Traditional Chinese medicine composition for detoxifying, dredging collaterals, eliminating paste and stabilizing sugar and preparation method of traditional Chinese medicine composition

Abstract

The invention relates to the field of traditional Chinese medicine compositions, and particularly discloses a traditional Chinese medicine composition for detoxifying, dredging collaterals, eliminating paste and stabilizing sugar and a preparation method thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 7 to 15g of rhizoma coptidis, 6 to 10g of wine-treated rhubarb, 15 to 30g of radix astragali seu hedysari, 12 to 15g of radix salviae miltiorrhizae, 12 to 20g of radix paeoniae rubra, 6 to 10g of radix bupleuri, 6 to 10 parts of herba artemisiae scopariae, 7 to 15 parts of fructus crataegi and 7 to 15 parts of pericarpium citri reticulatae. The detoxifying, collateral dredging, ointment eliminating and sugar stabilizing traditional Chinese medicine composition can achieve the effects of reducing blood sugar fluctuation and lipid deposition on the basis of achieving the effects of reducing blood sugar and improving the insulin resistance level, can effectively reduce the dosage of compound medicine for a patient so as to improve the compliance of the patient, is safe and effective, delays the occurrence of diabetes complicated diseases and complications, and has a wide application prospect. And the living quality of the patient is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine compositions, and more particularly, to a detoxification and meridian unblocking ointment and a method for preparing the same. BACKGROUND

[0002] In recent years, the prevalence of diabetes has shown a sharp upward trend year by year. The latest prediction of Lancet is that the number of patients will exceed 131 million in 2050. Diabetes remains a major global public health problem, seriously threatening human health, and is one of the main causes of disability and death. Among the many types of diabetes, type 2 diabetes (T2DM) occupies a dominant position. T2DM not only has a high incidence, but also has a complex disease, involving multiple physiological systems and metabolic links of the human body. Its pathogenesis is closely related to lifestyle, genetic factors, environmental factors, etc. With the acceleration of modern life rhythm and the change of people's lifestyle, the incidence of T2DM is high. The latest epidemiological survey shows that the prevalence of diabetes among people aged 18 and above in China is 11.2%, ranking first in the world in terms of the number of patients, most of whom are T2MD patients. The occurrence of T2MD brings great physical pain and psychological pressure to patients, and also increases the heavy economic burden on society. Abnormal blood glucose fluctuations, lipid metabolism disorders, and insulin resistance are important factors in the development of T2DM. These factors interact and influence each other, forming a complex pathological network. In T2DM patients, diurnal blood glucose fluctuations will be abnormally increased, with a fluctuation range of about 2.5-3 times that of normal people. This abnormal blood glucose fluctuation will cause damage to various organs of the patient's body. In addition, T2MD is also highly related to lipid metabolism disorders and insulin resistance (IR). Lipid metabolism disorders can be used as an early predictor of independent risk factors for T2DM. More than 60% of patients with abnormal lipid metabolism were found to have abnormal glucose metabolism, and the probability of developing diabetes was as high as 47.3%.

[0003] In response to T2DM, there are various means commonly used in clinic. On the one hand, insulin is used to supplement the insufficient insulin in the body of a patient, so as to reduce the blood glucose level. Insulin can promote the uptake and utilization of glucose by cells, and reduce the source of blood glucose. On the other hand, oral hypoglycemic drugs are also commonly used, such as sulfonylurea drugs which can stimulate the secretion of insulin by islet beta cells, biguanide drugs which can inhibit the output of glucose by the liver, and increase the uptake and utilization of glucose by peripheral tissues, etc. Some drugs can also achieve the purpose of controlling blood glucose by improving insulin resistance. In addition, doctors will also advise patients to control diet and exercise intervention. Diet control requires patients to reasonably match food, control the intake of carbohydrates, fats and proteins, and avoid the intake of high-sugar, high-fat and high-salt foods. Exercise intervention encourages patients to engage in appropriate physical exercise, such as walking, jogging, swimming, etc., to improve the metabolic capacity of the body and enhance the sensitivity of insulin.

[0004] However, these treatment methods have their own limitations to a certain extent, although they can control the blood glucose level of T2DM patients. For example, the simultaneous use of insulin and oral hypoglycemic drugs can cause gastrointestinal discomfort, increased fat deposition, drug resistance, hypoglycemia and cardiovascular risk in patients. Moreover, there is currently no single treatment drug for T2DM patients with abnormal blood glucose fluctuations and glucose and lipid metabolism disorders. The superimposed use of multiple drugs not only increases the economic burden of patients, but also affects the compliance of patients to a certain extent. In addition, the existing treatment methods have limited effect on improving liver excretion dysfunction, regulating the rise and fall of qi, eliminating pathological products, etc., and cannot fundamentally solve the problems of T2DM patients such as abnormal blood glucose fluctuations, glucose and lipid metabolism disorders, and insulin resistance. SUMMARY

[0005] To solve the above technical problems, the present application provides a detoxification and collateral-dredging ointment for stabilizing blood glucose and a preparation method thereof.

[0006] In a first aspect, the present application provides a detoxification and collateral-dredging ointment for stabilizing blood glucose, which adopts the following technical solution: A detoxification and collateral-dredging ointment for stabilizing blood glucose, the raw materials used include the following components by weight: Huanglian 7 parts, Chishao 12 parts, Jiudahuang 6 parts, Huangqi 15 parts, Danshen 12 parts, Yincheng 6 parts, Shanzha 7 parts, Chenpi 7 parts, Chaihu 6 parts.

[0007] In a more preferred embodiment, the raw materials used include the following components by weight: Huanglian 15 parts, Chishao 20 parts, Jiudahuang 10 parts, Huangqi 30 parts, Danshen 15 parts, Yincheng 10 parts, Shanzha 15 parts, Chenpi 15 parts, Chaihu 10 parts.

[0008] In another preferred embodiment, the raw materials used include the following weight parts of medicinal material components: Huanglian 11 parts, Chishao 16 parts, Jiudahuang 8 parts, Huangqi 20 parts, Danshen 14 parts, Yincheng 8 parts, Shanzha 10 parts, Chenpi 13 parts, Chaihu 9 parts.

[0009] In another preferred embodiment, the raw materials used include the following weight parts of medicinal material components: Huanglian 11 parts, Chishao 16 parts, Jiudahuang 8 parts, Huangqi 25 parts, Danshen 14 parts, Yincheng 8 parts, Shanzha 10 parts, Chenpi 10 parts, Chaihu 8 parts.

[0010] By adopting the above technical scheme, the detoxification and meridian dredging ointment traditional Chinese medicine composition stabilizing blood sugar of the present application is constructed based on the theory of "bitter and sour regulation" and "toxicity damaging liver meridians", and is composed of Huanglian, Chishao, Jiudahuang, Huangqi, Danshen, Yincheng, Shanzha, Chenpi and Chaihu, and these components are mixed according to a certain ratio, so as to fully exert the synergistic effect between the various medicinal material components. The traditional Chinese medicine composition not only can play a role in reducing blood sugar and regulating insulin resistance, but also can effectively stabilize blood sugar fluctuation (also referred to as stabilizing blood sugar), reduce lipid deposition, effectively regulate the sugar and lipid metabolism levels of T2DM patients combined with lipid metabolism disorder, reduce the subcutaneous fat and visceral fat area, reduce the degree of fatty liver, and significantly improve the insulin resistance level. Meanwhile, in combination with the researches on metabolomics and intestinal flora, it is confirmed that the traditional Chinese medicine composition can improve various types of metabolites and circulating bile acid levels. The clinical researches show that the detoxification and meridian dredging ointment traditional Chinese medicine composition stabilizing blood sugar of the present application combined with conventional western medicine treatment can not only improve the blood sugar fluctuation of T2DM patients, reduce lipid deposition, regulate sugar and lipid metabolism and IR level, but also can effectively improve the TCM symptom performance, and the overall clinical effect is better than that of pure western medicine.

[0011] In a second aspect, the present application provides a preparation method of a detoxification and meridian dredging ointment traditional Chinese medicine composition stabilizing blood sugar, which adopts the following technical scheme: The preparation method of the detoxification and meridian dredging ointment traditional Chinese medicine composition stabilizing blood sugar comprises the following steps: All the medicinal material components are mixed, and then are subjected to extraction, decoction, precipitation, filtration, so as to obtain the detoxification and meridian dredging ointment traditional Chinese medicine composition decoction.

[0012] In a preferred embodiment, the following steps are further included: The decoction of the Jiedu Tongluo Xiaogao stable sugar traditional Chinese medicine composition is vacuum reduced pressure concentrated to obtain an extract, then the extract is spray dried, the spray dried powder is collected, and then the spray dried powder is sieved, mixed uniformly and dry granulated, and then it is packaged and coated to obtain the Jiedu Tongluo Xiaogao stable sugar traditional Chinese medicine composition granules.

[0013] In a more preferred embodiment, the extraction ratio during extraction is 1: (93-147) and the extraction time is 0.5 hours.

[0014] In a more preferred embodiment, the decoction is performed twice, each for 1 hour, and each is filtered, and the two liquid extracts are combined and then subjected to subsequent processes such as precipitation.

[0015] In a more preferred embodiment, the vacuum reduced pressure concentration is performed at a temperature of 50-80℃.

[0016] In a more preferred embodiment, the relative density of the extract at 80℃ is 1.04-1.06.

[0017] In a more preferred embodiment, the spray drying is performed at an inlet air temperature of 185-195℃ and an outlet air temperature of 80-90℃.

[0018] By using the above technical solution, the Jiedu Tongluo Xiaogao stable sugar traditional Chinese medicine composition of the present application can be prepared into a decoction or granules. The granules are convenient to store, carry and take, can better maintain the stability and effectiveness of the drug ingredients, and are beneficial to play the effects of reducing blood sugar, improving insulin resistance level, reducing blood sugar fluctuation and lipid deposition. At the same time, the preparation method of the present application can fully extract the effective ingredients from medicinal materials and ensure the stability and consistency of the preparation process, so that the traditional Chinese medicine composition has excellent efficacy and quality.

[0019] In summary, the present application has the following beneficial technical effects: The Jiedu Tongluo Xiaogao stable sugar traditional Chinese medicine composition of the present application can not only reduce blood sugar and improve insulin resistance level, but also reduce blood sugar fluctuation and lipid deposition. It has been proved in clinical and basic experiments that it can effectively reduce the blood sugar and blood lipid levels of T2DM patients, regulate HOMA-IR index, reduce visceral fat area, subcutaneous fat area and degree of fatty liver, and regulate metabolites, bile acids and intestinal flora levels. Compared with existing single drugs, the Jiedu Tongluo Xiaogao stable sugar traditional Chinese medicine composition of the present application can achieve clinical efficacy while improving patient compliance, is safe and effective, delays the occurrence of diabetes complications, and improves the quality of life of patients. The preparation method of the present application can fully extract the effective ingredients from medicinal materials and ensure the stability and consistency of the preparation process, so that the traditional Chinese medicine composition has excellent efficacy and quality. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Effect of JTTF on FBG of T2DM mice.

[0021] Figure 2 Effect of JTTF on whole-day dynamic serum glucose of T2DM mice.

[0022] Figure 3 Effect of JTTF on MG, SD and LAGE levels of T2DM mice, wherein Figure 3 A: MG of mice in each group after treatment; Figure 3 B: SD of mice in each group after treatment; Figure 3 C: LAGE of mice in each group after treatment. n=3, *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 4 Effect of JTTF on glucose tolerance of T2DM mice; A: change of blood glucose value of mice in each group after glucose injection for 0-120 min; B: AUC of IPGTT of mice in each group. n=3, ***P<0.001.

[0024] Figure 5 Effect of JTTF on IR of T2DM mice; A: serum insulin of mice in each group; B: HOMA-IR of mice in each group; C: TyG of mice in each group. n=3, **P<0.01, ***P<0.001.

[0025] Figure 6 Effect of JTTF on blood lipids of T2DM mice; A: TC of mice in each group; B: TG of mice in each group. n=3, *P<0.05, **P<0.01, ***P<0.001.

[0026] Figure 7 Effect of JTTF on liver coefficient of T2DM mice; A: liver weight of mice in each group; B: liver coefficient of mice in each group. n=8, **P<0.01, ***P<0.001.

[0027] Figure 8 Effect of JTTF on HE staining of liver of T2DM mice; 20x scale is 50 μm, and 40x scale is 20 μm.

[0028] Figure 9 Effect of JTTF on mRNA expression of IR-related factors in liver of T2DM mice; n=3, *P<0.01, **P<0.01, ***P<0.001.

[0029] Figure 10 Effects of JTTF on the protein levels of IR-related factors in the livers of T2DM mice; A: protein bands of IR-related factors in the livers of mice in each group; B: protein levels of p-IRS1 / IRS1 in the livers of mice in each group; C: protein levels of p-PI3K / PI3K in the livers of mice in each group; D: protein levels of p-AKT / AKT in the livers of mice in each group; E: protein levels of GLUT2 in the livers of mice in each group. n=3, *P<0.01, **P<0.01, ***P<0.001.

[0030] Figure 11 Comparison of glucose and CV in multiple time periods before and after JTTF intervention; A: average blood glucose in multiple time periods before and after JTTF intervention, B: blood glucose CV in multiple time periods before and after JTTF intervention; data are mean+standard deviation, ## P<0.01, ### P<0.001.

[0031] Figure 12 Comparison of SD in multiple time periods before and after treatment; note: data are expressed as mean+standard deviation, # P<0.05, ## P<0.01, ### P<0.001. DETAILED DESCRIPTION

[0032] The present application is further described in detail below in conjunction with the accompanying drawings, examples and verification experiments.

[0033] The raw materials are commercially available and are conventional reagents in the art, unless otherwise specified: IRS1 antibody Wuhan San Ying p-IRS1 (Tyr895) antibody Wuhan San Ying PI3K p85a antibody Chengdu Zhengneng p-PI3K p85 / p55 antibody Chengdu Zhengneng AKT antibody Wuhan San Ying p-AKT (Ser473) antibody Wuhan San Ying GLUT2 antibody Beijing Boao Sen β-actin antibody Wuhan San Ying HRP-labeled goat anti-rabbit Wuhan San Ying HRP-labeled goat anti-mouse Wuhan San Ying The animal experiments and the pharmaceutical compositions used in the clinical trials of the present application are decocted according to existing conventional methods.

[0034] The spray-dried powder or lyophilized powder of the present application is also prepared by conventional methods. EXAMPLE

[0035] A preparation method of a detoxification and collateral network-activating and phlegm-removing ointment traditional Chinese medicine composition, comprising the following steps: S1, 7 parts of Huanglian, 12 parts of Chishao, 6 parts of Jidaohuang, 15 parts of Huangqi, 12 parts of Danshen, 6 parts of Yincheng, 7 parts of Shanzha, 7 parts of Chenpi, and 6 parts of Chaihu are mixed and put into a multifunctional extraction tank, 10L of pure water is used to soak for 0.5 hours, and then heated to boiling, decocted twice, each time for 1 hour, and filtered respectively, the liquid extracts of the two times are combined, and then precipitated and filtered; S2, then according to the vacuum concentration standard operating procedures, the filtrate is vacuum reduced pressure concentrated to the extract with a relative density of 1.04 (80℃) at a temperature of 50℃, then the extract is spray dried according to the spray drying standard operating procedures, the inlet air temperature is controlled to be 185℃, and the outlet air temperature is controlled to be 80℃, the spray dried powder is collected, the spray dried powder is mixed uniformly after being sieved through an 80-mesh sieve, and dry granulation is performed, then the aluminum-plastic aluminum composite film (BOPP / AL / CPE) is used to seal and package on four sides, and a granule of the detoxification and collateral network-activating and phlegm-removing ointment traditional Chinese medicine composition is obtained. Embodiment

[0036] A preparation method of a detoxification and collateral network-activating and phlegm-removing ointment traditional Chinese medicine composition, comprising the following steps: S1, 15 parts of Huanglian, 20 parts of Chishao, 10 parts of Jidaohuang, 30 parts of Huangqi, 15 parts of Danshen, 10 parts of Yincheng, 15 parts of Shanzha, 15 parts of Chenpi, and 10 parts of Chaihu are mixed and put into a multifunctional extraction tank, 10L of pure water is used to soak for 0.5 hours, and then heated to boiling, decocted twice, each time for 1 hour, and filtered respectively, the liquid extracts of the two times are combined, and then precipitated and filtered; S2, then according to the vacuum concentration standard operating procedures, the filtrate is vacuum reduced pressure concentrated to the extract with a relative density of 1.06 (80℃) at a temperature of 80℃, then the extract is spray dried according to the spray drying standard operating procedures, the inlet air temperature is controlled to be 195℃, and the outlet air temperature is controlled to be 90℃, the spray dried powder is collected, the spray dried powder is mixed uniformly after being sieved through an 80-mesh sieve, and dry granulation is performed, then the aluminum-plastic aluminum composite film (BOPP / AL / CPE) is used to seal and package on four sides, and a granule of the detoxification and collateral network-activating and phlegm-removing ointment traditional Chinese medicine composition is obtained.

[0037] Embodiments 1 and 2 are prepared into granules, the pharmaceutical composition of the present application can also be prepared into powder (spray dried powder or freeze-dried powder) by using the existing method, and can also be a decoction, a pill and other dosage forms.

[0038] Animal experiment part 1 Research method 1.1 Drug configuration and dosage The daily dose of mice was calculated according to the equivalent dose ratio of human body surface area (the drug dose per kilogram of mice was 9.1 times the human dose). The mass of JTTF lyophilized powder was weighed, and the mass of lyophilized powder taken by mice per day was calculated according to the amount of medicinal material contained. The clinical adult dose was taken as the medium dose, and 0.5 times and 2.0 times were taken as the low dose and the high dose, respectively. The low, medium and high doses of mice lyophilized powder were calculated to be 31.2 mg / 20 g, 62.2 mg / 20 g and 124.4 mg / 20 g, respectively. The daily dose of metformin hydrochloride for mice was 4.0 mg / 20 g, which was calculated by the adult dose of 1.5 g / d through body surface area conversion. The drug was prepared and used immediately each time, and the corresponding mass of drug powder was weighed and dissolved in normal saline according to the proportion of 0.1 mL of drug solution per 20 g of mouse body weight. The drug solution can be placed in a water bath to promote dissolution.

[0039] 1.2 Establishment of T2DM mouse model 120 mice were randomly selected, 16 of which were used as blank control group, and were fed with ordinary maintenance feed, and free to eat and drink. The rest of the mice were fed with 60% high-fat feed. The feed was purchased from Research Diets Company (item number: D12492), and the composition ratio was: protein accounted for 20%, carbohydrates accounted for 20%, and fat accounted for 60%. The addition of high-fat feed needs to be gradual, about every 3 days, adding to the ordinary feed at a ratio of 30%, 50%, 70% and 100%, so that the mice can better adapt to the high-fat feed. After 4-6 weeks of high-fat feeding, mice with body weight close to 30 g were injected intraperitoneally with streptozotocin (STZ). STZ was prepared into a 1% STZ solution with a concentration of 0.1 mol / L and a pH of 4.5 citric acid-sodium citrate buffer, and was stored at low temperature and protected from light, and was used within 30 min. The mice were fasted the night before injection, and were injected intraperitoneally with a concentration of 50 mg / kg for 3 consecutive days. The mice were closely observed after each injection, and were given feed and water 1 hour after injection. After 72 hours of continuous injection, the fasting tail vein glucose level of the mice was detected, and a fasting blood glucose level >11.10 mmol / L was considered to be a successful model. If it does not meet the standard, 50 mg / kg of STZ solution is supplemented, and the blood glucose level is detected after 72 hours.

[0040] 1.3 Experimental grouping and intervention The mice were divided into 6 groups, 16 mice in each group, and the rest of the successfully modeled mice were kept for standby.

[0041] ① Control group (Control): fed with ordinary feed and water, and normal saline was used for gavage; ② Model control group (Model): fed with high-fat feed and water, and normal saline was used for gavage; 3. JTTF low-dose group (JTTF-L): fed with high-fat feed and water, and gavaged with JTTF at a low dose (1.56 g / kg); 4. JTTF medium-dose group (JTTF-M): fed with high-fat feed and water, and gavaged with JTTF at a medium dose (3.11 g / kg); 5. JTTF high-dose group (JTTF-H): fed with high-fat feed and water, and gavaged with JTTF at a high dose (6.22 g / kg); 6. Metformin group (Met): fed with high-fat feed and water, and gavaged with Met at 200 mg / kg. Each group of mice was gavaged once a day for 8 weeks. The gavaging was gentle, and after the traditional Chinese medicine liquid was completely dissolved and uniformly mixed, it was gavaged.

[0042] 1.4 Mouse tail vein glucose detection The FBG of the mice was measured every two weeks, and intraperitoneal glucose tolerance tests (IPGTT) were performed at 8 weeks. A blood glucose meter, blood glucose test paper, surgical scissors, alcohol cotton, and sterile cotton balls were prepared. The mouse was gently placed on the cage cover, the tail end of the mouse and the surgical scissors were disinfected with alcohol cotton, and the blood glucose test paper was inserted while it was drying. The tail end was cut off about 0.1-0.2 cm with surgical scissors, and the mouse tail was slightly massaged and squeezed from the proximal to the distal end to form blood droplets, the test paper was used to absorb the blood droplets and read the glucose content, the mouse tail was pressed with a cotton ball and disinfected. IPGTT was performed immediately after the FBG was measured, and the mouse was injected intraperitoneally with 2 g / kg of 20% glucose solution, and the blood glucose values at 15 min, 30 min, 60 min, 90 min, and 120 min after injection were collected, respectively.

[0043] 1.5 Specimen collection After 8 weeks of drug intervention, the mice were sampled at ZT6 (12:00, noon), ZT12 (18:00, sunset), ZT18 (24:00, midnight) and ZT24 (6:00, sunrise) according to the Zeitgeber time (ZT). The body weight of the mice was measured, and the whiskers on both sides were cut off. The eyeballs were removed, and the blood was collected into an Eppendorf (Ep) tube and left to stand at room temperature for 60 min. The mouse chest was opened, and the right auricle was cut open to perfuse the left ventricle with pre-cooled physiological saline. The mouse was placed on ice, and after the liver turned white, the liver was quickly removed. After weighing, the liver was rinsed in physiological saline and fixed in 4% paraformaldehyde for histopathological examination. The remaining liver tissue was frozen in liquid nitrogen and stored in a -80°C refrigerator for molecular biology index detection. The blood was centrifuged at 3000 rpm for 15 min, and the supernatant was collected and stored in a -80°C refrigerator.

[0044] 1.6 Serum glucose and blood lipid detection The serum glucose was determined by glucose oxidase method. According to the instructions of the glucose test kit, pure water, standard and sample were added to the 96-well plate as blank, standard and sample wells, and then working solution was added for reaction. The 96-well plate was incubated in a 37°C constant temperature drying box for 10 min, and the optical density (OD) was measured at 505 nm using a multifunctional enzyme marker.

[0045] TC was determined by cholesterol oxidase-peroxidase-4-aminophenol (COD-PAP) method using a TC test kit. The operation was the same as that of the glucose test kit, and the OD value was finally determined at 500 nm wavelength.

[0046] TG was determined by glycerophosphate oxidase-peroxidase assay (GPO-PAP) method using a TG test kit. The operation was the same as that of the TC test kit.

[0047] 1.7 Serum insulin detection ​The mouse serum insulin content was detected by using a mouse insulin enzyme linked immunosorbent assay (Elisa) kit. The double antibody sandwich method was used to combine insulin and horseradish peroxidase (HRP) labeled insulin antibody, and then 3,3',5,5'-tetramethylbenzidine (TMB) was used for color development reaction. The specific operation steps were carried out according to the instruction manual. The OD value was measured at 450 nm, and the insulin concentration was calculated.

[0048] 1.8 Hematoxylin and eosin (HE) staining The liver tissue soaked in 4% paraformaldehyde was taken out, dehydrated with gradient concentration of ethanol solution, and transparentized with xylene, and then poured into paraffin. The section was cut with a thickness of 3-4 μm, spread on the glass slide, and placed in a 60°C environment for baking. After the section was deparaffinized, xylene and reverse gradient concentration of ethanol were used for rehydration. The glass slide was stained with hematoxylin, differentiated, and then stained with eosin, and then dehydrated, transparentized, and finally sealed with neutral resin. The section was placed on an inverted optical microscope, and the morphology of the liver tissue at different magnifications was observed and photographed.

[0049] 1.9 RT-qPCR 1.9.1 Total RNA extraction RNA was extracted from liver using Trizol method, the whole process was operated on ice, wearing a mask, gloves and lab coat, and the centrifuge was pre-cooled. Cut the green bean size (about 20 mg) liver tissue into an RNase-free Eppendorf tube, add 1 mL of pre-cooled Trizol to the tube in the fume hood, then add 1-2 sterile steel balls to each tube, put it into a high-throughput tissue grinder for 1 min of grinding, and observe the liquid. If the particles are obvious, grind again. After standing for 5 min, add 200 μL of chloroform substitute to each well, shake the Eppendorf tube to mix the liquid thoroughly, and stand again for 5 min. Centrifuge at 12000 rpm for 15 min. Carefully pipette the supernatant (about 400 μL) into a new Eppendorf tube, add an equal volume of pre-cooled isopropanol, invert the tube and stand for 10 min. Centrifuge at 12000 rpm for 15 min again. The RNA will be precipitated at the bottom of the tube, discard the supernatant, and add an equal volume of 75% ethanol to wash it. 75% ethanol is prepared with diethyl pyrocarbonate (DEPC) water. Gently tap the tube wall and centrifuge for 5 min, and wash again. Discard the liquid and dry the RNA precipitate in a clean bench. After there is no liquid in the tube, add an appropriate amount of DEPC to dissolve the RNA. Use a microspectrophotometer to measure the concentration of RNA, and adjust the concentration to 300-400 ng / μL, and store it in a -80°C refrigerator.

[0050] 1.9.2 cDNA reverse transcription According to the RNA concentration, calculate the volume of 1 μg RNA required. Select HiScript Ⅲ 1st Strand cDNA Synthesis Kit for reverse transcription. According to the product instructions, prepare the reaction system. First, remove genomic DNA, add 1 μg of RNA and 5× gDNA wiper Mix 2 μL, and add RNase-free ddH2O to 10 μL. Mix gently, then incubate in a PCR instrument at 42°C for 2 min. Then prepare the first strand cDNA synthesis reaction solution, add 10× RT Mix 2 μL, HiScript Ⅲ Enzyme Mix 2 μL, Oligo (dT) 20 VN 1 μL, Random hexamers 1 μL and RNase-free ddH2O 4 μL to the mixture from the previous step. Mix well, then react at 50°C for 15 min and 80°C for 5 s. The product is stored in a -20°C refrigerator for a short period of time.

[0051] 1.9.3 Real time qPCR reaction SYBR Green I intercalating fluorescent method qPCR reaction was used. Primer design and synthesis were performed by Shengwo Bioengineering Co., Ltd. The primer dry powder was first centrifuged at 4000 rpm for 1 min, ddH2O was added to configure 200 μM, and it was ready for use after being diluted to 2 μM. The mouse primer sequence is shown in Table 13. The reaction system was configured according to ChamQ SYBR qPCR Master Mix kit: 2x Mix 10 μL, upstream primer 2 μL, downstream primer 2 μL, cDNA 1 μL, ddH2O 5 μL. The reaction system was suspended in a sterile 96-well PCR plate, sealed, centrifuged, and placed into a real-time fluorescent quantitative PCR instrument. The reaction program was set as follows: 95℃ 3 min pre-denaturation, 95℃ 10 s, 60℃ 30 s for 40 cycles, 95℃ 15 s, 60℃ 60 s, 95℃ 15 s to complete the melting curve collection. The data was exported, and 2 -ΔΔCt Calculation results. The primer sequence used is shown in Table 1

[0052] 1.10 Western blotting First, mouse liver tissue protein was extracted. 30 mg of liver tissue was weighed into a sterile Ep tube, 300 μL of lysis solution was added, and radioimmoprecipitation assay (RIPA) lysis solution was used. The lysis solution already contains 1% protease inhibitor and 1% phosphatase inhibitor. Add 2 steel balls to the Ep tube and grind with a tissue grinder. Ultrasonic lysis of the tissue liquid on ice, 2 s each time, 5 s interval, repeat 6 times. Put it in a 4°C centrifuge at a speed of 12000 rpm for 15 min, and then aspirate the supernatant to obtain the protein stock solution. Select bicinchoninic acid assay (BCA) to determine the protein concentration. According to the BCA protein quantification kit instructions, prepare a gradient concentration of protein standard solution, and configure the working solution of reagent A and reagent B at 50:1, and dilute the protein stock solution by 50 times. Use a 96-well plate to detect, add 5 μL of protein standard solution or sample to each well, then add 195 μL of working solution, and incubate at 37°C for 30 min. Measure the OD value at 562 nm with a microplate reader, and obtain the protein concentration according to the protein standard curve, and calculate the required loading amount of 20 μg protein. Add 6x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) double-color protein loading buffer to the protein stock solution, mix well, and then heat in a metal bath for 10 min. After protein dispensing, store at -80°C.

[0053] Next, Western blotting was performed. According to the molecular weight of the target protein, the gel was prepared, and SDS-PAGE was used for electrophoresis. The protein separation process was judged according to the position of the rainbow marker, and then the protein was transferred to a polyvinylidene fluoride (PVDF) membrane using a wet transfer method. Select 5% skimmed milk powder to block the protein membrane for 1 hour, and then wash with tris-buffered saline tween-20 (TBST) before subsequent immunological reaction. Incubate the primary antibody at an appropriate concentration at 4°C overnight, wash the membrane 3 times for 10 min each time the next day, and then add the secondary antibody and slowly shake for 1 hour. Wash the membrane again, react the membrane with enhanced chemiluminescence (ECL) reagent, and develop on a high-sensitivity chemiluminescence imaging system. Use Image J to obtain the gray value of the band.

[0054] 1.11 Statistical analysis method GraphPad Prism 8.4.0 software was used for statistical analysis. Data were expressed as mean ± standard error, and each experiment was repeated 3 times or more. Normality test was performed using Shapiro-Wilk. If normal, ANOVA was used, if variance was equal, ordinary ANOVA test was used, if variance was not equal, Brown-Forsythe and Welch ANOVA tests were used. Nonparametric test was used for non-normal distribution. Holm-Sidak test was used for pairwise comparison. P<0.05 was considered statistically significant.

[0055] 2 Results of the study 2.1 General state and body weight During the experiment, the Control group mice were active, responsive, and had a moderate body type, smooth and lustrous hair, normal water intake, food intake, and excretion, and dry bedding. In the early stage after modeling, the mice were obese, liked to sleep, had oily hair, and had a large increase in water intake and food intake, and a significant increase in urine output, with yellow and wet bedding, accompanied by a sour smell. Later, the Model group mice had poor mental state and slow response, and their hair was dry and shed. After JTTF and Met intervention, the general condition of the mice improved compared with the Model group. From the 0th week of self-group intervention, every two weeks, the body weight of the mice was measured. As shown in FIG. 2C, before intervention, the average body weight of the Control group mice was 23.48 g, and the body weight of the T2DM mice was about 30 g. With the progress of drug intervention, the body weight of the JTTF group and the Met group gradually decreased, especially in the last 2 weeks before gavage, the body weight decreased significantly. The body weight of the Model group mice changed little. Comparison of the body weight of mice in each group at the 8th week showed that the body weight of the Model group was significantly higher than that of the Control group (P<0.001), and the body weight of the mice was significantly reduced after intervention with low, medium, and high doses of JTTF and Met (P<0.01). Figure 10 C, before intervention, the average body weight of the Control group mice was 23.48 g, and the body weight of the T2DM mice was about 30 g. With the progress of drug intervention, the body weight of the JTTF group and the Met group gradually decreased, especially in the last 2 weeks before gavage, the body weight decreased significantly. The body weight of the Model group mice changed little. Comparison of the body weight of mice in each group at the 8th week showed that the body weight of the Model group was significantly higher than that of the Control group (P<0.001), and the body weight of the mice was significantly reduced after intervention with low, medium, and high doses of JTTF and Met (P<0.01).

[0056] 2.2 Effect of JTTF on blood glucose in T2DM mice 2.2.1 FBG From the first day of intervention, the FBG level of the mice was detected every two weeks. After overnight fasting, the tail vein blood glucose of the mice was detected every morning at 9:00. As shown in FIG. 2D, the FBG level of the Control group mice was about 5.0 mmol / L, and the FBG level of the Model group mice was about 20.0 mmol / L. After JTTF and Met intervention, the FBG level of the mice decreased significantly (P<0.01). Figure 1As shown, the FBG of the Control group mice was low during the intervention period, about 6 mmol / L, and relatively stable. At week 0, the FBG of the T2DM mice was all higher than 11.1 mmol / L, and there was no statistical difference in FBG between the Model group and the Chinese medicine and Met groups. With the increase of the intervention time, the FBG of the Model group mice was still within the film forming standard, while the T2DM mice gradually reduced the FBG due to the use of JTTF and Met drugs of various doses, and the gap with the Model group gradually widened, and after week 4, it was basically lower than 10 mmol / L, which was significantly lower than the Model group (P<0.001).

[0057] 2.2.2 24-hour dynamic blood glucose After the end of gavage, the blood of mice in each group was collected every 6 hours from ZT6, and serum was obtained by centrifugation, and the glucose content at each time point was detected using a glucose kit. The results are shown in Figure 2 As shown, the change of 24-hour dynamic serum glucose fluctuation of each group of mice is shown. As can be seen from the figure, the blood glucose of the Control group mice at ZT12, i.e. 18:00, was the highest, reaching 6.80 mmol / L, and the blood glucose at ZT24, i.e. 6:00, was the lowest, reaching 2.88 mmol / L, and the overall fluctuation was small. The Model group not only had higher blood glucose than the Control group all day, but also had larger fluctuations, with the highest blood glucose at ZT6, i.e. 12:00, reaching 23.74 mmol / L, and the lowest blood glucose at ZT24, i.e. 6:00, reaching 13.62 mmol / L. After intervention with different doses of JTTF, the blood glucose of mice was decreased, and the fluctuation form was improved, and the blood glucose fluctuation rhythm tended to be similar to the Control group. The blood glucose value was also reduced after Met intervention.

[0058] 2.2.3 MG After obtaining the blood glucose data of mice in each group at different time points for 24 hours, the blood glucose fluctuation related indicators were further calculated and analyzed. The mean value of the blood glucose values of mice at 4 time points was calculated to obtain the whole day MG of mice. As shown in Figure 3 As shown in A, the MG of the Control group was 5.39 mmol / L, while the MG of the Model group was as high as 17.06 mmol / L, with a significant difference (P<0.001). After treatment with low, medium and high doses of JTTF, the MG of T2DM mice was significantly lower than that of the Model group (P<0.001), and the MG of the Met group was also significantly reduced (P<0.001).

[0059] 2.2.4 SD According to the whole day blood glucose data of each group of mice, the blood glucose SD of mice was calculated by using the STDVE function to evaluate the degree of the overall blood glucose deviation from the mean of mice. The results are shown in Figure 3 B, the SD of the Control group was lower, while the Model group was as high as 4.92 mmol / L, with significant difference (P<0.01). Compared with the Model group, the intervention of JTTF could reduce the SD in a concentration gradient (P<0.05), and the SD of the Met group was also decreased (P<0.05).

[0060] 2.2.5 LAGE According to the blood glucose data of mice at each time point, the maximum and minimum blood glucose values were found, and the difference between the two values was obtained, i.e. LAGE, which was used as a reference for evaluating the blood glucose variability of mice. As shown in Figure 3 C, the LAGE of the Control group was lower, while the Model and Control groups had statistical difference (P<0.05). Different doses of JTTF and Met intervention could significantly reduce the LAGE of T2DM mice (P<0.05).

[0061] 2.2.6 Glucose tolerance At the 8th week of intervention, IPGTT test was performed to understand the glucose tolerance of mice. 3 mice from each group were randomly selected for the test. The results are shown in Figure 4 A, the blood glucose of all mice increased linearly at 15 min after intraperitoneal injection of glucose. The Control group showed a blood glucose peak at 15 min, which rapidly decreased within 30 min, and the blood glucose basically returned to normal at 2 hours. The Model group showed a delayed blood glucose peak, up to about 30 mmol / L, and the blood glucose returned slowly, with difficulty in metabolic absorption of glucose within 2 hours. Different doses of JTTF, especially high dose, could significantly improve the glucose metabolism capacity of T2DM mice, with an earlier blood glucose peak and a reduced blood glucose rise. Met also improved the glucose tolerance of mice. The area under the curve (AUC) of each group of mice was calculated, and the results are shown in Figure 4 B shows that the AUC of the Chinese medicine group, Met group and Control group was significantly lower than that of the Model group (P<0.001).

[0062] 2.3 Effect of JTTF on IR of T2DM mice The serum insulin of each group of mice was detected, and the HOMA-IR index was calculated according to the FBG, and the TyG index was calculated according to the serum FBG and TG. The results are shown in Figure 5The insulin, HOMA-IR and TyG of the Model group were significantly increased compared with the Control group (P<0.01). After treatment, the insulin of the JTTF-M, JTTF-H and Met groups was significantly decreased compared with the Model group (P<0.01), and the HOMA-IR and TyG of the three different dose JTTF and Met groups were significantly decreased compared with the Model group (P<0.001).

[0063] 2.4 Effect of JTTF on blood lipid of T2DM mice The serum TC and TG of each group of mice were compared. As shown in Figure 6 , the TC and TG values of the Model group were significantly increased compared with the Control group (P<0.05, P<0.001). After treatment, the TC and TG of the JTTF-H group were significantly decreased compared with the Model group (P<0.01). The TC and TG levels were also significantly decreased after Met intervention (P<0.01, P<0.001).

[0064] 2.5 Effect of JTTF on liver coefficient and liver tissue morphology of T2DM mice 2.5.1 Liver coefficient After comparing the blood glucose of mice in the horizontal and vertical directions, since the liver is an important target organ for glucose and lipid metabolism and IR, the focus will be on the liver in the following observation of liver changes. First, compare the liver coefficients of mice in each group. The mice were weighed before sampling, and all liver tissues were quickly stripped and weighed on a small electronic scale. The results are shown in Figure 7 A, the liver weight of the Model group was significantly increased compared with the Control group (P<0.01), indicating that the liver of T2DM mice was pathologically enlarged or edematous. Met can significantly reduce the increase of liver weight (P<0.01), and the reduction of the traditional Chinese medicine group has no statistical significance. The liver coefficient is obtained according to the ratio of liver weight and body weight. As shown in Figure 7 B, after treatment, the liver coefficients of the JTTF-L, JTTF-M, JTTF-H and Met groups were significantly decreased compared with the Model group (P<0.01).

[0065] 3.5.2 Liver HE staining To further observe the pathological morphology of JTTF on liver tissue, the liver tissues of each group of mice were stained with HE. As shown in Figure 8It can be seen that the liver cells in the Control group were arranged in order, with clear edges, tight cytoplasm, regular liver cords and sinusoids, and obvious liver lobules. However, in the Model group, the mouse liver cells were severely swollen and even broken, with disordered cell arrangement, unclear boundaries, loose cytoplasmic structure, vacuolization, deformed liver cord and sinusoid arrangement, and unobvious liver lobules. With the continuous increase of the JTTF intervention dose, the swelling degree of the mouse liver cells gradually decreased, the cell arrangement gradually became regular, the cytoplasmic vacuolization gradually improved, and the pathological morphology of the liver tissue gradually improved. Met also improved the pathological morphology of the liver tissue, but the effect observed under the microscope was not as good as that of the JTTF-M and JTTF-H groups.

[0066] 3.6.1 mRNA expression of liver IR-related factors 3.6.1 mRNA expression of liver IR-related factors To more accurately reveal the effect of JTTF on the insulin signaling pathway in the liver of T2DM mice, representative pathway factors regulating IR were selected, and molecular biology methods were used to observe their changes. First, the relative expression of insulin receptor substrate 1 (Irs1), phosphatidylinositol 3-kinase (Pi3k), serine / threonine kinase B (Akt), and glucose transporter 2 (Glut2) gene mRNA in the liver of mice in each group was detected, with β-actin as the internal reference. The results are shown in Table 3. Figure 9 As shown in Table 3, compared with the Control group, the relative expression of Irs1, Pi3k, Akt, and Glut2 genes in the Model group was significantly reduced (P<0.05). After traditional Chinese medicine intervention, JTTF increased the expression of IR-related genes in a concentration-dependent manner. Compared with the Model group, JTTF-H significantly increased the expression of Irs1, Pi3k, Akt, and Glut2 genes (P<0.05), and JTTF-L and JTTF-M significantly increased the expression of Irs1 and Glut2 (P<0.05). The expression of Irs1 and Pi3k in the Met group was significantly increased compared with the Model group (P<0.05). Therefore, JTTF can significantly increase the expression of insulin signaling pathway-related genes in T2DM mice.

[0067] 2.6.2 Protein level of liver IR-related factors After observing the regulatory effect of JTTF on liver IR-related factors in T2DM mice at the transcriptional level, the next step is to further explore the effect at the protein translation level, using β-actin as an internal control. Figure 10 The results showed that, compared with the control group, the phosphorylated levels of IRS1, PI3K, and AKT were significantly reduced in the Model group, and GLUT2 protein was significantly decreased (P<0.05). Except for the JTTF-L group, which did not show a significant increase in phosphorylated PI3K, the levels of these proteins significantly rebounded after traditional Chinese medicine treatment compared with the Model group (P<0.05), and the levels of the above proteins in the Met group also increased significantly (P<0.05). Therefore, JTTF can significantly increase the expression of insulin signaling pathway-related factors in T2DM mice and improve liver insulin resistance (IR).

[0068] In summary, by constructing a type 2 diabetes mellitus (T2DM) mouse model, we observed that JTTF significantly reduced body weight, fasting blood glucose (FBG), and blood lipids in T2DM mice, decreased 24-hour MG, SD, and LAGE levels, and improved 24-hour glycemic variability. Simultaneously, the study found that JTTF improved glucose tolerance, HOMA-IR, and TyG index, reduced liver coefficient, and improved the pathological structure of liver tissue. Furthermore, RT-qPCR and Western blotting results showed that JTTF significantly reduced the mRNA and protein expression levels of liver IR-related factors in T2DM mice. This study preliminarily demonstrates the role of JTTF in improving glycemic variability and regulating liver IR in T2DM mice.

[0069] The applicant conducted a prospective randomized controlled clinical trial on 96 patients with type 2 diabetes mellitus (T2DM) admitted to the endocrinology outpatient clinic and ward of Guangzhou University of Chinese Medicine Shenzhen Hospital (Futian) in 2023-2024. Patients were randomly assigned to a treatment group and a control group. The control group received routine Western medicine treatment (metformin 0.5g tid), while the treatment group received the detoxifying, meridian-clearing, blood sugar-stabilizing traditional Chinese medicine composition prepared in Example 1 (extracted as a decoction in a Good Manufacturing Practice (GMP) workshop by Kangmei Pharmaceutical Co., Ltd. (Shenzhen, China, Kangmei Smart Pharmacy Co., Ltd.). Example 1 describes the dosage for one dose, taken twice daily, half an hour after breakfast and dinner). After 12 weeks of intervention, 79 patients completed the study. The results showed that after treatment with the detoxifying, meridian-clearing, blood sugar-stabilizing traditional Chinese medicine composition, patients' blood glucose fluctuations (see Example 1) were significantly reduced. Figure 11 The range decreased to 14.67%-24.13%, with a particularly noticeable decrease in the early morning and at night. SD fluctuations (see...) Figure 12The range was reduced to 1.01-2.18 mmol / L, especially in the morning and at night; the glycemic excursion amplitude (Mean Amplitude of Glycemic Excursions, MAGE, see Table 2) was significantly different between the control group (Z=-2.226, P=0.026); the maximum glycemic excursion amplitude (LAGE, see Table 3) after treatment with Jiedu Tongluoxiao plaster was statistically significant (t=2.317, P=0.026); the mean difference in daytime blood glucose (MODD, see Table 4) after treatment with Jiedu Tongluoxiao plaster was statistically significant (Z=-3.377, P=0.001), and the time in the target range of glucose (TIR, see Table 5) after treatment was statistically significant (Z=-2.205, P=0.027).

[0070] Table 2 Comparison of MAGE between the two groups of patients P 50 ( P 25 , P 75 )]

[0071] Note: Intra-group comparison, #P <0.05; inter-group comparison *P <0.05.

[0072] Table 3 Comparison of LAGE between the two groups of patients P 50 ( P 25 , P 75 )] Note: Intra-group comparison, #P <0.05. Table 4 Comparison of MODD between the two groups of patients P 50 ( P 25 , P 75 )] Note: Intra-group comparison, #P <0.05, ##P <0.01. Table 5 Comparison of TIR between the two groups of patients P 50 ( P 25 , P 75 )] Note: intra-group comparison, #P <0.05. The embodiments of the present application are preferred embodiments of the present application, and are not intended to limit the protection scope of the present application, and therefore: any equivalent changes made according to the structure, shape, principle of the present application should be covered within the protection scope of the present application.

Claims

1. A traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control, characterized in that, The raw materials used include the following medicinal components in parts by weight: Coptis chinensis 7-15 g, rhubarb 6-10 g, Astragalus membranaceus 15-30 g, Salvia miltiorrhiza 12-15 g, Paeonia lactiflora 12-20 g, Bupleurum chinense 6-10 g, Artemisia capillaris 6-10 parts, Crataegus pinnatifida 7-15 parts, and Citrus reticulata 7-15 parts.

2. The traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 1, characterized in that, The raw materials used include the following medicinal components by weight: 7 parts Coptis chinensis, 12 parts Paeonia lactiflora, 6 parts Rheum palmatum (processed with wine), 15 parts Astragalus membranaceus, 12 parts Salvia miltiorrhiza, 6 parts Artemisia capillaris, 7 parts Crataegus pinnatifida, 7 parts Citrus reticulata peel, and 6 parts Bupleurum chinense.

3. The traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 1, characterized in that, The raw materials used include the following medicinal components by weight: 15 parts Coptis chinensis, 20 parts Paeonia lactiflora, 10 parts Rheum palmatum (processed with wine), 30 parts Astragalus membranaceus, 15 parts Salvia miltiorrhiza, 10 parts Artemisia capillaris, 15 parts Crataegus pinnatifida, 15 parts Citrus reticulata peel, and 10 parts Bupleurum chinense.

4. A method for preparing the traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control as described in any one of claims 1-3, characterized in that, Includes the following steps: All medicinal components are mixed, then extracted, decocted, and the liquid and dregs are separated to obtain a decoction of traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization and blood sugar control.

5. The preparation method of the traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 4, characterized in that, It also includes the following steps: The decoction of the traditional Chinese medicine composition for detoxification, blood circulation dredging, blood sugar stabilization and dehydration is concentrated under vacuum to obtain an extract. The extract is then freeze-dried or spray-dried to collect the freeze-dried powder or spray-dried powder. The freeze-dried powder or spray-dried powder is then sieved, mixed evenly, and dry-granulated. Finally, it is encapsulated to obtain granules of the traditional Chinese medicine composition for detoxification, blood circulation dredging, blood sugar stabilization and dehydration.

6. The preparation method of the traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 5, characterized in that, The extraction ratio during extraction is 1:(93-147), and the extraction time is 0.5-2 hours.

7. The preparation method of the traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 5, characterized in that, The decoction is performed 2-3 times, each time for 1 hour, and then filtered separately. The liquid extracts from the two decoctions are combined and then subjected to subsequent processes such as precipitation.

8. The preparation method of the traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 5, characterized in that, The vacuum concentration temperature is 50-80℃.

9. The preparation method of the traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 5, characterized in that, The relative density of the extract at 80°C is 1.04-1.

06.

10. The method for preparing a traditional Chinese medicine composition for detoxification, meridian clearing, blood sugar stabilization, and blood sugar control according to claim 5, characterized in that, The inlet air temperature of the spray dryer is 185-195℃, and the outlet air temperature is 80-90℃.

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