrus aurantium, 20g of Loquat Leaf, 20g of Dendrobium officinale, 15g of Radix Ophiopogonis, 15g of Licorice, 20g of Achyranthes bidentata, 20g of Puerariae lobata) for four months, and blood creatinine was stable at about 450μmol / L.

[0106] Liu, female, 77 years old, has a history of chronic renal insufficiency for 12 years. She often suffers from fatigue, dizziness, frequent urination, and chest tightness when tired. Diagnosis: fatigue, dizziness, frequent urination, urgency, chest tightness, pale purple tongue, white and greasy fur, and slippery pulse. Western medicine diagnosis: chronic renal failure; Chinese medicine diagnosis: damp-heat damage to yin syndrome. The present invention (the medicine in Example 2, twice a day) was given, and the blood creatinine was maintained at 100-110 μmol / L during the 12-year follow-up.

[0107] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A Chinese medicine composition for treating kidney disease, characterized in that: The invention is composed of the following raw materials in parts by weight: 15-20 parts of raw rehmannia root, 10-20 parts of artemisia capillaris, 10-15 parts of scutellaria baicalensis, 10-15 parts of fructus aurantii, 15-20 parts of loquat leaf, 15-20 parts of dendrobium officinale, 15-20 parts of ophiopogon japonicus, 10-15 parts of perilla stem, 7-15 parts of amomum villosum, 15-20 parts of magnolia bark, and 15-20 parts of salvia miltiorrhiza, wherein raw rehmannia root and artemisia capillaris are monarch drugs, baicalensis baicalensis, fructus aurantii, loquat leaf, dendrobium officinale, ophiopogon japonicus, perilla stem and amomum villosum are ministerial drugs, and magnolia bark and salvia miltiorrhiza are adjuvant drugs.

2. The Chinese medicine composition for treating kidney disease according to claim 1, characterized in that: The invention is composed of the following raw materials in parts by weight: 15-20 parts of raw rehmannia root, 10-15 parts of artemisia capillaris, 10-15 parts of scutellaria baicalensis, 10-15 parts of fructus aurantii, 15-20 parts of loquat leaf, 15-20 parts of dendrobium, 15-20 parts of ophiopogon, 10-15 parts of perilla stem, 7-10 parts of amomum villosum, 15-20 parts of magnolia bark and 15-20 parts of salvia miltiorrhiza.

3. The Chinese medicine composition for treating kidney disease according to claim 2, characterized in that: The invention is composed of the following raw materials in parts by mass: 15 parts of raw rehmannia root, 15 parts of artemisia capillaris, 10 parts of scutellaria baicalensis, 15 parts of fructus aurantii, 15 parts of loquat leaf, 15 parts of dendrobium, 15 parts of ophiopogon japonicus, 15 parts of perilla stem, 7 parts of amomum villosum, 15 parts of magnolia bark, and 15 parts of salvia miltiorrhiza.

4. A drug for treating kidney disease, characterized in that: The invention is prepared from the Chinese medicine composition according to any one of claims 1 to 3 and pharmaceutically acceptable excipients.

5. The drug for treating kidney disease according to claim 4, characterized in that: The dosage form of the medicine is liquid preparation, granules or concentrated pills.

6. The drug for treating kidney disease according to claim 5, characterized in that: The auxiliary material is one or more of dextrin, starch, edible alcohol and flavoring agent, and the flavoring agent is aspartame or sucrose.

7. The method for preparing the drug for treating kidney disease according to claim 4, characterized in that: The method comprises the following steps: taking various raw materials of the traditional Chinese medicine composition, soaking them in water, performing a decoction extraction, filtering to obtain a medicine residue and a primary filtrate; adding water to the medicine residue for a secondary decoction extraction, filtering to obtain a secondary filtrate; combining the primary filtrate and the secondary filtrate, concentrating them, and adding auxiliary materials as needed to prepare the medicine.

8. The method for preparing a drug for treating kidney disease according to claim 7, characterized in that: During the first decoction extraction and the second decoction extraction, 5-10 times of water in weight of the total weight of the Chinese medicine composition is added, the decoction extraction time for each time is 60-90 minutes, and the water soaking time is 60-120 minutes.

9. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of medicines for treating kidney diseases.

10. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicament for treating kidney diseases caused by damp-heat damaging yin.

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