WO2023125719A1 - Veronica undulata extract as well as preparation method therefor and use thereof - Google Patents

Abstract

Disclosed in the present invention are a Veronica undulata extract as well as a preparation method therefor and the use thereof. The Veronica undulata extract contains iridoid glycoside active ingredients, and the iridoid glycoside active ingredients include verbascoside, catalposide and picroside II. The weight percentage of the iridoid glycoside active ingredients in the Veronica undulata extract is 40-90%. The mass ratio of the verbascoside to the catalposide is (3.0-5.0):1; and the mass ratio of the catalposide to the picroside II is 1:(0.1-3.0). In the present invention, the active ingredients of the Veronica undulata medicinal material extract are defined, the effective part having stable quality is prepared, the substance action basis of the drug efficacy is clear, and the medication safety is effectively improved.

Description

A kind of Shuikuan extract and its preparation method and application

This application claims the priority of the patent application PCT/CN2021/142190 with the filing date of 2021/12/28. This application cites the above patent application in its entirety.

technical field

The present invention relates to a kind of extract of bitter gourd and its preparation method and application.

Background technique

Heart failure, referred to as heart failure, refers to a syndrome mainly caused by circulatory dysfunction due to myocardial diastolic and/or systolic dysfunction and insufficient cardiac output to maintain tissue metabolism under the condition of adequate venous return. According to statistics, the number of heart failure patients in the world has reached 64.3 million, and 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 the residents aged ≥35 years, the prevalence of heart failure is 1.3%, that is, about 13.7 million people suffer from heart failure. And as the aging population continues to intensify in my country, the incidence of heart failure will surely increase further, increasing the burden of public health care.

In recent years, the advantages of conventional Chinese medicine combined with Western medicine in the treatment of heart failure have gradually emerged. 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 raw materials or crude extracts, and their active ingredients, ineffective ingredients, and toxic ingredients are not clear, 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 medicines as raw materials has entered the market, such as ginkgolide injection for cardiovascular and cerebrovascular diseases, salvianolic acid for injection, etc., but there is no medicine for heart failure at present. Oral modern Chinese patent medicine for treatment.

At present, there is a study on the extraction of active ingredients based on clinical prescriptions for the treatment of chronic heart failure ^[1] , a traditional Chinese medicine compound composed of seven raw materials of boiled antlers, safflower, psoralen, epimedium, dogwood, privet lucidum and agarwood , to extract 8 active ingredients of hydroxysafflower pigment, loganin, psoralen, isopsoralen, gallic acid, 5-hydroxymethylfurfural, tergumin, and agaroside, but there is no Remove the non-valid part, the above problem still exists. On the other hand, there are also component compositions with cardioprotective effects obtained from single medicinal materials ^[2] , such as compositions composed of Eucommia total flavonoids, Eucommia iridoids and Eucommia lignin, however, compared with such Composition, a single effective part is easier to define the material basis, the production cost and process difficulty are lower, and it is easier to carry out quality control.

It is of great social significance and broad market prospect to develop effective parts of traditional Chinese medicine with clear material basis, relatively clear mechanism of action, definite curative effect and high safety. It has been reported in the literature that A. japonicus contains a series of iridoids ^{[3, 4]} , among which pigetrin II can improve mitochondrial function by reducing the production of reactive oxygen species, thereby inhibiting hypoxia/reoxygenation-induced Cardiomyocyte apoptosis ^[5] , so it is worthy of further research on its active ingredients to obtain a safer and more effective anti-heart failure effective site.

Also disclosed in the prior art is an effective part of a traditional Chinese medicine for treating coronary heart disease and hyperlipidemia, a preparation method and a method for isolating active ingredients therefrom (CN104840451A, 2015.08.19), which takes Curcuma aromatica The dried rhizome of salisb.) is the medicinal part, which is extracted with an organic solvent, and the extract is separated and purified to obtain an effective part containing sesquiterpenoids. The content of the sesquiterpenoids in the effective part is greater than 50%. Show that curcumadiol, original curcumadiol, curcumadione and total extract all have significant therapeutic effect on myocardial ischemia, can significantly reduce the range of myocardial infarction in rat myocardial injury caused by ischemia, and have a significant effect on myocardial infarction in rats with coronary artery ligation. Ischemic injury has obvious protective effect. The patent involves 5 preparation methods, 4 of which involve organic solvents other than ethanol. In the production process, organic solvent residues need to be investigated, and the production cost and environmental protection cost are high. Another method for macroporous resin separation The content of the active ingredient in the obtained effective part is 54.06%, and there is still a large part of the material basis that has not been clarified.

The references are as follows:

[1] A method for extracting active ingredients of anti-chronic heart failure traditional Chinese medicine compound. Publication number CN113181201A, public date: 2021.07.30;

[2] A component composition of Eucommia ulmoides with cardioprotective effect and its preparation. Publication number CN104435067A, public date: 2015.03.25;

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

[4] Su B N, Zhu Q X, Jia Z J. Aquaticol, a novel bis-sesquiterpene from Veronica anagallis-aquatica[J]. Tetrahedron Letters, 1999, 40(2): 357-358;

[5] Li J, Yu S, Mo D, et al. Picroside II inhibits 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.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects that the drugs for the treatment of myocardial ischemia in the prior art either have poor therapeutic effects or have unclear material action basis, and provide a kind of extract of Shui Kuiping and its preparation method and application . The present invention clarifies the active ingredients of the extract of Scutellaria chinensis, prepares the effective part with stable quality, clarifies the material action basis of the medicinal effect, and effectively improves the safety of medication.

As for the extract of A. chinensis, the present invention prepares the effective fraction of iridoid glycosides from the ethanol extract of A. chinensis (for example, 75% ethanol extract of A. chinensis) by preparing a liquid phase, and utilizes high-speed countercurrent chromatography to extract Three active components, Verproside, Catalposide and Picroside II, were isolated, and their anti-heart failure efficacy and safety were verified in vivo and in vitro.

The present invention provides a kind of extract of Herba chinensis, which contains active ingredients of iridoid glycosides, and the active ingredients of said iridoid glycosides include verbascoside, catalpin and coptis pedis Glycoside II; Wherein:

The weight percentage of the iridoid glycosides active ingredient in the extract of Shuikupin is 40-90%;

The mass ratio of the verbascoside to the catalbin is (3.0-5.0): 1;

The mass ratio of the catalpaside and the tuberside II is 1: (0.1-3.0).

In the present invention, the extract of Aquatica can be the extract of the whole plant of Veronica anagallis-aquatica L. and Veronica undulata Wall., which are conventional plants in the field; preferably Specifically, the extract of Aquatica is the extract of the aerial parts of Veronica anagallis-aquatica L. and Veronica undulata Wall.

Wherein, the bitter sage can be the bitter sage produced in Yangbi County, Dali Prefecture, Yunnan Province, or the bitter scorpion produced in Dali City, Yunnan Province.

In the present invention, the extract of Sichoria chinensis can be obtained by the following method: eluting the crude extract of Sichoria syringae through column chromatography; wherein:

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

The mobile phase of described elution is water and acetonitrile.

Wherein, the crude extract of Piscarina can be conventional crude extract of Piscarina in the art, for example, the extract obtained by ethanol extraction with a volume fraction of 0-95% (eg, 70-80%, or 75%).

The crude extract of Sichoria chinensis can be obtained by the following method: mixing the herbal medicinal material Sichoria syringae with an extraction solvent, heating and extracting, and filtering to obtain a filtrate; the extraction solvent can have a volume fraction of 0-95 % ethanol.

The plant medicinal material Shuikuan can be subjected to pulverization and pretreatment and then extracted by heating. The crushing can be crushed to 30 mesh.

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

The ratio of the mass g of the botanical medicinal material Scutellaria chinensis to the volume mL of the extraction solvent may be a conventional ratio in the art, such as 1:(5-15), and for example 1:10.

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

The heating extraction method may be heating reflux extraction.

The number of times of the heat extraction can be one time or two times or more.

The heating extraction time may be 1-3 hours, such as 2 hours. When the number of times of the heating extraction is two times or more, the single time of the heating extraction may be 1-3 hours, for example, 2 hours.

The mesh number of the filtering screen can be 200 mesh.

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 may be vacuum drying.

Wherein, the crude extract of Aquapicus can be dissolved in water before being eluted by column chromatography. After the crude extract of the hydropicthiaceae is dissolved, the insoluble matter can be removed by filtration.

Wherein, the particle size of the adsorbent may be 5-15 μm, such as 10 μm.

Wherein, the column temperature of the column chromatography may be 20-30°C, such as 25°C.

Wherein, the column in the column chromatography can be a DAC50 column.

Wherein, the elution may be gradient elution.

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

At 0-10min, the volume of the mobile phase A is 95%;

In 10-20min, the volume of the mobile phase A is decreased from 95% to 90%;

In 20-60min, the volume of the mobile phase A was decreased from 90% to 50%.

When the above-mentioned gradient elution procedure is used, the eluate can be collected for 24-40 minutes.

Wherein, the flow rate of the mobile phase may be 50-90mL/min, such as 70mL/min.

Wherein, the eluate obtained after the elution can be post-treated according to conventional means in the art, for example, the acetonitrile is removed by concentration under reduced pressure, and then the effective fraction is obtained by freeze-drying.

In the present invention, preferably, the mass ratio of verbascoside to catalbin is (3.5-4.8):1, for example (3.56-4.75):1, further for example 3.56:1 or 4.75:1.

In the present invention, preferably, the mass ratio of the catalpaside and the pipericillin II is 1:(0.3-2.8), such as 1:(0.33-2.5), further such as 1:0.33 or 1:2.5.

In the present invention, preferably, the mass ratio of verbascoside, catalpaside and tuberside II is 4.75:1:2.5 or 3.56:1:0.33.

In the present invention, preferably, the mass ratio of the verbascoside to the catalpaside is (4.0-5.0): 1, and the mass ratio of the catalpaside to the puberisin II is 1: (0.3-3.0) .

In the present invention, preferably, the mass ratio of the verbascoside and the catalbin is (3.0-5.0): 1, and the mass ratio of the catalbin and the puberisin II is 1: (0.31-2.5) .

In the present invention, the mass ratio of verbascoside and catalbin may be (4.0-5.0):1 or (3.0-4.0):1, such as 4.75:1 or 3.56:1.

In the present invention, the mass ratio of the catalbinoside and berberine II may be 1:(0.31-3.0), for example 1:0.33 or 1:(2.0-3.0), for example 1:2.5.

In the present invention, the percentage by weight of the iridoid glycosides active ingredient in the extract of Splenicia saponica is preferably 40-80%, such as 40-50%, 50-80% or 50-70%, or For example 44% or 66%.

In the present invention, the percentage by weight of the verbascoside in the extract of Piedea chinensis can be 30-50%, such as 32%, 38%. The calculation of the weight percentage does not include the solvent contained in the extract of the bittern.

In the present invention, the weight percentage of the catalpa glycoside in the Spirulina chinensis extract may be 5-15%, such as 8% or 9%. The calculation of the weight percentage does not include the solvent contained in the extract of the bittern.

In the present invention, the weight percentage of the berberidin II in the extract of Piedea chinensis can be 1-40%, such as 3% or 20%.

Preferably, when the weight percent of the iridoid glycosides active ingredient in the extract of Splenicia chinensis is 50-80% (for example, 66%), the content of verbascoside in the extract of Splenicia chinensis The weight percentage can be 30-50% (for example 38%), the weight percentage of catalpaside in the described Shui Kuipian extract can be 5-15% (such as 8%), and the content of Pibeticoside II in the The weight percentage in the extract may be 10-30% (eg 20%).

Preferably, when the weight percent of the iridoid glycosides active ingredient in the extract of Splenicia chinensis is 40-80% (for example, 44%), the content of verbascoside in the extract of Splenicia chinensis The weight percentage can be 30-50% (for example 32%), the weight percentage of catalpaside in the described Shui Kuiping extract can be 5-15% (such as 9%), and the content of Pirutin II in the The weight percentage in the extract can be 1-20% (eg 3%).

The present invention also provides a preparation method of the extract of Scutellaria chinensis, which comprises the following steps: eluting the crude extract of Scutellaria chinensis by column chromatography; wherein:

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

The mobile phase of described elution is water and acetonitrile.

In the present invention, the extract of Aquatica can be the extract of the whole plant of Veronica anagallis-aquatica L. and Veronica undulata Wall., which are conventional plants in the field of Scrophulariaceae, preferably the aerial part of extracts.

In the present invention, the crude extract of the scutellaria can be obtained by the following method: mix the plant medicinal material succulents and the extraction solvent, extract by heating, and filter to obtain the filtrate; the extraction solvent can be the volume fraction 0-95% ethanol.

The plant medicinal material Shuikuan can be subjected to pulverization and pretreatment and then extracted by heating. The crushing can be crushed to 30 mesh.

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

The ratio of the mass g of the botanical medicinal material Scutellaria chinensis to the volume ml of the extraction solvent may be a conventional ratio in the art, for example 1:(5-15), and for example 1:10.

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

The heating extraction method may be heating reflux extraction.

The number of times of the heat extraction can be one time or two times or more.

The heating extraction time may be 1-3 hours, such as 2 hours. When the number of times of the heating extraction is two times or more, the single time of the heating extraction may be 1-3 hours, for example, 2 hours.

The mesh number of the filtering screen can be 200 mesh.

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 may be vacuum drying.

In the present invention, the crude extract of Aquapicus can be dissolved in water before being eluted by column chromatography. After the crude extract of Scutellaria chinensis is dissolved, insoluble matter can be removed by filtration.

In the present invention, the particle size of the adsorbent may be 5-15 μm, such as 10 μm.

In the present invention, the column temperature of the column chromatography may be 20-30°C, such as 25°C.

Wherein, the column in the column chromatography can be a DAC50 column.

In the present invention, the elution may be gradient elution.

Wherein, preferably, when the elution is a gradient elution, the mobile phase A is water, and the mobile phase B is acetonitrile; the total volume of the mobile phase A and B is 100%, and the gradient elution The procedure is as follows:

At 0-10min, the volume of the mobile phase A is 95%;

In 10-20min, the volume of the mobile phase A is decreased from 95% to 90%;

In 20-60min, the volume of the mobile phase A was decreased from 90% to 50%.

When the above-mentioned gradient elution procedure is used, the eluate can be collected for 24-40 minutes.

In the present invention, the flow rate of the mobile phase may be 50-90 mL/min, such as 70 mL/min.

In the present invention, the eluate obtained after the elution can be post-treated according to conventional means in the art, for example, the acetonitrile is removed by concentration under reduced pressure, and then the effective fraction is obtained by freeze-drying.

The present invention also provides an extract of Scutellaria chinensis, which is prepared by the above-mentioned method.

The present invention also provides an application of the extract of the quinces chinensis in the preparation of medicaments for treating and/or preventing heart failure.

The present invention also provides a method for treating and/or preventing heart failure, the method comprising administering a therapeutically effective amount of the extract of Shui Kuiping to a subject.

The term "heart failure" refers to a syndrome dominated by circulatory dysfunction due to myocardial diastolic and/or systolic dysfunction in the presence of adequate venous return, resulting in insufficient cardiac output to maintain tissue metabolic needs. For pulmonary congestion, vena cava congestion.

The term "therapeutically effective amount" refers to the amount required to alleviate the symptoms of heart failure in a subject. Individual adjustments in dosage will be required in each particular case. The dosage can vary widely, depending on many factors such as the severity of the condition being treated, the age and general health of the patient, other drugs the patient is being treated with, the route and form of administration, and the physician's preference and experience.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

The effective fraction of iridoid glycosides, which is mainly composed of three active components, was prepared from the extract (such as 75% ethanol extract) of the single Chinese medicinal material Shui Kuipong by preparing the liquid phase, and it was verified at the cell level in vitro The protective effect on cardiomyocyte oxygen-glucose deprivation damage was verified in the isoproterenol-induced heart failure mouse model and the left anterior descending coronary artery ligation heart failure mouse model, and the in vivo anti-heart failure drug effect was verified by subacute toxicity Experiments have verified its safety.

Description of drawings

Figure 1 shows the cell survival rates of the model group, the positive control group and the effective site group under the OGD model.

Figure 2 is the effect of the active part on the primary cardiomyocytes of neonatal SD rats; wherein, Figure A is the cell survival rate under the OGD model; Figure B is the beating frequency-time curve.

Figure 3 is a pathological section of the heart of the isoproterenol-induced model mice.

Fig. 4 is the Masson staining (15x) of the heart section of the mouse modeled after myocardial infarction surgery.

Fig. 5 is a view of the appearance of the important organs of the mice after continuous administration of the effective site at a dose of 6 g/kg for two weeks.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Embodiment 1: Preparation of effective fraction

Batch 1: The 75% ethanol extract of Veronica anagallis-aquatica L. was obtained by the following method: take about 3kg of the plant medicinal material Veronica anagallis-aquatica L. (produced in Yunnan Province Yangbi County, Dali Prefecture), crushed to 30 mesh, reflux extracted 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, and combined at 65°C ± 5 °C rotary steaming concentrated to a thick paste, vacuum dried and pulverized to obtain 715g of extract powder.

Take 300 g 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, 300 g, 10 μm). Water is used as mobile phase A, acetonitrile is used as mobile phase B; flow rate is 70mL/min, gradient elution program is shown in Table 1, detection wavelength is 254nm, column temperature is 25°C, and injection volume is 1L (injection volume refers to 1L 75% alcohol extract filtrate). Take the eluate for 24-40min, concentrate under reduced pressure until the acetonitrile is completely removed, and freeze-dry to obtain 42g of solid active fraction.

Table 1: Gradient elution program for active fraction preparation

time (min)	Mobile phase A(%)	Mobile phase B(%)
0	95	5
10	95	5
20	90	10
60	50	50

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. After freeze-drying, 38 g of solid active fractions were obtained.

Example 2: Separation of iridoid glycoside active ingredients

Configure ethyl acetate-n-butanol-water two-phase solvent, and separate the phases. The above phase was pumped into a semi-preparative countercurrent chromatography (TBE-300C, Shanghai Tongtian) mainframe as the stationary phase, and the lower phase was pumped into the mobile phase at a speed of 800 rpm and a flow rate of 3.0 mL/min for system equilibrium. When the balance reaches complete balance, dissolve the solid effective part sample of batch 1 with the lower phase solvent, and start to inject 300 mg each time. The sample is separated in the host, and the separated sample fraction is collected after detection by the detector.

As detected by HPLC, the purity of verbascoside was 95.2%, that of catalpaside was 99.2%, and that of berberine II was 95.5%.

Example 3: Structural identification of three active ingredients

The structures of the components separated in Example 2 were confirmed by nuclear magnetic resonance methods (hydrogen spectrum, carbon spectrum), mass spectrometry (ESI-high resolution) and other spectral methods. The structural characterization data are as follows.

Verbacoside ¹ H NMR (600MHz, DMSO-d ₆ ) δ 7.40(d, J=2.2Hz, 1H), 7.36(dd, J=8.3, 2.1Hz, 1H), 6.83(d, J=8.3Hz, 1H ), 6.42 (dd, J=5.9, 1.8Hz, 1H), 5.10 (d, J=9.5Hz, 1H), 5.04 (dd, J=8.2, 1.3Hz, 1H), 4.95 (dd, J=6.0, 4.4Hz, 1H), 4.62(d, J=7.8Hz, 1H), 3.92(d, J=13.3Hz, 1H), 3.74-3.70(m, 2H), 3.68(d, J=1.3Hz, 1H) , 3.44-3.41(m, 1H), 3.19(t, J=9.0Hz, 1H), 3.16(td, J=7.2, 3.5Hz, 1H), 3.07-3.04(m, 1H), 3.04-3.01(m , 1H), 2.54 (ddt, J=12.1, 6.1, 1.9Hz, 4H), 2.48 (dd, J=9.6, 7.7Hz, 1H); ¹³ C NMR (151MHz, DMSO-d ₆ ) 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.

Catalbin ¹ H NMR (600MHz, DMSO-d ₆ ) δ 7.88-7.83(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 C NMR (151MHz, DMSO-d ₆ ) δ 166.03, HR MS(ESI)m/z calcd for C ₂₂ H ₂₆ NaO ₁₂ ⁺ [M+Na] ⁺ 505.1316, found 505.1319.

Pibroside II ¹ H NMR (600MHz, DMSO-d ₆ ) δ 7.52(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-d ₆ ) δ 166.10, 148.05, 141.56, 124.34, 115.79 , 113.11, 102.30, 98.31, 93.41, 80.13, 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.

Example 4: Detection and analysis of the content of iridoid glycosides in the effective part

Accurately weigh the appropriate amount of verbascoside, catalpaside, and berberine II reference substance, add methanol to dissolve to make a 1mg/mL reference substance solution. Simultaneously, an appropriate amount of effective parts (the solid effective parts obtained after freeze-drying in Example 1 batch one) was accurately weighed and dissolved in methanol to make a 1 mg/mL test solution. Waters Alliance 2695 HPLC and Unitray C18 (150×4.6mm id, 5.0μm,

Huapu Innovation Technology Co., Ltd.), using 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°C, reference sample injection volume 1μL , The injection volume of the test sample is 10 μL, and the analysis results are taken three times to get the average value of the peak area. In the solid effective parts of batch one, the content of verbascoside was 38%, the content of catalpaside was 8%, and the content of berberine II was 20%.

Table 2: Analytical Gradient Elution Program

time (min)	Mobile phase A(%)	Mobile phase B(%)
0	85	15
twenty two	85	15

30

50

50

Table 3: Content determination of the three active ingredients in the active fraction of batch one

compound	Standard peak area	effective peak area	Content (mg/mL)
Verbacoside	956061	3611735	0.38
catalbin	3388043	2730279	0.08
Pirucetin II	1228229	2469599	0.20

Using the same iridoid glycoside content detection and analysis method as the solid effective part in batch one, in the solid effective part of batch two, the content of verbascoside was 32% (0.32mg/mL), and the content of catalpaside It is 9% (0.09mg/mL), and the content of berberine II is 3% (0.03mg/mL).

Example 5: The protective effect of active ingredients and effective fractions on H9c2(2-1) cells under the oxygen-glucose deprivation (OGD) model in vitro

5.1 Experimental method

The cultured rat cardiomyocytes H9c2 (2-1) were digested and counted and inoculated into a 96-well plate at an amount of 8000/well. After the cells grew to a density of 70-80%, the active ingredient mother solution (the active ingredient mother solution was Refers to the mother liquor obtained after the three compounds of actascoside, catalpaside, and berberine II were respectively dissolved) and the batch one solid effective part obtained after freeze-drying in Example 1, and the batch two solid effective part was diluted to the required The concentration was added to the well plate for pretreatment. After 12 hours, the medium of the modeling group was replaced with a PBS solution containing the corresponding concentration of active ingredients and solid effective parts. The types, concentrations, and solid effective parts of each modeling group The type and concentration of the parts were the same as those in the pretreatment (separate active ingredients and effective part dilutions were co-incubated with the cells from 12 hours before modeling to 6 hours after modeling), and the well plate was placed in In the hypoxic chamber, the air was replaced by N ₂ , and the hypoxic chamber was returned to the cell culture incubator to continue culturing for 6 hours. The cells in the control group continued to be cultured normally in the cell incubator. After modeling, the supernatant was discarded, and 100 μL of CCK-8 detection solution was added to each well, incubated in a 37°C incubator in the dark for 2 hours, and the absorbance value (OD value) at a wavelength of 450 nm was detected with a microplate reader.

5.2 Data processing

Cell survival rate (%)=[(OD _{OGD treatment group} -OD _{solvent control} )/(OD _{normal control group} -OD _{solvent control} )]×100%. GraphPad Prism 7 software was used to statistically analyze the experimental results, and the experimental results were expressed as mean ± standard error, and the comparison between groups was performed by Unpaired t test. *: Significant difference relative to the model group.

5.3 Experimental results

See Figure 1, Table 4-1, Table 4-2, and Table 4-3 for the effects of each active ingredient on the survival rate of cardiomyocytes under the OGD model. The results showed that the survival rate of the cells in the model group decreased to 36.4% compared with the normal control group, indicating that the OGD damage was serious and the model was successfully established. The three iridoid glycoside active ingredients can significantly improve the cell survival rate at a certain concentration, indicating that the three isolated compounds do have cardiomyocyte protection.

Table 4-1

drug type	Dosing concentration	Cell viability (%)
No drug administration, model group	/	36.36±0.77
Nicorandil	100μM	41.07±1.27**
Verbacoside	1μM	38.67±0.50
Verbacoside	25μM	43.49±1.77**
Verbacoside	50μM	47.07±1.33****
Verbacoside	100μM	50.03±0.59****
catalbin	1μM	38.03±0.28
catalbin	25μM	43.56±2.25**
catalbin	50μM	44.52±0.71****
catalbin	100μM	45.76±1.08****
Pirucetin II	1μM	43.76±1.89**
Pirucetin II	25μM	45.87±1.44***
Pirucetin II	50μM	49.95±1.30****
Pirucetin II	100μM	56.51±2.16****

Table 4-2

drug type	Dosing concentration	Cell viability (%)
No drug administration, model group	/	54.48±1.61
Nicorandil	100μM	60.57±0.95*
Effective parts (batch 1)	100μg/mL	69.29±2.83**
Effective parts (batch 1)	200μg/mL	73.56±1.86***
Effective parts (batch 1)	400μg/mL	74.66±2.23***
Effective part (batch 2)	100μg/mL	67.41±2.32**
Effective part (batch 2)	200μg/mL	72.24±0.94***
Effective part (batch 2)	400μg/mL	73.45±0.75***

Based on Table 4-1 and 4-2, according to: (cell survival rate of drug administration group-cell survival rate of model group)/(100-cell survival rate of model group), this index shows that compared with monomer group, effective The site group achieved a more excellent cardiomyocyte protection effect.

Table 4-3

Example 6: The protective effect of batch one solid active fraction on H9c2(2-1) cells under the in vitro oxygen-glucose deprivation (OGD) model

6.1 Experimental method

The cultured rat cardiomyocytes H9c2 (2-1) were digested and counted and inoculated into a 12-well plate at an amount of 80,000/well. After the cells grew to a density of 70-80%, the mother liquor of the effective part (in Example 1) The solid effective part of Batch 1 obtained after freeze-drying) was diluted to the required concentration and added to the well plate for pretreatment. After 12 hours, the medium of the modeling group was replaced with the PBS solution containing the corresponding concentration of effective part , and the orifice plate was placed in the hypoxic chamber, and the air was replaced by _N2 , and the hypoxic chamber was returned to the cell culture incubator to continue culturing for 6 hours. The cells in the control group continued to be cultured normally in the cell incubator.

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 the dark for 30 minutes in a cell incubator at 37°C; wash the cells with serum-free culture medium for 1-2 times, photographed using a fluorescence microscope (480nm wavelength excitation).

6.1.2 Detection of lactate dehydrogenase (LDH) content: After modeling, take the supernatant of each well into a 96-well plate, add 50 μL of LDH working solution in the dark, incubate at 37°C for 30 minutes in the dark, and add 50 μL to each well to stop solution, and use a microplate reader to detect the OD value at a wavelength of 490 nm.

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 min each time; fix the cells with pre-cooled methanol for 10 min, and absorb Remove methanol, add 0.5% crystal violet staining solution to cover the cells, and incubate for 10 minutes; recover the crystal violet staining solution, wash the stained cells with water until the dye solution is eluted; leave to dry at room temperature, and photograph under a microscope.

6.2 Data processing

LDH release factor=(OD _{test well} -OD _{corresponding to solvent control} )/(OD _{model group} -OD _{corresponding to solvent control} ); ROS fluorescence intensity and crystal violet staining area were quantified by ImageJ. GraphPad Prism 7 software was used to statistically analyze the experimental results, and the experimental results were expressed as mean ± standard error, and the comparison between groups was performed by Unpaired t test. #: Significant difference relative to the normal control group, *: Significant difference relative to the model group.

6.3 Experimental results

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 produced reactive oxygen species, and the cells in the 200μg/mL and 400μg/mL effective parts treatment groups produced significantly more reactive oxygen species than the model group The reduction, especially in the 400μg/mL effective fraction treatment group, indicated that the effective fraction could play a cytoprotective role by reducing the generation of reactive oxygen species. The actual concentrations of verbascoside, catalpaside and tuberside II in the diluted liquid of the solid effective part of batch 1 are shown in Table 5-2.

Table 5-1

Table 5-2

As shown in Table 6, the release of LDH in the model group after OGD treatment was significantly higher than that in the normal control group, while the release of LDH in cells treated with effective parts of various concentrations was significantly lower than that in the model group, reflecting the protective effect on cardiomyocytes.

Table 6

As shown in Table 7, the staining area of the model group was significantly reduced compared with the control group, indicating that the cell volume was significantly reduced, and the staining area of the group treated with the effective part was increased compared with the model group, and it was concentration-dependent. The effective part was 400 μg The number of cells in the /mL treatment group was the largest, that is, the protective effect on cells was the most significant.

Table 7

Example 7: The effect of the solid effective fraction of batch one on the primary cardiomyocytes of neonatal SD rats

7.1 Experimental method

Take out the heart of a newborn SD rat born 1-3 days ago, wash the blood with phosphate buffered saline; cut the heart into a mimetic tissue piece with ophthalmic scissors, add type II collagenase to digest in a 37°C water bath, repeat three times, and the tissue piece is completely digested After passing through a 200-mesh sieve; the sieved cell solution was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended with a special medium for cardiomyocytes and transferred to a culture dish and incubated in a 37°C incubator for 90 minutes. The solution was re-inoculated in another culture dish, and after adding 0.1 mM 5-BrdU, the culture was continued in a 37°C incubator. Change the fluid every 48 hours.

7.7.1 Detection of cell viability: Digest and count the extracted primary rat cardiomyocytes and inoculate them into 48-well plates at an amount of 16,000/well. After the cells grow for 48 hours, dissolve the solid effective parts of batch one After the mother liquor was diluted into the required concentration and added to the orifice plate for pretreatment, after 6 hours, the culture medium of the modeling group was replaced with the effective part containing the corresponding concentration (obtained after freeze-drying in batch one of Example 1). Solid effective part, in the liquid after the solid effective part is diluted, the actual concentration of verbascoside, catalpaside and tuberside II is shown in Table 5-2) in PBS solution, and the orifice plate is placed in the hypoxic chamber, N ₂ After replacing the air, put the anoxic chamber back into the cell culture incubator and continue culturing for 3 hours. After modeling, discard the supernatant, add 100 μL of CCK-8 detection solution to each well, incubate in a 37°C incubator in the dark for 4 hours, and detect the absorbance value (OD value) at 450nm wavelength with a microplate reader

7.1.2 Statistics of the number of cell beats: The extracted neonatal SD rat cardiomyocytes were evenly inoculated into six 35mm culture dishes, divided into two groups, cultured in a 37°C, 5% CO ₂ cell incubator for 24 hours, and continued to culture for 24 hours , until the cells evenly rejuvenated; the solid effective part group of batch one was added with the sample mother solution to make the final concentration 200 μg/mL, and the normal control group was added with the same amount of DMSO as a control; immediately after adding, observe and collect the cell beating video under the microscope (15s) , collect 5 cells per dish, and collect a total of 15 cells in each group; put the cells back into the incubator immediately after the shooting; follow the same operation as above to collect beating videos at 12h, 24h, 36h and 48h after administration, and count each video The number of cell beats.

7.2 Data processing

Cell survival rate (%)=[(OD _{OGD treatment group} -OD _{solvent control} )/(OD _{normal control group} -OD _{solvent control} )]×100%. GraphPad Prism 7 software was used to statistically analyze the experimental results, and the experimental results were expressed as mean ± standard error, and the comparison between groups was performed by Unpaired t test. #: Significant difference relative to the control group, *: Significant difference relative to the model group.

7.3 Experimental results

As shown in Figure 2A and Table 8, after 3 hours of OGD injury, the survival rate of primary cells in the model group dropped to 49.2%, and the modeling was successful. 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 has been confirmed that the effective part has a good protective effect on the OGD injury of primary cardiomyocytes, and indeed reduces cell death, because primary cardiomyocytes will not proliferate.

Table 8

drug type	Dosing concentration	Cell viability (%)
No drug administration, model group	/	49.15±1.31
Nicorandil	100μM	53.68±0.37*
Effective parts (batch 1)	100μg/mL	62.30±2.06**
Effective parts (batch 1)	200μg/mL	67.26±0.37***
Effective parts (batch 1)	400μg/mL	75.13±2.36***

As shown in Figure 2B and Table 9, as the culture time of primary cardiomyocytes in neonatal SD rats increases, the frequency of spontaneous beating of cardiomyocytes will naturally decrease. The effective site treatment can maintain the beating frequency of primary cardiomyocytes, and the rhythmic beating of cardiomyocytes is the power source of cardiac contraction and relaxation. Therefore, it can be speculated that the effective sites have the potential to maintain cardiac systolic and diastolic functions under adverse conditions.

Table 9

Example 8: Therapeutic Effects of Batch One Solid Active Parts on Isoproterenol (ISO)-Induced Heart Failure Mice 8.1 Experimental Method

The 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. Gastrointestinal administration (the solid effective part of batch one obtained after freeze-drying in embodiment 1 and the 75% ethanol extract in embodiment 1) every morning, matched group and model group give equal amount of drinking water, morning and afternoon subcutaneous injection of isoproterenol hydrochloride once, the dose of 1-2 days is 40mg/kg, the dose of 3-7 days is 20mg/kg, the dose of 8-14 days is 10mg/kg, the same amount is injected in the control group saline. On the 15th day, 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 to take heart slices, and the slices were stained with HE and Masson .

8.2 Data processing

Using Vevo software to take 3 cardiac cycles, measure systolic ventricular septal thickness (IVSs), diastolic ventricular septal thickness (IVSd), left ventricular systolic diameter (LVIDs), left ventricular diastolic diameter (LVIDd), left ventricular systolic Posterior wall thickness (LVPWs), left ventricular diastolic posterior wall thickness (LVPWd), calculated ejection fraction (EF), fractional shortening (FS), left ventricular end systolic volume (LVESV) and left ventricular end diastolic volume (LVEDV ), the area of fibrosis was quantified by imageJ, and the experimental results were statistically analyzed by GraphPad Prism 7 software. The experimental results were expressed as mean ± standard error, and the comparison between groups was performed by Unpaired t test. #: Significant difference relative to the control group, *: Significant difference relative to the model group.

8.3 Experimental results

As shown in the results of Table 10-1 and Table 11, the cardiac function of the mice in the model group was significantly lower than that of the normal mice, specifically manifested as a decrease in the ejection fraction and short-axis shortening rate, left ventricular end-systolic volume and left ventricular end-diastolic volume Increased, increased fibrosis area. Both the high and low dose administration groups of the effective part can significantly improve the cardiac dysfunction caused by isoproterenol hydrochloride, and the mice of the 75% ethanol extract group treated with the same dose as the high dose group of the effective part did not show drug effect, although The previous patent (application number: CN202011417172.0) discloses the new application of Chinese medicine water lettuce for anti-heart failure. The 0.6g/kg dose of 75% ethanol extract of water lettuce (namely Beishui bittern) behaves under the induction model of isoproterenol Significant drug effects have been achieved, but it can be seen that the induction dose and frequency of isoproterenol in this prior patent and the present invention are different. The present invention uses isoproterenol stimulation twice a day, and the total dose of single-day administration is higher. At the same time, considering that the content of active ingredients in the crude extract is affected by the harvest season and processing of medicinal materials, the active ingredients are not yet clear, and it is impossible to detect the content of active ingredients for quality control, resulting in a negative result in 75% of the ethanol extract in this experiment. However, in the present invention, the active ingredient in the effective part is clear, quality control can be carried out, the problem of unstable drug effect is overcome, the drug effect is better, and the drug effect is shown at a lower dose.

Combined with the results of pathological sections, as shown in Figure 3, the HE staining of the mouse heart sections in the model group showed obvious inflammatory infiltration (pointed by the arrow), and Masson staining showed severe cardiac fibrosis, resulting in obvious organic damage , the 0.15g/kg dose group slightly alleviated the inflammatory response and fibrosis, and the 0.6g/kg high-dose group alleviated the inflammatory response and fibrosis very well, showing better drug efficacy. The effect of the 75% ethanol extract group It is equivalent to the 0.15g/kg dosage group of the effective part (batch one).

Table 10-1: Indexes of Cardiac Function

Table 10-2

Table 11

Embodiment 9: The therapeutic effect of a batch of solid effective fractions on myocardial infarction surgery (MI) to construct heart failure mice

9.1 Experimental method

After the 8-10 week old C57 male mice were fed adaptively, the neck and left chest of the mice were depilated with isoflurane-assisted anesthesia, and then the mice were fixed in the supine position, the skin of the neck was cut, and the muscle layer was separated for use. The trachea was exposed, the tracheal tube was inserted through the mouth, and the ventilator was connected. After the spontaneous breathing of the mouse was synchronized with the ventilator, the mouse was adjusted to the right side lying position, and the operation area was disinfected with iodine. Hand incision, cut the skin, pectoralis major, expose the ribs, open the pleura, expand the chest with a thoracotomy, cut the pericardium to fully expose the heart, and use a non-invasive suture at the junction of the conus pulmonary artery and the left atrial appendage 2 to 3 mm below The left anterior descending coronary artery was ligated, and the pale myocardium and weakened pulsation below the ligation site were used as signs of successful modeling. After modeling, the adjacent ribs were sutured tightly and the chest wall was gently squeezed to discharge the gas in the chest cavity. The skin was sutured and disinfected with iodophor. In the sham group, sutures were threaded through the heart but not ligated. The mice in the operation group were randomly divided into 4 groups on the second day after operation (the operation group, the effective site 0.15g/kg group, and the effective site 0.6g/kg group (the solid effective product of batch one obtained after freeze-drying in Example 1) Site), positive drug LCZ696 0.06g/kg group), 9 mice in each group. The drug treatment group was intragastrically administered daily, and the sham operation group and the operation group were intragastrically administered the same volume of drinking water for 14 days. On the 15th day after the operation, 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. Sections were subjected to Masson staining.

9.2 Data processing

Use Vevo software to measure IVSs, IVSd, LVIDs and LVIDd, take the average value and calculate EF%, FS%, LVESV LVEDV, use GraphPad Prism 7 software to statistically analyze the experimental results, express the experimental results as mean ± standard error, and compare between groups The Unpairedt test was used, and the survival curve was tested by Log-rank. #: Significant difference relative to the control group, *: Significant difference relative to the model group.

9.3 Experimental results

As shown in the results in Table 13, the survival rate of the mice in the operation group was only 42.9% without drug treatment, and the heart and body were significantly higher than those in the sham operation group, and the heart function was significantly decreased. The low-dose administration group of the effective part failed to improve the survival rate of mice with myocardial infarction and failed to improve cardiac function, but the 0.6g/kg high-dose group significantly improved the survival rate (100%) of mice with MI surgery, and improved the survival rate of mice. Cardiac function, lower heart-to-body ratio, and the effect is better than positive drugs. Combined with the results of pathological sections, as shown in Figure 4 and Table 12, the 0.6g/kg high-dose group can also reduce cardiac fibrosis. Taken together, the effective parts show stronger efficacy than the chemical drugs used in the first-line clinical practice. The actual content of verbascoside, catalpaside and pubetroside II in the liquid after dilution of the effective part of the solid is shown in Table 10-2.

Table 12

Table 13: Survival rate, heart-to-body ratio and cardiac function indicators

Example 10: Subacute Toxicity Evaluation of Solid Active Parts of Batch Two

10.1 Experimental method

ICR mice were bred to a weight of about 25 g and then randomly divided into two groups, the normal control group (Control) and the effective part administration group (HL0877i-6g/kg), 10 in each group, half male and half male. Give mice gavage every day and give 6g/kg dosage effective part (the solid effective part of the batch two that obtains after freeze-drying in embodiment 1) (under this dose, verbascoside, catalpaside and tuberculoside II are the actual Concentration is successively 1.92g/kg, 0.54g/kg, 0.18g/kg), and the normal control group is fed with equal amount of drinking water. Continuous administration for 14 days. The toxicity and death of the mice were observed every day, and the daily body weight was recorded. On the 14th day of administration, the mice in each group were killed, dissected, and whether the important organs of the mice were lesions were observed with the naked eye. At the same time, the heart, liver, spleen, lung, and kidney were weighed, and the organ/body weight was calculated for the visceral body ratio.

10.2 Data processing

GraphPad Prism 7 software was used to statistically analyze the experimental results, and the experimental results were expressed as mean ± standard deviation, and the comparison between groups was performed by TWO-way ANOVA test.

10.3 Experimental results

Continuous administration was carried out for two weeks with 10 times the effective dose (6g/kg), during which no mice died and no obvious toxic reaction was observed. As shown in Figure 5 and Table 14, the daily body weight of the mice was no different from that of the normal control group. The mice were dissected on the 15th day, and the color, texture, and shape of important organs (heart, liver, spleen, lung, and kidney) were the same as those of the normal control group, and the ratio of organs to body weight was also not significantly different. It can be seen that the safety window of the effective part is more than 10 times, and the safety is good.

Table 14

Note: The number of days for calculating body weight in the above table is based on the day of administration as day 0, that is, day 1 is the first day after administration.

Example 11

Shuikuan extract: its preparation method is the same as the preparation method of the 75% ethanol extract in batch one in Example 1, and finally the solid extract is obtained by freeze-drying.

(2) To verify the protective effect of the above extracts on H9c2(2-1) cells under the oxygen-glucose deprivation (OGD) model in vitro

The experimental method and data processing are the same as in Example 5.

(3) Experimental results

See Table 15 for the effect of the extract of Shui Kuipong on the survival rate of cardiomyocytes under the OGD model. The results showed that the survival rate of the cells in the model group decreased to 33.95% compared with the normal control group, which indicated that the OGD damage was serious and the model was successfully established. At a certain concentration, the extract of Scutellaria can significantly improve the cell survival rate, indicating that the extract of Shuikulong does have a protective effect on cardiomyocytes.

Table 15

drug type	Dosing concentration	Cell viability (%)
No drug administration, model group	/	33.95±1.14
Nicorandil	100μM	41.35±1.14*
Water bitter gourd (batch 1)	10μg/mL	45.02±0.90**
Water bitter gourd (batch 1)	50μg/mL	48.39±1.13***
Water bitter gourd (batch 1)	100μg/mL	57.11±1.65***

Although the specific implementations of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these implementations without departing from the principle and essence of the present invention. Revise. Accordingly, the protection scope of the present invention is defined by the appended claims.

Claims (11)

1. An extract of Scutellaria chinensis, characterized in that, the extract of Scutellaria chinensis contains active ingredients of iridoid glycosides, and the active ingredients of iridoid glycosides include verbascoside, catalpaside and Coptis chinensis Glycoside II; Wherein:

   The weight percentage of the iridoid glycosides active ingredient in the extract of Shuikupin is 40-90%;

   The mass ratio of the verbascoside to the catalbin is (3.0-5.0): 1;

   The mass ratio of the catalpaside and the tuberside II is 1: (0.1-3.0).
2. The extract of Herba chinensis as claimed in claim 1, characterized in that, said extract of Herbia chinensis satisfies one or more of the following conditions:

   1. The extract of Aquatica is the extract of the whole plant of Veronica anagallis-aquatica (Veronica anagallis-aquatica L.) and Veronica undulata Wall., such as the extract of aerial parts;

   ②The extract of Sichoria chinensis is obtained by the following method: eluting the crude extract of Sichoria chinensis by column chromatography; wherein:

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

   The mobile phase of described elution is water and acetonitrile.
3. The extract of Scutellaria chinensis as claimed in claim 1 or 2, characterized in that, the extract of Splenicia chinensis satisfies one or more of the following conditions:

   ① The crude extract of Sichoria chinensis is obtained by the following method: mix the herbal medicinal material Sichoria sativa with an extraction solvent, heat, extract, and filter to obtain a filtrate; the extraction solvent can have a volume fraction of 0-95 % ethanol;

   2. The crude extract of the bitter plant is dissolved in water before being eluted by column chromatography, and the crude extract of the bitter plant is filtered to remove insoluble matter after dissolution;

   ③ The particle size of the adsorbent is 5-15 μm, such as 10 μm;

   ④The column temperature of the column chromatography is 20-30°C, such as 25°C;

   5. the column in the column chromatography is a DAC50 column;

   6. The elution is gradient elution;

   When the elution is a gradient elution, preferably, the mobile phase A is water, and the mobile phase B is acetonitrile; the total volume of the mobile phase A and B is 100%, and the gradient elution procedure is as follows :

   At 0-10min, the volume of the mobile phase A is 95%;

   In 10-20min, the volume of the mobile phase A is decreased from 95% to 90%;

   At 20-60min, the volume of the mobile phase A is decreased from 90% to 50%;

   ⑦The flow rate of the mobile phase is 50-90mL/min, such as 70mL/min; and

   8. The eluent obtained after the elution is first concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a solid effective fraction.
4. As at least one of the claims 1-3, the extract of Herba chinensis, is characterized in that, the preparation method of the crude extract of Herba chinensis satisfies one or more of the following conditions:

   ① The herbal medicinal material Shuikuan is subjected to crushing pretreatment and then extracted by heating; the crushing can be crushed to 30 mesh;

   ②The extraction solvent is ethanol with a volume fraction of 70-80%, such as ethanol with a volume fraction of 75%;

   ③ The ratio of the mass g of the botanical medicinal material Scutellaria chinensis to the volume ml of the extraction solvent is 1: (5-15), for example 1:10;

   ④ The temperature of the heating extraction is 92-98°C, such as 95°C;

   5. The heating and extraction method is heating and reflux extraction;

   ⑥The number of times of heating and extraction is one time or two times or more;

   ⑦The heating extraction time is 1-3h, such as 2h; when the heating extraction times are two times or more, the single heating extraction time can be 1-3h, such as 2h;

   8. The mesh number of the filter is 200 mesh; and

   ⑨The filtrate is concentrated and dried; the concentration can be condensed to a thick paste by rotary evaporation at 65°C±5°C; the drying can be vacuum drying.
5. As the extract of at least one of the claims 1-4, it is characterized in that, the extract of the described Sichorax satisfies one or more of the following conditions:

   ① The mass ratio of verbascoside to catalpaside is (3.5-4.8):1, for example (3.56-4.75):1, and for example 3.56:1 or 4.75:1;

   ②The mass ratio of the catalpaside and the puberisin II is 1:(0.3-2.8), for example 1:(0.33-2.5), for example 1:0.33 or 1:2.5;

   Preferably, the mass ratio of verbascoside, catalpaside and tuberside II is 4.75:1:2.5 or 3.56:1:0.33;

   ③ The weight percentage of the iridoid glycosides active ingredient in the extract of Splenicia chinensis is 40-80%, such as 40-50% or 50-80%, also such as 44% or 66%;

   ④The weight percentage of the verbascoside in the extract of the bittern is 30-50%, such as 32%, 38%;

   ⑤ The weight percentage of the catalpa glycoside in the Shuikuan extract is 5-15%, such as 8% or 9%;

   and ⑥ the percentage by weight of the pipericillin II in the extract of the water balsamina is 1-40%, such as 3% or 20%;

   Preferably, when the weight percent of the iridoid glycosides active ingredient in the Spirulina chinensis extract is 50-80%, the weight percent of verbascoside in the Splenicia chinensis extract can be 30% by weight. -50%, the percentage by weight of catalpa glycoside in the extract of Pelicia chinensis can be 5-15%, and the percentage by weight of berberine II in the extract of Pelicia chinensis can be 1-40%; for example, when When the weight percent of the iridoid glycosides active ingredient in the Spirulina chinensis extract is 66%, the weight percent of verbascoside in the Splenicia chinensis extract can be 38%, and catalpaside in the The percentage by weight in the extract of Kusheria chinensis can be 8%, and the percentage by weight of berberine II in the extract of Kussia chinensis can be 20%; for example, when the iridoid glycosides active ingredient is in When the percentage by weight in the extract of Splenicia chinensis is 44%, the percentage by weight of verbascoside in the extract of Splenicia chinensis can be 32%, and the percentage by weight of catalpaside in the extract of Splenicia chinensis can be is 9%, and the percentage by weight of Pipericillin II in the extract of Piperaceae can be 3%.
6. As at least one of the claims 1-5, the extract of Splenica chinensis, is characterized in that, the mass ratio of said verbascoside and said catalbin is (4.0-5.0): 1, and said catalbin and said catalbin The mass ratio of Pirucetin II is 1: (0.3-3.0);

   Alternatively, the mass ratio of the verbascoside to the catalbin is (3.0-5.0): 1, and the mass ratio of the catalbin to the puberisin II is 1: (0.31-2.5);

   Alternatively, the mass ratio of verbascoside to catalbin is (4.0-5.0):1 or (3.0-4.0):1, such as 4.75:1 or 3.56:1;

   Alternatively, the mass ratio of the catalbinoside and berberine II is 1:(0.31-3.0), such as 1:0.33 or 1:(2.0-3.0), also such as 1:2.5.
7. A kind of preparation method of the extract of bittern, it is characterized in that it comprises the following steps: the crude extract of bittern is eluted by column chromatography; wherein:

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

   The mobile phase of described elution is water and acetonitrile.
8. The preparation method of the extract of Aquacarpa as claimed in claim 7, characterized in that, the plant source of the extract of Aquacarpus is the same as the plant source of the extract of Aquacarpus described in claim 2;

   And/or, the preparation method of the extract of the Shui Kubota extract is the same as the preparation method of the Shui Kuying extract described in any one of claims 2-4.
9. A kind of bittern extract, which is prepared by the preparation method of the extract of bittern as claimed in claim 7 or 8.
10. A use of the extract of Shui Kuiping as described in any one of claims 1-6 and 9 in the preparation of a medicament for treating and/or preventing heart failure.
11. A method for treating and/or preventing heart failure, characterized in that the method comprises administering to a subject a therapeutically effective amount of the extract of Aquacarpus as claimed in any one of claims 1-6 and 9.

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