Plant extract for treating cardiac fibrosis as well as preparation method and application thereof

Abstract

The invention relates to the technical field of plant extraction, in particular to a plant extract for treating cardiac fibrosis and a preparation method and application thereof, and the plant extract is obtained by mixing and extracting astragalus membranaceus and sappan wood. The prepared plant extract can effectively inhibit the cardiac fibrosis process and can be used for preparing drugs for treating cardiac fibrosis. When the mass ratio of hematoxylon to astragalus membranaceus is 1: 1 and the concentration of the plant extract is 0.001 mg / mL, expression of fibrosis-related protein alpha-SMA and the like can be remarkably reduced, transcription of fibrosis-related gene COL1A is inhibited, collagen fiber deposition is reduced, and the hematoxylon extract is stable in physicochemical property and suitable for long-term storage and transportation and has outstanding clinical application value and industrialization prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and in particular to a plant extract for treating cardiac fibrosis, a preparation method thereof and an application thereof. Background Art

[0002] Cardiac fibrosis is a key pathological link in the development of various heart diseases. Its core characteristics are excessive collagen deposition in myocardial tissue, abnormal fibroblast activation, and myofibroblast transformation, leading to increased myocardial stiffness and ventricular remodeling, ultimately causing heart failure and even cardiac insufficiency, seriously threatening patients' lives and health. Clinical data show that the incidence of cardiac fibrosis after myocardial infarction is as high as 60%, making it one of the main causes of poor long-term prognosis in patients with myocardial infarction.

[0003] Currently, the drugs used clinically to improve cardiac fibrosis mainly include angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), and beta-blockers. Although these drugs can slow the progression of fibrosis to a certain extent, they have a single target and are prone to side effects such as hypotension and renal damage with long-term use. They also have limited effects on reversing established fibrotic tissue. In addition, although some new targeted drugs (such as TGF-β receptor antagonists) have shown anti-fibrotic potential in animal experiments, most have not yet entered the clinical application stage due to systemic toxicity and clinical safety issues.

[0004] Plant extracts, due to their multi-component and multi-target properties, have attracted considerable attention in the treatment of cardiovascular diseases. For example, Chinese invention patent application CN114945407A provides a plant extract composition for treating cardiovascular and metabolic diseases. It uses naringin and chlorogenic acid to treat or prevent dyslipidemia, cardiovascular disease, metabolic syndrome, and hypercholesterolemia. However, there is limited research on the combined use of Astragalus membranaceus (Fisch.) Bunge and Biancaea sappan (L.) Tod., and this combination suffers from unstable physicochemical properties. Summary of the Invention

[0005] In order to solve the problems in the prior art, the first aspect of the present invention provides a plant extract for treating cardiac fibrosis. The plant extract is obtained by mixed extraction of Astragalus and Sappan.

[0006] In one embodiment, the plant extract inhibits the expression of α-SMA and CO11A1 proteins.

[0007] The second aspect of the present invention provides a method for preparing the plant extract, comprising at least the following steps:

[0008] S1. Slice and dry Astragalus and Sappan wood respectively;

[0009] S2. Soaking the dried Astragalus and Sappan in ultrapure water for three times, filtering and combining the filtrates to obtain a crude extract;

[0010] S3. The crude extract is subjected to impurity removal, concentration, freeze-drying, and grinding to obtain the plant extract.

[0011] In one embodiment, in step S1, the Astragalus membranaceus is sliced ​​to obtain oblique slices with a thickness of 2-4 mm; and the Sappan wood is sliced ​​to obtain thin slices with a thickness of 2-3 mm.

[0012] In one embodiment, the drying conditions in step S1 are drying in an oven at 40° C. for 24-36 hours until the water content of Astragalus membranaceus is 8-12 wt % and the water content of Sappan wood is 10-14 wt %.

[0013] In one embodiment, the mass ratio of the dried Astragalus and Sappan in S2 is 1:1.

[0014] When the mass ratio of dried astragalus and sappan wood in the present invention is 1:1, the total flavonoid content of the plant extract fluctuates slightly and its chemical properties are stable after storage at room temperature for 3 months. The possible reason is that when the ratio of astragalus and sappan wood is 1:1, the flavonoid components and other active substances contained in them, such as astragaloside in astragalus and hematoxylin in sappan wood, may form a synergistic effect. At this ratio, the molecular interactions between the two types of components are more balanced, which can reduce the oxidation and degradation of a single component due to structural instability, thereby maintaining the stability of the total flavonoid content. In addition, at this ratio, impurities in astragalus and sappan wood may be more easily removed simultaneously during the extraction and impurity removal process, reducing the encapsulation or catalytic degradation of flavonoid components by impurities, and indirectly improving the chemical stability of the extract.

[0015] In one embodiment, the solid-to-liquid ratio of the dried astragalus and sappan wood to ultrapure water is 1 g:10 mL.

[0016] In one embodiment, the process extracted in step S2 is:

[0017] Step 1: Soak the dried Astragalus and Sappan wood in ultrapure water at room temperature and heat to boiling, then condense and reflux for 1-1.5 hours to collect the extract;

[0018] Step 2: filtering the extract to obtain a filter residue and a filtrate;

[0019] Step 3: Add ultrapure water to the filter residue and repeat the first and second steps.

[0020] In one embodiment, the soaking time is 10-14 hours.

[0021] In one embodiment, the impurity removal process in step S3 is: adding the crude extract to anhydrous ethanol for 12-20 hours, filtering through a 0.45 μm microporous filter membrane, discarding the filter residue, and retaining the filtrate to complete the impurity removal step.

[0022] In one embodiment, the volume ratio of the crude extract to anhydrous ethanol is 1:3.

[0023] In one embodiment, the concentration method in step S3 is: evaporating the filtrate after impurity removal at 60° C. to remove the solvent and concentrate it to 1 / 10 of the original volume.

[0024] In one embodiment, the freeze-drying conditions in step S3 are: -50°C, 0.01 mbar, freeze-drying for 24 hours.

[0025] A third aspect of the present invention provides a use of a plant extract in the preparation of a medicament for treating cardiac fibrosis.

[0026] In one embodiment, the concentration of the plant extract in the drug for treating cardiac fibrosis is less than 0.05 mg / mL, and can be exemplified as 0.01 mg / mL, 0.005 mg / mL, or 0.001 mg / mL.

[0027] Beneficial effects

[0028] 1. The plant extract prepared by the present invention can effectively inhibit the process of cardiac fibrosis. When the mass ratio of sappan wood to astragalus is 1:1 and the concentration of the plant extract is 0.001 mg / mL, it can significantly reduce the expression of fibrosis-related proteins such as α-SMA, inhibit the transcription of the fibrosis-related gene COL1A, and reduce collagen fiber deposition.

[0029] 2. The plant extract provided by the present invention uses total flavonoids as the active ingredient indicator. After storage at room temperature for 3 months, the total flavonoid content fluctuates little, the physical and chemical properties are stable, and it is suitable for long-term storage and transportation, providing convenience for clinical application and industrialization.

[0030] 3. The plant extract provided by the present invention has low toxicity and side effects, reliable safety, and a simple preparation process.

[0031] 4. Compared with existing imported anti-myocardial infarction and fibrosis drugs, the plant extract provided by the present invention uses natural plants as raw materials, has significantly reduced production costs, and has clear therapeutic effects and stability, and has outstanding clinical application value and industrialization potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Figure A is a schematic diagram of the subsequent experimental process for the treatment of 3T3 cells with Suhuang extract; Figure B shows the effect of different concentrations of Suhuang extract prepared in various Examples and Comparative Examples on 3T3 cell viability; Figure C shows the inhibitory effect of different concentrations of Suhuang extract prepared in various Examples and Comparative Examples on α-SMA expression; Figure G shows the inhibitory effect of Suhuang extract on COL1A transcription; Figure H shows the changes in collagen fiber content in cells detected by Sirius red staining. **P<0.01, *P<0.05, ***P<0.001.

[0033] Figure 2 A shows the experimental design for the inhibitory effect of plant extracts on cardiac fibrosis in mice with myocardial infarction; B shows cardiac ultrasound images of the three groups of mice; C and D show left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), respectively; E shows Masson staining of the heart; F shows the statistical results of myocardial infarction area; G shows a Western Blot image of a fibrosis-related protein (α-SMA) in cardiac tissue; H shows the quantitative analysis of α-SMA protein expression. **P<0.01, *P<0.05, ***P<0.001. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods in the examples where specific conditions are not specified were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents used where the manufacturer is not specified are all conventional products that can be purchased commercially.

[0035] Example 1

[0036] In a first aspect, this example provides a plant extract for treating cardiac fibrosis, wherein the plant extract is obtained by mixed extraction of astragalus and sappan wood.

[0037] The plant extract inhibits the expression of α-SMA and CO11A1 proteins.

[0038] The second aspect of this example provides a method for preparing the plant extract, comprising the following steps:

[0039] S1. Slice and dry Astragalus and Sappan wood respectively;

[0040] S2. Soaking the dried Astragalus and Sappan in ultrapure water for three times, filtering and combining the filtrates to obtain a crude extract;

[0041] S3. The crude extract is subjected to impurity removal, concentration, freeze-drying, and grinding to obtain the plant extract.

[0042] In step S1, the astragalus root is sliced ​​to obtain oblique slices with a thickness of 3 mm; the Sappan wood is sliced ​​to obtain thin slices with a thickness of 2 mm.

[0043] The drying condition in step S1 is to dry in an oven at 40° C. for 30 hours until the water content of Astragalus membranaceus reaches 10 wt % and the water content of Sappan wood reaches 12 wt %.

[0044] The process extracted in the S2 step is:

[0045] Step 1: Accurately weigh 12.5 g each of dried Sappan Wood and Astragalus, add 250 mL of ultrapure water, soak at room temperature for 12 hours, then heat to boiling, and condense and reflux for 1 hour to collect the extract;

[0046] Step 2: filtering the extract to obtain a filter residue and a filtrate;

[0047] Step 3: Add the filter residue to 250 mL of ultrapure water and repeat the first and second steps.

[0048] The impurity removal process in step S3 is as follows: adding the crude extract to three times the volume of anhydrous ethanol for 15 hours, filtering through a 0.45 μm microporous filter membrane, discarding the filter residue, and retaining the filtrate to complete the impurity removal step.

[0049] The concentration method in step S3 is as follows: evaporating the filtrate after impurity removal at 60° C. to remove the solvent and concentrate the filtrate to 1 / 10 of its original volume.

[0050] The freeze-drying conditions in step S3 are: -50°C, 0.01 mbar, freeze-drying for 24 hours.

[0051] The plant extract is obtained by grinding in step S3 and passing through a 200-mesh sieve.

[0052] The third aspect of this example provides a use of a plant extract in the preparation of a drug for treating cardiac fibrosis.

[0053] Comparative Example 1

[0054] The specific implementation of this example is the same as that of Example 1, except that the mass ratio of the dried Astragalus and Sappan in S2 is 1:2.

[0055] Comparative Example 2

[0056] The specific implementation of this example is the same as that of Example 1, except that the mass ratio of the dried Astragalus and Sappan in S2 is 2:1.

[0057] Performance Testing

[0058] 1. Stability test:

[0059] The total flavonoids were used as an indicator to detect the active ingredient content of the plant extract (abbreviated as Suhuang extract). The total flavonoids content was determined using the NaNO2-Al(NO3)3 method. The steps are as follows: Accurately weigh 5 mg of rutin standard and place it in a 25 mL volumetric flask, then dissolve it in 75% ethanol and adjust it to the scale to prepare a 0.2 mg / mL standard solution. Accurately pipette 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of rutin standard solution into a 25 mL volumetric flask, add 1.0 mL of 5% NaNO2 solution, shake well, let it stand for 5 minutes, add 1.0 mL of 10% Al(NO3)3 solution, shake well, let it stand for 5 minutes; then add 10 mL of 10% NaOH solution, adjust the volume to 25 mL with 75% ethanol, shake well, and let it stand for 15 minutes. Use 0 mL of rutin standard solution as the blank control, use rutin concentration as the vertical axis, and the absorbance value at 510 nm as the horizontal axis to draw a standard curve. Calculate the regression equation of the standard curve, Y = 5.043X + 0.0725, R 2 =0.9940, Y is the absorbance value, X is the concentration.

[0060] Accurately weigh 5 mg of sample and dilute it in 25 mL of 75% ethanol. Take 5 mL and place it in a 25 mL volumetric flask. Add 1.0 mL of 5% NaNO2 solution, shake well, let it stand for 5 minutes, add 1.0 mL of 10% Al(NO3)3 solution, shake well, let it stand for 5 minutes; then add 10 mL of 10% NaOH solution, dilute to 25 mL with 75% ethanol, shake well, and let it stand for 15 minutes. Measure the absorbance at 510 nm and substitute it into the standard curve to calculate the content of total flavonoids in each sample. The plant extracts prepared in Example 1 and Comparative Examples 1-2 were stored at room temperature (24°C) for 3 months and then the content of total flavonoids in the samples was measured. The test results are shown in Table 1.

[0061] Table 1

[0062]

[0063] 2. Plant extract concentration screening:

[0064] A certain amount of the plant extracts of each embodiment and comparative example was weighed and dissolved in DMSO to prepare solutions with concentrations of 0.1, 0.05, 0.01, 0.005, and 0.001 g / mL, respectively ( Figure 1 The ratio of 1:1 corresponds to the sample of Example 1 with a ratio of 1:1 for Sappan wood: Astragalus membranaceus, 1:2 corresponds to the sample of Comparative Example 1 with a ratio of 1:2 for Sappan wood: Astragalus membranaceus, and 2:1 corresponds to the sample of Comparative Example 2 with a ratio of 2:1 for Sappan wood: Astragalus membranaceus. 3T3 cells in the logarithmic growth phase were trypsinized and resuspended in DMEM medium containing 10% fetal bovine serum (FBS) to adjust the cell density to 1×105 cells / mL. The cell suspension was inoculated into a 96-well plate at 100 μL per well and placed in a saturated humidity, 37°C, 5% CO2 incubator for pre-culture until the cells adhered. After the cells adhered, the original culture medium was discarded, and the plates were washed with PBS. According to the experimental design, culture medium containing different concentrations (0.1, 0.05, 0.01, 0.005, 0.001 mg / mL) of Suhuang extract was added, 100 μL per well. Control groups were set up: blank culture medium (no cells), negative control (cells + culture medium without Suhuang extract), and positive control (cells + cell lysis solution Lysis Solution). Six replicate wells were set up for each group to avoid edge effects. After 24 hours of treatment, the original culture medium was discarded, and after washing with PBS, 100 μL of culture medium containing 10% CCK-8 solution was added to each well. Gently shake to mix and continue to culture for 1-4 hours. Use an enzyme reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm. See the results. Figure 1 B.

[0065] As can be seen from the figure, in the three groups with different ratios of Suhuang extract, the cell viability of the groups with final concentrations of 0.01, 0.005, and 0.001 mg / mL of Suhuang extract did not show significant changes compared with the control group, while the cell viability of the groups with final concentrations of 0.1 and 0.05 mg / mL of Suhuang extract decreased significantly. Therefore, the final concentrations of 0.01, 0.005, and 0.001 mg / mL of Suhuang extract were selected for subsequent screening.

[0066] 3. Screening of optimal plant extract ratio and concentration:

[0067] (1) Immunoblotting test:

[0068] A certain amount of plant extract from each example and comparative example was weighed and dissolved in DMSO to prepare extract solutions at concentrations of 0.01, 0.005, and 0.001 g / mL, with the corresponding ratios of Sappan:Astragalus of 1:1, 1:2, and 2:1. 3T3 cells were seeded in 12-well plates and cultured in DMEM supplemented with 10% FBS until 40% to 50% confluency. TGF-β (growth factor) (20 ng / mL) and various concentrations of the agent (0.01, 0.005, and 0.001 mg / mL dissolved in medium supplemented with 20 ng / mL TGF-β) were added and treated for 12 hours. Three biological replicates were performed for each group. A control group was treated with an equal volume of DMEM medium without TGF-β treatment. The cells were rinsed three times with PBS, and protein was extracted from the harvested cells using RIPA buffer containing a cocktail of protease and phosphatase inhibitors. SDS sample buffer was added, and the cells were heated at 95°C for 15 minutes to denature the proteins. Then, equal amounts of protein samples were separated on SDS-PAGE gels, transferred to PVDF membranes, and then subjected to Western blot analysis to explore the extent to which Suhuang extract inhibits the expression of fibrosis-related proteins in cells. Antibodies used for antibody incubation include HSP90 (4108S, CST), α-SMA (AB150301, Abcam), and GAPDH (2118S, CST). After incubation overnight at 4°C, HRP-conjugated secondary antibodies (Invitrogen) were used, and signals were visualized using SuperSignal West Femto substrate (Pierce) on a Chemidoc imaging system (Bio-Rad). Data analysis was performed using ImageJ software.

[0069] Figure 1 The CF results showed that the ability of the Suhuang extracts of Comparative Examples 1 and 2 to inhibit the expression of fibrosis proteins and the transcription levels of fibrosis-related genes was not as good as that of Example 1. The fibrosis was significantly inhibited in the group with a final concentration of Suhuang extract of 0.001 mg / mL and a ratio of Su wood to Astragalus of 1:1.

[0070] (2) qPCR experiments

[0071] 3T3 cells were seeded in 12-well plates and cultured in DMEM containing 10% FBS until 40% to 50% confluency. TGF-β (concentration: 20 ng / mL) and a 0.001 mg / mL concentration of Su wood: Astragalus root (1:1 ratio, as described in Example 1) were added for 12 and 24 hours. A control group received an equal volume of DMEM medium without TGF-β treatment. Three biological replicates were performed for each group. The medium was discarded, and the cells were washed twice with PBS. 300 μL of Trizol was added and pipetted evenly. The tubes were pipetted into 1.5 mL EP tubes, 100 μL of chloroform was added, and the tubes were shaken vigorously for 30 seconds and allowed to stand for 20 minutes. The tubes were centrifuged at 12,000 rpm at 4°C for 20 minutes. The supernatant was aspirated, and an equal volume of isopropanol was added. The tubes were inverted and allowed to stand on ice for 15 minutes. The tubes were centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was discarded. Add 1 mL of pre-cooled anhydrous ethanol to each tube and centrifuge at 12,000 rpm at 4°C for 10 minutes. Open the EP tube, pour off the ethanol, and let it stand until the anhydrous ethanol is completely evaporated and the RNA precipitate is translucent. Dissolve the RNA in 20 μL of RNase-free water and measure the concentration (Nanodrop) and purity (A260 / A280 ratio must be 1.8-2.0). To maintain RNA activity, all the above steps must be performed on ice. Take 0.5 μg of total RNA and perform reverse transcription using a reverse transcription kit (R433-01, Vazyme). The reaction system (10 μL) is as shown in Table 2, and the program is 50°C, 5 min → 85°C, 5 sec.

[0072] Reagents Usage 4×All-in-one Ultra qRT SuperMix 2.5 μL Template RNA 500ng <![CDATA[RNase-free H2O]]> to 10 μL

[0073] Using reverse-transcribed cDNA as the template, GAPDH as the internal reference gene, and COL1A as the target gene, the qPCR reaction system shown in Table 3 was configured, with three technical replicates for each sample. The reaction procedure was as follows: 4 cycles of 95°C for 10 min, followed by denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec, and extension at 72°C for 30 sec, repeated 40 times. Fluorescence signal was monitored. After completion of the reaction, the Ct value for each well was recorded and the expression level of the target gene was calculated.

[0074] Table 3

[0075] Reagents Usage SYBR Green Master Mix 5μL Forward primer (10 μM) 1 μL Reverse primer (10 μM) 1 μL cDNA template 1 μL <![CDATA[RNase-free H2O]]> 2μL

[0076] Test results see Figure 1 G. The results showed that the extract of Suhuang in Example 1 could significantly inhibit the expression of fibrosis-related gene COL1A at a concentration of 0.001 mg / mL, suggesting that it may inhibit the expression of related proteins by inhibiting the transcription level of fibrosis-related genes, thereby further reducing the degree of cell fibrosis.

[0077] (3) Cell Sirius Red Staining

[0078] 3T3 cells were seeded in 12-well plates and cultured in DMEM supplemented with 10% FBS until 40% to 50% confluency. TGF-β (20 ng / mL) and a 1:1 ratio of Sappan:Astragalus (Example 1) at a concentration of 0.001 mg / mL were added for 12 hours. A control group was treated with an equal volume of DMEM without TGF-β treatment. Three biological replicates were performed for each group. After discarding the culture medium, the cells were gently washed twice with PBS. 500 μL of 4% paraformaldehyde (PFA) was added to fix the cells for 15 minutes and then washed twice with PBS. 500 μL of 0.5% Sirius Red stain (solvent: 1% acetic acid solution) was added and stained at room temperature in the dark for 40 minutes. The residual stain was then washed twice with PBS to remove the remaining stain. 800 μL of 0.1 M hydrochloric acid was added for 2 minutes to dissolve unbound dye, and the hydrochloric acid solution was discarded. 500 μL of 0.1 M sodium hydroxide was added and shaken to desorb the dye bound to collagen. The supernatant was collected and the absorbance (OD value) was measured at a wavelength of 540 nm to quantify the collagen content.

[0079] Test results see Figure 1 H. The results showed that the treatment with the final concentration of Suhuang extract of 0.001 mg / mL and the ratio of Suhuang to Astragalus of 1:1 significantly reduced the content of collagen fibers in the cells and significantly inhibited the fibrosis process.

[0080] 4. Test on the inhibitory effect of plant extracts on cardiac fibrosis in mice with myocardial infarction:

[0081] Experimental design ( Figure 2 A):

[0082] Animal model: Twenty-four adult (12 weeks) male C57BL / 6J mice weighing 25-30 g were randomly divided into three groups;

[0083] Sham operation group: 4 rats, normal diet, shaved hair, no surgery.

[0084] Model group: 10 rats, acute myocardial infarction model was established by ligation of the left anterior descending coronary artery.

[0085] Experimental group: 10 mice, based on the model group, were given intraperitoneal injection of the Suhuang extract obtained in Example 1 (at a dose of 20 mg / kg) on ​​the 4th, 5th and 6th day after myocardial infarction surgery.

[0086] Experimental steps:

[0087] Acute myocardial infarction model establishment:

[0088] Experimental mice were fasted for 12 hours before surgery. Mice were placed in the induction chamber of the anesthesia machine, and the oxygen flow rate was adjusted (0.25 MPa, 1 L / min). The anesthetic (isoflurane) concentration was adjusted to 5%. Anesthesia was induced in approximately 1 minute, and the anesthetic concentration was then adjusted to 2%. The mouse was connected to a face mask and inhalation was continued. The mouse was fixed in the supine position, and the surgical area on the left chest was shaved and disinfected. A longitudinal incision approximately 1.5 cm long was made in the skin approximately 1-2 mm from the left edge of the sternum. A vertical mattress suture was placed at the incision to prepare a suture. The chest wall muscles were bluntly dissected layer by layer, and the thoracic cavity was quickly entered from the third or fourth intercostal space. The intercostal space was opened with hemostats, and the heart was gently squeezed with the left hand in time with the beating heart to eject it from the intercostal space. A 6-0 suture needle with thread was used to pierce the anterior descending coronary artery 1-2 mm below the inferior edge of the left atrial appendage and 0.5 mm adjacent to the pulmonary artery cone, and the ligature was applied with appropriate tightness. After ligation, the heart is gently returned to the chest cavity. The chest cavity is squeezed to expel air while the sutures remaining at the ligation incision are tightened to complete the procedure. During the procedure, the anesthetic concentration is gradually adjusted to zero. After removing the mask, the mouse is placed on a constant temperature pad for approximately 3-5 minutes for resuscitation.

[0089] Treatment process:

[0090] All three groups were fed normally after surgery. For the experimental group, the extract of Suhuang from Example 1 was injected intraperitoneally at 20 mg / kg for three consecutive days on the 4th, 5th and 6th days after surgery.

[0091] Cardiac ultrasound:

[0092] On the 7th day after surgery, cardiac ultrasound was performed on the three groups of mice to detect cardiac function. The mice were anesthetized with isoflurane gas (concentration 3%), and after the anesthesia was stable, the mice were fixed in a supine position on the operating table. The hair in the chest area was shaved to ensure that the skin was exposed, and conductive gel was applied to enhance the contact between the ultrasound probe and the skin. A high-frequency ultrasound probe (30MHz) was selected, and the gain, depth and frame rate were adjusted to ensure that the long axis and short axis sections of the left ventricle were clearly visible. In the parasternal left ventricular long axis section, the left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) were measured. M-mode ultrasound images were recorded in the left ventricular short axis section (papillary muscle level), the left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) were analyzed, and the heart rate, ventricular wall thickness and volume changes ( Figure 2 B).

[0093] Figure 2The results of C and D showed that the left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) of the model group mice were significantly decreased compared with the sham operation group, indicating that acute myocardial infarction had a negative impact on the cardiac function of mice. The left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) of the experimental group injected with the Suhuang extract of Example 1 were significantly increased compared with the model group, indicating that the injection of the Suhuang extract of Example 1 can rescue the adverse effects of acute myocardial infarction on cardiac function.

[0094] Heart Acquisition:

[0095] After cardiac ultrasound, the mice were sacrificed and their hearts harvested. The harvested hearts were perfused with PBS. Three hearts were randomly selected from each of the sham, model, and experimental groups, placed in RIPA, and stored at -80°C. The remaining hearts were embedded in OCT embedding medium and stored at -80°C for subsequent experiments.

[0096] Heart sections and Masson staining:

[0097] The embedded heart was sliced ​​from the apex using a rotary cryostat, with an interval of 180 μm between each slice, for a total of 16 slices, to evenly reflect the extent of infarction throughout the heart. Each slice was cut into 5 μm thickness, attached to a glass slide, fixed in 4% PFA at room temperature for 60 minutes, and rinsed with water. Masson staining was then performed, and the infarct area was observed under a microscope and statistically analyzed ( Figure 2 E, F).

[0098] From the staining results, it can be seen that the myocardial infarction area of ​​the experimental group injected with the Suhuang extract of Example 1 was significantly smaller than that of the model group. Statistical results showed that the injection of the Suhuang extract of Example 1 significantly reduced cardiac fibrosis after acute myocardial infarction.

[0099] Western Blot:

[0100] Heart samples stored at -80°C were thawed and broken using a tissue grinder. After adjusting the protein concentration to the appropriate range, SDS sample buffer was added and heated at 95°C for 15 minutes to denature the proteins. Equal amounts of protein samples were then separated on SDS-PAGE gels, transferred to PVDF membranes, and subjected to Western blot analysis to investigate the extent to which the Suhuang extract inhibited the expression of fibrosis-related proteins in cardiac tissue.

[0101] The results showed that the expression of fibrosis-related protein (α-SMA) in the heart tissue of the experimental group injected with the extract of Example 1 was significantly reduced ( Figure 2 G, H).

[0102] The above results collectively indicate that Suhuang extract has a significant protective effect on cardiac function after myocardial infarction. This protective effect may be achieved by inhibiting the expression of fibrosis-related protein (α-SMA) and reducing the area of ​​myocardial infarction.

Claims

1. A plant extract for treating cardiac fibrosis, characterized in that: The plant extract is obtained by mixed extraction of astragalus and sappan wood.

2. A method for preparing the plant extract according to claim 1, characterized in that: At least the following steps are included: S1. Slice and dry Astragalus and Sappan wood respectively; S2. Soaking the dried Astragalus and Sappan in ultrapure water for three times, filtering and combining the filtrates to obtain a crude extract; S3. The crude extract is subjected to impurity removal, concentration, freeze-drying, and grinding to obtain the plant extract.

3. The method for preparing the plant extract according to claim 2, wherein In step S1, the astragalus root is sliced ​​to obtain oblique slices with a thickness of 2-4 mm; the sappan wood is sliced ​​to obtain thin slices with a thickness of 2-3 mm.

4. The method for preparing the plant extract according to claim 3, wherein The drying conditions in step S1 are as follows: drying in an oven at 40° C. for 24-36 hours until the water content of the astragalus root is 8-12 wt %, and the water content of the sappan wood is 10-14 wt %.

5. The method for preparing the plant extract according to claim 2, wherein: The mass ratio of the dried Astragalus and Sappan in S2 is 1:

1.

6. The method for preparing the plant extract according to claim 5, characterized in that: The solid-to-liquid ratio of the dried astragalus and sappan wood to ultrapure water is 1 g:10 mL.

7. The method for preparing the plant extract according to claim 2, wherein: The process extracted in the S2 step is: Step 1: Soak the dried Astragalus and Sappan wood in ultrapure water at room temperature and heat to boiling, then condense and reflux for 1-1.5 hours to collect the extract; Step 2: filtering the extract to obtain a filter residue and a filtrate; Step 3: Add ultrapure water to the filter residue and repeat the first and second steps.

8. The method for preparing the plant extract according to claim 7, wherein: The soaking time is 10-14 hours.

9. The method for preparing the plant extract according to claim 2, wherein: The impurity removal process in step S3 is as follows: adding the crude extract to anhydrous ethanol for 12-20 hours, filtering through a 0.45 μm microporous filter membrane, discarding the filter residue, and completing the impurity removal step.

10. Use of the plant extract according to claim 1 in preparing a medicament for treating cardiac fibrosis.

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