Dai medicine composition as well as preparation method and application thereof

Abstract

The invention discloses a Dai medicine composition as well as a preparation method and application thereof, and belongs to the technical field of medicines. The Dai medicine composition comprises the following raw materials in parts by weight: 10-20 parts of a turmeric extract and 10-20 parts of amomum tsao-ko essential oil, the turmeric extract is an alcohol extract of turmeric, and the content of sweet myrrh curcumol in the turmeric extract is 5-10 wt.%. The invention discloses application of the Dai medicine composition in preparation of a medicine for preventing or / and treating acute myocardial infarction. The acute myocardial infarction comprises acute myocardial infarction under the condition of non-alcoholic fatty liver. Based on the Yaojiao theory of Dai medicine, the turmeric extract and the amomum tsao-ko essential oil are creatively matched, and the turmeric extract and the amomum tsao-ko essential oil are combined to supplement soil to achieve the effects of leveling four towers, clearing away fire and toxic materials and promoting blood circulation and ventilating. Tests show that the composition disclosed by the invention can relieve liver injury caused by non-alcoholic fatty liver disease, effectively inhibit left ventricular remodeling, improve cardiac functions and reduce acute myocardial infarction of mice under the condition of NAFLD (non-alcoholic fatty liver disease).

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and specifically relates to a Dai medicine composition and a preparation method and application thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes caused by factors other than alcohol and other clear liver-damaging factors. It is an acquired metabolic stress liver injury closely related to insulin resistance and genetic susceptibility. In recent years, the incidence and prevalence of non-alcoholic fatty liver disease have increased year by year, and it has become the most common chronic liver disease in the world. More and more studies have shown that there is a strong correlation between NAFLD and ischemic heart disease (IHD), the main adverse event of cardiovascular disease, and it may be an independent risk factor. IHD is the leading cause of death worldwide and the most common cause of death in patients with NAFLD. Many prospective or retrospective studies have pointed out that the prevalence of NAFLD is positively correlated with the long-term risk of ischemic heart disease. The risks of acute myocardial infarction and myocardial ischemia-reperfusion injury caused by myocardial ischemia will increase with the increase in the prevalence and severity of symptoms of NAFLD. As two important global public health diseases, the devastating impact of NAFLD on ischemic heart disease risk events is becoming increasingly apparent, which not only puts greater pressure on the public health system, but also further increases the economic and psychological burden on patients.

[0003] Therefore, providing a drug for preventing and treating acute myocardial infarction under the condition of non-alcoholic fatty liver has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0004] One of the purposes of the present invention is to provide a Dai medicine composition, which can not only alleviate liver damage caused by non-alcoholic fatty liver disease, but also prevent and treat acute myocardial infarction under the condition of non-alcoholic fatty liver disease (NAFLD).

[0005] The second object of the present invention is to provide a method for preparing the Dai medicine composition.

[0006] The third object of the present invention is to provide an application of the Dai medicine composition.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The invention discloses a Dai medicine composition, which comprises the following raw materials in parts by weight: 10-20 parts of turmeric extract and 10-20 parts of tsaoko essential oil. The turmeric extract is an alcohol extract of turmeric, and the content of sweet bisabolol curcumin in the turmeric extract is 5-10wt.%.

[0009] In some embodiments of the present invention, the Dai medicine composition comprises the following raw materials in parts by weight: 12-18 parts of turmeric extract and 12-18 parts of tsaoko essential oil.

[0010] In some embodiments of the present invention, the Dai medicine composition comprises the following raw materials in parts by weight: 10 parts of turmeric extract and 10 parts of tsaoko essential oil.

[0011] In some embodiments of the present invention, the method for preparing the turmeric extract comprises the following steps:

[0012] S1. Extraction and concentration: Take turmeric rhizomes, add alcohol solution to extract, and concentrate the extract to obtain an extract;

[0013] S2. Silica gel column separation: The extract obtained in step S1 was separated by a silica gel column, and the gradient elution was performed with a petroleum ether / ethyl acetate solvent of 8 to 12:1, 3 to 7:1, 1.5 to 3:1, and 0.5 to 1.2:1, respectively, and each fraction was subjected to thin layer chromatography using a GF254 silica gel plate, and the eluate of 0.5 to 1.2:1 was collected and concentrated to obtain a concentrate;

[0014] S3. Resin adsorption separation: The concentrated solution obtained in step S2 was applied to an MCI adsorption resin column, and gradient eluted with water, 15-25%, 35-45%, 55-65%, 75-85%, 95-100% methanol aqueous solution, and each fraction was subjected to thin layer chromatography using GF254 silica gel plate, and the eluate of 15-65% methanol aqueous solution was collected and concentrated to obtain the components separated by the resin;

[0015] S4. Semi-preparative HPLC separation: The resin-separated components obtained in step S3 are separated by semi-preparative HPLC, and the eluate containing bisabolol is collected, concentrated, and dried to obtain a turmeric extract.

[0016] Preferably, in step S1, methanol-water solution is used for reflux extraction, and more preferably, the concentration of methanol-water solution is 50-90%; more preferably 75%; the concentration of methanol-water solution is volume concentration;

[0017] Preferably, in step S2, gradient elution is performed with petroleum ether / ethyl acetate solvents in a ratio of 10:1, 5:1, 2:1, and 1:1, and the 1:1 eluate is collected;

[0018] Preferably, in step S3, gradient elution is performed with water, 20%, 40%, 60%, 80%, and 100% methanol aqueous solution in sequence, and the eluate of 20%-60% methanol aqueous solution is collected;

[0019] Preferably, in step S4, the chromatographic conditions of semi-preparative HPLC are as follows: octadecylsilane bonded silica gel is used as filler, methanol is used as mobile phase A, and water is used as mobile phase B for gradient elution; the gradient elution procedure is as follows:

[0020] 0min, 15-25% methanol, 75-85% water;

[0021] 15min, 40-60% methanol, 40-60% water;

[0022] Preferably, 0 min, 20% methanol, 80% water;

[0023] 15 min, 50% methanol, 50% water;

[0024] Preferably, the mobile phase flow rate is 0.8 to 1.2 mL / min, and the column temperature is 15 to 25°C, preferably 20°C;

[0025] More preferably, the eluate is collected from 0.5 to 2 minutes before the elution of bisabolol; and the collection of the eluate is stopped when the relative retention time is twice the elution time of bisabolol. More preferably, the detection wavelength is 230 nm and / or 254 nm.

[0026] In one embodiment of the present invention, the eluate is collected for 7 to 13 minutes.

[0027] In some embodiments of the present invention, the Amomum villosum essential oil is obtained by CO2 supercritical extraction;

[0028] Preferably, the preparation method of tsaoko essential oil comprises the following steps: weighing tsaoko powder, loading into an extraction kettle, extracting at a temperature of 50 to 60°C, a pressure of 25 to 35 MPa, an average CO2 flow rate of 15 to 25 g / min, and an extraction time of 1 to 1.5 h;

[0029] More preferably, the extraction temperature is 55°C, the pressure is 30 MPa, the average CO2 flow rate is 20 g / min, and the extraction time is 1.5 h;

[0030] Preferably, the extraction times are 1 to 3 times.

[0031] In some embodiments of the present invention, the composition is a preparation prepared by conventional methods using turmeric extract and tsaoko essential oil as raw materials and adding pharmaceutically acceptable excipients;

[0032] Preferably, the preparation is an oral preparation.

[0033] The preparation method of the Dai medicine composition disclosed in the present invention comprises the following steps: preparing turmeric extract and tsaoko essential oil in proportion, adding pharmaceutically acceptable auxiliary materials, mixing evenly, granulating, filling capsules, and obtaining capsules;

[0034] Preferably, the total amount of turmeric extract and tsaoko essential oil is 20-50 parts, and the auxiliary material is 70-50 parts;

[0035] Further preferably, the excipients used to prepare the capsules are selected from any two or three of xylitol, mannitol, sorbitol, lacto-oligosaccharides, fructo-oligosaccharides, isomalt and erythritol.

[0036] The preparation method of the Dai medicine composition disclosed in the present invention comprises the following steps: preparing turmeric extract and tsaoko essential oil in proportion, adding pharmaceutically acceptable auxiliary materials, mixing evenly, granulating, and tableting to obtain tablets;

[0037] Preferably, the total amount of turmeric extract and tsaoko essential oil is 20-50 parts, and the auxiliary material is 70-50 parts;

[0038] Further preferably, the excipients used to prepare the capsules are selected from any two or three of xylitol, mannitol, sorbitol, lacto-oligosaccharides, fructo-oligosaccharides, isomalt and erythritol.

[0039] The present invention discloses an application of the Dai medicine composition in the preparation of a drug for preventing and / or treating acute myocardial infarction; preferably, the acute myocardial infarction includes acute myocardial infarction under the condition of non-alcoholic fatty liver.

[0040] The present invention discloses an application of the Dai medicine composition in preparing a medicine for alleviating liver damage caused by non-alcoholic fatty liver disease.

[0041] "Ya Jie" is the Dai word for "antidote". The "Ya Jie theory" is a high-level summary of the Dai medical prevention thought. The treatment principles and medication methods emphasize "antidote before illness, antidote before treatment, treatment before antidote, and antidote and treatment at the same time". The Ya Jie theory believes that the liver digests food, transforms qi and blood, and excretes waste. It is not only the main place for "poison" accumulation, but also an important organ for "detoxification". If the liver function is abnormal, the "poison" will continue to accumulate until the "critical balance" is broken, and it will be manifested externally in some extrahepatic organs, and when "Pa Ya" (viral evil spirit) invades the human body, the "imbalance of the four towers" will be accelerated, leading to the occurrence of extrahepatic diseases. In order to treat extrahepatic diseases caused by abnormal liver function and restore the "balance of the four towers" of the human body, Dai medicine often uses drugs to remove liver "poison", restore the liver's "detoxification" function, improve liver dysfunction, and then prevent and treat extrahepatic diseases.

[0042] The turmeric described in the present invention is the dried rhizome of turmeric (Curcuma longa L.) of the genus Curcuma of the ginger family, and is called "haoming" in Dai medicine. It is a commonly used yajie drug in Dai medicine clinic. Dai medicine believes that turmeric is smelly, bitter, slightly spicy, flat and hot in nature, and can enter soil, water and wind towers, and has the effects of clearing heat and detoxifying, promoting blood circulation and relieving pain, and promoting qi and removing blood stasis.

[0043] The tsaoko described in the present invention is the dried mature fruit of the zingiberaceae plant Amomum tsaoko Crevost & Lem.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention is based on the theory of the elegant interpretation of Dai medicine, and creatively combines turmeric extract with tsaoko essential oil. The two medicines are used together to replenish the qi in the "earth tower", and play the role of balancing the four towers, clearing away heat and detoxifying, and promoting blood circulation and ventilation. Experiments have shown that the composition of the present invention can alleviate liver damage caused by non-alcoholic fatty liver disease, effectively inhibit left ventricular remodeling, improve heart function, and reduce acute myocardial infarction in mice under NAFLD conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Attached Figure 1 This is a graph showing the results of HPLC testing of turmeric extract;

[0047] Attached Figure 2 Figure 2 shows the pathological changes of liver tissues of mice in each group;

[0048] Attached Figure 3 The graphs show the changes in the levels of TC, TG, LDL, AST and ALT in the serum of mice in each group, where *P<0.05, **P<0.01;

[0049] Attached Figure 4 The results of TTC+Evans Blue staining of the hearts of mice in each group and the myocardial infarction rate of each group, where *P<0.05, **P<0.01;

[0050] Attached Figure 5 The graph shows the changes in CK and LDH levels in the serum of mice in each group, where *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0052] Unless otherwise specified, the "parts" described in the embodiments of the present invention refer to parts by mass.

[0053] Example 1

[0054] This embodiment discloses a method for preparing the turmeric extract of the present invention, which is as follows:

[0055] S1. Extraction and concentration: Take 1 kg of turmeric rhizomes, crush, add 3 times the weight of 75% methanol, reflux extraction three times, each time for 3 hours, filter, combine the filtrate, concentrate under reduced pressure at 50 ° C to obtain an extract;

[0056] S2. Silica gel column separation: The extract obtained in step S1 was separated by silica gel column, and the petroleum ether / ethyl acetate solvent was gradient eluted in 10:1, 5:1, 2:1, and 1:1, and the eluate was collected at 1:1 and concentrated under reduced pressure at 50°C to obtain the silica gel column separated extract;

[0057] S3. Resin adsorption separation: The extract was separated by silica gel column on MCI adsorption resin column, followed by gradient elution with water, 20%, 40%, 60%, 80%, 100% methanol aqueous solution, and the eluate of 20%-60% methanol aqueous solution was combined and concentrated to obtain the extract separated by resin;

[0058] S4. Semi-preparative HPLC separation: The resin-separated extract obtained in step S3 is separated by semi-preparative HPLC, and the eluate is collected for 7-13 min, concentrated, and dried to obtain a turmeric extract.

[0059] The conditions of semi-preparative HPLC in this embodiment are as follows:

[0060] Instrument: Agilent 1200 series HPLC system,

[0061] Chromatographic column: Extend-C column 5μm, 4.6×150mm,

[0062] Mobile phase: 20% → 50% methanol in water, flow rate 1.0 mL / min

[0063] The column temperature was 20°C.

[0064] The detection wavelengths are 230nm and 254nm.

[0065] The content of natural drugs may vary due to different origins, harvesting times, and processing conditions. The applicant used the method of this embodiment to extract and test multiple batches of turmeric, and found that the content of bisabolol in the obtained turmeric extract was 5-10wt.%.

[0066] Example 2

[0067] This embodiment discloses a method for preparing the tsaoko essential oil of the present invention, which is as follows:

[0068] Weigh 200g of dried Amomum villosum fruit, crush it to 60-80 mesh, and then put it into an extraction kettle. The extraction temperature is 55°C, the pressure is 30MPa, the average CO2 flow rate is 20g / min, and the extraction is 1h. Take out the volatile oil extract from the separation tank I and repeat the extraction 3 times. Combine the extracts, centrifuge, let stand, add anhydrous sodium sulfate to dry, filter, and obtain the Amomum villosum essential oil.

[0069] Example 3

[0070] This example discloses a method for preparing the composition of the present invention.

[0071] Take turmeric extract and tsaoko essential oil, mix them evenly in a mass ratio of 1:1 to obtain a mixture;

[0072] Take 50 parts of the mixture, add 50 parts of xylitol, mix well, wet granulate with 80% ethanol, dry, and fill into capsules. Each capsule weighs 0.25g, and adults take orally, 4 capsules each time, 3 times a day.

[0073] Example 4

[0074] This example discloses a method for preparing the composition of the present invention.

[0075] Take turmeric extract and tsaoko essential oil, mix them evenly in a mass ratio of 1:1 to obtain a mixture;

[0076] Take 50 parts of the mixture, add 35 parts of mannitol and sorbitol, mix well, wet granulate with 80% ethanol, dry, and tablet. The obtained tablets weigh 0.3g / tablet. Adults take orally, 4 tablets each time, 3 times a day.

[0077] Example 5

[0078] This example discloses a method for preparing the composition of the present invention.

[0079] Take 100 parts of turmeric extract and 200 parts of tsaoko essential oil, mix them evenly to obtain a mixture;

[0080] Take 50 parts of the mixture, add 50 parts of xylitol, mix well, wet granulate with 80% ethanol, dry, and fill into capsules. Each capsule weighs 0.25g, and adults take orally, 4 capsules each time, 3 times a day.

[0081] Example 6

[0082] This example discloses a method for preparing the composition of the present invention.

[0083] Take 200 parts of turmeric extract and 180 parts of tsaoko essential oil, mix them evenly to obtain a mixture;

[0084] Take 50 parts of the mixture, add 50 parts of xylitol, mix well, wet granulate with 80% ethanol, dry, and fill into capsules. Each capsule weighs 0.25g, and adults take orally, 4 capsules each time, 3 times a day.

[0085] Example 7

[0086] This example discloses a method for preparing the composition of the present invention.

[0087] Take 120 parts of turmeric extract and 100 parts of tsaoko essential oil, mix them evenly to obtain a mixture;

[0088] Take 50 parts of the mixture, add 50 parts of xylitol, mix well, wet granulate with 80% ethanol, dry, and fill into capsules. Each capsule weighs 0.25g, and adults take orally, 4 capsules each time, 3 times a day.

[0089] Example 5

[0090] This example discloses a method for preparing the composition of the present invention.

[0091] Take 180 parts of turmeric extract and 120 parts of tsaoko essential oil, mix them evenly to obtain a mixture;

[0092] Take 50 parts of the mixture, add 50 parts of xylitol, mix well, wet granulate with 80% ethanol, dry, and fill into capsules. Each capsule weighs 0.25g, and adults take orally, 4 capsules each time, 3 times a day.

[0093] Test Example 1

[0094] This test example discloses a pharmacodynamic test of the composition of the present invention on reducing acute myocardial infarction in mice under NAFLD conditions.

[0095] 1 Experimental Materials

[0096] Experimental animals: 8-9 weeks old C57BL / 6J male mice weighing 20±2g were provided by Sibeifu (Beijing) Biotechnology Co., Ltd., license number SCXK (Beijing) 2019-0010. The experimental procedures were in accordance with animal ethics.

[0097] Feed: Ordinary feed (Moldiets Mole Biology, batch number: M20223002); MCD high-fat feed (Moldites Mole Biology, batch number: M10421)

[0098] Reagents: Creatine kinase MB (CK-MB), lactate dehydrogenase (LDH), triglyceride (TG, 230122), total cholesterol (TC, batch number: 221218), low-density lipoprotein (LDL, batch number: 221019), alanine aminotransferase (AST, batch number: 221115) and aspartate aminotransferase (ALT, batch number: 221110) kits were purchased from Nanjing Jiancheng Bioengineering Institute; TCC (2,3,5-triphenyltetrazolium chloride), Evans Blue dye, hematoxylin, ponceau red, and aniline blue dye were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0099] 4% paraformaldehyde tissue specimen fixative and xylene (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0100] Instruments and equipment: electronic balance (Shanghai Yueping Scientific Instrument Co., Ltd., YP1002MAX100g); electronic analytical balance (Ohaus, USA, AR1140MAX110g); full-wavelength scanning multifunctional microplate reader (Thermo Fisher Scientific, USA, VariosKan Flash); desktop high-speed refrigerated centrifuge (Eppendorf, Germany, Eppendorf-5810R); ADInstrumentspowerlab small animal ECG data acquisition system (ADInstruments, ADInstrumentspowerlab8 / 35); small animal ultrasound imaging system (VisualSonics, Vevo3100); cryostat (Meikang, Germany, HM550); small animal ventilator (Harvard Apparatus, 687); small animal anesthesia machine (NORVAP, 13238).

[0101] 2 Experimental grouping: Adaptive feeding for 1 week (temperature 23±2℃, humidity 50%-60%, 12h / 12h dark and light cycle alternating, 4 mice per cage, free drinking water and food, bedding changed every 2 days), 60 mice were randomly selected, of which 10 mice in the blank control group were fed with ordinary feed; the remaining 50 mice were fed with MCD high-fat feed. After 4 weeks, the mice fed with MCD high-fat feed showed non-alcoholic fatty liver, and they were randomly divided into 5 groups (10 in each group), the drug was dissolved in saline containing 1% DMSO, and administered by gavage for 8 weeks:

[0102] (1) Blank control group (10 rats): fed with normal feed;

[0103] (2) Sham operation group (10 rats): MCD high-fat diet + intragastric administration of 0.1 mL / 10 g / d of saline containing 1% DMSO;

[0104] (3) Model group (10 rats): MCD high-fat diet + intragastric administration of 0.1 mL / 10 g / d of saline containing 1% DMSO;

[0105] (4) Dai medicine combination group (10 rats): MCD high-fat diet + oral administration of Dai medicine combination solution 200 μg / 10 g / d (dissolved in physiological saline containing 1% DMSO);

[0106] (5) Turmeric extract group (10 mice): MCD high-fat diet + intragastric administration of 200 μg / 10 g / d of turmeric extract solution (dissolved in saline containing 1% DMSO);

[0107] (6) Amomum villosum extract group (10 mice): MCD high-fat diet + oral administration of 200 μg / 10 g / d of Amomum villosum extract solution (dissolved in physiological saline containing 1% DMSO);

[0108] The turmeric extract of this test example is the same batch of turmeric extract prepared according to the method of Example 1.

[0109] The tsaoko essential oil in this test example is the same batch of tsaoko essential oil prepared according to the method of Example 2.

[0110] The Dai medicine composition solution of this test example is prepared by mixing turmeric extract and tsaoko essential oil (mass ratio 1:1) evenly and dissolving them in physiological saline containing 1% DMSO.

[0111] The turmeric extract solution of this test example was prepared by dissolving the turmeric extract in physiological saline containing 1% DMSO.

[0112] The amomum villosum extract solution of this test example is prepared by dissolving amomum villosum essential oil in physiological saline containing 1% DMSO.

[0113] 3 Experimental methods and detection indicators

[0114] 3.1 Acute myocardial infarction under NAFLD conditions: At the end of the 13th week, 30 g / L sodium pentobarbital was injected intraperitoneally for anesthesia. After fixation in the supine position, the animals were connected to an electrocardiogram monitor, endotracheal intubation and small animal ventilator. A longitudinal incision was made at 0.5 cm from the left sternal edge, and the third to fifth ribs were cut to fully expose the heart. After the heart was balanced for 30 minutes, the anterior descending coronary artery was ligated with a 5 / 0 line. The coronary artery occlusion induced acute myocardial ischemia. The ST segment elevation under the II lead of the electrocardiogram indicated successful ischemia. The sham operation group only threaded the line without ligation. After 30 minutes of ischemia, the apex of the heart turned white or cyanotic to the naked eye. The size of the myocardial infarction area is the most direct observation indicator for evaluating the effect of the drug. The mouse heart sections were stained with Evans blue and TTC double staining method and the myocardial infarction rate was calculated.

[0115] 3.2 Assessment of myocardial infarction area (TTC+Evans Blue staining): The left coronary artery was re-ligated, the aortic arch was separated, and the Evans Blue dye solution was injected into the heart from the aortic arch. The heart was removed and quickly frozen with dry ice. The heart was frozen and sectioned. The whole heart was evenly cut into 5 slices, each slice was about 1 mm thick, and the heart slices were placed in 37°C TTC dye solution for staining. The staining time was 3 minutes. After staining, the heart slices were placed in formalin and images were collected 24 hours later. In the stained heart slices, the non-infarction area myocardial tissue was blue, the area at risk (AAR) was red, and the grayish white was the infarction area (I). After collecting the image, the Image-Pro Plus image analysis software was used to calculate the area of ​​each part. The ratio of the infarction area (I) to the area at risk (AAR) was the percentage of myocardial infarction area. Four mice in each group were selected for HE staining to observe the changes in myocardial tissue structure.

[0116] 3.3 Ultrasound detection of left ventricular function: Remove the hair on the chest wall and upper abdomen with depilatory cream, lie supine on the ultrasound machine, and fix the mouse. Take the parasternal left ventricular long axis inflow tract section, and slightly tilt the probe to the left side of the mouse's chest to explore, so that the left atrium and ventricular inflow tract can be clearly seen, and the left ventricular width and length are both maximized, which is ideal, to obtain a B-mode ultrasound image, and then obtain an M-mode ultrasound image at the level of the mitral chordae tendineae. Measure heart rate (HR), cardiac output (CO), left ventricular mass (LV Mass), left ventricular volume at end diastole (LVVold), left ventricular volume at end systole (LV Vols), left ventricular ejection fraction (EF%), and fractional shortening (FS%) data.

[0117] 3.4 Changes in liver tissue structure (HE staining, Masson staining): Take a 3mm thick liver tissue sample, dehydrate with gradient alcohol 70%, 80%, 95%, 100% for 30 minutes each, two bottles of xylene for 20 minutes each, and two cylinders of paraffin wax for 30 minutes each, embed, slice 4μm, and bake the slices to complete the preparation. Then use xylene, hematoxylin and other dyes for HE staining; use hematoxylin, ponceau red, aniline blue and other dyes for Masson staining, and finally use neutral gum to seal the slices to complete the staining. After staining, perform a panoramic scan.

[0118] 3.5 Changes in serum biochemical indices of mice: Blood was collected by enucleation and placed in a 4°C refrigerator for 2 h, then at room temperature for 1-2 h, and then centrifuged at 3000 r / min for 10 min. The supernatant was aspirated and divided into 1.5 mL centrifuge tubes. Part of the serum was taken and used to detect the levels of lactate dehydrogenase (LDH) and creatine phosphatase (CK-MB), as well as triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein (LDL) using an automatic biochemical analyzer.

[0119] 4 Statistical analysis

[0120] GraphPad software and SPSS17.0 software were used for statistical analysis. The data of each group were expressed as mean±standard deviation (±SD). One-way analysis of variance was used to compare multiple groups of data, and t-test was used to compare between two groups. P<0.05 was considered to be statistically significant.

[0121] 5 Experimental results

[0122] 5.1 Effects of Dai medicine combination on non-alcoholic fatty liver disease (NAFLD) related indicators

[0123] (1) Changes in liver tissue structure: Figure 2 As shown in the figure, the liver structure of mice in the normal control group was clear and there was no obvious lesion. Compared with the normal control group, the liver cells of mice in the model group showed vacuolation, watery degeneration, and mild fibrosis and inflammatory infiltration. Compared with the model group, the vacuolation, fibrosis and inflammatory infiltration of liver cells in the Dai medicine composition group and the turmeric extract group were reduced, and the activity of the Dai medicine composition group was better.

[0124] (2) Changes in serum biochemical indicators of mice: Figure 3As shown in the results, total cholesterol (TC), serum triglycerides (TG), and low-density lipoprotein (LDL) are indispensable detection indicators for studying lipid metabolism. Compared with the model group, the Dai medicine composition group can reduce total cholesterol (TC), serum triglycerides (TG), and low-density lipoprotein (LDL) in mouse serum, especially the reduction of LDL content is the most obvious. The experimental results suggest that the Dai medicine composition can improve the dyslipidemia of mice caused by non-alcoholic fatty liver disease, and its activity is better than that of turmeric extract and grass fruit extract.

[0125] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are biochemical indicators that reflect liver cell damage. If the liver is damaged, the transaminase in the liver cells will enter the blood, and the levels of ALT and AST in the blood will increase. Compared with the model group, the Dai medicine combination group can reduce the AST and ALT levels. The experimental results suggest that the Dai medicine combination group can alleviate liver damage caused by non-alcoholic fatty liver disease, and its activity is better than that of turmeric extract and cardamom extract.

[0126] 5.2 Dai medicine combination improves acute myocardial infarction under NAFLD conditions

[0127] (1) The results of left heart ultrasound examination are shown in Table 1. Heart rate (HR) and cardiac output (CO) reflect cardiac function; left ventricular weight (LV Mass) is used to evaluate the degree of myocardial hypertrophy; left ventricular volume at end diastole (LV Vold), left ventricular volume at end systole (LVVols), left ventricular ejection fraction (EF%) and fractional shortening (FS%) reflect the function of the heart, especially the left ventricle. Compared with the model group, the Dai medicine composition of the present invention can slow down the increase in heart rate under NAFLD conditions, increase cardiac output, inhibit left ventricular remodeling (hypertrophy and dilatation), and improve cardiac function, and its activity is better than that of Amomum villosum extract and turmeric extract.

[0128] Table 1. Left ventricular remodeling under conditions of improving NAFLD

[0129]

[0130] In Table 1, △ P<0.05, *P<0.01, n=10.

[0131] (2) Myocardial infarction area assessment (TTC + Evans Blue staining) results: as shown in the attached Figure 4 As shown, the Dai medicine composition can significantly reduce the area of ​​acute myocardial infarction under NAFLD conditions. Compared with the myocardial infarction rate of the model group (59.37%), the myocardial infarction rate of the Dai medicine composition group (24.18%) decreased by 35.19%, and its activity was better than that of turmeric extract and amomum villosum extract.

[0132] (3) Changes in CK and LDH levels in mouse serum: Figure 5 As shown in the figure, CK and LDH are enzymes commonly present in the plasma of myocardial cells, called myocardial enzymes. When myocardial cells are damaged or necrotic, they will be released into the blood in large quantities. Elevated serum myocardial enzymes are laboratory indicators for evaluating and diagnosing myocardial infarction and the degree of myocardial cell damage. Compared with the model group, the Dai medicine composition can reduce the levels of LDH and CK in mouse plasma, alleviate myocardial cell damage caused by acute myocardial infarction under NAFLD conditions, and its activity is better than that of turmeric extract and tsaoko extract.

[0133] The Dai medicine composition of the present invention has a good protective effect on acute myocardial infarction in mice under non-alcoholic fatty liver disease (NAFLD) conditions. It not only improves liver steatosis, fibrosis and inflammation caused by NAFLD, but also inhibits left ventricular remodeling, improves cardiac function, and reduces the occurrence of myocardial infarction and myocardial cell damage. It can be used as a medicine to prevent or treat acute myocardial infarction under non-alcoholic fatty liver disease (NAFLD) conditions.

[0134] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that have no substantial significance on the main design concept and spirit of the present invention, which solve technical problems consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields should also be included in the patent protection scope of the present invention.

Claims

1. A Dai medicine composition, characterized in that: The invention comprises the following raw materials in parts by weight: 10-20 parts of turmeric extract and 10-20 parts of tsaoko essential oil. The turmeric extract is an alcohol extract of turmeric, and the content of sweet bisabolol in the turmeric extract is 5-10wt.%.

2. A Dai medicine composition according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 12-18 parts of turmeric extract and 12-18 parts of tsaoko essential oil.

3. A Dai medicine composition according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 10 parts of turmeric extract and 10 parts of tsaoko essential oil.

4. A Dai medicine composition according to any one of claims 1 to 3, characterized in that: The preparation method of the turmeric extract comprises the following steps: S1. Extraction and concentration: Take turmeric rhizomes, add alcohol solution to extract, and concentrate the extract to obtain an extract; S2. Silica gel column separation: The extract obtained in step S1 was separated by a silica gel column, and gradient elution was performed with a petroleum ether / ethyl acetate solvent of 8 to 12:1, 3 to 7:1, 1.5 to 3:1, and 0.5 to 1.2:1, and the eluate of 0.5 to 1.2:1 was collected and concentrated to obtain a concentrate; S3. Resin adsorption separation: The concentrated solution obtained in step S2 was applied to an MCI adsorption resin column, and gradient eluted with water, 15-25%, 35-45%, 55-65%, 75-85%, and 95-100% aqueous methanol solution, and the eluate of 15-65% aqueous methanol solution was collected and concentrated to obtain the resin-separated components; S4 semi-preparative HPLC separation: The resin separation fraction obtained in step S3 was separated by semi-preparative HPLC, and the eluate containing bisabolol turmeric alcohol was collected, concentrated, and dried to obtain a turmeric extract; Preferably, in step S1, methanol-water solution is used for reflux extraction; further preferably, the concentration of methanol-water solution is 50-90%; Preferably, in step S2, gradient elution is performed with petroleum ether / ethyl acetate solvents in a ratio of 10:1, 5:1, 2:1, and 1:1, and the 1:1 eluate is collected; Preferably, in step S3, gradient elution is performed with water, 20%, 40%, 60%, 80%, and 100% methanol aqueous solution in sequence, and the eluate of 20%-60% methanol aqueous solution is collected; Preferably, in step S4, the chromatographic conditions of semi-preparative HPLC are as follows: octadecylsilane bonded silica gel is used as filler, methanol is used as mobile phase A, and water is used as mobile phase B for gradient elution; the gradient elution procedure is as follows: 0min, 15-25% methanol, 75-85% water; 15min, 40-60% methanol, 40-60% water; Preferably, 0 min, 20% methanol, 80% water; 15 min, 50% methanol, 50% water; Preferably, the mobile phase flow rate is 0.8 to 1.2 mL / min, and the column temperature is 15 to 25°C, preferably 20°C; More preferably, the eluate is collected from 0.5 to 2 minutes before the elution of bisabolol; and the collection of the eluate is stopped when the relative retention time is twice the elution time of bisabolol. More preferably, the detection wavelength is 230 nm and / or 254 nm.

5. A Dai medicine composition according to any one of claims 1 to 3, characterized in that: The tsaoko essential oil is obtained by CO2 supercritical extraction; Preferably, the preparation method of tsaoko essential oil comprises the following steps: weighing tsaoko powder, loading into an extraction kettle, extracting at a temperature of 50 to 60°C, a pressure of 25 to 35 MPa, an average CO2 flow rate of 15 to 25 g / min, and an extraction time of 1 to 1.5 h; More preferably, the extraction temperature is 55°C, the pressure is 30 MPa, the average CO2 flow rate is 20 g / min, and the extraction time is 1.5 h; Preferably, the extraction times are 1 to 3 times.

6. A Dai medicine composition according to any one of claims 1 to 3, characterized in that: The composition is a preparation prepared by conventional methods using turmeric extract and tsaoko essential oil as raw materials and adding pharmaceutically acceptable excipients; Preferably, the preparation is an oral preparation.

7. The method for preparing the Dai medicine composition according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: preparing turmeric extract and tsaoko essential oil in proportion, adding pharmaceutically acceptable auxiliary materials, mixing evenly, granulating, and filling capsules to obtain capsules; Preferably, the total amount of turmeric extract and tsaoko essential oil is 20-50 parts, and the auxiliary material is 70-50 parts; Further preferably, the excipients used to prepare the capsules are selected from any two or three of xylitol, mannitol, sorbitol, lacto-oligosaccharides, fructo-oligosaccharides, isomalt and erythritol.

8. The method for preparing the Dai medicine composition according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: preparing turmeric extract and tsaoko essential oil in proportion, adding pharmaceutically acceptable auxiliary materials, mixing evenly, granulating, and tableting to obtain tablets; Preferably, the total amount of turmeric extract and tsaoko essential oil is 20-50 parts, and the auxiliary material is 70-50 parts; Further preferably, the excipients used to prepare the capsules are selected from any two or three of xylitol, mannitol, sorbitol, lacto-oligosaccharides, fructo-oligosaccharides, isomalt and erythritol.

9. Use of the Dai medicine composition according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating acute myocardial infarction; preferably, the acute myocardial infarction includes acute myocardial infarction under the condition of non-alcoholic fatty liver.

10. Use of the Dai medicine composition according to any one of claims 1 to 6 in the preparation of a medicament for alleviating liver damage caused by non-alcoholic fatty liver disease.

Images