Xijiaqi composition and its application in preparing medicine for improving heart failure after myocardial infarction

Abstract

The present invention discloses a Siegesbeckia scoparia composition and its use in preparing a medicine for improving heart failure after myocardial infarction. Experiments have shown that the Siegesbeckia scoparia composition, which is prepared in a mass ratio of 17-19 parts of astragalus extract, 2-4 parts of Siegesbeckia scoparia extract, and 1 part of Periploca cortex, can significantly improve the cardiac function of rats with heart failure after myocardial infarction, reduce the release of myocardial damage enzymes, restore myocardial tissue morphology, and alleviate myocardial fibrosis.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine composition and its application, in particular to a Siegesbeckia scoparia composition and its application in preparing a medicine for improving heart failure after myocardial infarction. Background Art

[0002] Chronic heart failure (CHF) is a clinical syndrome caused by various structural or functional cardiac diseases that impair ventricular filling and / or ejection capacity. It is the terminal stage of many cardiovascular diseases and a major cause of high cardiovascular mortality. There are 4.5 million patients with heart failure in my country, with approximately 500,000 new cases each year. With the aging population, the prevalence of CHF is increasing significantly. As the last frontier in cardiovascular disease prevention and control, CHF has become a major global public health issue, posing a serious threat to public health. Despite significant advances in modern medicine in the prevention and treatment of CHF, mortality and rehospitalization rates remain high, and the overall prognosis remains poor. Therefore, there is an urgent need to explore new therapies for the prevention and treatment of CHF.

[0003] Traditional Chinese medicine (TCM) boasts multi-pathway, multi-ingredient, multi-target, and safe effects, demonstrating significant efficacy in preventing and treating CHF. Clinical practice has demonstrated its advantages in improving CHF patients' clinical symptoms, enhancing quality of life, improving cardiac function, reversing ventricular remodeling, and slowing the progression of CHF, demonstrating its clear therapeutic value. According to TCM, CHF falls under the categories of "palpitations," "asthma," "phlegm and fluid retention," and "edema," representing a syndrome characterized by both underlying deficiency and superficial excess. The pathogenesis can be summarized as "deficiency," "stasis," and "dampness." Deficiency of heart qi and yang is the core pathogenesis (the underlying cause), while blood stasis, turbid phlegm, and fluid retention are the primary pathological products (the superficial symptoms). Treatment focuses on invigorating qi, unblocking the meridians, promoting blood circulation and removing stasis, and dispelling dampness and promoting diuresis.

[0004] Currently, there is no report on the use of a composition composed of Astragalus, Siegesbeckia and Periploca for preparing a drug for improving heart failure after myocardial infarction. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Siegesbeckia scoparia composition.

[0006] The second object of the present invention is to provide a use of a Siegesbeckia scoparia composition in the preparation of a drug for improving heart failure after myocardial infarction.

[0007] The technical solution of the present invention is summarized as follows:

[0008] The Siegesbeckia composite material is composed of astragalus root extract, siegesbeckia herba extract and perilla bark extract.

[0009] The mass ratio of the astragalus extract, the sisseed oil herb extract and the perennial root extract is (17-19): (2-4): 1.

[0010] The application of the above-mentioned Xijiaqi composition in the preparation of a medicine for improving heart failure after myocardial infarction.

[0011] Advantages of the present invention:

[0012] (1) Experiments have shown that the Xijiaqi combination can significantly increase the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) of rats with heart failure after myocardial infarction, thereby improving cardiac function.

[0013] (2) The Xijiaqi combination can significantly inhibit the release of myocardial injury enzymes, myocardial specific troponin T (cTnT) and heart failure marker brain natriuretic peptide (BNP), in rats with heart failure after myocardial infarction, and reduce myocardial damage.

[0014] (3) The Xijiaqi combination can significantly restore the myocardial tissue morphology and structure of rats with heart failure after myocardial infarction, and can significantly reduce myocardial fibrosis in rats with heart failure after myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 To investigate the effect of Xijiaqi combination on cardiac function in rats with heart failure after myocardial infarction.

[0016] Figure 2 To investigate the effect of Xijiaqi combination on myocardial injury enzyme profiles cTnT and BNP in rats with heart failure after myocardial infarction.

[0017] Figure 3 To investigate the effects of the Xijiaqi combination on the morphological structure of myocardial tissue in rats with heart failure after myocardial infarction.

[0018] Figure 4 To investigate the effects of Xijiaqi combination on myocardial fibrosis in rats with heart failure after myocardial infarction. DETAILED DESCRIPTION

[0019] The Astragalus extracts of each embodiment of the present invention were purchased from Beijing Kangrentang Pharmaceutical Co., Ltd. (Batch No.: 22025921), the Siegesbeckia scoparia extract was purchased from Beijing Kangrentang Pharmaceutical Co., Ltd. (Batch No.: 22010811), and the Periploca odorata extract was purchased from Beijing Kangrentang Pharmaceutical Co., Ltd. (Batch No.: 21004811). The sources of the extracts in the composition of the present invention are disclosed in order to better illustrate the present invention, but the sources of the extracts of the present invention are not limited.

[0020] Xijiaqi combination, abbreviated as XJQ; it can also be called Xijiaqi prescription.

[0021] Preparation of the Xijiaqi composition: the astragalus root extract, the Siegesbeckia scoparia extract and the Periploca rotundus extract are mixed evenly to obtain the composition.

[0022] Example 1

[0023] The Siegesbeckia composition consists of 18.06 grams of Astragalus membranaceus extract, 3.17 grams of Siegesbeckia scoparia extract and 1 gram of Periploca rotundus extract.

[0024] Example 2

[0025] The Siegesbeckia composition consists of 17 grams of Astragalus membranaceus extract, 4 grams of Siegesbeckia scoparia extract and 1 gram of Periploca rotunda extract by mass.

[0026] Example 3

[0027] The Siegesbeckia composition consists of 19 grams of Astragalus membranaceus extract, 2 grams of Siegesbeckia herb extract and 1 gram of Periploca rotundus extract by mass.

[0028] Example 4

[0029] The method for establishing a CHF model after myocardial infarction in rats includes the following steps:

[0030] Male SD rats, 8 weeks old, weighing 220 ± 10 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0031] (1) Rats were anesthetized with 2% sodium pentobarbital (60 mg / kg) intraperitoneally;

[0032] (2) Intubate the trachea with a 16G intravenous needle and connect to a small animal ventilator;

[0033] (3) The rat heart was exposed by thoracotomy at the 3rd and 4th intercostal spaces. The left anterior descending coronary artery (LADCA) was ligated with a 3 / 8 curved needle 2 mm below the intersection of the left atrial appendage and the pulmonary artery cone. Myocardial regional ischemia was confirmed by visual observation (whitening) and electrocardiographic monitoring (ST segment elevation and QRS widening).

[0034] (4) The intercostal muscles, muscles, and skin are sutured layer by layer from the inside out to close the chest cavity.

[0035] Inclusion criteria for rats with heart failure after myocardial infarction: 24 hours after surgery, the rats had 6-8 pathological Q waves on the 12-lead electrocardiogram.

[0036] Example 5

[0037] For grouping and dosing, the drugs were prepared as in Example 1, comprising the following steps:

[0038] (1) Thirty-six male SD rats were randomly divided into six groups according to the number of pathological Q waves on the electrocardiogram 24 hours after surgery, with six rats in each group:

[0039] ①Sham operation group;

[0040] ②Chronic heart failure group (CHF);

[0041] ③ Low dose of Xijiaqi combination (0.778 g / kg) + chronic heart failure group (XJQL+CHF);

[0042] ④ The middle dose of Xijiaqi combination (1.556 g / kg) + chronic heart failure group (XJQM+CHF);

[0043] ⑤ High dose of Xijiaqi combination (3.112 g / kg) + chronic heart failure group (XJQH+CHF);

[0044] ⑥ Perindopril (0.0008g / kg) + chronic heart failure group (PDPL+CHF)

[0045] The sham group underwent the same surgical procedure, but the LADCA was threaded without ligation.

[0046] The group receiving the Xijiaqi combination was given oral administration starting from 24 hours after surgery, once a day, for 6 consecutive weeks; the sham group and the CHF group were given an equal volume of normal saline by oral administration for 6 consecutive weeks.

[0047] (2) After 6 weeks, the cardiac function, myocardial injury enzyme spectrum, myocardial tissue morphology and myocardial fibrosis of rats in each group were detected.

[0048] Example 6

[0049] Effects of Xijiaqi combination on cardiac function in rats with heart failure after myocardial infarction.

[0050] Experimental Materials

[0051] Instrument: Vevo2100 ultra-high-resolution small animal ultrasound imaging system.

[0052] Experimental methods

[0053] Rats were anesthetized intraperitoneally with 1% sodium pentobarbital (4 ml / kg). After complete anesthesia, they were fixed supine on a custom-made mouse board. The chest skin was routinely prepared. Left ventricular short axis measurements were performed using the Vevo 2100 small animal ultrasound imaging system. Left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular anterior wall thickness at end-diastole, and left ventricular anterior wall thickness at end-diastole were performed. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated. Each parameter was measured over three consecutive cardiac cycles and the average value was calculated.

[0054] Experimental results

[0055] The left ventricular ejection fraction and left ventricular fractional shortening of rats in each group are shown in Table 1.

[0056] Table 1 LVEF and LVFS of rats in each experimental group

[0057]

[0058] Note: * P<0.05vs Sham group, # P<0.05 vs CHF group, n=6.

[0059] From Table 1 and Figure 1 The results of A showed that compared with the Sham group, the LVEF of the CHF group rats ( Figure 1 B) and LVFS( Figure 1 C) significantly decreased (P < 0.05); compared with the CHF group, different doses of the Xijiaqi composition of Example 1 of the present invention can improve LVEF and LVFS levels to varying degrees, among which the effects of the medium-dose and high-dose groups of the Xijiaqi composition on improving LVEF and LVFS are statistically different from those of the CHF group (P < 0.05); the results indicate that the Xijiaqi composition can significantly improve the cardiac function of rats with CHF after myocardial infarction.

[0060] Example 7

[0061] Effects of Xijiaqi combination on myocardial injury enzyme profiles cTnT and BNP in rats with heart failure after myocardial infarction.

[0062] Experimental Materials

[0063] (1) Reagents: Rat cardiac troponin T (cTnT) ELISA kit (DG96089Q), rat brain natriuretic peptide (BNP) ELISA kit (DG20595D).

[0064] (2) Instruments: 37°C constant temperature and humidity chamber, microplate reader (MULTISKAN MK3, Thermo, USA).

[0065] Experimental methods

[0066] (1) Serum sample collection and processing

[0067] Six weeks after surgery, whole blood was collected from the abdominal aorta of each group of rats using a blood collection needle. The specimens were centrifuged at 1500×g for 15 minutes at 4°C within 30 minutes after collection, and the supernatant was collected for detection.

[0068] (2) ELISA operation steps

[0069] ① Take the test kit out of the refrigerated environment and equilibrate it at room temperature;

[0070] ② Take out the required microporous enzyme strips from the aluminum foil bag after equilibration at room temperature for 20 minutes;

[0071] ③ Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well;

[0072] ④ Add 50 μL of the sample to be tested to the sample well, and do not add to the blank well;

[0073] ⑤Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard and sample wells, seal the reaction wells with a sealing film, and incubate in a constant temperature and humidity chamber at 37°C for 60 min.

[0074] ⑥ Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times;

[0075] ⑦ Add 50 μL of substrate color reagent A and B to each well and incubate at 37°C in the dark for 15 minutes;

[0076] ⑧ Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0077] (3) Calculation of experimental results

[0078] Use the OD value of the measured standard as the horizontal coordinate and the concentration value of the standard as the vertical coordinate to draw a standard curve on coordinate paper or using relevant software, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.

[0079] Experimental results

[0080] The levels of cTnT and BNP in the serum of rats in each group are shown in Table 2.

[0081] Table 2 The levels of cTnT and BNP in the serum of rats in each experimental group

[0082]

[0083] Note: P<0.05 vs Sham group, # P<0.05 vs CHF group, n=6.

[0084] The results in Table 2 show that compared with the Sham group, the cTnT ( Figure 2 A) and BNP( Figure 2 B) content was significantly increased (P < 0.05); compared with the CHF group, different doses of the Xijiaqi composition of Example 1 of the present invention can reduce the levels of cTnT and BNP in the serum of CHF rats to varying degrees (P < 0.05); this result shows that the Xijiaqi composition can significantly reduce myocardial damage in CHF rats after myocardial infarction.

[0085] Example 8

[0086] Effects of Xijiaqi combination on the morphological structure of myocardial tissue in rats with heart failure after myocardial infarction.

[0087] Experimental Materials

[0088] (1) Reagents: 4% paraformaldehyde, ethanol, xylene, Harris hematoxylin stain, and eosin stain.

[0089] (2) Instruments: Leica ASP300S fully automatic dehydrator, paraffin embedding machine, rotary paraffin slicer, optical microscope.

[0090] Experimental methods

[0091] Six weeks after surgery, rat hearts were removed from the chest and fixed in 4% paraformaldehyde for 48 hours. The hearts were then dehydrated in a fully automated dehydrator using 50%-70%-80%-95%-100% ethanol, cleared with xylene, and immersed in wax. The hearts were then embedded in a paraffin embedding machine to create tissue wax blocks. Finally, 5 μm thick serial paraffin sections were cut using a rotary paraffin sectioning machine. HE staining procedures were as follows:

[0092] ① Preparation: Place the slices to be stained with HE in a 60° oven for 60 minutes.

[0093] ② Dewaxing to hydration: Dewax the prepared paraffin sections in xylene for 15 minutes. Then, begin a gradient hydration process from high to low concentrations: soak in each solution tank for 2-5 minutes, starting with anhydrous ethanol, 95% ethanol, 90% ethanol, 70% ethanol, 60% ethanol, and finally 50% ethanol. Rinse with running water for 5 minutes after completing these steps.

[0094] ③ Staining: Place the dewaxed sections in a Harris hematoxylin staining solution for 10 minutes, rinse with running water for 1 minute, then differentiate with 1% hydrochloric acid ethanol for 30 seconds, wash thoroughly with tap water, bluing for 10 minutes, stain with 1% eosin solution for 3 minutes, and rinse with running water for 1 minute.

[0095] ④ Dehydration: Dehydrate step by step from low concentration to high concentration, soaking in 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and anhydrous ethanol for 2 minutes respectively;

[0096] ⑤ Transparency: Soak the dehydrated slices in xylene twice for 10 minutes each time, and finally seal the slices with neutral gum.

[0097] ⑥ Take photos: Observe the myocardial cell damage in the edge area of myocardial infarction under a microscope, and select an appropriate magnification to take photos and record them.

[0098] Experimental results

[0099] like Figure 3 As shown, the upper picture is an image taken under the condition of ×1 objective lens, and the lower picture is an image taken under the condition of ×20 objective lens. It can be seen that the morphological structure of the myocardial tissue of the rats in the Sham group is normal: the myocardial fibers are arranged neatly; the cell nuclei are clear, evenly colored and of uniform size, there is no inflammatory cell infiltration, and there is little fibrous tissue between cells. Compared with the Sham group, the myocardial infarction focus of the rats in the CHF group is obvious: the number of myocardial cells is reduced, the myocardial fibers are disordered, the myocardial tissue gaps are enlarged, the coloring is uneven, and the fibrous hyperplasia is obvious. Different concentrations of the Siegesbeckia composition of Example 1 of the present invention can alleviate myocardial damage caused by CHF to varying degrees and restore the myocardial tissue structure.

[0100] Example 9

[0101] Effects of Xijiaqi combination on myocardial fibrosis in rats with heart failure after myocardial infarction.

[0102] Experimental Materials

[0103] Reagents: Masson trichrome staining kit.

[0104] Experimental method: Masson staining

[0105] ① Preparation: Place the Masson-stained sections in a 60°C oven for 60 minutes.

[0106] ② Dewaxing to hydration: Dewax the prepared paraffin sections in xylene for 15 minutes. Then, begin a gradient hydration process from high to low concentrations: soak in each solution tank for 2-5 minutes, starting with anhydrous ethanol, 95% ethanol, 90% ethanol, 70% ethanol, 60% ethanol, and finally 50% ethanol. Rinse with running water for 5 minutes after completing these steps.

[0107] ③ Staining: Soak the sections in pre-prepared Weigert iron hematoxylin staining solution for 10 minutes, differentiate with acidic ethanol differentiation solution for 10 seconds, rinse with pure water for 10 seconds, return to blue with Masson blueing solution for 5 minutes, rinse with pure water for 1 minute, stain with Ponceau fuchsin for 8 minutes, rinse with weak acid solution (distilled water: weak acid solution = 2:1) for 1 minute, wash with phosphomolybdic acid solution for 2 minutes, rinse with weak acid solution for 1 minute, stain with aniline blue solution for 90 seconds, and wash with weak acid solution for 1 minute.

[0108] ④ Dehydration: Rapid dehydration in 95% ethanol solution once, and dehydration in anhydrous ethanol three times, each time for 5 seconds.

[0109] ⑤ Transparency: Soak the dehydrated slices in xylene twice for 2 minutes each time, and finally seal the slices with neutral gum.

[0110] ⑥ Take photos: Observe the myocardial fibrosis under a microscope and select an appropriate magnification to take photos and record them.

[0111] Experimental results

[0112] like Figure 4 As shown, the upper figure is an image taken under the condition of ×1 objective lens, and the lower figure is an image taken under the condition of ×20 objective lens, in which the blue-stained ones represent collagen fibers. It can be seen that very small amounts of collagen fibers can be seen in the interstitial spaces between the myocardial cells of the rats in the Sham group; the myocardial cells have normal morphological structure, and are densely and neatly arranged. In the anterior wall of the ventricle of the rats in the CHF group, a large area of myocardial infarction area was stained blue, the myocardial fibers were disorderly arranged, and the muscle cells were swollen and broken. Different concentrations of the Xijiaqi composition of Example 1 of the present invention can alleviate myocardial fibrosis to different degrees: with the increase of the dosage concentration, the arrangement of the myocardial cells gradually became neater and denser, the intercellular spaces narrowed, and the collagen fiber content decreased.

[0113] Experiments have shown that the Xijiaqi compositions prepared in Examples 2 and 3 can also significantly improve the cardiac function of rats with heart failure after myocardial infarction, reduce myocardial damage, restore myocardial tissue morphology and structure, and alleviate myocardial fibrosis.

Claims

1. A Xijiaqi composition, characterized in that The invention is composed of astragalus extract, siegesbeckia herba extract and perilla bark extract; the mass ratio of the astragalus extract, siegesbeckia herba extract and perilla bark extract is (17-19): (2-4):

1.

2. Use of the Xijiaqi composition according to claim 1 in the preparation of a drug for improving heart failure after myocardial infarction.

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