WO2022171096A1 - Use of traditional chinese medicine composition in preparation of drugs for preventing or treating diabetes and/or diabetic complications - Google Patents

Abstract

The use of a Jingfang preparation in the preparation of a drug or health care product for preventing or treating diabetes and/or diabetic complications. The Jingfang preparation is prepared from notopterygium rhizoma et radix, angelicae pubescentis radix, poria, saposhnikoviae radix, schizonepetae herba, chuanxiong rhizoma, platycodonis radix, bupleuri radix, peucedani radix, aurantii fructus and glycyrrhizae radix et rhizoma. The preparation not only has the effect of reducing blood sugar in a diabetic model rat and increasing the sugar tolerance of the diabetic rat, but can also prevent and improve various diabetic complications, such as diabetic cardiomyopathy and diabetic fundus diseases.

Description

Use of a traditional Chinese medicine composition in the preparation of a medicine for preventing or treating diabetes and/or diabetes complications technical field

The invention belongs to the technical field of medicine, in particular to the use of a traditional Chinese medicine composition in the preparation of medicines or health products for preventing or treating diabetes and/or diabetes complications.

Background technique

The prescription of Jingfang Granules comes from the Jingfangbaidu Powder contained in Zhang Shiche's "Recipes for Recovering Lives" in the Ming Dynasty. A total of 11 traditional Chinese medicine composition.

Jing Fangbaidu Powder is mildly warm and diffuses, which is good for dispersing wind and relieves the surface; Fangfeng Xinsan is sweet and mildly warm, it is a general medicine for treating wind, good for dispelling wind and dampness, and releasing pain relief. The two must have stronger medicinal power, and are used for treating exogenous wind-cold or damp pathogens, so they are the king medicine. Qianghuo is pungent, warm, bitter and dry, and it mainly enters the bladder meridian, which is good for eliminating wind-cold-damp pathogens above and on the surface; Duhuo-xin disperses bitterness and dryness, mainly enters the kidney meridian, and mainly disperses wind-cold-dampness pathogens in the inner and lower layers. Cold, and dispels rheumatism from the upper and lower body, and relieves arthralgia by clearing the joints. Chuanxiong Xin Wen Xing San is good for promoting blood circulation and promoting qi, dispelling wind and relieving pain. The combination of the three medicines can help the monarch medicine to disperse wind-cold, dispel wind-dampness, and relieve arthralgia, so it is the minister medicine. Chaihu Kuxiexin disperses mild cold, good at relieving the surface and relieving fever; Qianhuxin Sans bitterness and relieves cold, good at dispelling wind and dispelling lung; Campanulaceae Xinxin dispersing bitterness and calming, good at invigorating lung-qi, expelling phlegm and relieving cough; Poria cocos sweet It is good for invigorating the spleen and removing dampness; Citrus aurantium is good for dispelling bitterness and dispelling mild cold, good for regulating Qi and stagnation, and when Qi is smooth, dampness is dispersed. The combination of the five herbs can help the monarch and the ministers to relieve external cold, expel wind and overcome dampness, and relieve pain, so it is an adjuvant. Glycyrrhiza ganping, to reconcile various medicines, to make medicine. The combination of various medicines is pungent, warm and dry, and has the effect of dispelling external cold, dispelling wind and conquering dampness, so it is good for colds caused by exogenous wind-cold and dampness.

Ancient medical books record that Jingfangbaidu Powder is used to treat those who experience wind-heat, wind-cold, and rheumatism in the respiratory system, digestive system, and skin diseases. The Xin San and Peaceful Prescriptions. Modern physicians also use Jingfangbaidu Powder to treat a variety of infectious diseases, such as infantile influenza, acute viral upper respiratory tract infection, H1N1 influenza, mumps, chickenpox, and dengue fever. Jingfangbaidu Powder can not only treat various colds and prevent plagues, but also has a very significant effect on toothaches, breast nodules, skin diseases, headaches and body pains. At the same time, in 2021, Jingfang Granules will also be designated by many provinces and cities as the recommended drug for the prevention and treatment of new coronary pneumonia.

Diabetes mellitus is a group of clinical syndromes mainly characterized by chronic hyperglycemia caused by a combination of genetic and environmental factors. In the early stage of diabetes, patients may have no symptoms except high blood sugar. If not treated in time, persistent high blood sugar will gradually erode the blood vessels and nerves of the patient, causing lesions of various tissues and organs in the body, resulting in various serious diseases. acute and chronic complications.

Diabetic cardiomyopathy refers to a specific myocardial disease that occurs in diabetic patients but cannot be explained by hypertensive heart disease, coronary atherosclerotic cardiomyopathy, valvular heart disease and other cardiac diseases. Long-term hyperglycemia causes extensive focal necrosis of the myocardium caused by cardiac microvascular and myocardial metabolic disorders, which can eventually progress to heart failure, arrhythmia, and cardiogenic shock. Diabetic cardiomyopathy is one of the main complications of diabetic patients. The disease is mainly manifested by decreased cardiac function and myocardial hypertrophy. Long-term development can also lead to myocardial fibrosis and heart failure. The pathogenesis of diabetic cardiomyopathy includes myocardial cell metabolism disorder, myocardial cell calcium transport defect, myocardial interstitial fibrosis, coronary microvascular disease and cardiac autonomic neuropathy.

Diabetic retinopathy is divided into diabetic retinopathy and diabetic macular degeneration, of which diabetic retinopathy is the main manifestation. Diabetic retinopathy is the microvascular lesions of the retina caused by hyperglycemia in diabetic patients, the loss of capillary pericytes, the subsequent thinning of endothelial cells, and the damage of barrier function. disfunction. The early pathological changes of diabetic retinopathy mainly include the reduction or disappearance of retinal capillary pericytes, the formation of shadow pericytes and the appearance of acellular capillaries, endothelial cell proliferation, basement membrane thickening and blood-retinal barrier damage.

At present, the clinical treatment of diabetes and its complications mainly adopts symptomatic treatment and combined drugs, such as hypoglycemic, hypotensive, lipid-lowering, AR/AGEs inhibitors and renin-angiotensin-aldosterone system (RAAS) inhibitors and other drugs. Although the above-mentioned therapeutic drugs can alleviate the symptoms of diabetic complications to a certain extent, they cannot effectively prevent the further deterioration of the disease, and are prone to side effects such as electrolyte imbalance and hypercreatinine, which are not conducive to long-term clinical use. How to develop a drug that can effectively treat diabetes, delay and control the occurrence and development of diabetic complications, and has good efficacy and high safety is an urgent problem to be solved in the current medical field.

SUMMARY OF THE INVENTION

The invention provides the use of a traditional Chinese medicine composition in the preparation of medicines or health products for preventing or treating diabetes and/or diabetes complications.

The traditional Chinese medicine composition, namely the Jingfang composition, is mainly prepared from Qianghuo, Duhuo, Poria, Fangfeng, Nepeta, Chuanxiong, Platycodon grandiflorum, Bupleurum, Qianhu, Citrus aurantium, and licorice, and its composition ratio is commercially available. The prescription ratio of Jingfang granules or Jingfang mixture fluctuates within a range of 20%; further, it fluctuates within a range of 10%; in some products, it fluctuates within a range of 5%. If the weight ratio of Qianghuo and Duhuo is 1:1, and the upper and lower fluctuations are within the range of 20%, the actual weight ratio range can be (0.8-1.2):(0.8-1.2).

Specifically, the traditional Chinese medicine composition is mainly prepared from the following components:

Preferably, the traditional Chinese medicine composition is mainly prepared from the following components:

Further preferably, the traditional Chinese medicine composition is mainly prepared from the following components:

Further preferably, the traditional Chinese medicine composition is mainly prepared from the following components:

The present invention provides a preparation, namely Jingfang preparation, which is prepared from the above-mentioned traditional Chinese medicine composition by adopting the prior art. In some instances, the formulation also contains at least one pharmaceutically acceptable excipient. In some applications, the formulation contains no excipients other than water.

In some embodiments, the antiviral preparations of the present invention are granules, mixtures, pills, drop pills, syrups, powders, granules, tablets or capsules.

In an embodiment of the present invention, the formulation is Jingfang Granule, Jingfang Mixture, Jingfang Pill, Jingfang Dropping Pill, Jingfang Syrup, Jingfang Powder, Jingfang Granule, Jingfang Tablet or Jingfang Capsule.

In one embodiment of the present invention, the antiviral preparation can be used in combination with other drugs to treat or prevent diabetes and/or diabetic complications.

In one embodiment of the present invention, the antifungal preparation of the present invention is antifungal granules.

In one embodiment of the present invention, the Jingfang preparation of the present invention is Jingfang mixture.

In one embodiment of the present invention, the prescription and preparation method of the Jingfang granules of the present invention can refer to the Ministry of Health standard WS3-B-0328-90.

In one embodiment of the present invention, the prescription and preparation method of the Jingfang mixture of the present invention can refer to the Ministry of Health standard WS3-B-1377-93.

In one embodiment of the present invention, the Jingfang preparation of the present invention reduces fasting blood glucose.

In one embodiment of the present invention, the vitiligo preparation of the present invention increases glucose tolerance.

In one embodiment of the present invention, the diabetes of the present invention is selected from type 1 or type 2 diabetes.

In one embodiment of the present invention, the diabetic complications of the present invention are selected from diabetes complications caused by type 1 or type 2 diabetes.

In one embodiment of the present invention, the diabetic complication of the present invention is selected from diabetic cardiomyopathy or diabetic retinopathy.

In one embodiment of the present invention, the diabetic fundus disease of the present invention is diabetic retinopathy.

In one embodiment of the present invention, the diabetic retinopathy of the present invention is non-proliferative diabetic retinopathy, proliferative diabetic retinopathy.

The present invention also provides a preparation method of the above-mentioned Jing Fang preparation, and the preparation method mainly comprises the following steps:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: prepare a 10-40% ethanol solution with the Ligusticum chuanxiong and Citrus aurantium aqueous solution after the distillation obtained in step A, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum chinensis, Platycodon grandiflorum, Licorice Radix, step A gained after the distillation of Nepeta, Fangfeng, Qianghuo, Duhuo, the medicinal dregs of the fore hair, add water to decoct, and the decoction liquid is concentrated for subsequent use;

Step E: Mix the percolating liquid obtained in step C and the decoction liquid obtained in step D, concentrate, and add the volatile oil obtained in step A, and then obtain.

Preferably, the preparation method mainly comprises the following steps:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: prepare the 15-30% ethanol solution of the Ligusticum chuanxiong and Citrus aurantium aqueous solution after the distillation obtained in step A, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, Glycyrrhizae Radix, step A gained after the distillation of Nepeta, Fangfeng, Qianghuo, Duhuo, Huohu medicinal residues, add water to decoct, the decoction liquid is concentrated into thick paste, for subsequent use;

Step E: mix the percolating liquid obtained in step C and the thick paste obtained in step D, concentrate it into a clear paste, add the volatile oil obtained in step A, and then get it.

Further preferably, the preparation method mainly comprises the following steps:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract the volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: the aqueous solution of Chuanxiong and Citrus aurantium after the distillation obtained in step A is prepared into a 25% ethanol solution, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, Glycyrrhizae Radix, step A gained after the distillation of Nepeta, Fangfeng, Qianghuo, Duhuo, Huohu medicinal residues, add water to decoct, the decoction liquid is concentrated into thick paste, for subsequent use;

Step E: mix the percolating liquid obtained in step C and the thick paste obtained in step D, concentrate it into a clear paste, add the volatile oil obtained in step A, and then get it.

Compared with the prior art, the present invention has achieved remarkable technical effects:

(1) The present invention provides a new application of Jingfang preparation for lowering blood sugar, and it has been proved through experiments that it has a lowering effect on the blood sugar of the alloxan hyperglycemia rat model, and can increase the blood sugar of the alloxan hyperglycemia rat model. Glucose tolerance. Jingfang preparations are easy to take orally, have high safety, and have few side effects. , which can be used to prepare medicines for treating or preventing diabetes or medicines for lowering blood sugar or health care products.

(2) The Jingfang preparation of the present invention can reduce the levels of serum creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in diabetic cardiomyopathy model rats, and improve the damage status of diabetic cardiomyocytes; index, improving myocardial hypertrophy and left ventricular remodeling. The results of the pharmacodynamics experiment suggest that the Jingfang preparation of the present invention has exact curative effect on diabetic cardiomyopathy.

(3) The Jingfang preparation of the present invention can reduce the level of serum AGEs in diabetic retinopathy model rats, reduce the ratio of retinal endothelial cells to pericytes (E/P), improve the retinal ultrastructure of the fundus of diabetic rats, and relieve diabetic rats. The diameter of retinal capillaries is uneven, and the pericytes and endothelial cells are irregularly arranged, which significantly reduces the number of capillaries without endothelial cells. The experimental results show that the Jingfang preparation of the present invention can inhibit the loss of pericytes and the proliferation of endothelial cells, maintain the balance of pericytes and endothelial cells, maintain the blood-retinal barrier, improve retinal microvascular damage to a certain extent, and can be used for the treatment of diabetic retinopathy. Jingfang preparation can reduce the retinal normalized EB value, suggesting that Jingfang preparation can alleviate the increase of vascular permeability in patients with diabetic retinopathy, and has a protective effect on the blood-retinal barrier function.

Description of drawings

Accompanying drawing 1 is the PAS staining picture of the retinal digestion of each group of rats in Example 3, wherein A represents the normal control group, B represents the model group, C represents the positive control group, D represents the Jingfang low-dose group, E Indicates Jingfang high-dose group.

specific embodiment

The present invention is further clarified below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Modifications all fall within the scope defined by the appended claims of this application.

Example 1 Preparation of granules

prescription:

Preparation:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: the aqueous solution of Chuanxiong and Citrus aurantium after the distillation obtained in step A is prepared into a 25% ethanol solution, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, licorice, Nepeta, Fangfeng, Qianghuo, Duhuo, and the medicinal residues after the distillation of Step A, add water and decoct twice, each time for 1.5 hours, and combine the two decoction liquids, After filtration, concentrate into a thick paste for later use;

Step E: mix the percolate obtained in step C and the thick paste obtained in step D, stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in step A, and mix well, That's it.

Example 2 Preparation of granules

prescription:

The preparation method is the same as in Example 1.

Example 3 Tablet preparation

prescription:

Preparation:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: the aqueous solution of Chuanxiong and Citrus aurantium after the distillation obtained in step A is prepared into a 15% ethanol solution, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, licorice, Nepeta, Fangfeng, Qianghuo, Duhuo, and the medicinal residues after the distillation of Step A, add water and decoct twice, each time for 1.5 hours, and combine the two decoction liquids, After filtration, concentrate into a thick paste for later use;

Step E: mix the percolate obtained in step C and the thick paste obtained in step D, stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in step A, and mix well, Make into granules, add appropriate amount of auxiliary materials, mix well, and press into tablets.

Example 4 Oral liquid preparation

prescription:

Preparation:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: prepare a 10% ethanol solution with the Ligusticum chuanxiong and Citrus aurantium aqueous solution after the distillation obtained in step A, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, licorice, Nepeta, Fangfeng, Qianghuo, Duhuo, and the medicinal residues after the distillation of Step A, add water and decoct twice, each time for 1.5 hours, and combine the two decoction liquids, After filtration, concentrate into a thick paste for later use;

Step E: Mix the percolating liquid obtained in Step C and the thick paste obtained in Step D, let stand, filter and condense into a clear paste, add an appropriate amount of sucrose, mix well, add the volatile oil obtained in Step A, mix well, add water to 1000ml, that is, .

Example 5 Preparation of syrup

prescription:

Preparation:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: the aqueous solution of Chuanxiong and Citrus aurantium after the distillation obtained in step A is prepared into a 40% ethanol solution, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, licorice, Nepeta, Fangfeng, Qianghuo, Duhuo, and the medicinal residues after the distillation of Step A, add water and decoct twice, each time for 1.5 hours, and combine the two decoction liquids, After filtration, concentrate into a thick paste for later use;

Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into clear paste, add an appropriate amount of sucrose, mix well, add 500ml of volatile oil and simple syrup obtained in Step A, mix well, and let stand , filter, add water to 1000ml, that is.

Example 6 Preparation of capsules

prescription:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: the aqueous solution of Chuanxiong and Citrus aurantium after the distillation obtained in step A is prepared into a 30% ethanol solution, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, licorice, Nepeta, Fangfeng, Qianghuo, Duhuo, and the medicinal residues after the distillation of Step A, add water and decoct twice, each time for 1.5 hours, and combine the two decoction liquids, After filtration, concentrate into a thick paste for later use;

Step E: mix the percolate obtained in step C and the thick paste obtained in step D, stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in step A, and mix well, It is made into granules, dried, pulverized, and packed into capsules.

Example 7 Pill preparation

prescription:

Preparation:

Step A: Nepeta, Fangfeng, Qianghuo, Duhuo, Qianhu, Chuanxiong, and Citrus aurantium are respectively distilled to extract volatile oil for subsequent use, and the distilled medicinal residues, the distilled Chuanxiong, and the aqueous solution of Citrus aurantium are for subsequent use;

Step B: the aqueous solution of Chuanxiong and Citrus aurantium after the distillation obtained in step A is prepared into a 25% ethanol solution, for subsequent use;

Step C: mixing Poria cocos, Ligusticum chuanxiong and Citrus aurantium after the distillation obtained in step A, percolating and extracting with the ethanol solution obtained in step B, and the percolating liquid is for subsequent use;

Step D: with Bupleurum, Platycodon grandiflorum, licorice, Nepeta, Fangfeng, Qianghuo, Duhuo, and the medicinal residues after the distillation of Step A, add water and decoct twice, each time for 1.5 hours, and combine the two decoction liquids, After filtration, concentrate into a thick paste for later use;

Step E: mix the percolate obtained in step C and the thick paste obtained in step D, stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in step A, and mix well, Dry, pulverize, sieve, add 40-60 g of refined honey, make a suitable amount of water to make pellets, and dry to get it.

2. Pharmacological experiments

In order to verify the efficacy of the Jingfang preparation of the present invention in preventing or treating diabetes and/or diabetic complications, the inventors have carried out relevant pharmacodynamic test studies. It should be noted that the medicines selected in the following pharmacodynamic tests are the medicines obtained from the representative formulations of the present invention and the preparation methods thereof, and the medicines obtained from other formulations and preparation methods contained in the present invention are also obtained by the inventors. The experimental results show that the medicines obtained by other formulas and preparation methods have the same or similar effects, but due to space limitations, they are not exhaustive here.

The inventor wants to explain that the following experimental studies are carried out on the basis of the acute toxicity test and long-term toxicity test to prove the safety of the drug, and the doses in the experimental studies are all within the safe dose range.

Efficacy Example 1 Hypoglycemic effect of Jingfang preparation on diabetic model rats

1. Materials

Animal: SD rats, SPF grade; 100 rats, half male and half male; body weight 190-220 g; source: Jinan Pengyue Laboratory Animal Breeding Co., Ltd., production license number SCXK (Lu) 20190003.

Reagent: Alloxan, Sigma Company, USA, batch number: A7413-25G.

Jingfang Granules (with sugar), 15g per bag, Shandong New Times Pharmaceutical Co., Ltd.;

Jingfang Granules (sugar-free type)), 15g per bag, prepared with reference to the Ministry of Health standard WS3-B-0328-90, using dextrin instead of sucrose;

Jingfang Mixture, 10mL each, Lunan Houpu Pharmaceutical Co., Ltd.

2. Dosage

The recommended oral dose of Jingfang Granules (sugar-containing or sugar-free) is 15g, calculated on the basis of 60Kg body weight, and the equivalent dose is 0.25g/kg BW. There are two dose groups, low and high, which are 2.5g/kg BW and 5g/kg, respectively. Kg BW (equivalent to 10 times and 20 times the recommended human dose). The recommended oral dose of Jingfang Mixture is 10mL, calculated based on 60Kg body weight, the equivalent dose is 0.17mL/Kg BW, and two dose groups, low and high, are set, which are 1.7mL/Kg BW and 3.4mL/Kg BW respectively.

3. Method

3.1 Establishment of experimental alloxan hyperglycemia rat model

Take 100 rats, fasted for 24 hours (can not help water), each rat was intraperitoneally injected with alloxan (except the blank control group), the dose was 120mg/kg BW, the second administration after 24 hours, intraperitoneal injection Alloxan was administered at a dose of 100 mg/kg BW, and 25% glucose 1 mL/100 g BW was administered by gavage 2.5 hours and 5 hours after alloxan administration, respectively. Seventy-two hours after the last administration, the fasting blood glucose was tested, and those higher than 11.1 mmol/L were regarded as diabetic rats. The blank control group was given the same amount of distilled water.

3.2 Effects of Jingfang preparations on fasting blood sugar in rat model of alloxan hyperglycemia

The hyperglycemic rats were randomly divided into 7 groups, 10 rats in each group, and 10 normal rats were selected as blank control group; the administration methods and doses of each group were as follows:

Blank control group: 2 mL of distilled water was given by gavage;

Model group: 2 mL of distilled water was given by gavage;

Jingfang Granules (sugar-containing type)-high dose group: 5g/kg BW by gavage;

Jingfang Granules (sugar-containing type)-low dose group: 2.5g/kg BW by gavage;

Jingfang Granules (sugar-free)-high dose group: 5g/kg BW by gavage;

Jingfang Granules (sugar-free)-low dose group: 2.5g/kg BW by gavage;

Jingfang mixture-high dose group: 3.4mL/kg BW was given by gavage;

Jingfang mixture-low dose group: 1.7mL/kg BW was given by gavage.

The required dose of Jingfang granules was dissolved in 2 mL of distilled water, and the Jingfang mixture was diluted to 2 mL with distilled water, and administered by intragastric administration for 30 days, once a day, 2 mL each time. Blood was collected from the tail vein of each rat 6 hours after the last administration, and the fasting blood glucose was measured.

表示，比较组间差异，P<0.05表示差异有统计学意义，P<0.01，表示有显著差异。试验结果如表1所示。 Statistical method: Excel statistical software, measurement data using mean ± standard deviation

P<0.05 means that the difference is statistically significant, and P<0.01 means that there is a significant difference. The test results are shown in Table 1.

Table 1 Effects of Jingfang preparations on blood sugar in rats

Note: ▲P<0.05, ▲▲P<0.01 compared with the model group; *P<0.05, **P<0.01 compared with before administration; □P<0.05, □□P<0.01 compared with the blank control group.

It can be seen from Table 1 that compared with the blank control group, the fasting blood glucose value of the rats after modeling was significantly increased, and the difference was significant (□P<0.05, □□P<0.01), indicating that the hyperglycemic rat model was established. Compared with the model group, after continuous administration of Jingfang preparations for 30 days, the fasting blood glucose of rats in each dose group was lower than that of the model control group, and the difference was significant (▲P<0.05, ▲▲P<0.01), indicating that Jingfang preparations were effective for four. The oxopyrimidine hyperglycemia rat model has the effect of reducing fasting blood sugar. Compared with before administration, after administration, the blood glucose of rats in each dose group decreased, and the difference was significant (*P<0.05, **P<0.01).

3.3 Effect of Jingfang preparation on glucose tolerance in rat model of alloxan hyperemia

Hyperglycemia model rats were given continuous administration for 30 days (the model group was given the same volume of distilled water), and fasted for 6 hours after the last administration. After 2 hours, blood was collected from the tail vein to measure the blood glucose level. The effects of the test preparations on the glucose tolerance of the alloxan hyperglycemia rat model were observed. The test results are shown in Table 2.

Table 2 Effects of Jingfang preparations on oral glucose tolerance in rats

Note: Compared with the model group ▲P<0.05, ▲▲P<0.01.

It can be seen from Table 2 that, after continuous administration of different doses of Jingfang preparations for 30 days, the blood glucose values of rats in each dose group at 0h, 0.5h, and 2h after glucose administration were all lower than those in the model control group, and the difference was significant (▲P<0.05, ▲ ▲P<0.01), indicating that Jingfang preparation has the effect of increasing glucose tolerance in hyperglycemic rat model.

Efficacy Example 2 The effect of traditional Chinese medicine composition on diabetic cardiomyopathy model rats

1 material

1.1 Animals:

Male SD rats, SPF grade, body weight 150-180g, the animals were adaptively reared in the animal room for one week, during which they had free access to water and food, adequate light and ventilation, 12 hours of light, 12 hours of darkness, and the laboratory temperature was 20-26 ℃, relative humidity 46%±6%.

1.2 Drugs and Reagents

Jingfang extract, provided by Shandong New Times Pharmaceutical Co., Ltd., batch number: 8022009004;

Volatile oil, provided by Shandong New Times Pharmaceutical Co., Ltd., batch number: 8032009004;

Streptozotocin, purchased from BioFrox Company, batch number: V900890-1G.

2 Experimental process

2.1 Animal grouping, modeling and administration

The rats were adaptively fed for 1 week, and 10 normal rats were randomly selected as the normal control group. During the experiment, they were fed with normal rat chow. %, basal feed 81%), fed continuously for 8 weeks. Except for the normal control group, the rats in the other groups were injected with streptozotocin (STZ) 60 mg/kg at one time, and blood glucose was measured by the tail vein of the rats after 1 week. Model is successful. The 40 rats with successful modeling were selected to continue to be fed with high-sugar and high-fat diet for 4 weeks. Color Doppler ultrasonography indicated that the diabetic cardiomyopathy model was successfully established when cardiac dysfunction was detected. After 4 weeks, 40 rats were randomly divided into 4 groups: model group, Jingfang low-dose, middle-dose and high-dose groups, 10 rats in each group. Jingfang low-dose, middle-dose and high-dose groups were given Jingfang extract (containing volatile oil) 2g/kg, 4g/kg, 6g/kg by gavage every day, dissolved in purified water, and the model group and normal control group were given the same volume by gavage every day Purified water, administered continuously for 8 weeks. Jingfang extract (containing volatile oil) is prepared by mixing the clearing paste and volatile oil in the preparation process of Example 1 according to clearing paste: volatile oil = 1 kg: 1.8 mL.

2.2 Observation indicators

2.2.1 Determination of serum creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in rats

After 8 weeks of administration, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and blood was collected from the abdominal aorta. After the serum was collected, the serum creatine kinase isoenzyme (CK-MB) of the rats in each group was measured by flow cytometry. , Lactate dehydrogenase (LDH) content.

2.2.2 Determination of rat heart mass index HW/BW and left ventricular mass index LVW/BW

After blood collection, the rat heart tissue was quickly removed on ice, the excess tissue was freed, washed with pre-cooled PBS (phosphate buffered saline), blotted dry with filter paper, and the rat heart mass (HW) was weighed respectively. The left ventricle was isolated and left ventricular mass (LVW) was weighed, and the cardiac mass index HW/BW (heart mass/body mass) and left ventricular mass index LVW/BW (left ventricular mass/body mass) were calculated.

2.3 Statistical processing

的形式表示，组间比较采用单因素方差分析，两组间比较采用t检验。以P<0.05表示差异具有统计学意义。3.结果及结论 SPSS 22.0 software was used for statistical processing, and the experimental data were expressed as "mean ± standard deviation"

One-way ANOVA was used for comparison between groups, and t-test was used for comparison between two groups. The difference was statistically significant at P<0.05. 3. Results and Conclusions

3.1 The effect of traditional Chinese medicine composition on serum creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in streptozotocin-induced diabetic cardiomyopathy model rats

n＝10) Table 3 Effects of traditional Chinese medicine compositions on serum CK-MB and LDH levels in streptozotocin-induced diabetic cardiomyopathy model rats (

n=10)

Note: *P<0.05, **P<0.01 compared with the normal control group; ^# P<0.05, ^## P<0.01 compared with the model group.

As can be seen from Table 3, compared with the normal control group, the serum levels of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in the model group were significantly increased (P<0.01), indicating that the model group Myocardial injury occurred in rats; compared with the model group, the serum levels of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in Jingfang middle and high dose groups were significantly decreased (P<0.05). , P<0.01), indicating that the state of myocardial injury in diabetic cardiomyopathy model rats was significantly improved after the intervention of medium and high doses of traditional Chinese medicine compositions.

3.2 The effect of traditional Chinese medicine composition on the cardiac index of streptozotocin-induced diabetic cardiomyopathy model rats

n＝10) Table 4 The effect of traditional Chinese medicine composition on the cardiac index of streptozotocin-induced diabetic cardiomyopathy model rats (

n=10)

Note: *P<0.05, **P<0.01 compared with the normal control group; ^# P<0.05, ^## P<0.01 compared with the model group.

As can be seen from Table 4, compared with the normal control group, the cardiac mass index and left ventricular mass index of the rats in the model group were significantly increased (P<0.01), indicating that the rats in the model group presented significant myocardial hypertrophy and left ventricular remodeling; Compared with the model group, the visceral mass index and left ventricular mass index of Jingfang middle and high dose groups were significantly decreased (P<0.05, P<0.01), indicating that the traditional Chinese medicine composition of the present invention can improve the changes of cardiac structure in model rats.

Efficacy Example 3 The effect of traditional Chinese medicine composition on alloxan-induced diabetic fundus lesions in model rats

1 material

1.1 Animals:

SD rats, male, SPF grade, body weight 150-180g, the animals were adaptively raised in the animal room for one week, during which they had free access to water and food, adequate light and ventilation, 12 hours of light, 12 hours of darkness, and the laboratory temperature was 20-26 ℃, relative humidity 46%±6%.

1.2 Drugs and Reagents

Jingfang extract, provided by Shandong New Times Pharmaceutical Co., Ltd., batch number: 8022009004;

Volatile oil, provided by Shandong New Times Pharmaceutical Co., Ltd., batch number: 8032009004;

Alloxan, purchased from American Sigma Company, batch number: A7413-25G;

Metformin hydrochloride enteric-coated tablets, purchased from Guizhou Shengjitang Pharmaceutical Co., Ltd., batch number: 20210101;

2 Experimental process

2.1 Animal grouping, modeling and administration

The rats were reared adaptively for one week, during which they had free access to water and food, sufficient light and ventilation, 12 hours of light and 12 hours of darkness, the laboratory temperature was 20-26°C, and the relative humidity was 46%±6%.

After 7 days of adapting to the environment, all rats were given a comprehensive body weight score and were randomly divided into a normal group and a model group. The alloxan-induced diabetes model was used to establish the model. After that, each rat was intraperitoneally injected with alloxan solution at a dose of 200 mg/kg at one time, and the random blood sugar (without fasting) was detected 3 days after the injection. The concentration of more than 16.7 mmol/L was a qualified diabetic model rat, and continued to feed for 12 days. A diabetic retinopathy model was formed after weeks.

The diabetic rats were randomly divided into 4 gavage groups: model group, positive control group, Jingfang low-dose group, Jingfang high-dose group, 6 rats in each group, and 6 normal rats were selected as the normal control group, and the rats were fed daily. Once in the stomach, for 90 days, the body weight of the rats was measured every week to adjust the dosage. The administration and dosage of each group are as follows:

Normal control group: equal volume of purified water;

Model group: equal volume of purified water;

Positive control group: Metformin hydrochloride enteric-coated tablets, 12 mg/kg, dissolved in purified water;

Jingfang low-dose group: Jingfang extract (containing volatile oil), 2g/kg, dissolved in purified water;

Jingfang high-dose group: Jingfang extract (containing volatile oil), 6g/kg, dissolved in purified water;

Jingfang extract (containing volatile oil) is prepared by mixing the clearing paste and volatile oil in the preparation process of Example 1 according to clearing paste: volatile oil = 1 kg: 1.8 mL.

2.2 Observation indicators

2.2.1 Determination of retinal vascular morphology and endothelial cell/pericyte (E/P) ratio in rats

After the test, the left eyeballs of 6 rats in each group were immediately removed, immersed in 4% formaldehyde solution, and fixed at 4°C for 12 hours. After the fixation, the cells were rinsed three times with normal saline and placed in a petri dish, and retinal detachment was performed under a stereomicroscope. After retinal detachment, the retina was rinsed with normal saline, then placed in 1% Triton-100, incubated in a shaker at 37°C 40rpm for 1.5h, washed three times with PBS, and digested with 8% trypsin (prepared with 0.1M Tris HCl) at 37°C and 40rpm for 2h. Gently pipette with a plastic-tip dropper until the retina becomes transparent. After digestion, it was transferred into water, spread on glass slides, and dried in an oven at 60°C. After PAS staining, dehydration and mounting, the morphological changes of retinal blood vessels can be observed under an electron microscope. The results are shown in Figure 1. Observe the results of PAS staining of the rat retinal digested slides under a 400x microscope. Each specimen selects 3 areas of the same size to record capillary cells (including endothelial cells E and pericytes P) and calculate endothelial cells/pericytes (E /P) ratio, the results are shown in Table 5.

2.2.2 Determination of advanced glycation end products (AGEs) in rat retinal homogenate

6 rats in each group were taken to peel off the retinal tissue of the right eyeball for homogenization, rinse the retinal tissue with pre-cooled PBS to remove residual blood; Repeated grinding on ice to fully lyse the tissue cells; finally, the homogenate was centrifuged at 5000 g for 5-10 min, and the supernatant was taken to measure the level of AGEs in the retinal homogenate by ELISA.

2.3 Statistical processing

的形式表示，组间比较采用单因素方差分析，两组间比较采用t检验。以P<0.05表示差异具有统计学意义。 SPSS 22.0 software was used for statistical processing, and the experimental data were expressed as "mean ± standard deviation"

One-way ANOVA was used for comparison between groups, and t-test was used for comparison between two groups. The difference was statistically significant at P<0.05.

3. Results and Conclusions

3.1 The effect of traditional Chinese medicine composition on retinal blood vessel morphology and E/P ratio in rats

From the retinal slices, it can be found that the retinal blood vessels of the model group have uneven diameters, tortuous and dilated veins, irregular arrangement of pericytes and endothelial cells in the blood vessels, abnormal increase of shadow pericytes, and multiple endothelial cells without endothelial cells. capillaries. Compared with the model group, the retinal capillaries in the retina of the rats in the low-, high-dose and positive control groups of Jingfang were more uniform in diameter and thickness, perivascular cells and endothelial cells were arranged regularly, and capillaries without endothelial cells were significantly reduced. Compared with the positive control group, the high-dose Jingfang group had fewer capillaries without endothelial cells, less tortuous and dilated veins, and better effect. It can be seen that Jingfang preparation can improve the ultrastructure of retinal retina in diabetic rats, alleviate the uneven diameter of retinal capillaries, irregular arrangement of pericytes and endothelial cells in diabetic rats, and significantly reduce capillaries without endothelial cells. number of blood vessels.

n＝6) Table 5 Influence of traditional Chinese medicine composition on retinal E/P ratio of diabetic retinopathy model rats (

n=6)

Note: *P<0.05, **P<0.01 compared with the normal control group; ^# P<0.05, ^## P<0.01 compared with the model group.

It can be seen from Table 5 that compared with the normal control group, the retinal E/P ratio of the rats in the model group was significantly increased (P<0.01), indicating that the rats with diabetic retinopathy had a loss of pericytes and an increase in endothelial cells. Compared with the model group, the retinal E/P ratio of Jingfang low- and high-dose groups and the positive control group was significantly decreased (P<0.05, P<0.01). It shows that the traditional Chinese medicine composition of the present invention can inhibit the loss of pericytes and the proliferation of endothelial cells, maintain the balance of pericytes and endothelial cells, maintain the blood-retinal barrier, improve retinal microvascular damage to a certain extent, and can be used for the treatment of diabetic retinopathy.

3.2 The effect of traditional Chinese medicine composition on advanced glycation end products (AGEs) in retinal homogenate

n＝6) Table 6 The effect of traditional Chinese medicine composition on the level of AGEs in retinal homogenate (

n=6)

Note: *P<0.05, **P<0.01 compared with the normal control group; ^# P<0.05, ^## P<0.01 compared with the model group.

It can be seen from Table 6 that the level of retinal AGEs in diabetic retinopathy rats was significantly higher than that in the normal group (P<0.01). The low and high doses of Jingfang can reduce the level of AGEs in the retina of rats, and there was a significant difference compared with the model group (P<0.05, P<0.01). The experimental results show that the traditional Chinese medicine composition of the present invention can inhibit the level of AGEs, thereby improving the permeability of retinal microvessels.

Efficacy Example 4 The effect of traditional Chinese medicine composition on streptozotocin-induced diabetic fundus lesion model rats

1. Test material

1.1 Experimental animals:

SD rats, SPF grade, weighing 150-180 g, male. Animals were reared adaptively in the animal room for one week, during which they had free access to water and food, sufficient light and ventilation, 12 hours of light and 12 hours of darkness, the laboratory temperature was 20-26°C, and the relative humidity was 46%±6%.

1.2 Reagents:

Jingfang extract, provided by Shandong New Times Pharmaceutical Co., Ltd., batch number: 8022009004;

Volatile oil, provided by Shandong New Times Pharmaceutical Co., Ltd., batch number: 8032009004;

Metformin hydrochloride enteric-coated tablets, purchased from Guizhou Shengjitang Pharmaceutical Co., Ltd., batch number: 20210101;

Streptozotocin, purchased from Merck, lot number: V900890-1G;

Evans Blue (EB), purchased from sigma.

2. Test method and content

2.1 Modeling

30 SD rats were adaptively reared for one week, and 6 rats were selected as the normal group. After fasting for 8 h, the rest rats were injected with STZ solution at a dose of 70 mg/kg to prepare the diabetes model. The streptozotocin solution was prepared with 0.1 mmol/L, pH 4.5 citric acid-sodium citrate buffer solution immediately before use. The normal group was injected with an equal dose of citric acid-sodium citrate buffer. 72h after injection, the random blood glucose in the tail vein of the rats was detected, and the blood glucose value was ≥16.7 mmol/L for 3 consecutive days, which was the standard for successful modeling.

2.2 Grouping and Administration

The STZ-induced diabetic rats were randomly divided into 4 groups: model group, positive control group, Jingfang low-dose and high-dose groups, with 6 rats in each group. The rats in each group were intragastrically administered once a day for 90 consecutive days, and the body weight of the rats was measured weekly to adjust the dose. The administration and dosage of each group are as follows:

Normal group: equal volume of purified water;

Model group: equal volume of purified water;

Positive control group: metformin, 12 mg/kg/d, dissolved in purified water;

Jingfang low-dose group: Jingfang extract (containing volatile oil), 2g/kg, dissolved in purified water;

Jingfang high-dose group: Jingfang extract (containing volatile oil), 6g/kg, dissolved in purified water;

Jingfang extract (containing volatile oil) is prepared by mixing the clearing paste and volatile oil in the preparation process of Example 1 according to clearing paste: volatile oil = 1 kg: 1.8 mL.

2.2 Quantitative determination of retinal vascular leakage with Evans blue tracer

After the test, after the rats were anesthetized, the EB liquid was slowly injected through the tail vein at a dose of 45 mg/kg, and it was observed that the eyes and whole body skin of the rats turned blue instantly. After 120 minutes of circulation, the chest cavity was opened to expose the heart, and hemostatic clamp Holding the inferior vena cava, the left ventricle was perfused with the ventricular perfusate preheated to 37°C, and the right atrial appendage was cut at the same time. Immediately after perfusion, the eyeballs were removed, and the retinas were carefully dissociated, dried overnight, and weighed. The retina was incubated with 150 μL of formamide at 70° C. for 18 hours, then the extract was transferred into a centrifuge tube, and centrifuged at 4° C. and 6000 rpm/min for 90 min. Take 100 μL of the supernatant to measure its absorbance (A) value with a microplate reader, measure the absorbance values at two wavelengths of 620 nm and 740 nm, and take the difference between the two values, that is, the net absorbance value as the final absorbance value of the sample. Each sample was measured 3 times with an interval of 5 seconds, and the average value was finally taken, and then the EB concentration in the sample was calculated according to the absorbance difference of the EB standard solution in formamide. The EB (ng) content was normalized by retinal dry weight (mg) and the results were expressed as: ng/mg. See Table 7.

2.3 Statistical analysis

的形式表示，组间比较采用单因素方差分析，两组间比较采用t检验。以P<0.05表示差异具有统计学意义。 SPSS 22.0 software was used for statistical processing, and the experimental data were expressed as "mean ± standard deviation"

One-way ANOVA was used for comparison between groups, and t-test was used for comparison between two groups. The difference was statistically significant at P<0.05.

3. Test results and analysis

n＝6) Table 7 The influence of traditional Chinese medicine composition on standardized EB content (

n=6)

Note: *P<0.05, ^** P<0.01 compared with the normal group; ^# P<0.05, ^## P<0.01 compared with the model group.

Evans blue (EB) is a tetrasodium diazonium dye that binds tightly to plasma albumin in a 10:1 ratio after intravenous injection. When vascular permeability increases, this dye-protein complex leaks into surrounding tissue. Therefore, EB can quantitatively measure retinal vascular leakage. Table 7 shows that compared with the normal group, the EB value of the model group was significantly increased, with a statistical difference (P<0.01). Compared with the model group, the EB values of Jingfang low-dose, high-dose groups and positive control group were significantly lower, with statistical difference (P<0.05, P<0.01). It can be seen that the traditional Chinese medicine composition of the present invention can reduce the retinal normalized EB value, suggesting that the traditional Chinese medicine composition of the present invention can alleviate the increase of vascular permeability in patients with diabetic retinopathy, has a protective effect on the blood-retinal inner barrier function, and can be used for the treatment of diabetic retinopathy. treatment, the effect is remarkable.

Claims (10)

A use of a traditional Chinese medicine composition, characterized in that the traditional Chinese medicinal composition is mainly prepared from Qianghuo, Duhuo, Poria, Fangfeng, Nepeta, Chuanxiong, Platycodon grandiflorum, Bupleurum, Qianhu, Citrus aurantium, and Licorice; the The traditional Chinese medicine composition is used for preparing medicines or health products for preventing or treating diabetes and/or diabetic complications. The purposes of the traditional Chinese medicine composition as claimed in claim 1, is characterized in that, described traditional Chinese medicine composition is mainly prepared from the following components:

The purposes of the traditional Chinese medicine composition as claimed in claim 2 is characterized in that, described traditional Chinese medicine composition is mainly prepared from the following components:

The purposes of the traditional Chinese medicine composition as claimed in claim 3, is characterized in that, described Chinese medicinal composition is mainly prepared from the following components:

The purposes of the traditional Chinese medicine composition as claimed in claim 1, is characterized in that, described Chinese medicinal composition is granule, mixture, pill, drop pill, syrup, powder or granule. The use of the traditional Chinese medicine composition according to claim 5, wherein the traditional Chinese medicinal composition is Jingfang Granules or Jingfang Mixture. The use of the traditional Chinese medicine composition according to claim 1, wherein the diabetes is selected from type 1 or type 2 diabetes, and the diabetes complication is selected from chronic diabetes complications caused by type 1 or type 2 diabetes. The use of the traditional Chinese medicine composition according to any one of claims 1 to 7, wherein the diabetic complications are selected from diabetic cardiomyopathy or diabetic fundus lesions. The use of the traditional Chinese medicine composition according to claim 8, wherein the diabetic fundus disease is diabetic retinopathy. The use of the traditional Chinese medicine composition according to claim 9, wherein the diabetic retinopathy is non-proliferative diabetic retinopathy or proliferative diabetic retinopathy.

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