A pharmaceutical composition and its use

Abstract

The present invention discloses a pharmaceutical composition comprising more than 26 substances, obtained by mixing ethanol extracts of costus root, agarwood, jujube, terminalia chebula, nutmeg, kapok, and frankincense with travertine. The composition can improve myocardial ischemia in subjects; increase the ejection fraction of subjects with myocardial infarction; increase the fractional shortening of the heart; reduce the left ventricular end-diastolic diameter in subjects with myocardial infarction; reduce the left ventricular end-systolic diameter in subjects with myocardial infarction; shorten the sleep latency in subjects with insomnia; prolong the sleep duration in subjects; and upregulate the expression of GAD67 protein in the brain tissue of subjects.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine preparations and relates to a pharmaceutical composition and use thereof. Background Art

[0002] Bawei Chenxiang Wan is a water-based pill made from the classic Tibetan medicine Bawei Chenxiang Powder. The recipe dates back to the 8th century and was first recorded in the classic Tibetan medical treatise, the Four Medical Classics. It is now included in the first volume of the Ministry of Health's Drug Standards for Tibetan medicine. It is composed of the botanicals nutmeg, agarwood, jujube, frankincense, terminalia chebula, costus root, and kapok, and the mineral travertine. It clears heart heat and calms the mind, and is used for fever-related heart attacks and precordial pain. It can also open the mind and soothe the nerves, and is used to treat coma and delirium. Bawei Chenxiang Powder, included in the 2020 edition of the Chinese Pharmacopoeia, shares its ingredients but uses different dosages.

[0003] As a commonly used drug in Tibetan areas for the treatment of cardiovascular disease and associated insomnia, Bawei Chenxiang Wan (Eight Flavors Agarwood Pill) has demonstrated clinical efficacy. However, current basic research on the chemistry and pharmacology of Bawei Chenxiang Wan is relatively weak, limiting its further development and application. This is particularly evident in the single formulation and lack of quality standards for Bawei Chenxiang Wan, necessitating an urgent need for further basic research. This study designed and screened the optimal extraction route for the drug. The extracts obtained from three extraction routes were compared for their anti-myocardial ischemia and anti-insomnia effects, preliminarily exploring their anti-insomnia mechanism of action. Chromatographic analysis was also used to analyze the differences in chemical composition among the different extracts, laying the foundation for subsequent research on the active ingredients of Bawei Chenxiang Wan, improving quality standards, and developing dosage forms. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a pharmaceutical composition, wherein the active ingredients of the pharmaceutical composition include the following substances:

[0005] Terminalia chebula acid;

[0006] brevifoliol acid;

[0007] Quercetin-3-O-β-D-pyranoglucopyranoside;

[0008] Agarwood A;

[0009] Ellagic acid-4-O-rhamnoside;

[0010] 1,3,6-trigalloylglucose;

[0011] quercetin;

[0012] 3,3'-dimethylellagic acid;

[0013] 6,8-dihydroxy-2-(2-phenylethyl)chromone;

[0014] Arjuna;

[0015] Boswellin N;

[0016] 6,7-dimethoxy-2-[(2-(4-methoxyphenyl)ethyl]chromone;

[0017] 6,7-dimethoxy-2-(2-phenylethyl)chromone;

[0018] 2-(2-phenylethyl)chromone;

[0019] 6-methoxy-2-(2-phenylethyl)chromone;

[0020] Costunolide;

[0021] Dehydrocostus lactone;

[0022] Malabarone C;

[0023] Malabarone B;

[0024] Boswellin B;

[0025] In eugenol;

[0026] 11-Carbonyl-β-acetylboswellic acid;

[0027] 9,11-dehydroboswellic acid;

[0028] Boswellic acid;

[0029] acetyl boswellic acid;

[0030] Travertine.

[0031] In some embodiments, the 9,11-dehydroboswellic acid is one or both of 9,11-dehydro-α-boswellic acid and 9,11-dehydro-β-boswellic acid;

[0032] The boswellic acid is one or both of α-boswellic acid and β-boswellic acid;

[0033] The acetyl boswellic acid is one or both of acetyl-α-boswellic acid and acetyl-β-boswellic acid.

[0034] In some embodiments, the method for preparing the pharmaceutical composition comprises the following steps:

[0035] Mixing costus root, agarwood, jujube, terminalia chebula, nutmeg, kapok flower, and frankincense to obtain a mixture;

[0036] The mixture was boiled and refluxed with 50-80 v / v% ethanol aqueous solution to obtain a filtrate;

[0037] The filtrate is concentrated and then mixed with travertine and dried to obtain the pharmaceutical composition;

[0038] The weight ratio of costus root, agarwood, jujube, terminalia chebula, nutmeg, kapok flower, frankincense and travertine is 2-5:1-3:1-3:1-3:1-3:1-2:0.5-1.5:1.

[0039] In some embodiments, the terminalia chebula is stewed terminalia chebula fruit.

[0040] In some embodiments, the number of boiling and reflux is 1-3 times, and the time of each boiling and reflux is 1-3 hours. During the first boiling and reflux, the ethanol aqueous solution used for reflux is 5-15 times the weight of the raw material drug. During the non-first reflux and boiling, the ethanol aqueous solution used for reflux is 5-10 times the weight of the medicinal residue obtained by the previous boiling and reflux.

[0041] In some embodiments, the concentration is concentrated under reduced pressure at 35-45°C.

[0042] In some embodiments, the drying is performed under reduced pressure and vacuum at 45-55°C.

[0043] In some embodiments, the pharmaceutical composition contains substances with the following ion fragment signals:

[0044] The adduct ion is [M+H] + , the molecular weights of ion fragments include 357.04395±0.001, 341.13834±0.001, 311.12799±0.001, 251.10689±0.001, 271.11771±0.001, 233.15414±0.001, 231.13829±0.001, 471.34763±0.001, 307.26337±0.001, 513.35803±0.001;

[0045] The added ion is [M+Na] + , the molecular weights of ion fragments include 403.24615±0.001;

[0046] The adduct ion is [MH] -The molecular weights of the ion fragments include 291.01462±0.001, 477.06741±0.001, 363.10739±0.001, 447.05722±0.001, 635.08936±0.001, 301.03510±0.001, 329.03030±0.001, 281.08194±0.001, 503.33789±0.001, 357.17007±0.001, 341.17569±0.001, 453.33734±0.001, 455.35336±0.001, and 497.36371±0.001.

[0047] The second aspect of the present invention provides use of the pharmaceutical composition of the first aspect of the present invention in preparing a preparation for improving the health of a subject;

[0048] Improving the subject's health condition includes:

[0049] Improve myocardial ischemia in subjects;

[0050] Improve cardiac ejection fraction in subjects with myocardial infarction;

[0051] Increased cardiac fractional shortening in subjects with myocardial infarction;

[0052] Reduce left ventricular end-diastolic diameter in subjects with myocardial infarction;

[0053] Reduce left ventricular end-systolic diameter in subjects with myocardial infarction;

[0054] Shorten sleep latency in subjects with insomnia;

[0055] Prolong the subjects' sleep duration;

[0056] Upregulate the expression of GAD67 protein in the subjects' brain tissue.

[0057] In some embodiments, the subject is selected from a human and a mouse. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Statistical data of the effects of different drugs on cardiac function in AMI mice.

[0059] Figure 2 Representative echocardiograms of AMI mice in the sham-operated group.

[0060] Figure 3 Representative echocardiograms of AMI mice in the model group.

[0061] Figure 4 Representative echocardiograms of AMI mice in the water extraction group.

[0062] Figure 5 Representative echocardiograms of AMI mice in the ethanol extraction group.

[0063] Figure 6 Representative echocardiograms of AMI mice in the alcohol extraction followed by water extraction groups.

[0064] Figure 7 Representative echocardiograms of AMI mice in the fosinopril group.

[0065] Figure 8 These are representative echocardiograms of AMI mice in the original drug group of Bawei Chenxiang Pills.

[0066] Figure 9 HE staining photos of myocardial tissue in the sham operation group.

[0067] Figure 10 HE staining photos of myocardial tissue in the model group.

[0068] Figure 11 HE staining photos of myocardial tissue in the water extraction group.

[0069] Figure 12 These are HE staining photos of myocardial tissue in the ethanol extraction group.

[0070] Figure 13 HE staining photos of myocardial tissue in the ethanol extraction and water extraction groups.

[0071] Figure 14 HE staining photos of myocardial tissue in the fosinopril group.

[0072] Figure 15 This is a HE-stained photo of the myocardial tissue of the original drug group of Bawei Chenxiang Pills.

[0073] Figure 16 Statistics on the sleep-inducing effects of different drugs on PCPA insomnia mice. DETAILED DESCRIPTION

[0074] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0075] Materials and instruments not described in the present invention are conventional materials and instruments in the art, and operation details not described in the present invention are conventional operations in the art.

[0076] Graph Pad Prism 8.0 software was used for data statistical analysis. Experimental data were expressed as x ± SD. Two-sided Student t-test or one-way ANOVA was used for pairwise comparisons between groups or comparisons between multiple groups. P < 0.05 indicated statistical significance.

[0077] instrument

[0078] ZDUW temperature-controlled electric heating mantle, Linmao Technology (Beijing) Co., Ltd.; DZF-6090 reduced pressure vacuum drying oven, Shanghai Shenxian Constant Temperature Equipment Factory; R-210 rotary evaporator, Buchi, Switzerland; ALC-V8 animal ventilator, Shanghai Olcott Biotechnology Co., Ltd.; VevoTEM 2100 small animal ultrasound imaging system, Visual Sonics, Canada; multifunctional microplate reader, PerkinElmer, USA; 40714 Hilden Tissue LyserⅡ tissue disruptor, Qiagen, Germany; electrophoresis and electrotransfer kit, Bio-Rad, USA; chemiluminescence analyzer AI680, GE, USA; UPLC-Q-Exactive-Orbitrap-MS instrument, Thermo Fisher Scientific, USA.

[0079] Material

[0080] Fosinopril (lot number ACN3619, Shanghai Bristol-Myers Squibb Pharmaceuticals Co., Ltd.); diazepam (lot number 2104001, Tianjin Lisheng Pharmaceutical Co., Ltd.); 4-chloro-DL-phenylalanine (lot number A121S208585, Shanghai Yuanye Biotechnology Co., Ltd.); isoflurane (lot number 2023041501, Shandong Ante Animal Husbandry Technology Co., Ltd.); RIPA lysis buffer (lot number P0013B, Shanghai Biyuntian Biotechnology Co., Ltd.); SYRB dye-based fluorescence quantitative premix (lot number AQ601-01, Beijing Quanshijin Biotechnology Co., Ltd.); 10× electrophoresis buffer (lot number B1005, Beijing Prilai Gene Technology Co., Ltd.); 10× electrotransfer buffer (lot number B1006, Beijing Prilai Gene Technology Co., Ltd.); 10× wash buffer (lot number B1009, Beijing Prilai Gene Technology Co., Ltd.); Super ECL Plus supersensitive luminescent solution (lot number P1050, Beijing Pulilai Gene Technology Co., Ltd.); autofluorescence quencher (lot number G1221, Servicebio); PVDF membrane (lot number IPVH00010, Merck, Germany); BCA protein concentration assay kit (lot number P0012S, Shanghai Biyuntian Biotechnology Co., Ltd.); skim milk powder (lot number 190-12865, Wako Japan); primary antibody diluent (lot number NKB-201, TOYOBO); PageRuler pre-stained protein maker (lot number: 26616, Thermo Fisher Scientific, Inc.); Fermentas); PAGE gel rapid preparation kit (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.); antibodies: HRP-labeled goat anti-mouse secondary antibody IgG (lot number 7074P2, CST); anti-GAD67 mouse monoclonal antibody IgG (lot number: ab213508, Abcam); anti-GAPDH mouse monoclonal antibody IgG (lot number 10494-1-AP, Proteintech); anhydrous ethanol (lot number 20240301, Fuchen Chemical Reagent Co., Ltd.); mass spectrometry grade formic acid (lot number 205775, Thermo Fisher Scientific Inc.); mass spectrometry grade acetonitrile (lot number 221404, Thermo Fisher Scientific Inc.); distilled water (Hangzhou Wahaha Group Co., Ltd.).

[0081] experimental animals

[0082] Adult male ICR mice (weighing 36 ± 2 g, 8 weeks old) were used for the myocardial infarction model; male ICR mice (weighing 30 ± 2 g, 5 weeks old) were used for the insomnia model. All SPF-grade animals were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., with company qualification certificate number SCXK (Beijing) 2019-0010. They were housed in a barrier environment at the Animal Experimental Center of Beijing University of Chinese Medicine, at a standard room temperature of 25°C, under a 12-h fluorescent light cycle simulating daylight and nighttime, and fed a regular diet with free access to food and water. All animal experiments in this study were approved by the Ethics Committee of Beijing University of Chinese Medicine (Ethics Number: BUCM-4-2022080101-3025).

[0083] Example 1. Preparation of Chinese medicine composition

[0084] 1. Eight-flavor Agarwood Pills

[0085] The finished product and ingredients for Bawei Chenxiang Wan were provided by Tibet Ganlu Tibetan Medicine Co., Ltd. The ingredients were authenticated by Professor Zhang Yuan of Beijing University of Chinese Medicine. Herbal samples are stored at the Modern Research Center for Traditional Chinese Medicine, Beijing Institute of Chinese Medicine, Beijing University of Chinese Medicine. The formula information is shown in Table 1. Bawei Chenxiang Wan was prepared by weighing the ingredients according to the weight ratio of the formula, pulverizing them and mixing them evenly. An appropriate amount of honey was added, and the pills were prepared with water.

[0086] Table 1. Information on the Ministry-issued standard prescription for Bawei Chenxiang Pills

[0087]

[0088] 2. Water Extract (Extract Code: ST)

[0089] The medicinal materials are from the same sources as those of the Bawei Chenxiang Pills in Table 1. The preparation steps are as follows:

[0090] Take 35g of costus root, 20g of agarwood, 20g of jujube, 20g of terminalia chebula, 20g of nutmeg, 15g of kapok, and 10g of frankincense;

[0091] Add 10 times the weight of water to the medicinal material mixture, boil and reflux for 2 hours, and filter through gauze;

[0092] Add 8 times the weight of water to the residue, boil and reflux for 2 hours, and filter through gauze.

[0093] The two filtrates were combined and concentrated under reduced pressure at 40°C to a thick paste. 10 g of travertine was added and stirred evenly. The mixture was dried in a vacuum drying oven at 50°C and pulverized into a fine powder to obtain a pharmaceutical composition, codenamed ST. The paste yield was 19.51%.

[0094] 3. Ethanol extract (extract code: CT)

[0095] The medicinal materials are from the same sources as those of the Bawei Chenxiang Pills in Table 1. The preparation steps are as follows:

[0096] Take 35g of costus root, 20g of agarwood, 20g of jujube, 20g of terminalia chebula, 20g of nutmeg, 15g of kapok, and 10g of frankincense;

[0097] Add 70% ethanol aqueous solution 10 times the total weight of the medicinal materials to the medicinal material mixture, boil and reflux for 2 hours, and filter through gauze;

[0098] Add 70% ethanol aqueous solution (8 times the total weight of the medicinal materials) to the filter residue, boil and reflux for 2 hours, and filter through gauze;

[0099] The two filtrates were combined and concentrated under reduced pressure at 40°C to a thick paste. 10 g of travertine was added and stirred evenly. The mixture was dried in a vacuum drying oven at 50°C and pulverized into a fine powder to obtain a pharmaceutical composition, codenamed CT. The paste yield was 15.38%.

[0100] 4. Water extract after alcohol extraction (extract code: CST)

[0101] The medicinal materials are from the same sources as those of the Bawei Chenxiang Pills in Table 1. The preparation steps are as follows:

[0102] Take 35g of costus root, 20g of agarwood, 20g of jujube, 20g of terminalia chebula, 20g of nutmeg, 15g of kapok, and 10g of frankincense;

[0103] Add 70% ethanol aqueous solution 10 times the total weight of the medicinal materials to the medicinal material mixture, boil and reflux for 2 hours, and filter through gauze;

[0104] Add 70% ethanol aqueous solution (8 times the total weight of the medicinal materials) to the filter residue, boil and reflux for 2 hours, and filter through gauze;

[0105] Add 10 times the weight of water of the total weight of the medicinal materials to the second filtration residue, boil and reflux for 2 hours, filter with gauze, and obtain the medicinal residue water extract.

[0106] Combine the two alcohol extracts (filtrate) and the aqueous extract of the residue, concentrate under reduced pressure at 40°C to a thick paste, add 10 g of travertine, stir evenly, dry in a vacuum oven at 50°C, and grind into a fine powder to obtain the pharmaceutical composition, codenamed CST. The paste yield is 30.24%.

[0107] Example 2. Analysis of chemical composition differences of extracts

[0108] Preparation of test samples: Weigh 0.3000 g of CT, ST, or CST dry powder and add 10 mL of 70% ethanol, pure water, and 50% ethanol, respectively. After sonication until dissolved, sample the mixture and centrifuge at 13,000 rpm for 15 min. Remove the supernatant and filter it through 0.22 μm organic, aqueous, and organic filters, respectively. Place the supernatant into a clean injection vial for sample loading.

[0109] Chromatographic conditions: An ACQUITY UPLC HSS T3 column (S-1.8 μm, 2.1×100 mm) was used; the flow rate was 0.2 mL / min; the injection volume was 5 μL; the mobile phase was 0.1% formic acid aqueous solution (A)-acetonitrile (B); and the gradient elution was adopted: 0-10 min, 5-15% B; 10-20 min, 15% B; 20-50 min, 15-40% B; 50-60 min, 40-55% B; 60-80 min, 55-95% B.

[0110] Mass spectrometry conditions: HESI ion source, mass spectrometry detection in positive and negative ion modes; sheath gas 45 arb, auxiliary gas 15 arb, spray voltage 3.5 kV (+) / 2.8 kV (-); ion transfer tube temperature 320°C, auxiliary gas temperature 350°C; scan mode: Full MS / dd-MS 2 , Full MS resolution is 70000, dd-MS 2 The resolution was 17500, the quadrupole scan range was m / z 150 to 1500, the collision energy was a step energy of 20, 30, and 40 eV, and all other settings were default.

[0111] Software analysis: Data spectra were analyzed using Qual Browser of Xcalibur 3.0.63 (Thermo Fisher Scientific, USA).

[0112] The compositional information of each herb was collected through databases such as CNKI and TCMSP, and a chemical composition database was established. Following the aforementioned chromatographic method, sample injection and analysis were performed, yielding ion chromatograms in both positive and negative ion modes for the three different extraction pathways: CT, ST, and CST. Component analysis was performed for the characteristic 0-100 min time period for CT, ST, and CST. The results are shown in Tables 2, 3, and 4.

[0113] Table 2. ST (0-100 min) component identification

[0114]

[0115]

[0116] Table 3. CT (0-100 min) component identification

[0117]

[0118]

[0119]

[0120] Table 4. CST (0-100 min) component identification

[0121]

[0122]

[0123]

[0124] Example 3: Evaluation of anti-myocardial ischemic effect

[0125] Normal male ICR mice were anesthetized with an intraperitoneal injection of sodium pentobarbital in saline (50 mg / kg mouse body weight). The skin on the chest was prepared and the animals were connected to a ventilator. After thoracotomy, the left anterior descending coronary artery (LAD) was visualized. The LAD was ligated with a sterile suture needle and the chest was closed with sutures to create an acute myocardial ischemia (AMI) mouse model. Sixty mice were treated.

[0126] (1) Sham group: 10 mice in total were treated with suture threading at the same position as the model group mice, but without ligation. Other procedures were the same. No medication was given.

[0127] 24 hours after modeling, the mice in the model group were randomly divided into the following six groups:

[0128] (2) Model group (Mod group), no drug administration;

[0129] (3) Water extraction group (ST group), using ST obtained in Example 1, with a dose of 760 mg powder / kg mouse body weight);

[0130] (4) Alcohol extraction group (CT group), using the CT obtained in Example 1, with a dose of 620 mg powder / kg mouse body weight);

[0131] (5) Alcohol extraction followed by water extraction group (SCT group), using the CST obtained in Example 1 at a dose of 1040 mg powder / kg mouse body weight);

[0132] (6) Positive drug fosinopril group (Group F), using fosinopril at a dose of 10 mg drug / kg mouse body weight;

[0133] (7) Bawei Chenxiang Wan original drug group (W group), using the Bawei Chenxiang Wan selected in Example 1, with a dose of 3000 mg powder / kg mouse body weight);

[0134] The drugs were dissolved in water and administered orally to 10 mice in each of the 7 groups for 7 consecutive days, once at the same time every day.

[0135] After 7 days, echocardiography was used to detect and calculate the ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVED; d), and left ventricular end-systolic diameter (LVED; s) of the mice in each group; and heart tissues were collected for HE staining to examine the myocardial tissue pathology.

[0136] The statistical data on the effects of different drugs on cardiac function in AMI mice can be found in Figure 1 , compared with the Sham group, ## P<0.01, ### P<0.001; compared with the Mod group, *P<0.05, **P<0.01, ***P<0.001.

[0137] Representative echocardiograms of 7 groups of AMI mice are shown in Figure 2-Figure 8 In each figure, the upper image shows the short-axis papillary muscle section of the heart, and the lower image shows the fluctuations of the anterior and posterior walls of this section.

[0138] The ultrasound results on the 8th day after surgery showed that compared with the sham operation group, the LVID;d and LVID;s of the model group mice were significantly increased (P<0.001), and the EF and FS values ​​were significantly decreased (P<0.001), indicating that the left ventricular anterior wall was thinned, the ventricle was dilated, and the left ventricular systolic and diastolic functions were impaired, indicating that the myocardial infarction model was successfully established. Compared with the model group, the positive drug fosinopril group (F) could significantly increase EF (P<0.01) and reduce LVID;d and LVID;s values ​​(P<0.05, P<0.01); the Baweichenxiang pill group (W) could significantly increase EF and FS values ​​(P<0.001, P<0.01) and reduce LVID;d and LVID;s values ​​(P<0.001); the alcohol extract group (CT) could significantly increase EF and FS (P<0 .001, P<0.01), and reduced LVID;d and LVID;s values ​​(P<0.01, P<0.001). The alcohol-extracted water extract group (CST) significantly increased EF and FS (P<0.001, P<0.05) and reduced LVID;d and LVID;s values ​​(P<0.01, P<0.001). The water extract group (ST) increased EF and FS and reduced LVID;d and LVID;s values. Ultrasound results suggested that both the CT and CST groups had significant anti-myocardial ischemic effects.

[0139] HE staining photos of the seven groups are shown as follows Figure 9-15As shown, HE-stained sections of myocardial tissue were observed under an optical microscope at 12.5 times (see the small picture in the upper left corner) and 400 times (see the large picture). In the sham-operated group, myocardial cells were intact and arranged neatly, with clear myocardial cell striations and no obvious inflammatory cell infiltration. In the model group, obvious myocardial cell arrangement disorder was observed, with a large number of inflammatory cells infiltrating in the tissue and some myocardial cells degenerating. After administration of F, W, CT, and CST, it was observed that inflammatory cell infiltration was reduced and the degree of myocardial cell disorder was improved.

[0140] Example 4: Experiment on the effect of drugs on mouse sleep

[0141] 1. Evaluation of sleep-aiding effects in mice

[0142] Mouse grouping:

[0143] (1) Normal group (Con group), normal male ICR mice, no modeling, no drug administration, 10 mice;

[0144] Normal male ICR mice were intraperitoneally injected with 4-chloro-DL-phenylalanine (PCPA) in saline at a dose of 500 mg / kg mouse body weight for five consecutive days, with daily injections. The mice were observed for a successful PCPA insomnia model. Loss of circadian rhythm, dull fur, and slow weight gain were observed. Successful models were randomly divided into six groups of 10 mice each. Dosing began the day after model establishment.

[0145] (2) Model group (Mod group), no drug was given.

[0146] (3) positive drug diazepam group (DZP group), the dose was 1.5 mg drug / kg mouse body weight;

[0147] (4) Bawei Chenxiang Wan original drug group (W group), using the Bawei Chenxiang Wan selected in Example 1, with a dose of 3000 mg powder / kg mouse body weight;

[0148] (5) Water extraction group (ST group): ST obtained in Example 1 was used at a dose of 760 mg powder / kg mouse body weight;

[0149] (6) Alcohol extraction group: CT obtained in Example 1 was used at a dose of 620 mg of powder / kg of mouse body weight;

[0150] (7) Alcohol extraction followed by water extraction group (SCT group): CST obtained in Example 1 was used at a dose of 1084 mg powder / kg mouse body weight;

[0151] Each group had 10 mice, and the drugs were administered orally for 7 consecutive days, once a day. One hour after the last oral administration, the sleep-inducing effects of the three extracts were evaluated by oral administration of sodium pentobarbital saline solution (50 mg drug / kg mouse body weight). The sleep latency and sleep maintenance time of the mice were recorded.

[0152] 2. GAD67 protein expression in mouse brain tissue

[0153] For each group of mice, after sodium pentobarbital anesthesia, brain tissue from each group of mice was obtained and stored in an ultra-low temperature freezer at -80°C. About 10 mg of brain tissue was minced and placed in a centrifuge tube. RIPA lysis buffer and a protein phosphatase inhibitor cocktail (100:1 volume ratio) were added at 100 mg / mL. After adding one steel ball, a tissue disruptor was used at 30 times / s. After oscillation and disruption for 3 minutes, the tissue was centrifuged at 12,000 rpm at 4°C for 10 minutes, and the tissue homogenate supernatant was obtained. The protein content of the sample was determined according to the BCA assay kit instructions, with a quantification of 4 μg / μL. Loading buffer was added, and the cells were denatured at 95°C for 10 minutes. The cells were then stored at -20°C until further use. Equal amounts of protein were loaded and separated by SDS-PAGE. The proteins were then transferred to PVDF membranes and blocked with 5% skim milk for 2 h. Specific antibodies against GAD67 and GAPDH proteins were added, respectively. The membranes were incubated overnight at 4°C, washed with TBST five times for 5 min each time, incubated with goat anti-mouse secondary antibody at room temperature for 2 h, washed with TBST five times for 5 min each time, and developed with enhanced chemiluminescence (ECL). Grayscale analysis was performed using ImageJ software. The protein level was expressed as the ratio of GAD67 expression in the cortical tissue of each group to the internal control GAPDH expression.

[0154] The results are as follows Figure 16As shown in the data, compared with the normal group, the sleep latency of mice in the model group was significantly prolonged (P<0.01) and the sleep duration was significantly shortened (P<0.001), indicating that the PCPA insomnia mouse model was successfully established. Compared with the model group, the diazepam group (DZP) significantly shortened the sleep latency of mice (P<0.001) and prolonged the sleep duration of mice (P<0.001). The Bawei Chenxiang Pill group (W) did not shorten the sleep latency of mice (P>0.05), but significantly prolonged the sleep duration of mice (P<0.01). The CT group significantly shortened the sleep latency of mice (P<0.001) and prolonged the sleep duration of mice (P<0.01). The ST group significantly prolonged the sleep duration of mice (P<0.001). Both the CT and ST groups had significant sleep-inducing effects. In terms of protein expression, compared with the blank group, the GAD67 protein expression level in the cortical tissue of mice in the PCPA model group was significantly decreased; compared with the model group, after ST, CT, and W intervention, the expression of GAD67 protein was significantly increased (P<0.001), indicating that ST, CT, and W exert a sleep-inducing effect by upregulating the protein expression of GAD67.

[0155] These results demonstrate that CT and CST exhibit clear anti-myocardial ischemic effects, while ST and CT exhibit clear sleep-inducing effects. Furthermore, CT exhibits a more pronounced anti-myocardial ischemic effect, while ST exhibits a more pronounced sleep-inducing effect. These results suggest that extracts obtained by different extraction methods exhibit distinct differences in efficacy, and that the anti-AMI and sleep-inducing effects may be contributed by distinct pharmacological substances. Further comparative chemical composition studies of the different extracts identified 17 major chemical components in ST, 25 major chemical components in CT, and 27 major chemical components in CST. Furthermore, the sleep-inducing effect of ST is associated with increased expression of GAD67, a key enzyme required for GABA synthesis, providing guidance for further mechanistic studies.

[0156] Tibetan medicine theory posits that heart disease is primarily caused by internal and external factors, such as emotional disharmony, melancholy and depression, irritability and insomnia, anger and resentment, as well as eating disorders and excessive hunger. Symptoms include restlessness, stabbing pain, and heart heat. Tibetan medicine integrates cardiac physiology with the mind and psychology, similar to the traditional Chinese medicine concept that "the heart governs the spirit." Bawei Chenxiang Wan, a commonly used Tibetan medicine treatment for heart disease and associated insomnia, has the potential to treat both the heart and the brain. Therefore, this study comprehensively evaluated the efficacy of the drug in a mouse model of acute myocardial ischemia induced by ligation of the left anterior descending coronary artery and a mouse model of insomnia induced by intraperitoneal injection of para-chlorophenylalanine, providing insights for further exploring the potential of this "heart-brain" approach.

[0157] This study used intraperitoneal injection of PCPA to establish a mouse model of insomnia. PCPA blocks 5-hydroxytryptamine synthesis by inhibiting tryptophan hydroxylation. Serotonin deficiency leads to a decrease in the number of inhibitory neurotransmitter GABAergic neurons, their synapses, and clusters, thus inducing insomnia. GAD67 protein is involved in regulating GABA synthesis. Previous studies on the mechanisms of Bawei Chenxiang Wan revealed that GAD67 protein was highly expressed in the Bawei Chenxiang Wan-treated group, so this protein was selected for preliminary exploration of the sleep-inducing mechanisms of different Bawei Chenxiang Wan extracts. Further systematic research is needed to elucidate the in-depth sleep-inducing mechanisms, anti-myocardial ischemic mechanisms, and the combined mechanisms of sleep-inducing and anti-myocardial ischemic effects of different Bawei Chenxiang Wan extracts. This will clarify the rich connotations of Bawei Chenxiang Wan's "heart-brain treatment" and the multi-component, multi-mechanistic characteristics of the compound.

[0158] The original formulation of Bawei Chenxiang Wan (Bawei Chenxiang Wan) was prepared from the raw powder of the medicinal material. To investigate the changes in efficacy after extraction compared to the original formulation, the mouse dosage calculated from the clinically effective dose of Bawei Chenxiang Wan (3g / 70kg) was used as the raw material dosage. The dosages of the three extracts were calculated based on the extraction rate. Since the mineral travertine cannot be extracted by organic solvents, the three extracts were converted to plant extracts based on the extraction rate of the botanical drug. These extracts were then combined with the travertine at the original dosage of the formulation. The resulting dosages were: Bawei Chenxiang Wan (3000mg / kg), ethanol extract (620mg / kg), water extract (760mg / kg), and ethanol-water extract (1084mg / kg).

[0159] Comparing the efficacy of different extracts allows for optimal extraction routes to be identified. Comparing their components allows for a valuable exploration of the drug's active ingredients. Alcohol and water extracts differ significantly in polarity distribution, naturally leading to differences in their efficacy. For example, in this study, Bawei Chenxiang Wan's CT exhibited a predominance of anti-myocardial ischemia effects, while ST exhibited a sleep-inducing effect. Furthermore, attempts were made to combine alcohol and water extractions to achieve a more comprehensive extract composition, aiming to achieve an ideal combination of efficacy.

[0160] Based on the indications of Bawei Chenxiang Wan, this study clarified the pharmacological advantages of three extraction routes using a dual pharmacodynamic evaluation system in mouse models of acute myocardial ischemia and insomnia. Component analysis results suggest that chromones and terpenoids primarily contribute to the anti-ischemic effect, while tannins and phenolic acids primarily contribute to the sleep-inducing effect. Furthermore, this sleep-inducing effect is associated with regulating GAD67 protein expression. These results provide a scientific basis for elucidating the pharmacological components of Bawei Chenxiang Wan, establishing quality standards, and rationalizing its clinical use, laying a solid foundation.

[0161] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for improving myocardial ischemia and sleep; The preparation method of the pharmaceutical composition comprises the following steps: Mixing costus root, agarwood, jujube, terminalia chebula, nutmeg, kapok flower, and frankincense to obtain a mixture; The mixture was boiled and refluxed with 50-80 v / v% ethanol aqueous solution to obtain a filtrate; The filtrate is concentrated and then mixed with travertine and dried to obtain the pharmaceutical composition; The weight ratio of costus root, agarwood, jujube, stewed terminalia chebula, nutmeg, kapok flower, frankincense and travertine is 2-5:1-3:1-3:1-3:1-3:1-2:0.5-1.5:

1.

2. The use according to claim 1, characterized in that The active ingredients of the pharmaceutical composition include the following substances: Terminalia chebula acid; brevifoliol acid; Quercetin-3-O- β -D-pyranoglucopyranoside; Agarwood A; Ellagic acid-4-O-rhamnoside; 1,3,6-trigalloylglucose; quercetin; 3,3'-dimethylellagic acid; 6,8-dihydroxy-2-(2-phenylethyl)chromone; Arjuna; Boswellin N; 6,7-dimethoxy-2-[2-(4-methoxyphenyl)ethyl]chromone; 6,7-dimethoxy-2-(2-phenylethyl)chromone; 2-(2-phenylethyl)chromone; 6-methoxy-2-(2-phenylethyl)chromone; Costunolide; Dehydrocostus lactone; Malabarone C; Malabarone B; Boswellin B; In eugenol; 11-Carbonyl- β -Acetylboswellic acid; 9,11-dehydroboswellic acid; Boswellic acid; acetyl boswellic acid; Travertine.

3. The use according to claim 2, characterized in that The 9,11-dehydroboswellic acid is 9,11-dehydro- α -Boswellic acid and 9,11-dehydro- β - One or both of the boswellic acids; The boswellic acid is α - Boswellia sour and β - One or both of the boswellic acids; The acetyl boswellic acid is acetyl- α -Boswellic acid and acetyl- β - One or both of the boswellic acids.

4. The use according to claim 1, wherein The number of boiling and reflux is 1-3 times, and the time of each boiling and reflux is 1-3 hours. During the first boiling and reflux, the ethanol aqueous solution used for reflux is 5-15 times the weight of the raw material drug. During the non-first reflux and boiling, the ethanol aqueous solution used for reflux is 5-10 times the weight of the drug residue obtained by the previous boiling and reflux.

5. The use according to claim 1, characterized in that The concentration is carried out under reduced pressure at 35-45°C.

6. The use according to claim 1, wherein The drying is carried out under reduced pressure and vacuum at 45-55°C.

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