Pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition, which comprises more than 26 substances, and is prepared by mixing ethanol extracts of radix aucklandiae, agilawood, fructus choerospondiatis, fructus chebulae, myristica fragrans, kapok and frankincense with travertine. The composition can improve myocardial ischemia of a subject; the cardiac ejection fraction of a myocardial infarction subject is increased; the cardiac minor axis shortening rate of a myocardial infarction subject is increased; reducing the inner diameter of the left ventricular end diastole of the myocardial infarction subject; reducing the inner diameter of the heart left ventricular systolic end of the myocardial infarction subject; the sleep latency period of an insomnia subject is shortened; the sleep maintenance time of the testee is prolonged; and the expression of the brain tissue GAD67 protein of the subject is up-regulated.

Description

Technical Field

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

[0002] Bawei Chenxiang Pills are made from the classic Tibetan medicine Bawei Chenxiang Powder. The recipe was created in the 8th century and was first recorded in the classic Tibetan medicine masterpiece "Four Medical Classics". It is now included in the first volume of Tibetan medicine "Medical Standards Issued by the Ministry of Health". It is composed of plant medicines such as nutmeg, agarwood, jujube, frankincense, terminalia chebula, costus root, kapok and mineral medicine travertine. It clears the heart heat and calms the mind. It is used for fever attacking the heart and precordial pain. It can also open the mind and calm the nerves. It is used to treat coma and delirium. Bawei Chenxiang Powder included in the "Chinese Pharmacopoeia" (2020 edition) has the same ingredients as it, but the dosage is different.

[0003] As a commonly used medicine for the treatment of cardiovascular diseases and associated insomnia symptoms in Tibetan areas, Bawei Chenxiang Pills have definite therapeutic effects in clinical applications. However, the basic research on the chemistry and pharmacology of Bawei Chenxiang Pills is relatively weak, which limits its further development and application, especially in the single form of the existing preparations of Bawei Chenxiang Pills and the lack of quality standards. It is urgent to carry out relevant basic research. This study designed and screened the optimal extraction route of the drug, compared the anti-myocardial ischemia and anti-insomnia effects of the extracts obtained by the three extraction routes, and preliminarily explored the anti-insomnia mechanism of action. At the same time, chromatographic technology was used to analyze the differences in chemical components in different extracts, laying a foundation for the subsequent research on the effective substances, quality standard improvement and dosage form development of Bawei Chenxiang Pills. 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] Chebulic acid;

[0006] Breviphenolic 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] Due to 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] Mix costusroot, agarwood, jujube, terminalia chebula, nutmeg, kapok and frankincense to obtain a mixture;

[0036] The mixture is 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, 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 boiling and refluxing are performed 1-3 times, and the time of each boiling and refluxing is 1-3 hours. During the first boiling and refluxing, 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.

[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 added 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 added 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] Increase the fractional shortening of the heart 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] Shortened sleep latency in subjects with insomnia;

[0055] Prolong the subjects' sleep duration;

[0056] Up-regulate 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 operation 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 alcohol extraction group.

[0063] Figure 6 Representative echocardiograms of AMI mice in the alcohol extraction and 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] Fig. 9 HE staining photos of myocardial tissue in the sham operation group.

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

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

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

[0070] Fig.13 These are HE staining photos of myocardial tissue in the alcohol extraction and water extraction groups.

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

[0072] Fig.15 This is a HE staining photo of the myocardial tissue in the original drug group of Bawei Chenxiang Pills.

[0073] Fig.16 The results are statistically presented for the sleep-inducing effects of different drugs on PCPA insomnia mice. DETAILED DESCRIPTION

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

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

[0076] The present invention uses Graph Pad Prism 8.0 software for data statistical analysis, the experimental data are expressed as x±SD, and the two-sided Student t-test or one-way ANOVA is used to compare two groups or multiple groups, and P<0.05 indicates statistical significance.

[0077] instrument

[0078] ZDUW temperature-adjustable 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; chemiluminometer AI680, GE, USA; UPLC-Q-Exactive-Orbitrap-MS instrument, Thermo Fisher Scientific, USA.

[0079] Material

[0080] Fosinopril (lot number ACN3619, Shanghai Bristol-Myers Squibb Pharmaceutical 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 method fluorescence quantitative premix reagent (lot number AQ601-01, Beijing Quanshijin Biotechnology Co., Ltd.); 10× electrophoresis buffer (lot number B1005, Beijing Pulilai Gene Technology Co., Ltd.); 10× electrotransfer buffer (lot number B1006, Beijing Pulilai Gene Technology Co., Ltd.); 10× washing buffer (lot number B1009, Beijing Pulilai Gene Technology Co., Ltd.); Super ECL Plus ultra-sensitive luminescent liquid (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 determination 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 Fermentas); PAGE gel rapid preparation kit (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.); Antibodies: HRP-labeled goat anti-mouse secondary antibody IgG (Lot No. 7074P2, CST); anti-GAD67 mouse monoclonal antibody IgG (Lot No. ab213508, Abcam); anti-GAPDH mouse monoclonal antibody IgG (Lot No. 10494-1-AP, Proteintech); Anhydrous ethanol (Lot No. 20240301, Fuchen Chemical Reagent Co., Ltd.); Mass spectrometry grade formic acid (Lot No. 205775, Thermo Fisher Scientific Co., Ltd.); Mass spectrometry grade acetonitrile (Lot No. 221404, Thermo-Fisher, USA); Distilled water (Hangzhou Wahaha Group Co., Ltd.).

[0081] Experimental animals

[0082] Adult male ICR mice (weight 36 ± 2g, 8 weeks old) were used for myocardial infarction model; male ICR mice (weight 30 ± 2g, 5 weeks old) were used for insomnia model; all SPF grade animals were ordered from Sibeifu (Beijing) Biotechnology Co., Ltd., and its company certificate number is SCXK (Beijing) 2019-0010. They were kept in cages in the barrier environment of the Animal Experiment Center of Beijing University of Chinese Medicine, with a standard room temperature of 25°C, 12h fluorescent light simulating a cycle of day and night, and fed with regular feed, with free access to food and water. The animal experiments in this study have been 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 medicine and prescription materials of Bawei Chenxiang Pills were provided by Tibet Ganlu Tibetan Medicine Co., Ltd. The materials were identified by Professor Zhang Yuan of Beijing University of Chinese Medicine. The samples of the materials were stored in the Modern Research Center of Traditional Chinese Medicine, Beijing Institute of Chinese Medicine, Beijing University of Chinese Medicine. The formula information is shown in Table 1. The preparation method of Bawei Chenxiang Pills is as follows: weigh each medicinal material according to the weight ratio of the formula, mix them evenly after crushing, add appropriate amount of honey, and make pills 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, and the preparation steps are as follows:

[0090] Take 35g of costusroot, 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 of the total weight of the medicinal materials to the residue, boil and reflux for 2 hours, and filter through gauze;

[0093] The two filtrates were combined, concentrated under reduced pressure at 40°C to a thick paste, 10 g of travertine was added and stirred evenly, dried in a reduced pressure vacuum drying oven at 50°C, and crushed into fine powder to obtain a pharmaceutical composition, code 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, and the preparation steps are as follows:

[0096] Take 35g of costusroot, 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 raw materials to the medicinal material mixture, boil and reflux for 2 hours, and filter with gauze;

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

[0099] The two filtrates were combined, concentrated under reduced pressure at 40°C to a thick paste, 10 g of travertine was added and stirred evenly, dried at 50°C in a reduced pressure vacuum drying oven, and crushed into fine powder to obtain a pharmaceutical composition, code 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, and the preparation steps are as follows:

[0102] Take 35g of costusroot, 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 raw materials to the medicinal material mixture, boil and reflux for 2 hours, and filter with gauze;

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

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

[0106] Combine the two alcohol extracts (filtrate) and the residue water extract, concentrate under reduced pressure at 40°C to a thick paste, add 10g of travertine and stir evenly, dry in a reduced pressure vacuum oven at 50°C, and grind into fine powder to obtain a 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 0.3000g of CT, ST, and CST dry powder, add 10mL of 70% ethanol, pure water, and 50% ethanol respectively, ultrasonicate until dissolved, take samples, centrifuge at 13000rpm for 15min, take supernatant, filter the supernatant through 0.22μm organic, water, and organic filter membranes respectively, and then place in a clean injection vial for sample loading.

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

[0110] Mass spectrometry conditions: HESI ion source, mass spectrometry detection in positive and negative ion modes; sheath gas 45arb, auxiliary gas 15arb, spray voltage 3.5kV(+) / 2.8kV(-); ion transfer tube temperature 320℃, auxiliary gas temperature 350℃; scan mode: Full MS / dd-MS 2 , Full MS resolution is 70000, dd-MS 2 The resolution is 17500; the quadrupole scanning range is m / z150 to 1500. The collision energy is step energy: 20, 30, 40 eV; the rest of the settings are default.

[0111] Software analysis: Qual Browser of Xcalibur 3.0.63 (Thermo Fisher Scientific, USA) was used for data spectral analysis.

[0112] The component information of each single herb was collected through databases such as CNKI and TCMSP, and a chemical component database was established. According to the above chromatographic method conditions, the ion flow diagrams of the three different path extracts of CT, ST, and CST in positive and negative ion modes were obtained, and the components of the characteristic 0-100min period of CT, ST, and CST were analyzed. The results are shown in Tables 2, 3, and 4.

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

[0114]

[0115]

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

[0117]

[0118]

[0119]

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

[0121]

[0122]

[0123]

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

[0125] Normal male ICR mice were anesthetized by intraperitoneal injection of sodium pentobarbital saline solution (injection dose of 50 mg / kg mouse body weight), the skin on the chest was prepared, and the animal was connected to a ventilator. After the thoracotomy, the left anterior descending coronary artery (LAD) was observed, and the left anterior descending coronary artery was ligated with a sterile suture needle and the chest was sutured and closed to obtain acute myocardial ischemia (AMI) mouse model animals, a total of 60.

[0126] (1) Sham group: Compared with the mice in the model group, the mice in the sham group were threaded at the same position without ligation, and other operation steps were the same, with a total of 10 mice. No drug 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 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 CST obtained in Example 1, with 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 Pills original drug group (W group), using the Bawei Chenxiang Pills selected in Example 1, with a dose of 3000 mg powder / kg mouse body weight);

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

[0135] After 7 days, echocardiography was used to detect and calculate the cardiac ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVED; d), and left ventricular end-systolic diameter (LVED; s) of mice in each group; heart tissues were obtained 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 figure shows the papillary muscle section of the heart in the short axis, and the lower figure 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 mice in the model group were significantly increased (P<0.001), and the EF and FS values ​​were significantly decreased (P<0.001), indicating that the anterior wall of the left ventricle was thinned, the ventricle was dilated, the systolic and diastolic functions of the left ventricle were impaired, and the myocardial infarction model was successfully established. Compared with the model group, the positive drug fosinopril group (F) can significantly increase EF (P<0.01), reduce LVID; d value, LVID; s value (P<0.05, P<0.01); the Bawei Chenxiang Pill group (W) can significantly increase EF and FS values ​​(P<0.001, P<0.01), and reduce LVID; d value, LVID; s value (P<0.001); the alcohol extract group (CT) can significantly increase EF and FS (P<0 .001, P<0.01), and reduced LVID;d value and LVID;s value (P<0.01, P<0.001); the water extract group (CST) after alcohol extraction can significantly increase EF and FS (P<0.001, P<0.05), and reduce LVID;d value and LVID;s value (P<0.01, P<0.001); the water extract group (ST) can increase EF and FS, and reduce LVID;d value and LVID;s value. Ultrasound results show that both CT and CST groups have significant anti-myocardial ischemia effects.

[0139] The HE staining photos of the seven groups are 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 operation group, myocardial cells were intact and neatly arranged, with clear myocardial cell striations, and no obvious inflammatory cell infiltration was observed; in the model group, obvious myocardial cell disorder was observed, a large number of inflammatory cells infiltrated in the tissue, and some myocardial cells were degenerated; after administration of F, W, CT, and CST, it was observed that the inflammatory cell infiltration was reduced and the degree of disordered myocardial cell arrangement was improved.

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

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

[0142] Mouse grouping:

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

[0144] Normal male ICR mice were intraperitoneally injected with 4-chloro-DL-phenylalanine (PCPA) saline solution at a dose of 500 mg / kg mouse body weight for 5 consecutive days, once a day. The mice were observed for disappearance of circadian rhythm, dull hair, and slow weight gain, indicating that the PCPA insomnia mouse model was successfully established. The mice with successful modeling were randomly divided into the following six groups, with 10 mice in each group. Drug administration began the next day after modeling was completed.

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

[0148] (5) Water extraction group (ST group), using ST obtained in Example 1, with a dose of 760 mg powder / kg mouse body weight;

[0149] (6) Alcohol extraction group, using CT obtained in Example 1, with a dose of 620 mg powder / kg mouse body weight;

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

[0151] Each group had 10 mice, and the drugs were administered by intragastric gavage for 7 consecutive days, once a day. One hour after the last gavage, the sleep-inducing effect of the three extracts was evaluated by intragastric gavage of sodium pentobarbital saline solution (50 mg drug / kg mouse body weight), and 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, the brain tissue of each group of mice was taken and stored in an ultra-low temperature refrigerator at -80℃. Take about 10mg of brain tissue, cut it into pieces and place it in a centrifuge tube, add RIPA lysis buffer and protein phosphatase inhibitor mixture (100:1 volume ratio) at 100mg / mL, add 1 steel ball and use tissue crusher 30 times / s, shake and crush for 3min, centrifuge at 4℃, 12000r / min for 10min, and take the tissue homogenate supernatant. According to the instructions of the BCA method kit, the sample protein content was detected, 4μg / μL was quantified, loading buffer was added, denatured at 95℃ for 10min, and stored at -20℃ for later use. An equal amount of protein was loaded and separated by SDS-PAGE. The protein was then transferred to a PVDF membrane and blocked with 5% skim milk for 2 h. Specific antibodies for GAD67 and GAPDH proteins were added, respectively, and the membrane was incubated overnight at 4°C. The membrane was washed with TBST 5 times for 5 min each time, incubated with goat anti-mouse secondary antibody at room temperature for 2 h, washed with TBST 5 times for 5 min each time, and developed with enhanced chemiluminescence (ECL). Grayscale analysis was performed using ImageJ software, and the ratio of GAD67 expression in the cortical tissue of each group to the internal reference GAPDH expression was used to represent the protein level.

[0154] The results are as follows Fig.16As shown in the figure, compared with the normal group, the sleep latency of mice in the model group was significantly prolonged (P<0.01), and the sleep maintenance time 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) could significantly shorten the sleep latency of mice (P<0.001) and prolong the sleep maintenance time 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 maintenance time of mice (P<0.01); the CT group significantly shortened the sleep latency of mice (P<0.001) and prolong the sleep maintenance time of mice (P<0.01); the ST group significantly prolonged the sleep maintenance time of mice (P<0.001); both the CT and ST groups had significant sleep-aiding effects. In terms of protein expression, compared with the blank group, the expression level of GAD67 protein 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 play a sleep-inducing role by upregulating the protein expression of GAD67.

[0155] The above results show that CT and CST have clear anti-myocardial ischemic effects, and ST and CT have clear sleep-inducing effects; in addition, CT is more significant in anti-myocardial ischemic efficacy, and ST is more significant in sleep-inducing efficacy. This result suggests that the extracts obtained by different extraction methods have obvious differences in efficacy, and the anti-AMI and sleep-inducing effects may be contributed by different pharmacological substances. Further comparative studies on the chemical components of different extracts were conducted, and finally 17 major chemical components were identified in ST; 25 major chemical components were identified from CT; and 27 major chemical components were identified from CST. In addition, the sleep-inducing effect of ST is related to increasing the expression of GAD67, a key enzyme required for GABA synthesis, which provides a direction for subsequent in-depth mechanism research.

[0156] Tibetan medicine theory believes that heart disease is mainly caused by internal and external factors such as emotional disharmony, sadness and depression, irritability and insomnia, anger and resentment, as well as eating disorders and excessive hunger, mainly including types such as heart uneasiness, heart pain, and heart heat. Tibetan medicine has the characteristic of unifying the understanding of heart physiology with mind and psychology, which is similar to the view of traditional Chinese medicine that "heart governs the mind". Bawei Chenxiang Pills is a commonly used drug in Tibetan medicine for the treatment of heart disease and associated insomnia symptoms. It has the potential to "treat the heart and brain together". Therefore, this study selected the mouse acute myocardial ischemia model prepared by ligation of the left anterior descending coronary artery and the mouse insomnia model prepared by intraperitoneal injection of p-chlorophenylalanine to comprehensively evaluate the efficacy of the drug, providing inspiration for further exploring the development and application of the potential efficacy of "treating the heart and brain together".

[0157] In this study, PCPA was intraperitoneally injected to prepare a mouse insomnia model. PCPA blocks the synthesis of 5-hydroxytryptamine by inhibiting tryptophan hydroxylation. The lack of serotonin leads to a decrease in the number of inhibitory neurotransmitter GABA neurons and their synapses and clusters, thereby inducing insomnia. GAD67 protein is involved in regulating the synthesis of GABA. In the previous exploration of the mechanism of Bawei Chenxiang Pills, it was found that GAD67 protein was well expressed in the Bawei Chenxiang Pills administration group, so it was selected to conduct a preliminary exploration of the sleep-aiding mechanism of different extracts of Bawei Chenxiang Pills. However, the in-depth sleep-aiding mechanism, anti-myocardial ischemia mechanism, and the common mechanism of sleep-aiding and anti-myocardial ischemia of different extracts of Bawei Chenxiang Pills still require subsequent systematic research to clarify the rich connotation of Bawei Chenxiang Pills' "heart and brain treatment" and the multi-component and multi-mechanism action characteristics of the compound.

[0158] The original preparation of Bawei Chenxiang Pills was made of the original powder of the medicinal material. In order to investigate the changes in the efficacy after extraction compared with the original preparation, the mouse dosage converted from the clinical effective dose of Bawei Chenxiang Pills (3g / 70kg) was used as the amount of the original medicinal material, and the dosage of the three extracts was obtained by conversion according to the extraction rate. Since the mineral medicine travertine is not extracted by organic solvents, the three extracts were converted into plant extracts according to the extraction rate of plant medicines, and then combined with travertine with the original prescription dosage for administration. Finally, the dosages obtained were Bawei Chenxiang Pills group (3000mg / kg), alcohol extract group (620mg / kg), water extract group (760mg / kg), and alcohol-water extract group (1084mg / kg).

[0159] By comparing the efficacy of different extracts, the optimal extraction path can be screened, and then combined with the comparison of their components, the pharmacological substances of the drug can be usefully explored. There are significant differences in the polarity distribution of drug alcohol extracts and water extracts, and their efficacy is naturally different. For example, in this study, the CT of Bawei Chenxiang Pills focuses on anti-myocardial ischemia and ST focuses on sleep-inducing efficacy. We also tried to combine alcohol extraction and water extraction to make the extract components more comprehensive in order to obtain an ideal combination of efficacy.

[0160] Based on the indications of Bawei Chenxiang Pills, this study clarified the efficacy advantages of the three extraction routes through a dual efficacy evaluation system of mouse acute myocardial ischemia model and mouse insomnia model. The results of component analysis showed that chromones and terpenoids mainly contributed to the anti-myocardial ischemia effect, tannins and phenolic acid components mainly contributed to the sleep-inducing effect, and the sleep-inducing effect was related to the regulation of GAD67 protein expression. The above results provide a scientific basis for clarifying the efficacy of Bawei Chenxiang Pills, establishing quality standards, and rational clinical medication, and lay a solid foundation.

[0161] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A pharmaceutical composition, the active ingredients of which include the following substances: Chebulic acid; Breviphenolic 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; Due to eugenol; 11-Carbonyl-β-acetylboswellic acid; 9,11-Dehydroboswellic acid; Boswellic acid; Acetyl boswellic acid; Travertine.

2. The pharmaceutical composition according to claim 1, characterized in that The 9,11-dehydroboswellic acid is one or both of 9,11-dehydro-α-boswellic acid and 9,11-dehydro-β-boswellic acid; The boswellic acid is one or both of α-boswellic acid and β-boswellic acid; The acetyl boswellic acid is one or both of acetyl-α-boswellic acid and acetyl-β-boswellic acid.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that The preparation method of the pharmaceutical composition comprises the following steps: Mix costusroot, agarwood, jujube, terminalia chebula, nutmeg, kapok and frankincense to obtain a mixture; The mixture is 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, terminalia chebula, nutmeg, kapok, frankincense and travertine is 2-5:1-3:1-3:1-3:1-3:1-2:0.5-1.5:

1.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The terminalia chebula is the stewed terminalia chebula fruit.

5. The pharmaceutical composition according to claim 3, characterized in that 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 medicine. During the non-first reflux and boiling, the ethanol aqueous solution used for reflux is 5-10 times the weight of the medicine residue obtained by the last boiling and reflux.

6. The pharmaceutical composition according to claim 3, characterized in that The concentration is carried out under reduced pressure at 35-45°C.

7. The pharmaceutical composition according to claim 3, characterized in that The drying is carried out under reduced pressure and vacuum at 45-55°C.

8. The pharmaceutical composition according to any one of claims 1 to 7, characterized in that The pharmaceutical composition contains substances with the following ion fragment signals: The added 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; The added ion is [M+Na] + , the molecular weights of ion fragments include 403.24615±0.001; The added 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.

9. Use of the pharmaceutical composition according to any one of claims 1 to 8 in preparing a preparation for improving the health status of a subject; Improving the subject's health condition includes: Improve myocardial ischemia in subjects; Improve cardiac ejection fraction in subjects with myocardial infarction; Increase the fractional shortening of the heart in subjects with myocardial infarction; Reduce left ventricular end-diastolic diameter in subjects with myocardial infarction; Reduce left ventricular end-systolic diameter in subjects with myocardial infarction; Shortened sleep latency in subjects with insomnia; Prolong the subjects' sleep duration; Up-regulate the expression of GAD67 protein in the subjects' brain tissue.

10. The use according to claim 9, characterized in that The subject is selected from humans and mice.

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