Dai medicine composition containing sweet myrrh curcumol as well as preparation method and application of Dai medicine composition

Abstract

The invention discloses a Dai medicine composition containing sweet myrrh curcumol as well as a preparation method and application of the Dai medicine composition, and belongs to the technical field of medicines. The Dai medicine composition disclosed by the invention is prepared from sweet myrrh curcumol, a fructus viticis extract and a pepper extract, wherein the mass ratio of the sweet myrrh curcumol to the fructus viticis extract to the pepper extract is (1 to 49.5 to 49.5) to (10 to 45 to 45); the fructus viticis extract is obtained by taking fructus viticis as a raw material through carbon dioxide supercritical extraction; the pepper extract is obtained by taking pepper as a raw material through carbon dioxide supercritical extraction. The invention discloses application of the Dai medicine composition in preparation of a medicine for preventing or / and treating acute myocardial infarction. According to the invention, the lipid metabolism disorder of mice caused by NAFLD can be improved, and the acute myocardial infarction area of mice under the condition of NAFLD can be reduced. The raw materials are natural in source, the formula and preparation are simple, the chemical structures of the main components are clear, quality control is facilitated, the application range of the Dai medicine is expanded, and the application value of the Dai medicine is improved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, specifically relates to the field of Dai medicine technology, and in particular relates to a Dai medicine composition containing bisabolol and curcuminol, and a preparation method and application thereof. Background Art

[0002] Dai medicine is an important branch of traditional Chinese medicine, mainly spread in Yunnan Province, China. Dai medicine believes that the human body is composed of four towers: "wind, fire, water, and earth". These four towers interact with each other to maintain the balance of the human body. "Yajie" is the Dai language YaGei. In Dai language, "Ya" refers to the state of illness or discomfort; "jie" means to relieve or treat. "Yajie" is translated as antidote. "Yajie theory" is a high-level summary of Dai medicine's preventive thinking. The treatment principles and medication methods emphasize "relieve before illness, resolve first and then treat, treat first and then resolve, and use both resolve and treat together". The Yajie 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, "poison" will continue to accumulate until the "critical balance" is broken, and it will be manifested externally in some / 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 "four towers balance" of the human body, Dai medicine often uses drugs to remove liver "toxins", restore the liver's "detoxification" function, improve liver dysfunction, and thus prevent and treat extrahepatic diseases.

[0003] The liver-heart axis theory proposed by modern medicine supports the main viewpoints of the "Yajie theory" and provides a new overall dynamic research perspective for the study of Yajie drugs to improve liver dysfunction and prevent and treat extrahepatic diseases. The liver-heart axis theory believes that there is an interaction between the liver and the heart, and their dysfunctions also affect each other.

[0004] In recent years, the incidence and prevalence of non-alcoholic fatty liver disease (NAFLD) have been increasing year by year, affecting about 25% of the world's population. More and more studies have shown that there is a strong positive correlation between NAFLD and the main adverse event of cardiovascular disease: ischemic heart disease (IHD). IHD is the leading cause of death worldwide and the most common cause of death in patients with NAFLD. Several prospective or retrospective studies have shown that compared with non-NAFLD populations, NAFLD patients have a 64% higher risk of cardiovascular disease (including fatal and non-fatal), and the more severe the NAFLD, the higher the incidence of cardiovascular events. This positive correlation increases with the worsening of the severity of NAFLD. As two important global public health diseases, the devastating impact of NAFLD on IHD events is becoming increasingly apparent, not only bringing greater pressure to the public health system, but also further deepening the economic and psychological burden on patients. In addition to weight control, finding active ingredients from multi-target ethnic medicines for adjuvant therapy is a common research strategy to reduce the public health threats related to the "liver-heart axis".

[0005] Purpose of the Invention

[0006] One of the purposes of the present invention is to provide a Dai medicine composition containing bisabolol and curcuminol, which can be used to prevent or treat acute myocardial infarction under the condition of non-alcoholic fatty liver disease (NAFLD).

[0007] The second object of the present invention is to provide a method for preparing the Dai medicine composition.

[0008] The third object of the present invention is to provide an application of the Dai medicine composition.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The invention discloses a Dai medicine composition containing bisabolol curcumin, which comprises bisabolol curcumin, vitex rotundifolia extract and pepper extract, wherein the mass ratio of the three is 1:49.5:49.5-10:45:45;

[0011] The Vitex rotundifolia extract is obtained by using Vitex rotundifolia as a raw material and supercritical extraction with carbon dioxide;

[0012] The pepper extract is obtained by taking pepper as raw material and extracting it through supercritical carbon dioxide.

[0013] In some embodiments of the present invention, the purity of the bisabolol curcumin is greater than or equal to 98%.

[0014] In some embodiments of the present invention, the bisabolol curcumin is obtained by extraction and purification using turmeric as a raw material.

[0015] In some embodiments of the present invention, the method for preparing the Vitex rapa extract comprises the following steps:

[0016] Weigh Vitex rotundifolia seeds, put them into an extraction kettle, first perform static extraction for 20-30 minutes, then release the pressure and perform dynamic extraction for 30-120 minutes, preferably 60 minutes;

[0017] Preferably, the static extraction conditions of Vitex rotundifolia are: temperature 30-35°C, pressure 25-30MPa;

[0018] Preferably, the dynamic extraction conditions of Vitex rotundifolia are: temperature 30-40°C, pressure 15-20MPa, CO2 flow rate 60-100L / h, more preferably 80L / h;

[0019] After the extraction is completed, centrifuge and take the supernatant to obtain the Vitex rotundifolia extract.

[0020] In some embodiments of the present invention, the method for preparing the pepper extract comprises the following steps:

[0021] Weigh pepper and put it into an extraction kettle. First, perform static extraction for 20-30 minutes, then release the pressure and perform dynamic extraction for 30-120 minutes, preferably 60 minutes.

[0022] Preferably, the static extraction conditions of pepper are: temperature 35-40°C, pressure 30-35MPa;

[0023] Preferably, the dynamic extraction conditions of pepper are: temperature 40-45°C, pressure 20-25MPa, CO2 flow rate 60-100L / h, more preferably 80L / h;

[0024] After the extraction is completed, centrifuge and take the supernatant to obtain the pepper extract.

[0025] In some embodiments of the present invention, the composition is a preparation prepared by using sweet bisabolol curcumin, vitex rotundifolia extract and pepper extract as raw materials, adding pharmaceutically acceptable excipients and following conventional methods;

[0026] Preferably, the preparation is an oral preparation.

[0027] The preparation method of the Dai medicine composition containing bisabolol and curcumin disclosed in the present invention comprises the following steps:

[0028] Prepare sweet bisabolol, vitex rotundifolia extract and pepper extract according to weight proportions, mix them evenly, add pharmaceutically acceptable excipients, mix them evenly, granulate them, and fill them into capsules to obtain capsules;

[0029] Preferably, the total amount of sweet bisabolol, vitex rotundifolia extract and pepper extract is 20 to 50 parts, and the auxiliary material is 50 to 70 parts;

[0030] Further preferably, the excipients used to prepare the capsule are selected from any two or three of xylitol, mannitol, sorbitol, lacto-oligosaccharides, fructo-oligosaccharides, isomalt, and erythritol.

[0031] In some embodiments of the present invention, the steps include: preparing sweet bisabolol, vitex rotundifolia extract and pepper extract in parts by weight, mixing them evenly, adding pharmaceutically acceptable excipients, mixing them evenly, granulating, and tableting to obtain tablets;

[0032] Preferably, the total amount of turmeric extract and tsaoko essential oil is 20-50 parts, and the auxiliary material is 70-50 parts;

[0033] Preferably, the total amount of sweet bisabolol, vitex rotundifolia extract and pepper extract is 20 to 50 parts, and the auxiliary material is 50 to 70 parts;

[0034] More 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.

[0035] The invention discloses an application of a Dai medicine composition containing bisabolol and curcuminol in preparing a medicine 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.

[0036] In some embodiments of the present invention, the Dai medicine composition containing bisabolol and curcumin is used in the preparation of a drug for alleviating liver damage caused by non-alcoholic fatty liver disease.

[0037] The structural formula of Bisacurone described in the present invention is as follows:

[0038]

[0039] The preparation method of the sweet bisabolol curcumin of the present invention is a prior art and can be prepared by referring to the method recorded in patent CN114736182A.

[0040] In some embodiments of the present invention, the method for preparing bisabolol curcumin comprises the following steps:

[0041] S1. Extraction and concentration: Take turmeric rhizomes, add alcohol solution to extract, and concentrate the extract to obtain an extract;

[0042] S2. Silica gel column separation: The extract obtained in step S1 was separated by a silica gel column, and gradient eluted with petroleum ether / ethyl acetate solvents of 8 to 12:1, 3 to 7:1, 1.5 to 3:1, and 0.5 to 1.2:1, respectively, collecting the eluate of 0.5 to 1.2:1, and concentrating to obtain a concentrate;

[0043] 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;

[0044] 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;

[0045] S5. Apply the turmeric extract to a gel column, elute with methanol, collect the eluate, and dry it to obtain bisabolol with a purity greater than or equal to 98%.

[0046] 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;

[0047] 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;

[0048] 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;

[0049] 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:

[0050] 0-8min, 20% methanol, 80% water;

[0051] 8-13min, 20% methanol in water → 100% methanol;

[0052] 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;

[0053] 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.

[0054] Preferably, the eluate is collected for 7 to 9 minutes.

[0055] 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.

[0056] The Vitex roxburghii described in the invention is the fruit of the Dai medicine tube bottom, that is, the dried mature fruit of the Verbenaceae plant Vitextrifolia var.simplicifolia Cham. or Vitex trifolia L.

[0057] The pepper described in the present invention is the fruit of the Dai medicine Piper nigrum, that is, the dried nearly mature or mature fruit of the pepper Piper nigrum L. of the Piperaceae family.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The present invention obtains a myrrhane-type sesquiterpenoid compound Bisacurone from turmeric rhizomes, and uses the compound in combination with a Dai medicine Guandi fruit (Vitex rotundifolia) extract and a Pinan fruit (Piper nigrum) extract according to the Yajie theory. The Guandi fruit has a light taste, bitter and cool nature; it enters the wind and water towers, clears heat and detoxifies, relieves swelling and relieves pain, removes dampness and relieves itching, and strengthens tendons and bones. The Pinan fruit has a smelly taste, spicy and hot nature; it enters the fire tower, removes toxins and strengthens the stomach, and relieves adverse reactions and stops vomiting. The three medicines are used together to match cold and heat, and to promote astringency and smoothness, and together play the functions of balancing the four towers, clearing heat and detoxifying, promoting stagnant qi, and activating qi and blood. The present invention can improve lipid metabolism disorders in mice caused by NAFLD, and reduce the area of ​​acute myocardial infarction in mice under NAFLD conditions. The raw materials of the present invention are natural, the formula and preparation are simple, and the chemical structure of the main components is clear, which is conducive to quality control, expands the application scope of Yajie Dai medicines, and improves the application value of Yajie Dai medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Attached Figure 1 The present invention is a flow chart of the preparation of bisabolol of Example 1.

[0061] Attached Figure 2 The results of TC, TG and LDL content in the serum of each group of mice are shown in Figure 2, where *P<0.05, **P<0.01.

[0062] Attached Figure 3 This is a principal component analysis diagram. PC1 represents the first principal component, PC2 represents the second principal component, and the percentage represents the explanation rate of the principal component to the data set. Each point in the diagram represents a sample. Samples in the same group are represented by the same color. Group represents the group. NC is the normal control group; MI is the model group; CL is the Dai medicine combination group.

[0063] Attached Figure 4 This is the OPLS-DA score graph. The horizontal axis represents the predicted principal component. The horizontal axis direction shows the gap between groups; the vertical axis represents the orthogonal principal component. The vertical axis direction shows the gap within the group. The percentage represents the explanation rate of the component for the data set. Each point in the figure represents a sample. Samples in the same group are represented by the same color. Group is the grouping. NC is the normal control group; MI is the model group; CL is the Dai medicine combination group.

[0064] Attached Figure 5 This is a cluster heat map of differential lipid content. The horizontal direction is the sample name, the vertical direction is the differential lipid information, Group is the grouping, and different colors are the colors filled with different values ​​obtained after different relative content standardization (red represents high content, green represents low content). NC is the normal control group; MI is the model group; CL is the Dai medicine combination group.

[0065] Attached Figure 6 This is the KEGG metabolism and signaling pathway diagram of differential sphingolipid components. Red indicates that the lipid content is significantly upregulated in the experimental group, blue indicates that the lipid is detected but no significant change occurs, and green indicates that the lipid content is significantly downregulated in the experimental group.

[0066] Attached Figure 7 It is the KEGG enrichment map of differential lipids; the horizontal axis represents the Rich Factor corresponding to each pathway; the vertical axis is the pathway name (sorted by P-value); the color of the point reflects the size of the P-value, the redder the point, the more significant the enrichment; the size of the point represents the number of enriched differential lipids.

[0067] Attached Figure 8 The results of TTC+Evans Blue staining of the hearts of mice in each group and the myocardial infarction rate of each group are shown, where *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0068] 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.

[0069] Unless otherwise specified, the "parts" described in the embodiments of the present invention refer to parts by mass.

[0070] Example 1

[0071] This embodiment discloses the preparation method of sweet bisabolol of the present invention, and its process flow is as shown in the attached Figure 1 As shown, the specific steps are as follows:

[0072] Take the dried rhizome of turmeric, crush it, add 3 times the weight of 75% methanol, and reflux extract three times (3h / time). Combine the filtrate and concentrate under reduced pressure at 50℃. The concentrated extract is separated by silica gel column and gradient eluted with petroleum ether / ethyl acetate (10:1, 5:1, 2:1, 1:1). Use GF 254 Each fraction was detected by thin layer chromatography on a silica gel plate, and the fractions at the 1:1 elution position were combined and concentrated under reduced pressure at 50°C, and then separated again using an MCI adsorption resin column, and eluted with distilled water and methanol in a gradient manner (methanol 0% → 20% → 40% → 60% → 80% → 100%). 254 The fractions were subjected to thin layer chromatography on silica gel plates, and the fractions eluted with 40% methanol were combined, concentrated under reduced pressure at 50° C., and then separated by semi-preparative HPLC (Agilent 1200 series HPLC system, Extend-C18 column 5 μm, id4.6×150 mm, flow rate 1.0 mL / min, gradient elution program 20% methanol aqueous solution (0-8 min), 20% methanol aqueous solution→100% methanol (8-13 min), column temperature 20° C., wavelength 230 nm and 254 nm), and the effluent corresponding to the chromatographic peak with the largest response value was collected, and then loaded onto a Sephadex LH-20 gel column, eluted with methanol, and the eluate was collected. The fractions were concentrated under reduced pressure at 50° C. and dried. The product was confirmed to be Bisacurone by structure, and its purity was greater than 98%.

[0073] Example 2

[0074] This embodiment discloses a method for preparing the Vitex rotundifolia extract of the present invention, which is as follows:

[0075] Weigh 100 g of dried Vitex rotundifolia, dry at 45° C., crush to 40-60 mesh, and then load into an extraction kettle, perform static extraction for 30 min (temperature 30-35° C., pressure 25-30 MPa), then release the pressure and perform dynamic extraction for 60 min (temperature 30-40° C., pressure 15-20 MPa, CO2 flow rate 80 L / h), release the extract from the separation kettle, centrifuge at 1000 rpm for 5 min, add anhydrous sodium sulfate to dry, and filter to obtain Vitex rotundifolia extract.

[0076] Example 3

[0077] The invention relates to a method for preparing a pepper extract. The method comprises the following steps: weighing 100 g of dried pepper, drying the pepper extract at 45° C., crushing the pepper extract to 40-60 mesh, and then loading the crushed pepper extract into an extraction kettle. The crushed pepper extract is subjected to static extraction for 30 min (temperature 35-40° C., pressure 30-35 MPa), and then releasing the pressure to perform dynamic extraction for 60 min (temperature 40-45° C., pressure 20-25 MPa, and CO2 flow rate 80 L / h). The extract is discharged from the separation kettle, centrifuged at 1000 rpm for 5 min, dried by adding anhydrous sodium sulfate, and filtered to obtain a pepper extract.

[0078] Example 4

[0079] This embodiment discloses a method for preparing the composition of the present invention, which is as follows:

[0080] Take sweet bisabolol, vitex rotundifolia extract and pepper extract, and mix them evenly in a mass ratio of 1:49.5:49.5 to obtain a mixed raw material.

[0081] Take 20 parts of mixed raw materials, 25 parts of xylitol and 25 parts of oligolactose, mix them evenly, wet granulate them with 80% ethanol, dry them, and fill them into capsules. Each capsule weighs 0.25g, and adults take it orally, 1 capsule each time, 3 times a day.

[0082] Example 5

[0083] This embodiment discloses a method for preparing the composition of the present invention, which is as follows:

[0084] Take sweet bisabolol, vitex rotundifolia extract and pepper extract, and mix them evenly in a mass ratio of 10:45:45 to obtain a mixed raw material.

[0085] Take 20 parts of mixed raw materials, 25 parts of xylitol and 25 parts of erythritol, mix them evenly, wet granulate them with 80% ethanol, dry them, and fill them into capsules. Each capsule weighs 0.25g, and adults take it orally, 1 capsule each time, 3 times a day.

[0086] Example 6

[0087] This embodiment discloses a method for preparing the composition of the present invention, which is as follows:

[0088] Take sweet bisabolol, vitex rotundifolia extract and pepper extract, and mix them evenly in a mass ratio of 5:47.5:47.5 to obtain a mixed raw material.

[0089] Take 50 parts of the mixed raw materials, 35 parts of oligofructose and 35 parts of isomalt, mix them evenly, wet granulate them with 80% ethanol, dry them, and fill them into capsules. Each capsule weighs 0.25g, and adults take it orally, 1 capsule each time, 3 times a day.

[0090] Example 7

[0091] This embodiment discloses a method for preparing the composition of the present invention, which is as follows:

[0092] Take sweet bisabolol, vitex rotundifolia extract and pepper extract, and mix them evenly in a mass ratio of 5:45.5:49.5 to obtain a mixed raw material.

[0093] Take 20 parts of the mixed raw materials, 25 parts of mannitol and 25 parts of sorbitol, mix them evenly, wet granulate them with 80% ethanol, dry them, and press them into tablets. Each tablet weighs 0.25 g. Adults can take it orally, 1 tablet each time, 3 times a day.

[0094] Example 8

[0095] This embodiment discloses a method for preparing the composition of the present invention, which is as follows:

[0096] Take sweet bisabolol, vitex rotundifolia extract and pepper extract, and mix them evenly in a mass ratio of 8:47:45 to obtain a mixed raw material.

[0097] Take 30 parts of the mixed raw materials, 25 parts of mannitol and 25 parts of sorbitol, mix them evenly, wet granulate them with 80% ethanol, dry them, and tablet them. Each tablet weighs 0.22 g. Adults can take it orally, 1 tablet each time, 3 times a day.

[0098] Test Example 1

[0099] This test example discloses an experiment on the effect of the composition of the present invention on improving acute myocardial infarction in mice under NAFLD conditions.

[0100] 1 Experimental Materials

[0101] 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.

[0102] Feed: Ordinary feed (Moldies, batch number: M20223002); MCD high-fat feed (Moldies, batch number: M10421)

[0103] Reagents: Triglyceride (TG, 230122), total cholesterol (TC, batch number: 221218), and low-density lipoprotein (LDL, batch number: 221019) kits were purchased from Nanjing Jiancheng Bioengineering Institute; TCC (2,3,5-triphenyltetrazolium chloride) and Evans Blue stain (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0104] 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, USA, VariosKan Flash); desktop high-speed refrigerated centrifuge (Eppendorf, Germany, Eppendorf-5810R); cryo-tome (HM550, Germany Mecon); small animal ventilator (Harvard Apparatus, 687); small animal anesthesia machine (NORVAP, 13238); ultra-high performance liquid chromatography tandem mass spectrometry (Sciex, the Netherlands, ExionLC AD UPLC–QTRAP); C30 chromatographic column (Thermo Accucore, id2.1x100 mm, 2.6μm);

[0105] 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, 10 mice in the normal control group were fed with ordinary feed; the remaining 50 mice were fed with MCD high-fat feed. After 4 weeks, the MCD high-fat feed group was randomly divided into 5 groups and gavage was administered for 8 weeks:

[0106] (1) Normal control group (10 rats): fed with normal feed;

[0107] (2) Sham operation group (10 rats): MCD high-fat diet + intragastric administration of 0.1 mL / 10 g / d of normal saline containing 1% DMSO;

[0108] (3) Model group (10 rats): MCD high-fat diet + intragastric administration of 0.1 mL / 10 g / d of saline containing 1% DMSO;

[0109] (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);

[0110] (5) Bisacurone group (10 mice): MCD high-fat diet + oral administration of 10 μg / 10 g / d of Bisacurone solution (dissolved in saline containing 1% DMSO);

[0111] (6) Vitex rotundifolia and Piper nigrum extract group (10 mice): MCD high-fat diet + intragastric administration of a mixture of Vitex rotundifolia and Piper nigrum extract (mass ratio 1:1) 200 μg / 10 g / d (dissolved in saline containing 1% DMSO);

[0112] The Dai medicine composition of this test example is prepared by uniformly mixing sweet bisabolol, vitex rotundifolia extract, and pepper extract in a mass ratio of 1:47.5:47.5.

[0113] The Bisacurone of this test example was prepared according to the method of Example 1; the Vitex rotundifolia extract of this test example was prepared according to the method of Example 2; and the Piper nigrum extract of this test example was prepared according to the method of Example 3.

[0114] 3 Experimental methods and detection indicators

[0115] 3.1 Acute myocardial infarction in mice 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 mice were connected to an electrocardiogram monitor, endotracheal intubation and connected to a small animal ventilator. A longitudinal incision was made 0.5 cm at the edge of the left sternum, and the third to fifth ribs were cut open 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 ST segment elevation under lead II of the electrocardiogram indicated successful ischemia. After 30 minutes of ischemia, the apex of the heart turned white or cyanotic. The myocardial infarction rate was calculated by double staining with Evans blue and TTC.

[0116] 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 sliced. 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 area of ​​each part was calculated using Image-Pro Plus image analysis software. The ratio of the area of ​​the infarction area (I) to the area at risk (AAR) was the percentage of myocardial infarction area.

[0117] 3.3 Changes in serum biochemical indicators of mice: Blood was collected by enucleation, and the collected blood was placed in a 4°C refrigerator for 2 hours, then placed at room temperature for 1-2 hours, and then centrifuged at 3000r / min for 10 minutes. The supernatant was aspirated and divided into 1.5mL centrifuge tubes and stored at -80°C. Part of the serum was taken and the contents 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) were detected using an automatic biochemical analyzer.

[0118] 3.4 Statistical analysis of animal experiments

[0119] GraphPad software and Prism 8.0 statistical software were used for statistical analysis. The data of each group were expressed as mean±SD. ANOVA was used for comparison of multiple groups, and t-test was used for comparison between two groups. p<0.05 was considered to be statistically significant.

[0120] 3.5 Serum broad targeted lipidome data detection: Six samples of mouse sera obtained according to the method in 3.3 were randomly selected from the normal control group, model group and Dai medicine combination group for broad targeted lipidome detection. Take 50 μL of each serum and add 1 mL of lipid extract (methyl tert-butyl ether: methanol = 3:1, V / V) containing a mixed internal standard (12:0Lyso PC: Cer (d18:1 / 4:0): PC (13:0 / 13:0) = 1:1:1), and vortex for 15 min; add 200 μL of water, vortex for 1 min, and centrifuge at 12000 r / min for 10 min at 4°C; after centrifugation, transfer 200 μL of the supernatant to the corresponding numbered centrifuge tube and concentrate to complete dryness; add 200 μL of lipid reconstitution solution (acetonitrile: isopropanol = 1:1, V / V), vortex for 3 min, centrifuge at 12000 r / min for 3 min, and transfer the supernatant for LC-MS / MS analysis. Mobile phase: Phase A, acetonitrile / water (60 / 40, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate); Phase B, acetonitrile / isopropanol (10 / 90, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate); mobile phase gradient: 0-2 min A / B (80:20-70:30, V / V), 2-4 min A / B (70:30-40:60, V / V), 4-9 min A / B (40:60-15:85, V / V), 9-15.5 min A / B (15:85-5:95, V / V), 15.5-17.5 min A / B (5:95, V / V), flow rate 0.35 mL / min; column temperature 45 °C; injection volume 2 μL. The electrospray ion source temperature was 500°C, the mass spectrometer voltage was 5500V in positive ion mode, -4500V in negative ion mode, ion source GS1 45psi, GS2 55psi, curtain gas 35psi, and collision-induced ionization parameters were set to Medium. In the triple quadrupole, each ion pair was scanned and detected according to the optimized declustering voltage and collision energy to obtain the original mass spectrometry data of all detected substances, including retention time RT (Retention time) and parent ion pairs.

[0121] 3.6 Broadly targeted lipidomics data processing and analysis

[0122] Based on the commercial lipidome database LipidView provided by AB SCIEX, the original mass spectrometry data of the samples obtained by the method described in 3.5 were processed using the software Analyst1.6.3. The signal intensity (CPS) of the characteristic ions of each detected substance was screened out using the multiple reaction monitoring (MRM) mode of the triple quadrupole mass spectrometer, and the mass spectrometry file of the sample was opened with MultiQuant software. The peak area of ​​the mass spectrum peaks of all substances was integrated and corrected. The peak area (Ara) of each chromatographic peak represented the relative content of the corresponding substance. Finally, all chromatographic peak area integration data were exported and saved.

[0123] (1) Principal Component Analysis (PCA): PCA was performed on samples (including quality control samples) to obtain a preliminary understanding of the overall lipid differences between groups of samples and the variability between samples within a group. PCA used the built-in statistical prcomp function of R software (wwwr-project.org / ), and set the prcomp function parameter scale = True, indicating that UV (unit variance scaling) processing was performed on the data (formula: Where μ is the mean and σ is the standard deviation). The processed data conforms to the standard normal distribution, that is, the mean is 0 and the standard deviation is 1.

[0124] (2) Orthogonal partial least squares discriminant analysis (OPLS-DA): Analyze lipid group data according to the OPLS-DA model, draw score graphs for each group, and further show the differences between groups. After OPLS-DA performs log2 transformation on the original data, it performs centralization processing (formula: x′=x-μ, where X is the sample quantitative information matrix and Y is the sample group information matrix. The variable information related to Y is the prediction principal component, and the variable information unrelated to Y is the orthogonal principal component). OPLS-DA model verification: The prediction parameters of the OPLS-DA evaluation model are R 2 X, R 2 Y and Q 2 , where R 2 X and R 2 Y represents the explanation rate of the constructed model for the X and Y matrices, and Q represents the predictive ability of the model. The closer these three indicators are to 1, the more stable and reliable the model is. 2 When >0.5, it can be considered as an effective model. 2 When p > 0.9, the model is excellent. Generally speaking, the model is best when p < 0.05.

[0125] (3) Differential lipid screening: Based on the OPLS-DA model, the OPLSR.Anal function of the MetaboAnalystR package in the R software was used to analyze and obtain the variable importance projection (VIP). Differential lipids were initially screened according to the following screening criteria:

[0126] For comparison of two groups:

[0127] 1). Select lipids with VIP>1. The VIP value indicates the influence of the inter-group difference of the corresponding lipids in the classification and discrimination of each group of samples in the model. It is generally believed that lipids with VIP>1 have significant differences.

[0128] 2) Select lipids with P-value < 0.05 (Student's t test). If there is a statistically significant difference in lipids between different groups, the difference is considered significant.

[0129] For multiple group comparisons:

[0130] 1). Select lipids with VIP>1. The VIP value indicates the influence of the inter-group difference of the corresponding lipids in the classification and discrimination of each group of samples in the model. It is generally believed that lipids with VIP>1 have significant differences.

[0131] 2). Select lipids with P-value < 0.05 (ANOVA). If there is a statistically significant difference in lipids between different groups, the difference is considered significant.

[0132] In order to observe the change of relative lipid content, we used UV (Unit Variance Scaling) to process the original relative content of differential lipids identified by the screening criteria (formula: Where μ is the mean and σ is the standard deviation. The processed data conforms to the standard normal distribution, that is, the mean is 0 and the standard deviation is 1), and then the cluster heat map is drawn by the R software ComplexHeatmap package.

[0133] (4) KEGG functional annotation of differential lipids: The KEGG (Kyoto Encyclopedia of Genes and Genomes) database is the main public database for integrated metabolic pathway queries. It provides metabolic pathways for carbohydrates, nucleotides, amino acids, and biodegradation, and provides comprehensive annotations for enzymes that catalyze each step of the reaction. Based on the differential lipid results, the KEGG database was used to annotate the serum lipids of different groups of mice to display the metabolic pathways containing differential lipids.

[0134] (5) KEGG enrichment analysis of differential lipids: Based on the results of significantly differential lipids, KEGG pathway enrichment analysis was performed, where the Rich Factor is the ratio of the number of differential lipids in the corresponding pathway to the total number of lipids annotated to the pathway. The larger the value, the greater the degree of enrichment. P-value is the hypergeometric test p value, and the calculation formula of the hypergeometric distribution is as follows:

[0135]

[0136] Among them, N represents the number of lipids with KEGG annotations in all lipids, n represents the number of differential lipids in N, M represents the number of lipids in a KEGG pathway in N, and m represents the number of differential lipids in a KEGG pathway in M. The closer the P-value is to 0, the more significant the enrichment. The size of the point in the figure represents the number of significantly differential lipids enriched in the corresponding pathway. The top 20 pathways ranked by P-value are selected and displayed from small to large.

[0137] Quality control samples and data quality control: Quality control samples are prepared by mixing sample extracts. (1) By overlapping and analyzing the total ion current graphs (TIC graphs) of mass spectrometry analysis of different quality control QC samples, the repeatability of lipid extraction and detection, i.e., technical repeatability, can be determined. (2) Pearson correlation analysis is performed on QC samples. The higher the correlation (the closer r is to 1), the better the stability of the entire detection process and the higher the data quality. (3) An internal standard of known concentration is added to the QC sample. The smaller the response difference of the internal standard (CV≤15%), the more stable the detection process and the higher the data quality. (4) The CV value, i.e., the coefficient of variation, is the ratio of the standard deviation of the original data to the mean of the original data, which can reflect the degree of data dispersion. The empirical cumulative distribution function (ECDF) can be used to analyze the frequency of CV of substances less than the reference value. The higher the proportion of substances with lower CV values ​​in QC samples, the more stable the experimental data; the proportion of substances with CV values ​​less than 0.5 in QC samples is higher than 85%, indicating that the experimental data is relatively stable; the proportion of substances with CV values ​​less than 0.3 in QC samples is higher than 75%, indicating that the experimental data is very stable.

[0138] Blank samples are interspersed throughout the entire experiment, and their peak conditions can reflect whether there are any residual substances during the detection process. If no obvious peaks of the internal standards are detected in the blank samples, it means that there are few residual substances and the cross-contamination between samples is within a controllable range.

[0139] 4 Experimental results

[0140] 4.1 Dai medicine composition improves lipid metabolism disorders in mice caused by NAFLD

[0141] (1) Changes in serum biochemical indicators of mice Figure 2 As shown in the figure: 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 Vitex rotundifolia and Piper methysticum fruit extracts, and slightly better than that of Bisacurone ( Figure 2 ).

[0142] (2) Serum broad targeted lipidome results Figure 3-Figure 7 As shown. Principal component analysis of serum broadly targeted lipidome ( Figure 3 ) and OPLS-DA( Figure 4 ) analysis showed significant differences among the groups, with nearly 500 metabolites detected ( Figure 5 ). KEGG pathway enrichment results showed that the Dai medicine composition of the present invention may affect multiple lipid metabolism processes related to NAFLD, such as sphingolipid metabolism, glycerophospholipid metabolism, glycerolipid metabolism, arachidonic acid metabolism, and linolenic acid metabolism ( Figure 7 ). Combined with literature analysis, the sphingolipid metabolism pathway may be the main potential metabolic pathway reflecting the communication of the "liver-heart axis". The Dai medicine composition may improve the activity of acute myocardial infarction under NAFLD conditions by acting on this pathway ( Figure 6 ).

[0143] 4.2 Dai medicine combination improves acute myocardial infarction under NAFLD conditions

[0144] Myocardial infarction area assessment (TTC + Evans Blue staining) results are as follows Figure 8 The results show that the Dai medicine composition of the present invention can significantly reduce the acute myocardial infarction area under NAFLD conditions. The myocardial infarction rate of the Dai medicine composition group of the present invention is 17.99%, which is 39.09% lower than the myocardial infarction rate of 57.08% in the model group, and the activity is better than that of Vitex and Piper methysticum extracts, and slightly better than that of Bisacurone.

[0145] 5 Experimental Conclusion

[0146] The Dai medicine composition of the present invention may alleviate acute myocardial infarction in mice under the condition of NAFLD by improving lipid metabolism disorder caused by NAFLD. The Dai medicine extract can be used to prevent or treat acute myocardial infarction under the condition of non-alcoholic fatty liver disease (NAFLD).

[0147] 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 containing sweet bisabolol and curcuminol, characterized in that: It includes sweet bisabolol, vitex rotundus extract and pepper extract, and the mass ratio of the three is 1:49.5:49.5 to 10:45:45; The Vitex rotundifolia extract is obtained by using Vitex rotundifolia as a raw material and supercritical extraction with carbon dioxide; The pepper extract is obtained by taking pepper as raw material and extracting it through supercritical carbon dioxide.

2. The Dai medicine composition containing sweet bisabolol and curcumin according to claim 1, characterized in that: The purity of the sweet bisabolol curcumin is greater than or equal to 98%.

3. The Dai medicine composition containing sweet bisabolol and curcumin according to claim 1, characterized in that: The sweet bisabolol is obtained by extracting and purifying turmeric as a raw material.

4. The Dai medicine composition containing sweet bisabolol and curcumin according to any one of claims 1 to 3, characterized in that: The preparation method of the Vitex rotundifolia extract comprises the following steps: Weigh Vitex rotundifolia seeds, put them into an extraction kettle, first perform static extraction for 20-30 minutes, then release the pressure and perform dynamic extraction for 30-120 minutes, preferably 60 minutes; Preferably, the static extraction conditions of Vitex rotundifolia are: temperature 30-35°C, pressure 25-30MPa; Preferably, the dynamic extraction conditions of Vitex rotundifolia are: temperature 30-40°C, pressure 15-20MPa, CO2 flow rate 60-100L / h, more preferably 80L / h; After the extraction is completed, centrifuge and take the supernatant to obtain the Vitex rotundifolia extract.

5. The Dai medicine composition containing sweet bisabolol and curcumin according to any one of claims 1 to 3, characterized in that: The preparation method of the pepper extract comprises the following steps: Weigh pepper and put it into an extraction kettle. First, perform static extraction for 20-30 minutes, then release the pressure and perform dynamic extraction for 30-120 minutes, preferably 60 minutes. Preferably, the static extraction conditions of pepper are: temperature 35-40°C, pressure 30-35MPa; Preferably, the dynamic extraction conditions of pepper are: temperature 40-45°C, pressure 20-25MPa, CO2 flow rate 60-100L / h, more preferably 80L / h; After the extraction is completed, centrifuge and take the supernatant to obtain the pepper extract.

6. The Dai medicine composition containing sweet bisabolol and curcumin according to any one of claims 1 to 3, characterized in that: The composition is a preparation prepared by using sweet bisabolol, vitex rotundifolia extract and pepper extract as raw materials, adding pharmaceutically acceptable excipients and following conventional methods; Preferably, the preparation is an oral preparation.

7. The method for preparing the Dai medicine composition containing bisabolol and curcumin according to any one of claims 1 to 6, characterized in that: The steps include: Prepare sweet bisabolol, vitex rotundifolia extract and pepper extract according to weight proportions, mix them evenly, add pharmaceutically acceptable excipients, mix them evenly, granulate them, and fill them into capsules to obtain capsules; Preferably, the total amount of sweet bisabolol, vitex rotundifolia extract and pepper extract is 20 to 50 parts, and the auxiliary material is 50 to 70 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 containing bisabolol and curcumin according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: preparing sweet bisabolol, vitex rotundus extract and pepper extract according to weight proportions, mixing them evenly, adding pharmaceutically acceptable auxiliary materials, mixing them 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; Preferably, the total amount of sweet bisabolol, vitex rotundifolia extract and pepper extract is 20 to 50 parts, and the auxiliary material is 50 to 70 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 containing bisabolol and curcuminol 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 containing bisabolol and curcuminol 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