CN100427115C - Effective part and preparation method of traditional Chinese medicine paeonol for treating cardiovascular disease - Google Patents

Abstract

The invention provides the effective components of traditional Chinese medicine paeonol, the content of the active components paeoniflorin is 15-35%, and the content of paeoniflorin b is 30-50%. The invention provides a preparation method. The paeonol medicinal material is pulverized, then heated and extracted, the extract is concentrated into an extract, dissolved, separated by an ODS-C18 column, and the eluent is concentrated and dried to obtain a sample, which is then used for preparative liquid chromatography. Continue to separate the obtained effective components, add pharmaceutical additives to prepare pharmaceutical preparations according to the preparation method allowed by pharmacy. The method of the invention uses a normal-phase silica gel column to effectively remove impurities such as sugar, protein, amino acid, etc., and increases the content of active ingredients. At the same time, it adopts preparative chromatography to quickly and accurately obtain active ingredients. The effective components of paeonol provided by the invention have a protective effect on myocardial ischemia, and its chemical composition is simple and clear, which is easier to control the quality of medicines in production and can be used in the preparation of medicines for treating and preventing cardiovascular diseases.

Description

Effective part and preparation method of traditional Chinese medicine paeonol for treating cardiovascular disease

technical field

The present invention relates to the method for extracting effective component from Chinese medicine, relate to the method for extracting effective component from Chinese medicine paeonol in particular, and this effective component mainly contains paeonidanin (Paeonidanin), paeonidanin b (Mudanpioside b) , and the present invention also relates to the application of the effective component in the preparation of medicaments for treating cardiovascular diseases.

Background technique

Coronary heart disease and angina pectoris are the "first killers" that endanger human health. In recent years, with the aging of my country's population and changes in people's work, life, diet structure and environment, the incidence of coronary heart disease and other cardiovascular and cerebrovascular diseases has also increased year by year. increase, seriously threatening the physical and mental health of the people. Many active substances in natural products have anti-myocardial ischemia and hypoxia effects, and some of them have been developed into new drugs for the treatment of coronary heart disease and angina pectoris, such as the drug Di'ao Xinxuekang for the treatment of coronary heart disease, which is composed of 8 steroidal saponins extracted from traditional Chinese medicine Made of pure traditional Chinese medicine preparation; Xindakang is a pure natural medicine extracted and processed from seabuckthorn, and its active ingredient is total flavonoids of seabuckthorn. Therefore, finding effective substances with anti-myocardial ischemia and hypoxia physiological activities from natural products is one of the effective ways to discover and develop new drugs. my country's medicinal biological resources are very rich, and its physiologically active substances are a natural treasure house for the research and discovery of new drug lead chemicals and the development of new drugs. At present, my country extracts active substances from natural products and develops new drugs for the treatment of coronary heart disease with good safety and low toxicity. The new drug for coronary heart disease and angina pectoris has important application value and broad development prospect.

Cortex Moutan, also known as Cortex Moutan, is the root bark of the dicotyledonous plant medicine Ranunculaceae plant peony (Paeoniasuffruticosa Andr.). Distributed in Hebei, Henan, Shandong, Sichuan, Shaanxi, Gansu and other places. It is cultivated all over the country. The medicinal materials are mainly produced in Anhui, Sichuan, Gansu, Shaanxi, Hubei, Hunan, Shandong, Guizhou and other places. In addition, Yunnan and Zhejiang are also produced. It is slightly cold in nature, bitter in taste, cool, enters the heart, liver, and kidney channels, and has the effects of clearing heat, cooling blood, harmonizing blood, and eliminating blood stasis. Danpi processing was first seen in the "Jizhu" written by Tao Hongjing of the Liang Dynasty in my country. It is believed that: "All promote breaking, remove the heart." "Compendium of Materia Medica" and other medical books record "Danpi wood heart is not used as medicine, it needs to be removed." Research shows that : Cortex paeonol contains paeonol, paeoniflorin, hydroxypaeoniflorin, benzoylpaeoniflorin, benzoylhydroxypaeoniflorin, paeoniflorigenone, 6-hydroxycoumarin (6-hydroxycoumarin) and gallic acid (GallicAcid), and benzoic acid, phytosterols, sucrose, glucose, arabinose, etc. The volatile oil contains benzoic acid (Benzoic Acid), tetradecane (Tetradecane), 2-hydroxy-4-methoxy-acetophenone (2-hydroxy-4-methoxy acetophenone), 2,6-ditert-butyl p- Benzoquinone (2,6-ditert-butyl-p-Benzoquinone), Diethyl Phthalate (Diethyl Phthalate), Isopropyl Myristate (Isopropyl Myristate) and other compounds (Chen Yuejiao et al., Guangzhou Food Industry Science and Technology, 2002 , 18(4):36-37). Wu Shaohua et al. isolated the following five compounds from paeonol for the first time: Betulinic Acid, Betulin, Oleanolic Acid, 3β, 23-dihydroxy-30-norolean- 12, 20(29)-dien-28-oic acid, 3-O-methylpaeonisuffral (Wu Shaohua et al., Chinese Herbal Medicine, 2002, 33(8): 679-680).

Cortex paeonol has the effect of reducing the degree of injury to experimental myocardial ischemia, and can reduce myocardial oxygen consumption and increase coronary flow. It is believed that cortex paeonol can regulate blood circulation and dredge blood vessels, so it has a protective effect on myocardial ischemia (Ma Yuling et al., Shanxi Medical Journal, 1984, 13(4): 212-214).

Contents of the invention

The object of the present invention is to provide a kind of Chinese medicine paeonol effective component, and this effective component has paeonidanin (Paeonidanin) and mudanpioside b (Mudanpioside b).

In the effective components of the traditional Chinese medicine paeonol obtained by the method of the invention, the content of Paeonidanin (Paeonidanin) is 15-35%, and the content of Mudanpioside b (Mudanpioside b) is 30-50%.

Among the preferred effective components of traditional Chinese medicine paeonol, the content of Paeonidanin (Paeonidanin) is 18-33%, and the content of Mudanpioside b (Mudanpioside b) is 32-48%.

Among the best effective components of traditional Chinese medicine paeonol, the content of Paeonidanin (Paeonidanin) is 20-30%, and the content of Mudanpioside b (Mudanpioside b) is 35-45%.

Another object of the present invention is to provide the preparation method of the effective component of traditional Chinese medicine paeonol, realized by the following technical solutions:

Add ethyl acetate and ethanol after pulverizing the paeonol medicinal material, heat and extract to obtain extract I, discard it, add ethanol to the dregs, heat and extract to obtain extract II, concentrate the extract II into extract, dissolve the extract with methanol, Use ODS-C18 column to separate it, first use low-concentration methanol as the mobile phase to obtain eluent I, then change to high-concentration methanol as the mobile phase to obtain eluent II, and obtain the sample after concentration and drying; Chromatography continues to separate the obtained samples; preparative chromatographic separation conditions: the chromatographic column is a semi-preparative column, the mobile phase is water and acetonitrile, and gradient elution.

The preferred method for preparing the effective components of traditional Chinese medicine paeonol is realized by the following technical scheme: add ethyl acetate and ethanol (1:0.8-1.2) after pulverizing the paeonol medicinal material, heat and reflux for 0.8-1.2 hours, extract 1-3 times, Combine the filtrates to obtain the extract I, discard it, add 60% to 80% ethanol to the dregs, heat and reflux for 0.8 to 1.2 hours, extract 1 to 3 times, combine the filtrates to obtain the extract II, and concentrate the extract II to obtain an extract , dissolve the extract with 40%-60% methanol, and use ODS-C18 column to separate it. First, use 3%-8% methanol as the mobile phase to obtain eluent I, and then replace it with 50%-70% methanol as the mobile phase. Phase, to obtain eluent II, concentrated and dried to obtain a sample; continue to separate the sample obtained by preparative liquid chromatography; separation conditions for preparative chromatography: the chromatographic column is a semi-preparative column, the mobile phase is water and acetonitrile, gradient elution, flow rate It is 2.8～3.2ml/min, and the column temperature is room temperature.

The best method for preparing the effective components of traditional Chinese medicine paeonol is realized by the following technical scheme: take paeonol medicinal material, grind it, add ethyl acetate and ethanol (1:1), heat and reflux for 1 hour, extract twice, and combine the filtrates To obtain the extract I, add 70% ethanol to the dregs, heat and reflux for 1 hour, extract twice, combine the filtrates to obtain the extract II, concentrate the extract II into an extract, dissolve the extract with 50% methanol, and use ODS-C18 column To separate it, first use 5% methanol as mobile phase to obtain eluent I, then change 60% methanol as mobile phase to obtain eluent II, and obtain sample after concentration and drying; Sample: Separation conditions of preparative chromatography: the chromatographic column is a Hanbang semi-preparative column (Lichrospher C18; 10.0mm×250mm, 5μm), the mobile phase is water (A) and acetonitrile (B), and the gradient elution procedure is as follows: at 0 minutes , mobile phase A is 90% aqueous solution, mobile phase B is 10% acetonitrile solution; in 8 minutes, mobile phase A is 81% aqueous solution, mobile phase B is 19% acetonitrile solution; in 60 minutes, mobile phase A It is 65% aqueous solution, and the mobile phase B is 35% acetonitrile solution; the flow rate is 3ml/min, and the column temperature is room temperature. The sample is dissolved in 100% ethanol, separated by preparative liquid chromatography, and the solution is collected in a period of 7.7 to 10.7 minutes, and the solution is concentrated and dried to obtain effective components.

Another object of the present invention is to provide the effective components of the traditional Chinese medicine Paeoniae Alba and add pharmaceutical additives to prepare pharmaceutical preparations according to the preparation method allowed by pharmacy.

Another object of the present invention is to provide the application of the effective components of the traditional Chinese medicine paeonol and its preparation in the preparation of medicines for treating and preventing cardiovascular diseases.

The present invention is characterized in that: the extraction and separation method uses a normal phase silica gel column, which can effectively remove sugar, protein, amino acid and other impurities, and increase the content of active ingredients, and at the same time, adopts preparative chromatography in the method, and can quickly and accurately obtain active ingredients. The effective components of paeonol provided by the invention have a protective effect on myocardial ischemia, and its chemical composition is simple and clear, and it is easier to clarify its mechanism of action in pharmacological research, and it is easier to control the quality of medicines in production.

Description of drawings

Fig. 1 is the HPLC analysis chart of the effective component of traditional Chinese medicine paeonol of the present invention.

Detailed ways

The essence of the present invention will be further described in detail below in conjunction with the embodiments of the present invention, which are only used to illustrate the present invention and do not limit the present invention.

Example 1 The preparation process of the effective components of traditional Chinese medicine paeonol

After pulverizing 200g paeonol medicinal material, add ethyl acetate and ethanol, heat and extract to obtain extract I, discard it. Add ethanol to the medicinal dregs, heat and extract to obtain extract II, concentrate the extract II into 42g of extract, dissolve the extract with methanol, and use ODS-C18 column to separate it. First, use low-concentration methanol as the mobile phase to obtain elution Solution I, then change high-concentration methanol as the mobile phase to obtain eluent II, and obtain 20.16 g of the sample after concentration and drying; continue to separate the sample obtained by preparative liquid chromatography; separation conditions for preparative chromatography: the chromatographic column is a semi-preparative column, The mobile phase is water and acetonitrile, gradient elution. The sample was dissolved in 100% ethanol, separated by preparative liquid chromatography, and the solution was collected in a period of 7.7-10.7 minutes. The solution was concentrated and dried to obtain 1.32 g of effective components.

Example 2 The preparation process of the effective components of traditional Chinese medicine paeonol

Add ethyl acetate and ethanol (1:0.8-1.2) after pulverizing 500g paeonol medicinal material, heat and reflux for 0.8-1.2 hours, extract 1-3 times, combine the filtrates to obtain extract I, discard it. Add 60%-80% ethanol to the dregs, heat and reflux for 0.8-1.2 hours, extract 1-3 times, combine the filtrates to obtain the extract II, concentrate the extract II into 106g of extract, dissolve with 40%-60% methanol Extract, use ODS-C18 column to separate it, first use 3% ~ 8% methanol as mobile phase to obtain eluent I, then change 50% ~ 70% methanol as mobile phase to obtain eluent II, concentrate After drying, 50.62g of the sample was obtained; the sample obtained by continuing to separate by preparative liquid chromatography; the separation conditions of the preparative chromatography: the chromatographic column is a semi-preparative column, the mobile phase is water and acetonitrile, gradient elution, the flow rate is 2.8 ~ 3.2ml/min , the column temperature is room temperature. The sample was dissolved in 100% ethanol, separated by preparative liquid chromatography, and the solution was collected in a period of 7.7-10.7 minutes. The solution was concentrated and dried to obtain 3.53 g of effective components.

Example 3 The preparation process of the effective components of traditional Chinese medicine paeonol

Take 250 g of paeonol medicinal material, grind it, add ethyl acetate and ethanol (1:1), heat to reflux for 1 hour, extract twice, and combine the filtrates to obtain extract I. Add 70% ethanol to the dregs, heat and reflux for 1 hour, extract twice, combine the filtrates to obtain the extract II, concentrate the extract II into 55g of extract, dissolve the extract with 50% methanol, and use ODS-C18 column to separate it , first use 5% methanol as the mobile phase to obtain eluent I, then change to 60% methanol as the mobile phase to obtain eluent II, and obtain 26.48 g of the sample after concentration and drying. The separation of the resulting sample was continued by preparative liquid chromatography. Separation conditions for preparative chromatography: the chromatographic column is a Hanbang semi-preparative column (Lichrospher C18; 10.0mm×250mm, 5μm), the mobile phase is water (A) and acetonitrile (B), the elution gradient is shown in Table 2, and the flow rate is 3ml/ min, the column temperature is room temperature. The sample was dissolved in 100% ethanol, separated by preparative liquid chromatography, and the solution was collected in a period of 7.7-10.7 minutes. The solution was concentrated and dried to obtain 1.83 g of effective components.

Example 4 The HPLC analysis of the effective components of traditional Chinese medicine paeonol

The present invention provides that the content of Paeonidanin (Paeonidanin) in the effective components of paeonol is 20-30%, and the content of Mudanpioside b (Mudanpioside b) is 35-45%. The content determination and fingerprints are as follows:

(1) Content Determination of Paeonidanin (Paeonidanin) and Mudanpiosideb (Mudanpiosideb) in the above paeonol extract (high performance liquid chromatography)

Chromatographic conditions Chromatographic column Agilent Zorbax SB-C18 column (4.6mm * 150mm, 5 μ m); Adopt gradient elution, mobile phase A phase is 0.2% glacial acetic acid aqueous solution, mobile phase B phase is the acetonitrile solution containing 0.2% glacial acetic acid; Gradient The elution procedure was as follows: at 0 minutes, mobile phase A was 90% 0.2% glacial acetic acid in water, mobile phase B was 10% 0.2% glacial acetic acid in acetonitrile; at 10 minutes, mobile phase A was 70% 0.2% Glacial acetic acid aqueous solution, mobile phase B is 30% 0.2% glacial acetic acid in acetonitrile solution; 30 minutes, mobile phase A is 50% 0.2% glacial acetic acid aqueous solution, mobile phase B is 50% 0.2% glacial acetic acid in acetonitrile solution ; flow rate 0.5mL·min ^-1 ; full detection wavelength; column temperature 30°C; ELSD conditions: drift tube temperature 105°C; nitrogen flow rate 1.8L/min

Preparation of the test solution Weigh about 1.83mg of this product, dissolve it in a 1ml volumetric flask with methanol solution, dilute to the mark, shake well, and obtain.

Determination method Accurately draw 5 μ l of the test solution, inject it into a liquid chromatograph, measure it, and obtain it.

(2) HPLC-ELSD fingerprint of above-mentioned paeonol extract

For the determination method , see (1) Determination of Paeonidanin and Mudanpioside b in the above paeonol extract (high performance liquid chromatography). Record the chromatographic time as 30 minutes.

Analysis Results The HPLC-ELSD analysis spectrum of the effective components of paeonol cortex is shown in Figure 1. Nos. 1 and 2 in Figure 1 are Paeonidanin and Mudanpioside b, respectively. The relative retention times of the two peaks were 3.82 (Paeonidanin) and 4.78 (Mudanpioside b) in turn. The peak area of Paeonidanin accounted for 20% of the total peak area, and the peak area of Mudanpioside b accounted for 40% of the total peak area.

The structural formula of paeonidanin (Paeonidanin, 9-O-methyl-4-one-albiflorin) of the present invention is:

The structural characteristic of Moutanpioside b (Mudanpioside b) of the present invention is:

Embodiment five Chinese medicine paeonol active component preparation

Mix 1 g of the active components of paeonol in Example 3 with 10.5 g of polyethylene glycol-6000, heat and melt, and then transfer the material to drip irrigation, drop the drug solution into liquid paraffin at 6-8°C, and remove the oil. , made 800 dropping pills.

Example 6 Chinese medicine paeonol active ingredient preparation

Take the paeonol active ingredient 1g of Example 3, glucose 5.5g, sodium thiosulfate 0.9g and distilled water 1ml, mix the above components evenly, freeze-dry, and pack 600 tubes to obtain final product.

Example 7 Chinese medicine paeonol active ingredient preparation

Take 1.5g of balsamic oil, add it to 13ml of saturated hydroxypropyl β-cyclodextrin, stir to dissolve, filter, and dry the filter leaves at low temperature, the clathrate powder of baldwood oil and hydroxypropyl β-cyclodextrin. In addition to the clathrate powder of the above-mentioned balm oil and hydroxypropyl β-cyclodextrin, take the paeonol extract 1g, mannitol 5.5g, edetate calcium sodium 0.9g and distilled water 2ml, the above-mentioned composition After mixing evenly, freeze-dry and pack into 500 tubes to obtain the product.

Embodiment 8 The pharmacological experiment of the effective component of traditional Chinese medicine paeonol

Pharmacological Model: Cardiomyocyte Ischemia and Hypoxia Model in Neonatal Rats

The primary cardiomyocytes were cultured. After the SD suckling mice were sacrificed 1-3 days after birth, the heart was taken out, washed with PBS solution, and then reduced to pieces of about 1 mm ³ . Digest with 0.125% trypsin (Sigma, USA) + 0.05% collagenase II (Gibco, USA) at 37 degrees for 3-4 times. After 1.5 hours of separation of fibroblasts by the differential attachment method, the cell suspension was transferred to a 48-well plate (about 4×10 ⁵ cells per well). Cells cultured with DMEM (Gibco, USA) plus 10% fetal bovine serum (Falcon, USA) for 3-6 days were used for drug efficacy screening. Replace with serum-free medium 1 day before screening. For cardiomyocytes cultured for 3 to 6 days, add drug-containing culture medium after washing with PBS. Place in a 95% _N2 and 5% _CO2 incubator hypoxic for 6 hours. Then reoxygenate for 3 h in a _CO incubator. The cell supernatant was taken to determine the LDH content.

Dosing regimen The extracted effective components of paeonol cortex were made into 10 ^-4 g/mL test solution with sugar-free Hanks solution, and a small amount of DMSO could be added to help dissolve the fat-soluble components (the final concentration of DMSO should be controlled below 0.2%). The final concentration of each component in the culture medium was controlled at 10 ^-5 g/mL.

Determination of lactate dehydrogenase content The content of lactate dehydrogenase in the cell culture medium can indicate the degree of cell damage. Higher levels of lactate dehydrogenase leaked into the supernatant indicated significant cell damage. Lactate dehydrogenase assay kit was purchased from Nanjing Jiancheng Bioengineering Institute. The measurement method is: take 30 microliters of cell supernatant, add 50 microliters of substrate buffer and 10 microliters of lactate dehydrogenase coenzyme, shake at 37 degrees for 15 minutes, and then add 50 microliters of 2,4-dinitrophenylhydrazine , 37 degrees shaking for 15 minutes. Parallel blanks. Take 50 microliters of the reaction solution and add 200 microliters of 0.4mol/L sodium hydroxide solution, let it stand for 3 to 4 minutes, and measure the photometric value at 450nm with a microplate reader.

Pharmacodynamic results

All data are expressed as mean ± variance. Statistical analysis was performed by one-way analysis of variance and T test. Compared with the model group, P<0.05 was considered to be significantly effective. The efficacy results are shown in Table 1. According to the test results of lactate dehydrogenase (LDH) in cardiomyocytes, the effective components of paeonol cortex have a very significant effect on reducing the release of LDH.

Table 1

Without further elaboration, it is believed that one skilled in the art can, using the preceding disclosure, utilize it to its fullest extent. Accordingly, the foregoing preferred specific embodiments should be understood as illustrative only and not limiting the scope of the invention in any way.

Claims (5)

1. a Chinese medicine cortex moutan valid target for the treatment of cardiovascular disease has paeonol glycoside and paeonoside b, it is characterized in that: the content of paeonol glycoside is 18～33%, and the content of paeonoside b is 32～48%.

2. the Chinese medicine cortex moutan valid target of treatment cardiovascular disease according to claim 1 is characterized in that: in this cortex moutan valid target, the content of paeonol glycoside is 20～30%, and the content of paeonoside b is 35～45%.

3. according to the preparation method of the Chinese medicine cortex moutan valid target of the arbitrary described treatment cardiovascular disease of claim 1-2, it is characterized in that being achieved through the following technical solutions: with adding ethyl acetate and ethanol after the Cortex Moutan pulverizing medicinal materials is 1: 0.8～1.2, reflux 0.8～1.2 hour, extract 1～3 time, merging filtrate gets extracting solution I, abandon it, in medicinal residues, add 60%～80% ethanol, reflux 0.8～1.2 hour is extracted 1～3 time, filtrate merge extracting solution II, II is condensed into extractum with extracting solution, with 40%～60% dissolve with methanol extractum, adopt the ODS-C18 post that it is separated, at first use 3%～8% methanol as mobile phase, get eluent I, change 50%～70% methanol then as mobile phase, get eluent II, get sample behind the concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography, wherein the separation condition of preparative hplc is the chromatographic column adopting semi-preparative column, mobile phase is water A and acetonitrile B, and when the gradient elution program was 0 minute, mobile phase A was that 90% aqueous solution, Mobile phase B are 10% acetonitrile solution; In the time of 8 minutes, mobile phase A is that 81% aqueous solution, Mobile phase B are 19% acetonitrile solution; In the time of 60 minutes, mobile phase A is that 65% aqueous solution, Mobile phase B are 35% acetonitrile solution; Flow velocity is 2.8～3.2ml/min, and column temperature is a room temperature, and sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 7.7～10.7min, and solution obtains effective site behind concentrate drying.

4. according to the Chinese medicine cortex moutan valid target of the arbitrary described treatment cardiovascular disease of claim 1-2, it is characterized in that: cortex moutan valid target adds medical additive and makes pharmaceutical formulation according to the preparation method that pharmaceutics allows.

5. according to the application in preparation treatment and angiocardiopathy preventing medicine of the Chinese medicine cortex moutan valid target of the arbitrary described treatment cardiovascular disease of claim 1-2.

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