Application of traditional Chinese medicine extract in preparation of anti-sepsis cardiomyopathy medicine

Abstract

The traditional Chinese medicine extract is an alkaloid component extracted from traditional Chinese medicine uncaria, and a preparation method of the traditional Chinese medicine extract comprises the following steps: taking traditional Chinese medicine uncaria powder, carrying out heating reflux extraction for three times by using ethanol, merging extracting solutions, and carrying out filtering, washing and drying at the temperature of less than 70 DEG C to obtain the traditional Chinese medicine extract. And performing vacuum concentration to obtain extract, and performing freeze drying. The traditional Chinese medicine extract disclosed by the invention is applied to the preparation of anti-sepsis cardiomyopathy medicines, can obviously improve cardiac functions, relieve myocardial damage, reduce inflammatory response and effectively inhibit sepsis cardiomyopathy, and has a good medicinal prospect.

Description

Technical Field

[0001] The invention relates to application of a traditional Chinese medicine extract in preparing an anti-septic cardiomyopathy drug. Background Art

[0002] Sepsis-induced cardiomyopathy (SIC) is a cardiac dysfunction caused by sepsis. It is an acute heart injury characterized by impaired left ventricular systolic and diastolic function accompanied by decreased ejection fraction. It is the most serious complication of sepsis and one of the main causes of death. About half of sepsis patients develop sepsis cardiomyopathy. Septic cardiomyopathy has a high mortality rate and complex pathological mechanisms, mainly including myocardial inhibition, excessive inflammation, mitochondrial dysfunction, myocardial cell apoptosis, oxidative stress and calcium ion imbalance. At present, there is no treatment measure supported by evidence-based medicine. Traditional anti-infection and vasoactive drug supportive therapy cannot reduce the high mortality rate of sepsis patients and has large side effects. Therefore, finding safe and effective anti-septic cardiomyopathy drugs can improve the prognosis of sepsis patients and reduce their mortality, which has important clinical application value.

[0003] In recent years, the positive therapeutic effects of Chinese herbal extracts on septic cardiomyopathy have attracted widespread attention. For example, tanshinones can reduce sepsis-induced myocardial dysfunction, astragaloside IV can improve myocardial contractility and reduce oxidative stress damage in sepsis models, and baicalin can reduce myocardial tissue inflammation and fibrosis in sepsis models. The above Chinese herbal extracts have certain therapeutic effects in the prevention and treatment of sepsis, but their effects on improving cardiac function, reducing myocardial damage and / or reducing inflammatory response are limited. Therefore, it is necessary to find a more effective drug for anti-septic cardiomyopathy. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an application of a Chinese medicine extract in the preparation of an anti-septic cardiomyopathy drug.

[0005] Technical solution: The present invention discloses an application of a traditional Chinese medicine extract in the preparation of an anti-septic cardiomyopathy drug, wherein the traditional Chinese medicine extract is an alkaloid component extracted from the traditional Chinese medicine Uncaria rhynchophylla.

[0006] The preparation method of the Chinese medicine extract comprises the following steps: taking Chinese medicine Uncaria powder, extracting it three times with ethanol by heating and refluxing, combining the extracts, concentrating to extract under reduced pressure at a temperature below 70°C, and freeze-drying the extract.

[0007] Wherein, the ethanol is 95% ethanol.

[0008] Wherein, the septic cardiomyopathy is the cardiac function damage caused by sepsis.

[0009] Wherein, the cardiac function damage is cardiac diastolic and / or systolic dysfunction.

[0010] The Chinese herbal extract reduces myocardial fiber disorder and rupture caused by sepsis.

[0011] The Chinese herbal extract reduces the elevated levels of CK-MB, LDH, IL-1β and TNF-α in serum caused by sepsis.

[0012] The Chinese medicine extract is administered by intragastric administration, with a dosage of 30 to 60 mg / kg, and is used in an anti-septic cardiomyopathy drug in the form of an oral preparation.

[0013] Principle of the invention: Uncaria rhynchophylla is a plant of the genus Uncaria of the Rubiaceae family. Most of the dried hooked stems and branches are used as medicine. It was first recorded in "Ming Yi Bie Lu" and is a commonly used Chinese medicine for the treatment of liver wind movement, epilepsy convulsions, high fever convulsions, colds and convulsions, children crying, pregnancy eclampsia, headache and dizziness. The chemical components in Uncaria rhynchophylla are complex and diverse, mainly including alkaloids, flavonoids and triterpenes, among which monoterpenoid indole alkaloids are the characteristic components and main active ingredients of Uncaria rhynchophylla plants, including rhynchophylline, isorhynchophylline, dehydrorhynchophylline, isodehydrorhynchophylline, hairy rhynchophylline, dehydrorhynchophylline and sutuzinyl methyl ether. Modern pharmacological studies have shown that Uncaria rhynchophylla and its main active alkaloid components have multiple activities such as antihypertensive, antiarrhythmic, anti-atherosclerotic, anti-tumor, analgesic, sedative, and neuroprotective.

[0014] The Chinese medicinal extract of the invention is extracted based on Uncaria rhynchophylla and its active alkaloids, and is applied in the preparation of an anti-septic cardiomyopathy drug, and can respectively increase the left ventricular ejection fraction (EF) and the shortening fraction (FS) abnormally reduced by LPS by 46.2% and 59.3%, significantly enhance cardiac function, improve the disordered arrangement and rupture of myocardial fibers caused by sepsis, and reduce the serum CK-MB level abnormally increased by LPS by 44.7%, effectively alleviating myocardial damage; reduce the proportion of myocardial CD68 positive cells, and respectively reduce the serum IL-1β and TNF-α levels abnormally increased by LPS by 45.5% and 47.9%, significantly reducing the inflammatory response; therefore, the Chinese medicinal extract of the invention has the potential to protect the cardiovascular system, and is expected to be applied to the treatment of septic cardiomyopathy.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the Chinese medicine extract of the present invention is used in the preparation of anti-septic cardiomyopathy drugs, which significantly enhances cardiac function; improves the disordered arrangement and breakage of myocardial fibers caused by sepsis, and effectively reduces myocardial damage; reduces the proportion of myocardial CD68 positive cells, significantly reduces the inflammatory response, and effectively inhibits septic cardiomyopathy, which has good medicinal prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The TIC total ion current diagram of the Uncaria rhynchophylla extract of the present invention in positive and negative ion modes;

[0017] Figure 2 This is a picture of the cardiac ultrasound results of mice;

[0018] Figure 3 The left ventricular ejection fraction (EF) and shortening fraction (FS) of mice are shown in Figure 1. A is EF, B is FS, and * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0019] Figure 4 HE staining results of heart tissues of mice in each group;

[0020] Figure 5 The results of CD68 positive cell staining in the heart tissue of mice in each group; A is the CD68 positive cell staining diagram, B is the statistical diagram of the proportion of CD68 positive cells, in the figure, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001;

[0021] Figure 6 The results of serum LDH and CK-MB levels in mice are shown in Figure 1. A is LDH, B is CK-MB, and ** represents p<0.01, and **** represents p<0.0001.

[0022] Figure 7 The results of serum TNF-α and IL-1β levels in mice are shown in Figure 1. A represents TNF-α, B represents IL-1β, and ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0024] Experimental Materials:

[0025] Experimental animals: Healthy ICR mice (male, weighing 24-26 g), clean grade, provided by the Comparative Medicine Center of Yangzhou University.

[0026] Experimental drugs: Chinese medicine Uncaria rhynchophylla powder was provided by Qingfeng Pharmaceutical.

[0027] Experimental reagents: physiological saline: purchased from Anhui Shuanghe Pharmaceutical; lipopolysaccharide (LPS): Sigma, USA; PBS, paraformaldehyde: Jiangsu Keygen Biotechnology Co., Ltd.; LDH kit: Nanjing Jiancheng Bioengineering Institute; mouse CK-MB, IL-1β, TNF-α detection kit: Hunan Aifang Biotechnology Co., Ltd.

[0028] Experimental instruments: Ultra-high resolution small animal ultrasound imaging system Vevo 3100LT: FUJIFILM Visual Sonics; SpectraMax M2e microplate reader: Moleclar Devices, USA.

[0029] Example 1

[0030] The Chinese medicine extract of the invention comprises the following steps: extracting Chinese medicine Uncaria rhynchophylla powder by heating and refluxing with 95% ethanol for three times, combining the extracts, pumping them into an external circulation concentration unit with a temperature lower than 70 DEG C and concentrating them under reduced pressure to obtain an extract, and freeze drying the extract.

[0031] Example 2

[0032] The HPLC / QTOF-MS component identification method of Uncaria rhynchophylla extract is:

[0033] Preparation of reference solution: Accurately weigh 10 mg each of rhynchophylline, isorhynchophylline, dehydrorhynchophylline and isodehydrorhynchophylline reference substances, place in a 10 mL volumetric flask, dissolve with acetonitrile, dilute to the scale, and filter with a 0.22 μm microporous filter membrane to obtain the reference solution.

[0034] Preparation of the test solution: Take 0.2 g of freeze-dried powder of the extract, place it in a 50 mL conical flask, add 5 mL of 75% methanol, ultrasonically treat for 45 min, cool, add 75% methanol and then centrifuge, and filter with a 0.22 μm microporous filter membrane to obtain the test solution.

[0035] Chromatographic and mass spectrometry conditions: chromatographic column HiQsil C8 (250 mm × 4.6 mm, 5 μm), mobile phase consisting of organic phase acetonitrile (A) and aqueous phase (B), wherein aqueous phase B contained 10 mM ammonium acetate, gradient elution program was set: 0-5 min, 12% A; 5-15 min, 12%-22% A; 15-30 min, 22%-32% A; 30-45 min, 32%-40% A; 45-50 min, 40%-100% A; flow rate: 0.9 mL / min, injection volume: 10 μL, column temperature: 30°C, detection wavelength: 245 nm.

[0036] Quantitative chromatographic conditions: HPLC / QTOF-MS analysis was performed using an Agilent LC1200-MS / QTOF 6520 mass spectrometer. Chromatographic separation was performed using an Agilent 1200 high performance liquid chromatograph (Agilent Technologies, Germany). Chromatographic conditions: chromatographic column HiQsil C8 (250 mm × 4.6 mm, 5 μm), mobile phase consisting of organic phase methanol-acetonitrile (7:3, v / v) (A) and aqueous phase (B), wherein aqueous phase B contained 10 mM ammonium acetate, gradient elution program was set: 0-145 min, 15-55% A; 145-150 min, 15% A; flow rate: 0.3 mL / min, injection volume: 10 μL, column temperature: 30 °C.

[0037] Mass spectrometry conditions: The mass spectrometry detection conditions are positive and negative ion analysis mode, electrospray ionization (ESI) mode, primary mass spectrometry scanning detection with a detection range of 100-1700 m / z, a drying gas temperature of 325°C, a flow rate of 8 L / min, a sheath gas temperature of 400°C, a capillary voltage of 3.5 kV, and a fragment voltage of 120 V.

[0038] HPLC-Q-TOF-MS was used to quickly identify the active ingredients in the extract of Uncaria rhynchophylla, and 35 compounds were identified, including rhynchophylline, dehydrorhynchophylline, isodehydrorhynchophylline, isorhynchophylline, dehydrorhynchophylline, rhynchophylline, dehydrorhynchophylline N-oxide, and sutuzinol methyl ether. Two existing reference compounds, rhynchophylline and isorhynchophylline, were compared with the reference compounds. The positive and negative ion chromatograms of HPLC-Q-TOF-MS of Uncaria rhynchophylla extract are shown in the figure. Figure 1 .

[0039] The information data of chemical components such as retention time, mass-to-charge ratio, error, molecular formula, compound name and fragment information are shown in Table 1.

[0040] Table 1. Information of 35 components in URA identified based on LC-QQQ MS

[0041]

[0042]

[0043]

[0044] a :Identification by reference

[0045] Example 3

[0046] Establishment and grouping of mouse septic cardiomyopathy model

[0047] 46 male ICR mice (8 weeks old, weighing 24-26g), clean grade, were adaptively fed for 5 days before the formal start. The feeding temperature was 20-25°C, the relative humidity was 50%-60%, the circadian rhythm was 12h, and the mice were free to drink water and eat. The animal experiment was approved by the Animal Ethics Committee of China Pharmaceutical University. The mice were randomly divided into a control group (Control) of 10 mice, a model group (LPS) of 12 mice, a low-dose group of the Chinese medicine extract of the present invention (URA-L) of 12 mice, and a high-dose group of the Chinese medicine extract of the present invention (URA-H) of 12 mice. The control group and the model group were gavaged with normal saline (0.15mL / 10g) for 7 consecutive days; the low-dose group of the Chinese medicine extract of the present invention was gavaged for 7 consecutive days at a dose of 30mg / kg; the high-dose group of the Chinese medicine extract of the present invention was gavaged for 7 consecutive days at a dose of 60mg / kg. One hour after administration on the last day, the control group was intraperitoneally injected with PBS, and each mouse in the remaining groups was intraperitoneally injected with LPS (10 mg / kg). The treatment lasted for 12 hours, and the status of the mice was monitored in time.

[0048] Determination of cardiac function indexes:

[0049] After 12 hours of treatment, an appropriate amount of depilatory cream was evenly applied to the heart of the mice for depilation, so that the skin of the heart was completely exposed. After the mice were anesthetized with 1% isoflurane, they were fixed on the operating board with their chests facing up, and an appropriate amount of coupling agent for detection was applied to the heart. The probe was placed on the chest of the mice, and the ultra-high-resolution small animal ultrasound imaging system Vevo3100LT was used to select two-dimensional M-mode ultrasound for echocardiography. Three cardiac cycles were measured continuously, and the data were processed with Vevo LAB3.2.6 software to obtain the left ventricular ejection fraction (EF) and shortening fraction (FS).

[0050] from Figures 2-3 It can be seen that compared with the control group (Control), the EF and FS of the model group (LPS) mice were significantly reduced (p < 0.01), and the cardiac function was significantly impaired; compared with the LPS group, the EF and FS of the low-dose group of the Chinese herbal extract of the present invention (URA-L) and the high-dose group of the Chinese herbal extract of the present invention (URA-H) were significantly improved (p < 0.05, p < 0.01), indicating that pretreatment with the Chinese herbal extract of the present invention can improve the cardiac function of mice with septic cardiomyopathy.

[0051] Example 4

[0052] HE staining:

[0053] After 12 hours of treatment, the mouse hearts were collected and fixed in 4% formalin for 24 hours. After dehydration, tissue transparency, paraffin embedding, sectioning, dewaxing, hematoxylin-eosin (HE) staining, dehydration and sealing, the myocardial cell morphology was observed under an optical microscope.

[0054] from Figure 4 It can be seen that the myocardial texture of the control group (Control) mice is clear, and the myocardial tissue cells are arranged neatly and regularly, while the model group (LPS) can see obvious myocardial damage, irregular arrangement of myocardial fibers, multiple ruptures, and clearly visible necrotic myocardial fibers, showing extensive cytoplasmic vacuolation, interstitial edema and partial nuclear dissolution. After pretreatment with the Chinese medicine extract of the present invention, compared with the LPS group, the degenerated and necrotic myocardial fibers in the URA-L and URA-H groups were significantly reduced, the cell texture tended to be clear, the myocardial cells were gradually arranged neatly, no obvious inflammatory infiltration was observed in the interstitium, and the vacuolar and nuclear loss phenomena were reduced.

[0055] Example 5

[0056] Immunohistochemical staining:

[0057] The paraffin-embedded heart tissue was deparaffinized, incubated in a 3% hydrogen peroxide solution in the dark for 25 minutes, and then rinsed three times with 1× PBS. Then 3% bovine serum albumin was added dropwise to the tissue chemical circle and sealed for 30 minutes. The sections were incubated with CD68 primary antibody at 4°C overnight, then incubated with goat anti-rabbit IgG (H+L) HRP at room temperature for 50 minutes and washed three times. Then 3,3'-diaminobenzidine (DAB) colorimetric solution was added dropwise, and the color development time was controlled under a microscope. The positive color was brown-yellow, and the sections were rinsed with tap water to stop the color development. The nuclei were restained with hematoxylin, and then the sections were dehydrated, sealed, and placed under an upright microscope for observation.

[0058] LPS can induce the accumulation of CD68 positive cells in cardiac tissue. Figure 5 The figure is the result of CD68 positive cell staining in the heart tissue of each group of mice in the present invention. As shown in the figure, the proportion of CD68 positive cells in the LPS group was significantly increased (p<0.0001), while the levels in the URA-L and URA-H groups were significantly decreased (p<0.001), indicating that the pretreatment of the Chinese herbal extract of the present invention can reduce myocardial inflammation in mice with septic cardiomyopathy, and the high-dose group of the Chinese herbal extract of the present invention has a better improvement effect than the low-dose group of the Chinese herbal extract of the present invention.

[0059] Example 6

[0060] Serum CK-MB, LDH, IL-1β, TNF-α levels:

[0061] After 12 hours of treatment, the eyeballs of the mice in the above groups were removed for blood collection, and the serum was collected to measure the levels of mouse serum creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) according to the instructions of the kit.

[0062] Serum LDH and CK-MB levels are reliable indicators for judging myocardial damage. Figure 6 It can be seen that compared with the Control group, the serum LDH and CK-MB levels of mice in the LPS group were significantly increased (p<0.0001), indicating myocardial damage; compared with the LPS group, the serum LDH and CK-MB levels of mice in the URA-H and URA-L groups were significantly decreased (p<0.01), and the CK-MB index in the URA-H group was more significantly reduced than that in the URA-L group, indicating that the pretreatment of the Chinese herbal extract of the present invention can reduce the myocardial damage of mice with septic cardiomyopathy, and the high-dose group of the Chinese herbal extract of the present invention has a better improvement effect than the low-dose group of the Chinese herbal extract of the present invention.

[0063] TNF-α and IL-1β are typical pro-inflammatory cytokines. Figure 7 It can be seen that compared with the Control group, the serum TNF-α and IL-1β levels of mice in the LPS group were significantly increased (p<0.0001); compared with the LPS group, the serum TNF-α and IL-1β levels of mice in the URA-H and URA-L groups were significantly decreased (p<0.01), and the decrease in the URA-H group was more obvious than that in the URA-L group, indicating that pretreatment with the Chinese herbal extract of the present invention can significantly reduce the level of serum inflammatory factors in mice with septic cardiomyopathy and reduce the inflammatory response, and the high-dose group of the Chinese herbal extract of the present invention has better effect than the low-dose group of the Chinese herbal extract of the present invention.

[0064] Statistical analysis: One-way analysis of variance (ANOVA) and Tukey test were used to test the statistical differences of the samples using IBM SPSS Statistics 26.0, and p < 0.05 was considered statistically significant.

[0065] The present invention investigates the effect of the Chinese herbal extract on septic cardiomyopathy based on ICR mice, and the main investigation indicators include mouse cardiac function, myocardial cell morphology, serum CK-MB, LDH, TNF-α and IL-1β levels. The results show that the Chinese herbal extract can significantly improve cardiac function, reduce myocardial damage, and reduce inflammatory response, and effectively inhibit septic cardiomyopathy, has good medicinal prospects, and provides a new drug for preventing and treating septic cardiomyopathy.

Claims

1. Use of a Chinese medicine extract in the preparation of an anti-septic cardiomyopathy drug, characterized in that: The Chinese medicine extract is an alkaloid component extracted from the Chinese medicine Uncaria rhynchophylla.

2. The use according to claim 1, characterized in that: The preparation method of the Chinese medicine extract comprises the following steps: taking Chinese medicine Uncaria powder, heating and refluxing extraction with ethanol for three times, combining the extracts, concentrating under reduced pressure to extract at a temperature below 70°C, and freeze-drying the extract.

3. The use according to claim 1, characterized in that: The ethanol is 95% ethanol.

4. The use according to claim 1, characterized in that: The septic cardiomyopathy is the cardiac function damage caused by sepsis.

5. The use according to claim 4, characterized in that: The cardiac function damage is cardiac diastolic and / or systolic dysfunction.

6. The use according to claim 1, characterized in that: The Chinese medicinal extract can reduce the disordered arrangement and rupture of myocardial fibers caused by sepsis.

7. The use according to claim 1, characterized in that: The traditional Chinese medicine extract reduces the contents of CK-MB, LDH, IL-1β and TNF-α in serum which are elevated due to sepsis.

8. The use according to claim 1, characterized in that: The Chinese medicinal extract is administered by intragastric administration, with a dosage of 30 to 60 mg / kg, and is used in an anti-septic cardiomyopathy drug in the form of an oral preparation.

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