A water calamus extract having efficacy of treating and / or preventing heart failure, and preparation method and application thereof

Abstract

The invention discloses a water schizonepeta extract and a preparation method and application thereof. The water schizonepeta extract contains iridoid glycosides as active ingredients, and the iridoid glycosides as active ingredients contain verbascoside, catalpa glycoside and picroside II; the weight percentage of the iridoid glycosides in the water schizonepeta extract is 40-90%; the mass ratio of verbascoside to catalpa glycoside is (3.0-5.0):1; the mass ratio of catalpa glycoside to picroside II is 1:(0.1-3.0). The present invention clarifies the active ingredients of the water schizonepeta medicinal material extract, prepares an effective part with stable quality, clarifies the material action basis of the drug effect, and effectively improves the safety of medication.

Description

[0001] This application claims priority to Chinese patent application CN202111627147X, filed on December 28, 2021. This application cites the full text of the above Chinese patent application. Technical Field

[0002] The invention relates to a water caltrop extract and a preparation method and application thereof. Background Art

[0003] Heart failure, referred to as HF, refers to a syndrome characterized by circulatory dysfunction caused by insufficient cardiac output to maintain tissue metabolism due to myocardial diastolic and / or systolic dysfunction under the condition of adequate venous return. According to statistics, the number of heart failure patients worldwide has reached 64.3 million, of which the prevalence of heart failure in developed countries is about 1-2%. According to the latest epidemiological survey in my country, the prevalence of heart failure has increased by 44% in the past 15 years. Among residents aged ≥35 years, the prevalence of heart failure is 1.3%, that is, about 13.7 million people suffer from heart failure. As the aging of the population in my country continues to intensify, the incidence of heart failure is bound to increase further, increasing the burden of public health and medical care.

[0004] In recent years, the advantages of traditional Chinese medicine combined with Western medicine in the conventional treatment of heart failure have gradually become apparent. For example, Qili Qiangxin Capsule was included in the Chinese medicine treatment plan for the first time in the "Guidelines for the Diagnosis and Treatment of Heart Failure in China 2018". However, most of the commonly used Chinese patent medicines or compound medicines are based on original medicinal materials or crude extracts. Their effective ingredients, ineffective ingredients and toxic ingredients are unclear, and there are problems such as large prescription doses, inconvenient administration and low safety. In order to solve the above problems, a class of modern Chinese patent medicines with the main active ingredients of traditional Chinese medicine as raw materials have entered the market, such as Ginkgolide Injection for cardiovascular and cerebrovascular diseases and Salvia miltiorrhiza Polyphenolic Acid for Injection, but there are currently no oral modern Chinese patent medicines for the treatment of heart failure.

[0005] At present, there is a study on the extraction of effective ingredients based on clinical prescriptions for the treatment of chronic heart failure. [1] , by boiling antlers, safflower, psoralea corylifolia, epimedium, cornus officinalis, Ligustrum lucidum and agarwood, 8 active ingredients including hydroxysafflower pigment, loganin, psoralen, isopsoralen, gallic acid, 5-hydroxymethylfurfural, teruncle glycoside and agarwood tetraol were extracted from the Chinese medicine compound, but the ineffective parts were not removed, and the above problems still exist. On the other hand, there are also component combinations with myocardial protective effects obtained from single medicinal materials. [2] For example, a composition consisting of total flavonoids from Eucommia ulmoides, eucommia ulmoides terpenes and eucommia ulmoides lignin. However, compared with this type of composition, a single effective part is easier to identify the material basis, has lower production costs and process difficulty, and is easier to control quality.

[0006] The development of effective parts of traditional Chinese medicine with a clear material basis, relatively clear mechanism of action, definite efficacy and high safety has important social significance and broad market prospects. [3,4] Picroside II can improve mitochondrial function by reducing the production of reactive oxygen species, thereby inhibiting hypoxia / reoxygenation-induced cardiomyocyte apoptosis. [5] , so it is worth further studying its active ingredients to obtain safer and more effective anti-heart failure effective parts.

[0007] The prior art also discloses an effective part of a traditional Chinese medicine for treating coronary heart disease and hyperlipidemia, a preparation method and a method for separating effective ingredients therefrom (CN104840451A, 2015.08.19), wherein the dried rhizome of Curcuma aromatica salisb. of the Curcumaceae family is used as the medicinal part, extracted with an organic solvent, and the extract is separated and purified to obtain an effective part containing sesquiterpenoid components, wherein the content of sesquiterpenoid components in the effective part is greater than 50%, and pharmacological experimental studies have shown that curcumadiol, protocurcumol, curcumadione and the total extract all have significant effects on treating myocardial ischemia, can significantly reduce the range of myocardial infarction area in rats with myocardial damage caused by ischemia, and have a significant protective effect on myocardial ischemic damage in rats with coronary artery ligation. This patent involves five preparation methods, four of which involve organic solvents other than ethanol. The organic solvent residues need to be investigated during the production process, and the production and environmental costs are high. Another method of macroporous resin separation obtains an active ingredient content of 54.06% in the effective part, and there is still a large part of the material basis that has not been clarified.

[0008] References are as follows:

[0009] [1] A method for extracting the active ingredients of a traditional Chinese medicine compound for treating chronic heart failure. Publication No. CN113181201A, Publication Date: 2021.07.30;

[0010] [2] A composition of Eucommia ulmoides components with myocardial protective effect and its preparation. Publication No. CN104435067A, Publication Date: 2015.03.25;

[0011] [3]Harput US, Varel M, Nagatsu A, et al. Acylated iridoid glucosides from Veronica anagalis-aquatica[J]. Phytochemistry, 2004, 65(14):2135-2139;

[0012] [4]Su BN, Zhu QX, Jia Z J. Aquaticol, a novel bis-sesquiterpene from Veronica anagalis-aquatica[J]. Tetrahedron Letters, 1999, 40(2): 357-358;

[0013] [5]Li J, Yu S, Mo D, et al. PicrosideⅡinhibits hypoxia / reoxygenation-induced cardiomyocyte apoptosis by ameliorating mitochondrial function through a mechanism involving a decrease in reactive oxygen species production. [J]. International Journal of Molecular Medicine, 2015, 35(2): 446-452. Summary of the invention

[0014] The technical problem to be solved by the present invention is to overcome the defects of the drugs for treating myocardial ischemia in the prior art that either the therapeutic effect is poor or the material action basis is unclear, and to provide a water caltrop extract and its preparation method and application. The present invention clarifies the active ingredients of the water caltrop extract, prepares an effective part with stable quality, clarifies the material action basis of the drug effect, and effectively improves the safety of medication.

[0015] For the extract of Iris polyphylla, the present invention prepares the effective part of iridoid glycosides from the ethanol extract of Iris polyphylla (for example, 75% ethanol extract of Iris polyphylla) by preparing liquid phase, and separates three active ingredients, namely, Verproside, Catalposide, and Picroside II, therefrom by high-speed countercurrent chromatography technology, and verifies its anti-heart failure efficacy and safety in vivo and in vitro.

[0016] The present invention provides a water calamus extract, wherein the water calamus extract contains iridoid glycosides as active ingredients, wherein the iridoid glycosides as active ingredients contain verbascoside, catalpa glycoside and picroside II; wherein:

[0017] The weight percentage of the iridoid glycoside active ingredient in the water spinach extract is 40-90%;

[0018] The mass ratio of the verbascoside to the catalpa glycoside is (3.0-5.0):1;

[0019] The mass ratio of the catalpa glycoside to the picroside II is 1:(0.1-3.0).

[0020] In the present invention, the water thorn extract can be an extract of the whole herb of the conventional Plantaginaceae plants Veronica anagallis-aquatica L. and Veronica undulata Wall.; preferably, the water thorn extract is an extract of the above-ground part of the Plantaginaceae plants Veronica anagallis-aquatica L. and Veronica undulata Wall.

[0021] Wherein, the northern water caltrop can be the northern water caltrop produced in Yangbi County, Dali Prefecture, Yunnan Province, or the northern water caltrop produced in Dali City, Yunnan Province.

[0022] In the present invention, the Ipomoea aquatica extract can be prepared by the following method: the crude extract of Ipomoea aquatica is eluted by column chromatography; wherein:

[0023] The adsorbent in the column chromatography is octadecylsilane bonded silica gel;

[0024] The mobile phase for elution is water and acetonitrile.

[0025] The crude extract of I. polyphylla can be a conventional crude extract of I. polyphylla in the art, such as an extract obtained by extraction with ethanol having a volume fraction of 0-95% (such as 70-80%, and another example of 75%).

[0026] The crude extract of Ipomoea aquatica can be prepared by the following method: mixing the plant medicinal material Ipomoea aquatica with an extraction solvent, extracting by heating, and filtering to obtain a filtrate; the extraction solvent can be ethanol with a volume fraction of 0-95%.

[0027] The herbal medicine Ipomoea aquatica can be pre-treated by pulverization and then extracted by heating. The pulverization can be pulverized into 30 meshes.

[0028] The extraction solvent may be ethanol with a volume fraction of 70-80%, for example, ethanol with a volume fraction of 75%.

[0029] The ratio of the mass g of the botanical medicinal material Ilex polyphylla to the volume mL of the extraction solvent can be a conventional ratio in the art, such as 1:(5-15), and another example is 1:10.

[0030] The temperature of the heating extraction may be 92-98°C, such as 95°C.

[0031] The heating extraction method can be heating reflux extraction.

[0032] The heating extraction may be performed once or twice or more.

[0033] The heating extraction time may be 1-3 hours, for example 2 hours. When the heating extraction is performed twice or more, the single heating extraction time may be 1-3 hours, for example 2 hours.

[0034] The mesh number of the filtration screen can be 200 meshes.

[0035] The filtrate can also be concentrated and dried according to conventional means in the art. The concentration can be concentrated by rotary evaporation at 65°C±5°C to a thick paste. The drying can be vacuum drying.

[0036] Wherein, the crude extract of Ilex schizonepeta can be dissolved in water before eluting by column chromatography. After being dissolved, the crude extract of Ilex schizonepeta can be filtered to remove insoluble matter.

[0037] Wherein, the particle size of the adsorbent may be 5-15 μm, for example, 10 μm.

[0038] Wherein, the column temperature of the column chromatography can be 20-30°C, for example 25°C.

[0039] Wherein, the column in the column chromatography can be a DAC50 column.

[0040] Wherein, the elution can be gradient elution.

[0041] Preferably, when the elution is gradient elution, mobile phase A is water and mobile phase B is acetonitrile; taking the total volume of the mobile phases A and B as 100%, the procedure of the gradient elution is as follows:

[0042] At 0-10 min, the volume of mobile phase A was 95%;

[0043] In 10-20 minutes, the volume of the mobile phase A is gradually reduced from 95% to 90%;

[0044] During 20-60 min, the volume of mobile phase A was gradually reduced from 90% to 50%.

[0045] When the above-mentioned gradient elution procedure is used, the eluent may be collected for 24-40 minutes.

[0046] Wherein, the flow rate of the mobile phase may be 50-90 mL / min, for example 70 mL / min.

[0047] The eluate obtained after the elution can be post-processed according to conventional means in the art, for example, first concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a solid effective fraction.

[0048] In the present invention, preferably, the mass ratio of the verbascoside to the catalpa glycoside is (3.5-4.8):1, for example (3.56-4.75):1, and for example 3.56:1 or 4.75:1.

[0049] In the present invention, preferably, the mass ratio of the catalpa glycoside to the picroside II is 1:(0.3-2.8), for example 1:(0.33-2.5), for example 1:0.33 or 1:2.5.

[0050] In the present invention, preferably, the mass ratio of verbascoside, catalpa glycoside and picroside II is 4.75:1:2.5 or 3.56:1:0.33.

[0051] In the present invention, preferably, the mass ratio of the verbascoside to the catalpa glycoside is (4.0-5.0):1, and the mass ratio of the catalpa glycoside to the picroside II is 1:(0.3-3.0).

[0052] In the present invention, preferably, the mass ratio of the verbascoside to the catalpa glycoside is (3.0-5.0):1, and the mass ratio of the catalpa glycoside to the picroside II is 1:(0.31-2.5).

[0053] In the present invention, the mass ratio of verbascoside to catalpa glycoside may be (4.0-5.0):1 or (3.0-4.0):1, for example 4.75:1 or 3.56:1.

[0054] In the present invention, the mass ratio of picroside to picroside II may be 1:(0.31-3.0), such as 1:0.33 or 1:(2.0-3.0), and also such as 1:2.5.

[0055] In the present invention, the weight percentage of the iridoid glycoside active ingredient in the water spinach extract is preferably 40-80%, such as 40-50%, 50-80% or 50-70%, and also such as 44% or 66%.

[0056] In the present invention, the weight percentage of verbascoside in the Ipomoea aquatica extract can be 30-50%, such as 32% or 38%. The calculation of the weight percentage does not include the solvent contained in the Ipomoea aquatica extract.

[0057] In the present invention, the weight percentage of the catalpa glycoside in the Ipomoea aquatica extract may be 5-15%, such as 8% or 9%. The calculation of the weight percentage does not include the solvent contained in the Ipomoea aquatica extract.

[0058] In the present invention, the weight percentage of the picroside II in the Ipomoea aquatica extract may be 1-40%, such as 3% or 20%.

[0059] Preferably, when the weight percentage of the iridoid glycoside active ingredient in the water spinach extract is 50-80% (e.g. 66%), the weight percentage of verbascoside in the water spinach extract may be 30-50% (e.g. 38%), the weight percentage of catalpa glycoside in the water spinach extract may be 5-15% (e.g. 8%), and the weight percentage of picroside II in the water spinach extract may be 10-30% (e.g. 20%).

[0060] Preferably, when the weight percentage of the iridoid glycoside active ingredient in the water spinach extract is 40-80% (e.g. 44%), the weight percentage of verbascoside in the water spinach extract may be 30-50% (e.g. 32%), the weight percentage of calathea glycoside in the water spinach extract may be 5-15% (e.g. 9%), and the weight percentage of picroside II in the water spinach extract may be 1-20% (e.g. 3%).

[0061] The present invention also provides a method for preparing the Iris polyphylla extract, which comprises the following steps: subjecting the crude extract of Iris polyphylla to column chromatography and eluting; wherein:

[0062] The adsorbent in the column chromatography is octadecylsilane bonded silica gel;

[0063] The mobile phase for elution is water and acetonitrile.

[0064] In the present invention, the water hyacinth extract can be an extract of the whole herb of Veronica anagallis-aquatica L. and Veronica undulata Wall., both of which are conventional plants of the Scrophulariaceae family, preferably an extract of the aerial part.

[0065] In the present invention, the crude extract of Ipomoea aquatica can be prepared by the following method: mixing the botanical material Ipomoea aquatica with an extraction solvent, extracting by heating, and filtering to obtain a filtrate; the extraction solvent can be ethanol with a volume fraction of 0-95%.

[0066] The herbal medicine Ipomoea aquatica can be pre-treated by pulverization and then extracted by heating. The pulverization can be pulverized into 30 meshes.

[0067] The extraction solvent may be ethanol with a volume fraction of 70-80%, for example, ethanol with a volume fraction of 75%.

[0068] The ratio of the mass g of the botanical medicinal material Ilex polyphylla to the volume ml of the extraction solvent can be a conventional ratio in the art, such as 1:(5-15), and another example is 1:10.

[0069] The temperature of the heating extraction may be 92-98°C, such as 95°C.

[0070] The heating extraction method can be heating reflux extraction.

[0071] The heating extraction may be performed once or twice or more.

[0072] The heating extraction time may be 1-3 hours, for example 2 hours. When the heating extraction is performed twice or more, the single heating extraction time may be 1-3 hours, for example 2 hours.

[0073] The mesh number of the filtration screen can be 200 meshes.

[0074] The filtrate can also be concentrated and dried according to conventional means in the art. The concentration can be concentrated by rotary evaporation at 65°C±5°C to a thick paste. The drying can be vacuum drying.

[0075] In the present invention, the crude extract of Ilex polyphylla can be dissolved in water before being eluted by column chromatography. After being dissolved, the crude extract of Ilex polyphylla can be filtered to remove insoluble matter.

[0076] In the present invention, the particle size of the adsorbent may be 5-15 μm, for example, 10 μm.

[0077] In the present invention, the column temperature of the column chromatography can be 20-30°C, for example 25°C.

[0078] Wherein, the column in the column chromatography can be a DAC50 column.

[0079] In the present invention, the elution may be gradient elution.

[0080] Wherein, preferably, when the elution is gradient elution, mobile phase A is water and mobile phase B is acetonitrile; taking the total volume of the mobile phases A and B as 100%, the procedure of the gradient elution is as follows:

[0081] At 0-10 min, the volume of mobile phase A was 95%;

[0082] In 10-20 minutes, the volume of the mobile phase A is gradually reduced from 95% to 90%;

[0083] During 20-60 min, the volume of mobile phase A was gradually reduced from 90% to 50%.

[0084] When the above-mentioned gradient elution procedure is used, the eluent may be collected for 24-40 minutes.

[0085] In the present invention, the flow rate of the mobile phase may be 50-90 mL / min, for example 70 mL / min.

[0086] In the present invention, the eluate obtained after the elution can be post-treated according to conventional means in the art, for example, first concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a solid effective fraction.

[0087] The invention also provides a water calamus extract, which is prepared by the method.

[0088] The present invention also provides a use of the Ipomoea aquatica extract in preparing a medicine for treating and / or preventing heart failure.

[0089] The present invention also provides a method for treating and / or preventing heart failure, comprising administering a therapeutically effective amount of the Ipomoea aquatica extract to a subject.

[0090] The term "heart failure" refers to a syndrome characterized by circulatory dysfunction caused by insufficient cardiac output to maintain tissue metabolic needs due to myocardial diastolic and / or systolic dysfunction in the presence of adequate venous return, which is mainly manifested as pulmonary congestion and vena cava congestion.

[0091] The term "therapeutically effective amount" refers to the amount required to relieve the symptoms of heart failure in an individual. The dosage is adjusted to the individual requirements in each specific case. The dosage can vary within a wide range, depending on many factors such as the severity of the condition to be treated, the age and general health of the patient, other drugs the patient is taking for treatment, the route and form of administration, and the preference and experience of the physician.

[0092] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0093] The reagents and raw materials used in the present invention are commercially available.

[0094] The positive and progressive effects of the present invention are:

[0095] The effective fraction of cyclopentane glycosides mainly composed of three active ingredients was obtained from the extract of the single Chinese medicinal material Sophora japonica (e.g. 75% ethanol extract) by preparing the liquid phase. The protective effect on oxygen-glucose deprivation injury of cardiomyocytes was verified at the in vitro cell level, the in vivo anti-heart failure efficacy was verified in the isoproterenol-induced heart failure mouse model and the left anterior descending coronary artery ligation heart failure mouse model, and its safety was verified by subacute toxicity experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1It is the cell survival rate of the model group, positive control group and effective part group under the OGD model.

[0097] Figure 2 Figure 3 is the effect of the effective part on primary cardiomyocytes of newborn SD rats; Figure A is the cell survival rate under the OGD model; Figure B is the beat number-time curve.

[0098] Figure 3 Heart pathological sections of mice induced by isoproterenol.

[0099] Figure 4 Masson staining of heart sections from mice undergoing myocardial infarction surgery (15x).

[0100] Figure 5 This is the appearance of important organs of mice after continuous administration of 6g / kg for two weeks as the effective part. DETAILED DESCRIPTION

[0101] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0102] Example 1: Preparation of effective fraction

[0103] Batch 1: The 75% ethanol extract of Veronica anagallis-aquatica L. was prepared by the following method: about 3 kg of the plant medicinal material Veronica anagallis-aquatica L. (produced in Yangbi County, Dali Prefecture, Yunnan Province) was taken, crushed through 30 mesh, and refluxed with 10 times the amount of 75% ethanol (g / ml) at 95°C for 3 times, each time for 2 hours, the extract was filtered through 200 mesh, combined and concentrated by rotary evaporation at 65°C±5°C to a thick paste, and then crushed to obtain 715 g of extract powder after vacuum drying.

[0104] Take 300g of the 75% ethanol extract powder of Veronica anagallis-aquatica L., dissolve it in water and filter to remove insoluble matter; the filtrate is prepared on a DAC50 column (C18, 300g, 10μm). Water is used as mobile phase A and acetonitrile is used as mobile phase B; the flow rate is 70mL / min, the gradient elution program is shown in Table 1, the detection wavelength is 254nm, the column temperature is 25℃, and the injection volume is 1L (the injection volume of 1L refers to the injection of 1L of 75% ethanol extract filtrate). Collect the eluate of 24-40min, concentrate it under reduced pressure until the acetonitrile is completely removed, and freeze-dry to obtain 42g of solid effective fraction.

[0105] Table 1: Gradient elution program for effective fraction preparation

[0106] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 95 5 20 90 10 60 50 50

[0107] Batch 2: The plant medicinal material Veronica anagallis-aquatica L. was produced in Dali City, Yunnan Province, and the other conditions were the same as batch 1. 38 g of solid effective parts were obtained by freeze drying.

[0108] Example 2: Isolation of iridoid glycosides

[0109] Prepare ethyl acetate-n-butanol-water two-phase solvent and separate the phases. The upper phase is pumped into the semi-preparative countercurrent chromatography (TBE-300C, Shanghai Tongtian) main unit as the stationary phase, and the lower phase is pumped into the mobile phase at a speed of 800rpm and a flow rate of 3.0mL / min to balance the system. When the balance reaches complete equilibrium, the solid effective part sample of batch one is dissolved with the lower phase solvent, and the injection is started, with 300mg injected each time. The sample is separated in the main unit, and the separated sample fractions are collected after detection by the detector.

[0110] According to HPLC detection, the purity of verbascoside was 95.2%, the purity of catalpa glycoside was 99.2%, and the purity of picroside II was 95.5%.

[0111] Example 3: Structural identification of three active ingredients

[0112] The structure of the components separated in Example 2 was confirmed by spectral methods such as nuclear magnetic resonance (H spectrum, C spectrum), mass spectrometry (ESI-high resolution), etc. The structural characterization data are as follows.

[0113] verbascoside 11H NMR (600 MHz, DMSO-d6) δ 7.40 (d, J = 2.2 Hz, 1H), 7.36 (dd, J = 8.3, 2.1 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.42 (dd, J = 5.9, 1.8 Hz, 1H), 5.10 (d, J = 9.5 Hz, 1H), 5.04 (dd, J = 8.2, 1.3 Hz, 1H), 4.95 (dd, J = 6.0, 4.4 Hz, 1H), 4.62 (d, J = 7.8 Hz, 1H), 3.92 (d, J = 13.3 Hz, 1H), 3.74–3.70 (m, 2H), 3.68 (d, J = 1.3 Hz, 1H), 3.44–3.41 (m, 1H), 3.19 (t, J = 9.0 Hz, 1H), 3.16 (td, J = 7.2, 3.5 Hz, 1H), 3.07–3.04 (m, 1H), 3.04–3.01 (m, 1H), 2.54 (ddt, J = 12.1, 6.1, 1.9 Hz, 4H), 2.48 (dd, J = 9.6, 7.7 Hz, 1H); 13 13C NMR (151 MHz, DMSO-d6) δ 166.12, 151.40, 145.65, 141.61, 122.63, 120.40, 116.82, 115.87, 102.25, 98.32, 93.43, 79.96, 77.93, 76.89, 73.90, 70.74, 66.23, 61.87, 58.95, 58.70, 42.28, 35.66; HRMS (ESI) m / z calcd for C 22 H 26 NaO 13 + [M+Na] + 521.1266, found 521.1272.

[0114] Catalpol 11H NMR(600MHz,DMSO-d6)δ7.88–7.83(m,2H),6.89–6.85(m,2H),6.43(dd,J=6.0,1.9Hz,1H),5.11(d,J=9.6Hz,1H),5.06(dd,J=8.0,1.4Hz,1H),4.96(dd,J=6.0,4.4Hz,1H),4.62(d,J=7.8Hz,1H),3.92(d,J=13.2Hz,1H),3.75–3.68(m,3H),3.43(dd,J=11.9,6.9Hz,1H),3.22–3.14(m,2H),3.04(dt,J=12.9,8.8Hz,2H),2.56(tdd,J=8.0,4.5,2.0Hz,1H),2.48(dd,J=9.6,7.7Hz,1H); 13 13C NMR(151MHz,DMSO-d6)δ166.03,162.93,141.60,132.19,120.17,115.95,102.25,98.33,93.42,80.05,77.94,76.89,73.90,70.74,66.26,61.87,58.93,58.67,42.26,35.61;HRMS(ESI)m / z calcd for C 22 H 26 NaO 12 + [M+Na] + 505.1316,found 505.1319.

[0115] Picroside II 1H NMR (600MHz, DMSO-d6) δ7.52(dd,J=8.3,2.0Hz,1H),7.46(d,J=2.1Hz,1H),6.87(d,J=8.3Hz,1H),6.43(dd,J=6.0 ,1.9Hz,1H),5.11(d,J=9.7Hz,1H),5.06(dd,J=8.1,1.3Hz,1H),4.97(dd,J=6.0,4.4Hz,1H),4.62(d,J=7.8Hz,1H ),3.92(d,J=13.2Hz,1H),3.82(s,3H),3.75–3.69(m,3H),3.43(dd,J=11.9,7.0Hz,1H),3.20(t,J=8.9Hz,1H),3. 16(ddd,J=9.3,6.9,2.1Hz,1H),3.07–3.01(m,2H),2.58(tdd,J=8.0,4.5,2.0Hz,1H),2.48(dd,J=9.6,7.7Hz,1H); 13 C NMR(151MHz,DMSO-d6)δ166.10,148.05,141.56,124.34,115.79,113.11,102.30,98.31,93.41,8 0.13,77.94,76.89,73.90,70.74,66.29,61.88,58.91,58.69,56.11,42.28,35.62; HRMS(ESI)m / z calcd for C 23 H 28 NaO 13 + [M+Na] + 535.1422, found 535.1429.

[0116] Example 4: Detection and analysis of the content of iridoid glycosides in the effective fraction

[0117] Weigh an appropriate amount of verbascoside, catalpa glycoside, and picroside II reference substances accurately, and dissolve them in methanol to prepare a 1 mg / mL reference solution. At the same time, weigh an appropriate amount of the prepared effective fraction (the solid effective fraction obtained after freeze-drying in batch 1 of Example 1) and dissolve it in methanol to prepare a 1 mg / mL test solution. Waters Alliance 2695 high performance liquid chromatography and Unitray C18 (150×4.6mm id, 5.0μm, Huapu Xinchuang Technology Co., Ltd.), with water as mobile phase A, acetonitrile as mobile phase B, flow rate 1mL / min, gradient elution program as shown in Table 2, detection wavelength 254nm, column temperature 25℃, reference sample injection volume 1μL, test sample injection volume 10μL, three analysis results each and the peak area average. The solid effective part of batch one was measured: verbascoside content was 38%, catalpa glycoside content was 8%, and picroside II content was 20%.

[0118] Table 2: Analytical gradient elution program

[0119]

[0120]

[0121] Table 3: Determination of the contents of three active ingredients in the effective part of batch 1

[0122] Compound Standard peak area Peak area of ​​effective part Content (mg / mL) verbascoside 956061 3611735 0.38 Catalpa glycoside 3388043 2730279 0.08 Picroside II 1228229 2469599 0.20

[0123] The same iridoid glycoside content detection and analysis method as that in the solid effective part of batch one was used to measure the content of verbascoside in the solid effective part of batch two: the content of verbascoside was 32% (0.32 mg / mL), the content of catalpa glycoside was 9% (0.09 mg / mL), and the content of picroside II was 3% (0.03 mg / mL).

[0124] Example 5: Protective effects of active ingredients and effective fractions on H9c2(2-1) cells in an in vitro oxygen glucose deprivation (OGD) model

[0125] 5.1 Experimental Methods

[0126] After digestion and counting of cultured rat cardiomyocytes H9c2 (2-1), the cells were inoculated into 96-well plates at a number of 8000 / well. After the cells grew to a density of 70-80%, the active ingredient mother solution (active ingredient mother solution refers to the mother solution obtained by dissolving three compounds of verbascoside, catalpa glycoside, and picroside II) and the solid effective fractions of batch 1 and batch 2 obtained after freeze-drying in Example 1 were diluted to the required concentration and added to the well plate for pretreatment. After 12 hours, the culture medium of the modeling group was replaced with a PBS solution containing the corresponding concentration of active ingredients and solid effective fractions. The type and concentration of the active ingredients of each modeling group, as well as the type and concentration of the solid effective fraction, were the same as those during pretreatment (the separate active ingredient and effective fraction dilutions were incubated with the cells from 12 hours before modeling to 6 hours after modeling), and the well plate was placed in an anoxic chamber. After N2 replaced the air, the anoxic chamber was returned to the cell culture incubator for continued culture for 6 hours. The cells of the control group continued to be cultured normally in the cell culture incubator. After modeling, the supernatant was discarded, 100 μL of CCK-8 detection solution was added to each well, and the cells were incubated in a 37°C incubator in the dark for 2 hours. The absorbance value (OD value) at a wavelength of 450 nm was detected using an enzyme marker.

[0127] 5.2 Data processing

[0128] Cell survival rate (%) = [(OD OGD处理组 -OD 溶剂对照 ) / (OD 正常对照组 -OD 溶剂对照 )] × 100%. The experimental results were statistically analyzed using GraphPadPrism 7 software, and the experimental results were expressed as mean ± standard error. Unpaired t test was used for inter-group comparison. *: significant difference relative to the model group.

[0129] 5.3 Experimental Results

[0130] The results of the effects of each active ingredient on the survival rate of cardiomyocytes under the OGD model are shown in Figure 1 , Table 4-1, Table 4-2, Table 4-3. The results showed that the cell survival rate in the model group was lower than that in the normal control group to 36.4%, indicating that OGD damage was serious and the model was successfully established. The three iridoid glycoside active ingredients could significantly increase the cell survival rate at a certain concentration, indicating that the three isolated compounds did have a protective effect on myocardial cells.

[0131] Table 4-1

[0132] Types of drugs Dosing concentration Cell survival rate (%) No drug, model group / 36.36±0.77 Nicorandil 100μM 41.07±1.27** verbascoside 1μM 38.67±0.50 verbascoside 25μM 43.49±1.77** verbascoside 50μM 47.07±1.33**** verbascoside 100μM 50.03±0.59**** Catalpa glycoside 1μM 38.03±0.28 Catalpa glycoside 25μM 43.56±2.25** Catalpa glycoside 50μM 44.52±0.71**** Catalpa glycoside 100μM 45.76±1.08**** Picroside II 1μM 43.76±1.89** Picroside II 25μM 45.87±1.44*** Picroside II 50μM 49.95±1.30**** Picroside II 100μM 56.51±2.16****

[0133] Table 4-2

[0134] Types of drugs Dosing concentration Cell survival rate (%) No drug, model group / 54.48±1.61 Nicorandil 100μM 60.57±0.95* Effective part (batch 1) 100 μg / mL 69.29±2.83** Effective part (batch 1) 200 μg / mL 73.56±1.86*** Effective part (batch 1) 400 μg / mL 74.66±2.23*** Effective part (batch 2) 100 μg / mL 67.41±2.32** Effective part (batch 2) 200 μg / mL 72.24±0.94*** Effective part (batch 2) 400 μg / mL 73.45±0.75***

[0135] Based on Tables 4-1 and 4-2, according to: (cell survival rate of the drug administration group - cell survival rate of the model group) / (100 - cell survival rate of the model group), this indicator shows that compared with the monomer group, the effective part group achieved a better cardiomyocyte protection effect.

[0136] Table 4-3

[0137]

[0138] Example 6: Protective effect of the solid active fraction of batch 1 on H9c2 (2-1) cells in an in vitro oxygen glucose deprivation (OGD) model

[0139] 6.1 Experimental methods

[0140] After the cultured rat cardiomyocytes H9c2 (2-1) were digested and counted, they were inoculated into 12-well plates at a number of 80,000 / well. After the cells grew to a density of 70-80%, the effective fraction mother solution (solid effective fraction of batch 1 obtained after freeze drying in Example 1) was diluted to the required concentration and added to the well plate for pretreatment. After 12 hours, the culture medium of the modeling group was replaced with a PBS solution containing the corresponding concentration of effective fractions, and the well plate was placed in an anoxic chamber. After N2 replaced the air, the anoxic chamber was returned to the cell culture incubator and continued to be cultured for 6 hours. The cells of the control group continued to be cultured normally in the cell culture incubator.

[0141] 6.1.1 Detection of reactive oxygen species (ROS) content: After modeling, discard the supernatant, add DCFH-DA working solution to cover the cells, and incubate in a 37°C cell culture incubator in the dark for 30 min; wash the cells 1 to 2 times with serum-free culture medium, and use a fluorescence microscope to photograph (480 nm wavelength excitation).

[0142] 6.1.2 Detection of lactate dehydrogenase (LDH) content: After modeling, take the supernatant from each well in a 96-well plate, add 50 μL of LDH working solution under light-proof conditions, incubate at 37°C in the dark for 30 min, add 50 μL of stop solution to each well, and use an enzyme-labeled instrument to detect the OD value at a wavelength of 490 nm.

[0143] 6.1.3 Crystal violet staining: After modeling, discard the supernatant, place the cells to be stained on ice, wash the cells twice with pre-cooled PBS, 3-5 minutes each time; fix the cells with pre-cooled methanol for 10 minutes, remove the methanol, add 0.5% crystal violet stain to cover the cells, and incubate for 10 minutes; recover the crystal violet stain, wash the stained cells with clean water until the stain is completely washed off; dry at room temperature and photograph under a microscope.

[0144] 6.2 Data processing

[0145] LDH release multiple = (OD 测试孔 -OD对应溶剂对照 ) / (OD 模型组 -OD 对应溶剂对照 ); ROS fluorescence intensity and crystal violet staining area were quantified using ImageJ. GraphPad Prism 7 software was used to statistically analyze the experimental results, and the experimental results were expressed as mean ± standard error. Unpaired t test was used for inter-group comparison. #: significant difference relative to the normal control group, *: significant difference relative to the model group.

[0146] 6.3 Experimental Results

[0147] As shown in Table 5-1, the cells in the control group basically did not generate reactive oxygen species, while the cells treated with OGD all generated reactive oxygen species, and the reactive oxygen species generated by the cells in the 200μg / mL and 400μg / mL effective fraction treatment groups were significantly less than those in the model group, especially the 400μg / mL effective fraction treatment group, indicating that the effective fraction can play a cell protective role by reducing the generation of reactive oxygen species. The actual concentrations of verbascoside, catalpa glycoside and picroside II in the liquid after the solid effective fraction of batch one was diluted are shown in Table 5-2.

[0148] Table 5-1

[0149]

[0150] Table 5-2

[0151]

[0152] As shown in Table 6, after OGD treatment, the release of LDH in the model group was significantly increased compared with the normal control group, while the release of LDH in cells treated with effective parts of each concentration was significantly decreased compared with the model group, reflecting the protective effect on cardiomyocytes.

[0153] Table 6

[0154]

[0155] As shown in Table 7, the staining area of ​​the model group was significantly reduced compared with the control group, indicating that the cell amount was significantly reduced. The staining area of ​​the group treated with the effective part increased compared with the model group and showed concentration dependence. The number of cells in the group treated with the effective part at 400 μg / mL was the largest, that is, the protective effect on cells was the most significant.

[0156] Table 7

[0157]

[0158] Example 7: Effects of the solid effective fraction of batch 1 on primary cardiomyocytes of newborn SD rats

[0159] 7.1 Experimental methods

[0160] The heart of a newborn SD rat of 1-3 days old was taken out, and the blood was washed with phosphate buffered saline; the heart was cut into minced tissue blocks with ophthalmic scissors, and then type II collagenase was added to digest in a 37°C water bath, and the digestion was repeated three times. After the tissue blocks were completely digested, they were passed through a 200-mesh sieve; the sieved cell solution was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, the cells were resuspended in a special culture medium for cardiomyocytes and transferred to a culture dish and cultured in a 37°C incubator for 90 minutes, the suspension was aspirated and re-inoculated in another culture dish, and 0.1mM 5-BrdU was added and continued to be cultured in a 37°C incubator. The medium was changed every 48 hours.

[0161] 7.7.1 Cell viability test: After the extracted primary rat cardiomyocytes were digested and counted, they were inoculated into a 48-well plate at a number of 16,000 / well. After the cells grew for 48 hours, the mother liquor after the solid effective part of batch one was dissolved was diluted to the required concentration and added to the well plate for pretreatment. After 6 hours, the culture medium of the modeling group was replaced with a PBS solution containing the corresponding concentration of effective part (the solid effective part obtained after freeze-drying in batch one of Example 1, the actual concentrations of verbascoside, catalpa glycoside and picroside II in the liquid after the solid effective part was diluted are shown in Table 5-2), and the well plate was placed in an anoxic chamber. After N2 replaced the air, the anoxic chamber was returned to the cell culture incubator and continued to be cultured for 3 hours. After the modeling was completed, the supernatant was discarded, 100 μL of CCK-8 detection solution was added to each well, and the cells were incubated in a 37°C incubator in the dark for 4 hours, and the absorbance value (OD value) at a wavelength of 450nm was detected by an enzyme marker.

[0162] 7.1.2 Counting of cell beating times: The extracted neonatal SD rat cardiomyocytes were evenly inoculated into 6 35 mm culture dishes and divided into two groups. They were cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours, and the medium was changed and cultured for another 24 hours until the cells were evenly beating again. The solid effective part group of batch one was added with sample mother solution to make the final concentration of 200 μg / mL, and the normal control group was added with an equal amount of DMSO as a control. After the addition, the cell beating videos (15 s) were immediately observed and collected under a microscope. Five cells were collected from each dish, and a total of 15 cells were collected from each group. After the filming was completed, the cells were immediately returned to the incubator. Beating videos were collected at 12h, 24h, 36h and 48h after administration according to the above operation, and the number of cell beatings in each video was counted.

[0163] 7.2 Data Processing

[0164] Cell survival rate (%) = [(OD OGD处理组 -OD 溶剂对照 ) / (OD 正常对照组 -OD 溶剂对照)] × 100%. The experimental results were statistically analyzed using GraphPadPrism 7 software, and the experimental results were expressed as mean ± standard error. Unpaired t test was used for inter-group comparison. #: significant difference relative to the control group, *: significant difference relative to the model group.

[0165] 7.3 Experimental Results

[0166] like Figure 2 A. As shown in Table 8, after 3 hours of OGD injury, the survival rate of primary cells in the model group dropped to 49.2%, and the model was successfully established. After treatment with different concentrations of effective parts, the survival rate of cells was significantly improved, and it showed obvious concentration dependence. The effective part 400μg / mL group increased the cell survival rate to 75.1%. It was confirmed that the effective part had a good protective effect on OGD injury of primary cardiomyocytes and indeed reduced cell death, because primary cardiomyocytes do not proliferate.

[0167] Table 8

[0168] Types of drugs Dosing concentration Cell survival rate (%) No drug, model group / 49.15±1.31 Nicorandil 100μM 53.68±0.37* Effective part (batch 1) 100 μg / mL 62.30±2.06** Effective part (batch 1) 200 μg / mL 67.26±0.37*** Effective part (batch 1) 400 μg / mL 75.13±2.36***

[0169] like Figure 2 B. As shown in Table 9, as the culture time of primary cardiomyocytes of newborn SD rats increases, the frequency of autonomous beating of cardiomyocytes will naturally decrease. Treatment with effective parts can enable primary cardiomyocytes to maintain a higher frequency of beating, and the rhythmic beating of cardiomyocytes is the power source of cardiac contraction and relaxation. Therefore, it can be inferred that the effective parts have the potential to maintain cardiac contraction and relaxation function under adverse conditions.

[0170] Table 9

[0171]

[0172] Example 8: Therapeutic effect of the solid effective fraction of batch 1 on isoproterenol (ISO)-induced heart failure mice

[0173] 8.1 Experimental methods

[0174] 7-week-old C57 male mice were divided into 4 groups (normal control group, model group, effective fraction 0.15g / kg group and effective fraction 0.6g / kg group), with 10 mice in each group. Every morning, the solid effective fraction of batch 1 obtained after freeze drying in Example 1 and the 75% ethanol extract in Example 1 were administered by gavage, and the control group and the model group were given an equal amount of drinking water. Isoproterenol hydrochloride was subcutaneously injected once in the morning and afternoon, with a dose of 40 mg / kg on the 1st to 2nd day, 20 mg / kg on the 3rd to 7th day, and 10 mg / kg on the 8th to 14th day. The control group was injected with an equal amount of normal saline. On the 15th day, the Visual-Sonics Vevo3100 small animal high-resolution micro-ultrasound imaging system was used to evaluate the cardiac structure and function of each group of experimental mice. On the 16th day, the mice were dissected to obtain the heart and heart sections were made, and the sections were stained with HE and Masson.

[0175] 8.2 Data Processing

[0176] Vevo software was used to measure systolic interventricular septal thickness (IVSs), diastolic interventricular septal thickness (IVSd), left ventricular systolic inner diameter (LVIDs), left ventricular diastolic inner diameter (LVIDd), left ventricular systolic posterior wall thickness (LVPWs), and left ventricular diastolic posterior wall thickness (LVPWd) in three cardiac cycles. Ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) were calculated. ImageJ was used to quantify the fibrosis area. GraphPadPrism 7 software was used to statistically analyze the experimental results. The experimental results were expressed as mean ± standard error. Unpaired t test was used for comparison between groups. #: significant difference relative to the control group, *: significant difference relative to the model group.

[0177] 8.3 Experimental Results

[0178] As shown in the results of Tables 10-1 and 11, the cardiac function of the mice in the model group was significantly reduced compared with that of normal mice, which was specifically manifested by decreased ejection fraction and short-axis shortening rate, increased left ventricular end-systolic volume and left ventricular end-diastolic volume, and increased fibrosis area. Both the high-dose and low-dose groups of the effective part can significantly improve the cardiac function impairment caused by isoproterenol hydrochloride. The mice in the 75% ethanol extract group treated with the same dose as the high-dose group of the effective part did not show any efficacy. Although the prior patent (application number: CN202011417172.0) disclosed a new use of Chinese medicine water lettuce for anti-heart failure, in which the 75% ethanol extract of water lettuce (i.e., Sophora flavescens) at a dose of 0.6 g / kg showed significant efficacy under the isoproterenol-induced model, it can be seen that the prior patent and the present invention The induction dose and frequency of isoproterenol in the present invention are different. The present invention adopts isoproterenol stimulation twice a day, and the total daily dosage is higher. At the same time, considering that the content of effective ingredients in the crude extract is affected by the harvesting season and processing of medicinal materials, the effective ingredients are not yet clear, and the content of effective ingredients cannot be detected for quality control, resulting in a negative result in the 75% ethanol extract in this experiment. The active ingredients in the effective part of the present invention are clear and can be quality controlled, which overcomes the problem of unstable efficacy and has better efficacy, and exhibits efficacy at a lower dose.

[0179] Combined with the results of pathological sections, such as Figure 3 As shown, HE staining of the mouse heart sections in the model group showed obvious inflammatory infiltration (indicated by the arrows), and Masson staining showed severe cardiac fibrosis, causing obvious organic damage. The 0.15g / kg dose group slightly alleviated the inflammatory response and fibrosis, while the 0.6g / kg high-dose group significantly alleviated the inflammatory response and fibrosis, showing better efficacy. The effect of the 75% ethanol extract group was equivalent to that of the 0.15g / kg dose group of the effective part (batch one).

[0180] Table 10-1: Cardiac function indexes

[0181]

[0182]

[0183] Table 10-2

[0184]

[0185] Table 11

[0186]

[0187] Example 9: Therapeutic effect of the solid effective fraction of batch 1 on heart failure mice induced by myocardial infarction (MI) surgery

[0188] 9.1 Experimental Methods

[0189] After 8-10 weeks of adaptive feeding of C57 male mice, the neck and left chest of the mice were depilated with isoflurane-assisted anesthesia. The mice were then fixed in a supine position, the neck skin was cut open, the muscle layer was separated to expose the trachea, the endotracheal tube was inserted through the mouth, and the ventilator was connected. After the mice spontaneously breathed and synchronized with the ventilator, the mice were adjusted to the right side lying position, the surgical area was disinfected with iodine, the second to third intercostal space was used as a hand incision, the skin and pectoralis major were cut in sequence, the ribs were exposed, the pleura was opened, the chest was expanded with a chest opener, the pericardium was cut open to fully expose the heart, and the left anterior descending branch of the coronary artery was ligated with a non-invasive suture 2-3 mm below the junction of the pulmonary artery cone and the left atrial appendage. The myocardial color became pale and the pulsation weakened below the ligation site as a sign of successful modeling. After modeling, the adjacent ribs were tightly sutured, the chest wall was gently squeezed to expel the gas in the chest cavity, the skin was sutured, and disinfected with iodine. The suture of the sham operation group passed through the heart but was not ligated. The mice in the surgical group were randomly divided into 4 groups (surgical group, effective part 0.15g / kg group, effective part 0.6g / kg group (solid effective part of batch 1 obtained after freeze drying in Example 1), positive drug LCZ696 0.06g / kg group) on the second day after surgery, with 9 mice in each group. The drug treatment group was gavaged every day, and the sham operation group and the surgical group were gavaged with an equal volume of drinking water for 14 days. On the 15th day after surgery, the Visual-Sonics Vevo 3100 small animal high-resolution micro-ultrasound imaging system was used to evaluate the cardiac structure and function of the experimental mice in each group. On the 16th day, the mice were dissected and the hearts were weighed to calculate the heart-body ratio, and the heart sections were made for Masson staining.

[0190] 9.2 Data Processing

[0191] Vevo software was used to measure IVSs, IVSd, LVIDs and LVIDd, and the average values ​​were taken to calculate EF%, FS%, LVESV and LVEDV. GraphPad Prism 7 software was used to statistically analyze the experimental results. The experimental results were expressed as mean ± standard error. Unpaired t test was used for inter-group comparison, and Log-rank test was used for survival curve. #: significant difference relative to the control group, *: significant difference relative to the model group.

[0192] 9.3 Experimental Results

[0193] As shown in the results of Table 13, the survival rate of mice in the surgery group was only 42.9% without drug treatment, and the heart body ratio was significantly increased compared with the sham surgery group, and the heart function was significantly reduced. The low-dose administration group of the effective part failed to improve the survival rate of mice with myocardial infarction and failed to improve heart function, but the high-dose group of 0.6g / kg significantly increased the survival rate of mice with MI surgery (100%), improved the heart function of mice, and reduced the heart body ratio, and the effect was better than that of positive drugs. Combined with the results of pathological sections, as shown in Figure 4 As shown in Table 12, the high-dose group of 0.6 g / kg can also reduce cardiac fibrosis. Overall, the effective fraction showed a stronger efficacy than the chemical drugs used in the clinical frontline. The actual contents of verbascoside, catalpa glycoside and picroside II in the liquid after the solid effective fraction was diluted are shown in Table 10-2.

[0194] Table 12

[0195]

[0196] Table 13: Survival rate, heart-body ratio and cardiac function indexes

[0197]

[0198]

[0199] Example 10: Subacute toxicity evaluation of solid effective fraction of batch 2

[0200] 10.1 Experimental Methods

[0201] ICR mice were raised to a body weight of about 25g and randomly divided into two groups, a normal control group (Control) and an effective part administration group (HL0877i-6g / kg), with 10 mice in each group, half male and half female. Mice were gavaged with 6g / kg effective part (solid effective part of batch 2 obtained after freeze drying in Example 1) every day (at this dose, verbascoside, catalpa glycoside and picroside II were actually 1.92g / kg, 0.54g / kg, and 0.18g / kg, respectively), and the normal control group was gavaged with an equal amount of drinking water. Continuous administration for 14 days. The toxic reactions and deaths of mice were observed every day, and the daily body weight was recorded. On the 14th day of administration, mice in each group were killed, dissected, and the important organs of mice were observed with the naked eye to see if they were lesions. At the same time, the heart, liver, spleen, lungs, and kidneys were weighed, and the organ / body weight was calculated to calculate the organ-body ratio.

[0202] 10.2 Data Processing

[0203] GraphPad Prism 7 software was used to analyze the experimental results. The experimental results were expressed as mean ± standard deviation, and the two-way ANOVA test was used for comparison among the groups.

[0204] 10.3 Experimental Results

[0205] The drug was administered continuously for two weeks at a dose 10 times the effective dose (6 g / kg). No mice died during the experiment and no obvious toxic reactions were observed. Figure 5As shown in Table 14, the daily body weight of mice was the same as that of the normal control group. On the 15th day, the mice were dissected, and the color, texture, and morphology of the important organs (heart, liver, spleen, lung, and kidney) were the same as those of the normal control group, and there was no significant difference in the ratio of organs to body weight. It can be seen that the safety window of the effective part is more than 10 times, and the safety is good.

[0206] Table 14

[0207]

[0208]

[0209] Note: The days for calculating body weight in the above table are based on the day of drug administration as Day 0, that is, Day 1 is the first day after drug administration.

[0210] Embodiment 11

[0211] Ipomoea aquatica extract: its preparation method is the same as the preparation method of the 75% ethanol extract of batch 1 in Example 1, and finally a solid extract is obtained by freeze-drying.

[0212] (2) Verification of the protective effect of the above extract on H9c2(2-1) cells under an in vitro oxygen-glucose deprivation (OGD) model. The experimental method and data processing were the same as those in Example 5.

[0213] (3) Experimental results

[0214] The results of the effect of water hyacinth extract on the survival rate of cardiomyocytes under the OGD model are shown in Table 15. The results showed that the cell survival rate of the model group was reduced to 33.95% compared with the normal control group, indicating that the OGD damage was serious and the model was successfully established. Water hyacinth extract can significantly increase the cell survival rate at a certain concentration, indicating that water hyacinth extract does have a protective effect on cardiomyocytes.

[0215] Table 15

[0216] Types of drugs Dosing concentration Cell survival rate (%) No drug, model group / 33.95±1.14 Nicorandil 100μM 41.35±1.14* Water hyacinth (batch 1) 10 μg / mL 45.02±0.90** Water hyacinth (batch 1) 50 μg / mL 48.39±1.13*** Water hyacinth (batch 1) 100 μg / mL 57.11±1.65***

Claims

1. A water spinach extract having the efficacy of treating and / or preventing heart failure, characterized in that: The water spinach extract contains iridoid glycosides as active ingredients, and the iridoid glycosides as active ingredients include verbascoside, catalpa glycoside and picroside II; wherein: The weight percentage of the iridoid glycoside active ingredient in the water spinach extract is 40-90%; The mass ratio of the verbascoside to the catalpa glycoside is (4.0-5.0):1; The mass ratio of the catalpa glycoside to the picroside II is 1:(2.0-3.0); The Ipomoea aquatica extract is prepared by the following method: the crude extract of Ipomoea aquatica is eluted by column chromatography; wherein: The adsorbent in the column chromatography is octadecylsilane bonded silica gel; The mobile phase for the elution is water and acetonitrile; The elution is a gradient elution; mobile phase A is water, and mobile phase B is acetonitrile; based on the total volume of the mobile phases A and B being 100%, the procedure of the gradient elution is as follows: At 0-10 min, the volume of mobile phase A was 95%; At 10-20 min, the volume of mobile phase A was decreased from 95% to 90%; At 20-60 min, the volume of mobile phase A was decreased from 90% to 50%; The crude extract of I. polyphylla is prepared by the following method: mixing the botanical material I. polyphylla with an extraction solvent, extracting by heating, and filtering to obtain a filtrate; wherein: The extraction solvent is ethanol with a volume fraction of 70-80%.

2. The Ipomoea aquatica extract according to claim 1, characterized in that The water caltrop extract is water caltrop ( Veronica anagallis - aquatica L.) or Ipomoea aquatica ( Veronica undulata Wall.) 3. The Ipomoea aquatica extract according to claim 1, characterized in that The water caltrop extract is water caltrop ( Veronica anagallis - aquatica L.) or Ipomoea aquatica ( Veronica undulata Wall.) Extract from the aerial parts.

4. The Ipomoea aquatica extract according to claim 1, characterized in that The crude extract of I. polyphylla is dissolved in water before being eluted by column chromatography, and the crude extract of I. polyphylla is filtered to remove insoluble matter after being dissolved.

5. The Ipomoea aquatica extract according to claim 1, characterized in that The particle size of the adsorbent is 5-15 μm.

6. The Ipomoea aquatica extract according to claim 5, characterized in that The particle size of the adsorbent is 10 μm.

7. The Ipomoea aquatica extract according to claim 1, characterized in that The column temperature of the column chromatography is 20-30°C.

8. The Ipomoea aquatica extract according to claim 7, characterized in that The column temperature of the column chromatography was 25°C.

9. The Ipomoea aquatica extract according to claim 1, characterized in that The column used in the column chromatography is a DAC50 column.

10. The Ipomoea aquatica extract according to claim 1, characterized in that The flow rate of the mobile phase is 50-90 mL / min.

11. The Ipomoea aquatica extract according to claim 10, characterized in that The flow rate of the mobile phase was 70 mL / min.

12. The Ipomoea aquatica extract according to claim 1, characterized in that The eluate obtained after the elution is first concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a solid effective fraction.

13. The Ipomoea aquatica extract according to claim 1, characterized in that The botanical medicinal material Ipomoea aquatica is pre-treated by pulverization and then extracted by heating.

14. The Ipomoea aquatica extract according to claim 13, characterized in that The pulverizing is pulverizing to 30 meshes.

15. The Ipomoea aquatica extract according to claim 1, characterized in that The extraction solvent is 75% ethanol by volume.

16. The Ipomoea aquatica extract according to claim 1, characterized in that The ratio of the mass g of the botanical medicinal material Ilex polygoni multiflori to the volume ml of the extraction solvent is 1:(5-15).

17. The Ipomoea aquatica extract according to claim 16, characterized in that The ratio of the mass g of the botanical medicinal material Ilex polyphylla to the volume ml of the extraction solvent is 1:

10.

18. The Ipomoea aquatica extract according to claim 1, characterized in that The temperature of the heating extraction is 92-98°C.

19. The Ipomoea aquatica extract according to claim 18, characterized in that The temperature of the heating extraction is 95°C.

20. The Ipomoea aquatica extract according to claim 1, characterized in that The heating extraction method is heating reflux extraction.

21. The Ipomoea aquatica extract according to claim 1, characterized in that The heating extraction is performed once or twice or more.

22. The Ipomoea aquatica extract according to claim 1, characterized in that The heating extraction time is 1-3h.

23. The Ipomoea aquatica extract according to claim 22, characterized in that The heating extraction time is 2 hours.

24. The Ipomoea aquatica extract according to claim 22, characterized in that When the heating extraction is performed twice or more, the single heating extraction time is 1-3 hours.

25. The Ipomoea aquatica extract according to claim 24, characterized in that The single time of the heating extraction is 2 hours.

26. The Ipomoea aquatica extract according to claim 1, characterized in that The mesh number of the filtration screen is 200 meshes.

27. The Ipomoea aquatica extract according to claim 1, characterized in that The filtrate is concentrated and dried.

28. The Ipomoea aquatica extract according to claim 27, characterized in that The concentration is performed by rotary evaporation at 65°C±5°C until a thick paste is obtained.

29. The Ipomoea aquatica extract according to claim 27, characterized in that The drying is vacuum drying.

30. The Ipomoea aquatica extract according to claim 1, characterized in that The mass ratio of the verbascoside to the catalpa glycoside is 4.75:

1.

31. The Ipomoea aquatica extract according to claim 1, characterized in that The mass ratio of the catalpa glycoside to the picroside II is 1:2.

5.

32. The Ipomoea aquatica extract according to claim 1, characterized in that The mass ratio of verbascoside, catalpa glycoside and picroside II is 4.75:1:2.

5.

33. The Ipomoea aquatica extract according to claim 1, characterized in that The weight percentage of the iridoid glycoside active ingredient in the water spinach extract is 50-80%.

34. The Ipomoea aquatica extract according to claim 33, characterized in that The weight percentage of the iridoid glycoside active ingredient in the water spinach extract is 66%.

35. The Ipomoea aquatica extract according to claim 1, characterized in that The weight percentage of verbascoside in the water spinach extract is 30-50%.

36. The Ipomoea aquatica extract according to claim 35, wherein The weight percentage of verbascoside in the watercress extract is 38%.

37. The Ipomoea aquatica extract according to claim 1, characterized in that The weight percentage of the catalpa glycoside in the water spinach extract is 5-15%.

38. The Ipomoea aquatica extract according to claim 37, characterized in that The weight percentage of the catalpa glycoside in the water spinach extract is 8%.

39. The Ipomoea aquatica extract according to claim 1, wherein The weight percentage of the picroside II in the Ipomoea aquatica extract is 20%.

40. The Ipomoea aquatica extract according to claim 1, characterized in that When the weight percentage of the iridoid glycoside active ingredient in the water spinach extract is 66%, the weight percentage of verbascoside in the water spinach extract is 38%, the weight percentage of catalpa glycoside in the water spinach extract is 8%, and the weight percentage of picroside II in the water spinach extract is 20%.

41. A method for preparing the Ipomoea aquatica extract according to any one of claims 1 to 40, characterized in that: The method comprises the following steps: subjecting the crude extract of Ilex polygoni multiflori to column chromatography and eluting; wherein: The crude extract of I. polyphylla is prepared by the following method: mixing the botanical material I. polyphylla with an extraction solvent, extracting by heating, and filtering to obtain a filtrate; The adsorbent in the column chromatography is octadecylsilane bonded silica gel; The mobile phase for the elution is water and acetonitrile; The elution is a gradient elution; mobile phase A is water, and mobile phase B is acetonitrile; based on the total volume of the mobile phases A and B being 100%, the procedure of the gradient elution is as follows: At 0-10 min, the volume of mobile phase A was 95%; At 10-20 min, the volume of mobile phase A was decreased from 95% to 90%; At 20-60 min, the volume of mobile phase A was decreased from 90% to 50%; The extraction solvent is ethanol with a volume fraction of 70-80%.

42. The method for preparing the Ipomoea aquatica extract according to claim 41, wherein: The plant source of the water calamus extract is the same as the plant source of the water calamus extract described in claim 2 or 3.

43. The method for preparing the Ipomoea aquatica extract according to claim 41, wherein: The preparation method of the Ipomoea aquatica extract is the same as the preparation method of the Ipomoea aquatica extract according to any one of claims 1 to 29.

44. Use of the Ipomoea aquatica extract according to any one of claims 1 to 40 in the preparation of a medicament for treating and / or preventing heart failure.

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