Application of limonin in preparation of medicine for improving left ventricular diastolic function

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of limonin in preparation of a medicine for improving the left ventricular diastolic function. The CAS number of the limonin is 180-71-8, the molecular formula of the limonin is C26H30O8, and the limonin in the medicine is an effective component. Tests find that after ejection fraction reserved heart failure (HFpEF) model group mice are subjected to limonin high, medium and low dose administration, compared with the model group without administration, the weight of the heart and lung of the mice is remarkably reduced, left ventricular diastolic dysfunction and centripetal cardiac hypertrophy of the mice can be remarkably improved, and the curative effect of the mice is improved. Compared with the positive drug empagliflozin, the drug effect indexes have no obvious difference. Besides, HE and Masson staining finds that compared with control group mice, obvious myocardial interstitial fibrosis occurs in the model group mice, and myocardial fibrosis can be obviously improved after limonin is high, medium and low and empagliflozin is administrated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of limonin in preparing a medicine for improving left ventricular diastolic function. Background Art

[0002] With the development of social economy and changes in residents' lifestyles, the incidence of cardiovascular diseases has continued to increase, and has become the leading cause of death among urban and rural residents. Heart failure is the terminal stage of various cardiovascular diseases, and the 5-year survival rate is only about 50% (Ma Liyuan, Wang Zengwu, Fan Jing, et al. Interpretation of the key points of "China Cardiovascular Health and Disease Report 2022" [J]. Chinese General Practice, 2023, 26(32): 3975-94.). Large-scale clinical survey results show that among heart failure patients in my country, heart failure with preserved ejection fraction (HFpEF) accounts for the highest proportion (ZHOU J, LI X, DONG Y, et al. Abstract 10820: Real-world Characteristics of Patients With Heart Failure in China: Baseline Results From The Retrospective Non-interventional Nationwide Study (REPRESENT-HF) [J]. Circulation, 2019, 140 (Suppl_1): A10820-A.), but the effective treatment of HFpEF is still limited. A large amount of clinical experimental data show that traditional heart failure drugs, such as ACEI, ARB, ARNI, etc., do not improve the prognosis of HFpEF. The latest heart failure clinical guidelines only list sodium-glu-cosecotransporter 2 inhibitor (SGLT2i) drugs (such as empagliflozin) as a Class A recommendation (2022 AHA / ACC / HFSA Guideline for the Management of Heart Failure [J]. J Card Fail, 2022, 28 (5): e1-e167.; MCDONAGH TA, METRA M, ADAMO M, et al. 2023 Focused Update of the 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure [J]. Eur Heart J, 2023, 44(37): 3627-39.), patients still face the disadvantage of very limited treatment options, and HFpEF does not have a significantly better prognosis than heart failure with reduced ejection fraction (HFrEF) because of a higher ejection fraction. On the contrary, the two have similar hospitalization rates and mortality rates (LAM CSP, GAMBLE GD, LING LH, et al. Mortality associated with heart failure with preserved vs. reduced ejection fraction in a prospective international multi-ethnic cohort study [J]. Eur Heart J, 2018, 39 (20): 1770-80.; MISHRA S, KASSD A. Cellular and molecular pathobiology of heart failure with preserved ejection fraction [J]. Nat Rev Cardiol, 2021, 18 (6): 400-23.). Therefore, it is urgent to discover new HFpEF treatment drugs and regimens. .

[0003] The invention patent application with application publication number CN 105503998 A discloses a limonoid compound (molecular formula C 26 H 30 O7) Use for myocardial protection. The prior art also records that: Evodia rutaecarpa has analgesic effect, body temperature raising effect, anti-ulcer, bidirectional regulation of intestinal motility and liver protection and choleretic effect on the digestive system, bidirectional regulation of uterine smooth muscle and bronchus, blood pressure lowering, thrombolysis, blood flow increase, vasodilation and other effects on the cardiovascular system, among which alkaloids and limonoid compounds are its main active ingredients. However, the limonoid compounds described in the prior art do not disclose that the limonoid compounds with antihypertensive function have therapeutic effects on heart failure with preserved ejection fraction, and the antihypertensive drugs in the prior art cannot treat heart failure with preserved ejection fraction.

[0004] In addition, due to the complexity of the HFpEF phenotype, there are many difficulties in the development of preclinical animal models. Currently, researchers mostly use a double or multiple hit model of hypertension and obesity to simulate the clinical manifestations of HFpEF as much as possible. Among them, the most common method is to use nitric oxide synthase inhibitors (L-NAME) to induce hypertension and high-fat diet to induce obesity. Related studies have shown that in this animal model, hypertension or obesity caused by L-NAME and high-fat diet alone cannot lead to the occurrence of HFpEF. Only when the two are superimposed will an obvious HFpEF phenotype appear (Gabriele G. Schiattarella, et al. Nitrosative stress drives heart failure with preserved ejection fraction [J]. Nature, 2019, 568 (7752): 351-356. DOI: 10.1038 / s41586-019-1100-z.). It can be seen from this that hypertension alone cannot lead to the occurrence of HFpEF, and there is no record in the prior art that antihypertensive drugs can treat HFpEF. Summary of the invention

[0005] In view of the severe situation that more effective therapeutic drugs are urgently needed for clinical HFpEF, the present invention has found through rigorous animal experiments that limonin (CAS: 1180-71-8) has a significant effect on improving left ventricular diastolic function, and its efficacy is not inferior to that of empagliflozin.

[0006] The present invention specifically adopts the following technical solutions:

[0007] The present invention has found that limonin can improve left ventricular diastolic function, that is, treat heart failure with reduced ejection fraction. Therefore, the present invention provides the use of limonin in the preparation of a drug for improving left ventricular diastolic function, wherein the limonin has a CAS number of 180-71-8 and a molecular formula of C 26 H 30 O8, chemical structure is

[0008] The limonin in the drug is an effective ingredient, and the drug also includes pharmaceutical excipients, which include polyethylene glycol (15)-hydroxystearate ( HS15) and normal saline. When preparing the drug, limonin is first dissolved in the auxiliary material HS15, then add physiological saline to dilute to the target concentration and mix well.

[0009] The beneficial effects of the present invention are:

[0010] The present invention found that oral administration of 100 mg / kg, 50 mg / kg, and 25 mg / kg of limonin can significantly improve the left ventricular diastolic function of HFpEF mice, and effectively reduce concentric myocardial hypertrophy and myocardial fibrosis, providing a new idea for the development of new therapeutic drugs for HFpEF. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The blood pressure, body weight and heart weight of each group of mice in Example 1. On the ordinate in the figure, SBP and DBP represent systolic blood pressure and diastolic blood pressure respectively, BW represents body weight, and HW / TL represents heart weight / tibia length.

[0012] Figure 2 The cardiac function of each group of mice in Example 1. On the ordinate in the figure, d and s after LVAW, LVID, and LVPW represent diastolic (diastolic period) and systolic (systolic period), respectively.

[0013] Figure 3 The results of ultrasound detection of cardiac function of mice in each group in Example 2. On the vertical axis, HW / TL represents heartweight / tibia length, LW / TL represents lung weight / tibia length; d and s after LVAW, LVID, and LVPW represent diastolic (diastolic period) and systolic (systolic period), respectively. On the horizontal axis, 100Li, 50Li, and 25Li represent the high-dose limonin group, the medium-dose limonin group, and the low-dose limonin group, and EMA represents the empagliflozin group.

[0014] Figure 4 These are the results of HE and Masson staining of the hearts of mice in each group in Example 2. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] The materials used in the examples are as follows:

[0017] 1. Animals: SPF-grade C57BL / 6J mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and housed in a barrier environment at the Animal Experiment Center of Beijing University of Chinese Medicine.

[0018] 2. Reagents and drugs: Limonin (HPLC ≥ 98%, Shanghai Yuanye Biotechnology Co., Ltd.), HS15 (Sigma, USA), L-NAME (Sigma, USA), high-fat diet (60% fat energy supply, Changzhou Shuyi Shuer Biotechnology Co., Ltd.), low-fat and low-sugar control diet (Changzhou Shuyi Shuer Biotechnology Co., Ltd.).

[0019] Example 1

[0020] This example evaluated the establishment of an animal model of HFpEF.

[0021] SPF-grade C57BL / 6J mice were divided into 4 groups, the CON group was a blank control group, the L-NAME group and the HFD group were a single nitric oxide synthase inhibitor and a high-fat diet group, respectively, and the Model group was a superposition group of L-NAME and HFD. Mice in the blank control group were given normal drinking water and a low-fat, low-sugar control diet. Mice in the L-NAME group were given a low-fat, low-sugar control diet, and the nitric oxide synthase inhibitor L-NAME (0.5 g / L) was added to the drinking water. The HFD group was given normal drinking water and a high-fat diet with 60% fat energy. Mice in the Model group were given a high-fat diet with 60% fat energy, and the nitric oxide synthase inhibitor L-NAME (0.5 g / L) was added to the drinking water. The experiment found that using the modeling method of the Model group, mice can show obvious HFpEF phenotypes after 5 to 6 weeks. In order to ensure that the HFpEF phenotype is more significant in this embodiment, the modeling time is extended to the 16th week. After modeling, blood pressure measurement and cardiac function tests were performed on mice in each group.

[0022] (1) Measurement of blood pressure in mice:

[0023] use The multi-channel non-invasive sphygmomanometer measures the systolic and diastolic blood pressure and heart rate of awake mice by the tail cuff method. First, the mice were placed in a cylindrical holder on a 37°C temperature-controlled platform. After 10 minutes of adaptation, the measurement was performed in a non-stressed state. The tail cuff was inflated and deflated 15 times for each measurement, and at least 8 stable blood pressure values ​​were selected from 3 consecutive measurements for analysis.

[0024] (2) Mouse cardiac function test:

[0025] After modeling, cardiac function was tested using the Vevo 2100 small animal ultrasound imaging system. After hair removal on the first day, anesthesia was induced with 4% to 5% isoflurane, fixed on a 37°C constant temperature physiological platform, and maintained with 1% to 2% isoflurane. The heart rate was maintained at 500 to 550 beats / min for systolic function testing and 410 to 450 beats / min for diastolic function testing. Coupling agent was applied to the chest, and the parasternal short axis section and apical four-chamber heart section were taken using the MS400 probe to test cardiac systolic function and diastolic function, respectively. The left ventricular internal diameter (LVID), left ventricular anterior wall thickness (LVAW) and left ventricular posterior wall thickness (LVPW) in the short-axis view were measured using M-mode ultrasound, and the ejection fraction (EF) and fractional shortening (FS) were calculated. The early and end-diastolic blood flow (E, A) of the mitral valve in the four-chamber view was recorded using the Pulsed-wave mode, and the early and end-diastolic velocity (e', a') of the mitral annulus was recorded using the Doppler imaging mode, and the diastolic function indices such as E / A, E / e', and e' / a' were calculated.

[0026] like Figure 1 As shown, in terms of blood pressure, compared with the blank control group, the blood pressure of mice in the L-NAME group and the Model group was significantly increased (P<0.01), while long-term high-fat diet had no significant effect on the blood pressure of mice; in terms of body weight, L-NAME drinking had no significant effect on the body weight of mice, and the body weight of mice in the HFD group and the Model group was significantly increased compared with the blank control group (P<0.01); in terms of organ index, only the heart weight of mice in the HFD group and the Model group was significantly increased compared with the blank control group (P<0.01), among which the heart weight of mice in the Model group was the heaviest, significantly higher than that in the HFD group (P<0.01), and there was no significant change in the heart weight using L-NAME alone.

[0027] like Figure 2 As shown in the data, compared with the blank control group, there was no significant difference in the ejection fraction of each group of mice. Only the mice in the Model group showed the most severe diastolic dysfunction and concentric myocardial hypertrophy, which was manifested by a significant increase in E / e' and a significant decrease in E / A and e' / a' (P<0.01). The mice in the L-NAME and HFD groups had only mild diastolic dysfunction and concentric myocardial hypertrophy, which were significantly lighter than those in the Model group (P<0.01).

[0028] In summary, the combination of L-NAME and HFD can induce more severe HFpEF, while the hypertension or obesity induced by L-NAME and HFD alone, respectively, has limited effect on cardiac function, which fully demonstrates that in this animal model, the interaction between hypertension and obesity promotes the progression of HFpEF. Hypertension or obesity alone cannot induce the occurrence of HFpEF, which also partially explains why antihypertensive drugs or lipid-lowering drugs alone cannot improve HFpEF in clinical experiments.

[0029] Example 2

[0030] 1. Animal model establishment and grouping:

[0031] SPF-grade C57BL / 6J mice were divided into a model group, a drug-treated group, and a blank control group. The mice in the model group and the drug-treated group were given a high-fat diet with 60% fat energy to induce obesity in the mice, and the nitric oxide synthase inhibitor L-NAME (0.5 g / L) was added to the drinking water to induce hypertension in the mice. After 5 to 6 weeks of modeling, the mice showed the phenotype of HFpEF, and the modeling lasted for a total of 12 weeks; the mice in the blank control group were given normal drinking water and a low-fat and low-sugar control diet.

[0032] Six weeks after modeling, mice were grouped by random block method according to weight, cardiac function and blood pressure to ensure that the model group and each drug group had the same cardiac function, weight and blood pressure. A total of 6 groups were divided, namely blank control group, model group, high-dose limonin group, medium-dose limonin group, low-dose limonin group and empagliflozin group, with 10 mice in each group.

[0033] 2. Dosage regimen: Dosage after modeling:

[0034] The medium dose of limonin was 50 mg / kg, the high dose and low dose were 100 mg / kg and 25 mg / kg respectively; empagliflozin was 10 mg / kg, and the administration volume was 0.01 ml / g; the blank control group and the model group were given an equal volume of saline and excipients. HS15 was administered from the 6th week of modeling for a total of 6 weeks, once a day.

[0035] In order to solve the problem of poor water solubility and low absorption rate of limonin, the limonin administration group used in this embodiment adopts a new type of non-ionic surfactant pharmaceutical excipient Compared with excipients such as Tween-80, HS15 has better solubilization and absorption-promoting effects, which helps to improve bioavailability. The details are as follows:

[0036] Accurately weigh HPLC grade limonin (≥98%) and dissolve it in the excipients first. HS15, then add physiological saline to dilute to an auxiliary material concentration of 20%, vortex mix and set aside.

[0037] 3. Mouse cardiac function test:

[0038] After 15 weeks of administration, the Vevo 2100 small animal ultrasound imaging system was used to detect cardiac function. The detection method was the same as in Example 1.

[0039] 4. Hematoxylin-eosin (HE) staining and Masson staining:

[0040] The hearts of mice in each group were fixed with 4% paraformaldehyde overnight, dehydrated and embedded in the usual way, and then 4 μm paraffin sections were made and dewaxed. HE and Masson staining were performed, and after dehydration with gradient ethanol, the sections were sealed with neutral gum, and the morphological changes of the heart tissues of mice in each group were observed under an optical microscope.

[0041] 5. Results

[0042] like Figure 3 As shown in the figure, ultrasound results showed that after 12 weeks of modeling, compared with the control group, the heart and lung weights of the model group mice were significantly increased (P<0.01), and severe left ventricular diastolic dysfunction and concentric myocardial hypertrophy occurred, which were specifically manifested by significantly increased E / e' and E / A, significantly decreased e' / a', significant thickening of the anterior and posterior walls of the left ventricle, and significantly decreased systolic and diastolic end-diastolic diameters (P<0.05 or 0.01), which were consistent with the manifestations of HFpEF. After high, medium and low doses of limonin were administered, the heart and lung weights of mice were significantly reduced compared with the model group (P<0.01), and all of them could significantly improve the left ventricular diastolic dysfunction and concentric myocardial hypertrophy of mice (P<0.05 or 0.01). There were no significant differences in the efficacy indicators compared with the positive drug empagliflozin.

[0043] like Figure 4 As shown, the results of HE and Masson staining showed that compared with the control group mice, the model group mice had obvious myocardial interstitial fibrosis, and the administration of high, medium, low levels of limonin and empagliflozin significantly improved myocardial fibrosis.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of limonin in the preparation of a drug for improving left ventricular diastolic function, characterized in that: The molecular formula of the limonin is C 26 H 30 O8, chemical structure is Limonin is the active ingredient in the medicine.

2. The use according to claim 1, characterized in that: The medicine also includes pharmaceutical excipients, which include polyethylene glycol (15)-hydroxystearate and physiological saline.

3. The use according to claim 2, characterized in that: When preparing the drug, limonin is first dissolved in the auxiliary material polyethylene glycol (15)-hydroxystearate, and then physiological saline is added to dilute to the target concentration and mixed.

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