Preparation method and application of pedunculoic acid and pedunculoic acid sodium salt

Abstract

The invention discloses a preparation method and application of pedunculoic acid and pedunculoic acid sodium salt. The applicant prepares the pedunculate sodium with good solubility and drug effect. Pharmacodynamic experiment results show that: for myocardial ischemia caused by coronary artery ligation of rats, the long pedunculate sodium can reduce the content of myocardial enzyme in serum, improve electrocardiogram performance caused by myocardial ischemia and relieve cardiac index increase caused by myocardial ischemia; therefore, the pedunculoside sodium has a protective effect on myocardial ischemia caused by coronary artery ligation of rats. The invention has important significance for preventing and treating cardiovascular diseases, such as coronary heart disease, myocardial ischemia and other common diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and more specifically, to a preparation method and application of ilicic acid and sodium ilicicate. Background Art

[0002] Cardiovascular and cerebrovascular diseases pose a great threat to humans. Countries around the world attach great importance to the prevention and treatment of cardiovascular and cerebrovascular diseases. Traditional Chinese medicine treats coronary heart disease with the principles of "tonifying qi and strengthening the heart", "aromatic resuscitation", "promoting blood circulation to remove blood stasis", and "combining supplementation and dredging", and has unique advantages in the treatment of coronary heart disease. However, most of the drugs used are compound preparations, with unclear active ingredients, and quality control is only limited to index components, which restricts the development of traditional Chinese medicine.

[0003] Jiubiying is the bark of Ilex rotunda Thunb. of the Aquifoliaceae family. It was first recorded in "Lingnan Caoyao Lu" and is mainly produced in Guangxi and Guangdong. It is cold in nature and bitter in taste, and has the effects of clearing heat and detoxifying, reducing swelling and relieving pain, etc. It is used for colds, tonsillitis, sore throat, acute gastroenteritis, rheumatic bone pain; externally for traumatic injuries, carbuncles and furuncles, external bleeding, burns and scalds. The 2020 edition of the first volume of the Chinese Pharmacopoeia includes this variety. Its chemical constituents mainly include triterpenoid saponins, flavonoid glycosides, phenols, tannins, etc., and the content of its saponin components is relatively high.

[0004] Pharmacodynamics shows that this drug has obvious pharmacological activities of anti-myocardial ischemia, improving coronary circulation, and reducing the area of myocardial infarction. The effective component ilicoside for treating coronary heart disease has been screened out, but its solubility and drug-forming properties are poor. Summary of the Invention

[0005] In view of this, the present invention provides a preparation method and application of ilicic acid and sodium ilicicate to solve the technical problems encountered in the background art.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A preparation method of ilicic acid, and its synthetic route is as follows:

[0008]

[0009] The preparation method is as follows:

[0010] (1) Weigh NaOH in a beaker, add anhydrous methanol, and stir until NaOH dissolves;

[0011] (2) Place ilicoside in a round-bottomed flask, and add NaOH methanol solution;

[0012] (3) Place the round-bottomed flask in an oil bath and reflux overnight;

[0013] (4) After monitoring the reaction by TLC and ensuring it is complete, cool the reaction solution to room temperature;

[0014] (5) Adjust the pH. The reaction solution gradually becomes turbid and turns into a rice paste. Filter the reaction solution through a Buchner funnel to obtain the solid, and after drying, longipedunculic acid is obtained.

[0015] Preferably, in step (1), the mass - to - volume ratio of NaOH to absolute ethanol is 1 g:(12.5 - 25) mL.

[0016] Preferably, in step (2), the mass - to - volume ratio of ilicicolin H to the NaOH methanol solution is 100 mg:(1 - 2) mL.

[0017] Preferably, in step (3), the reflux temperature is 60 °C.

[0018] Preferably, in step (5), adjusting the pH means adding dilute hydrochloric acid dropwise to the reaction solution to adjust the pH to 1.5 - 2.5.

[0019] Another object of the present invention is to provide a method for preparing sodium longipedunculicate, and the synthetic route is as follows:

[0020]

[0021] The preparation method includes:

[0022] 1) Weigh NaOH and longipedunculic acid and place them in a round - bottom flask;

[0023] 2) Add absolute ethanol as a solvent;

[0024] 3) Place the round - bottom flask in an oil - bath, after reflux reaction, cool the reaction solution to room temperature;

[0025] 5) Add chloroform dropwise to the reaction solution until the solution gradually becomes turbid;

[0026] 6) Place the turbid reaction solution in a refrigerator for cooling crystallization, and filter it, and wash it 2 - 3 times with chloroform.

[0027] Preferably, in step 1), the mass ratio of NaOH to longipedunculic acid is 4:5.

[0028] Preferably, in step 3), the reflux is carried out at 100 °C for 30 min.

[0029] Preferably, in step 6), the time for cooling crystallization is 24 h.

[0030] Another object of the present invention is to provide the application of the sodium longipedunculate prepared by the above - mentioned method for preparing sodium longipedunculate in medicine.

[0031] As can be seen from the above technical solutions, compared with the prior art, the present invention has the following beneficial effects:

[0032] The applicant prepared sodium ilicic acid with good solubility and pharmacodynamic effects through experiments. The results of pharmacodynamic experiments show that sodium ilicic acid can reduce the content of myocardial enzymes in the serum, improve the electrocardiogram manifestations caused by myocardial ischemia, and reduce the increase in cardiac index caused by myocardial ischemia for myocardial ischemia caused by coronary artery ligation in rats. Therefore, sodium ilicic glycoside has a protective effect on myocardial ischemia caused by coronary artery ligation in rats. It is of great significance for the prevention and treatment of cardiovascular diseases such as common diseases like coronary heart disease and myocardial ischemia. Brief Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0034] Figure 1 It is a scatter plot of fluorescence intensity at different concentrations of sodium ilicic acid;

[0035] Figure 2 It is the flight mass spectrometry data of sodium ilicic acid: [M+H]+ found: 512.37;

[0036] Figure 3 It is the 300HZ 1 1H-NMR data of sodium ilicic acid. Detailed Embodiments

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0038] Example 1

[0039] This example provides a preparation method of ilicic acid, and its synthetic route is as follows:

[0040]

[0041] The preparation method is as follows:

[0042] (1) Weigh 1 g of NaOH in a beaker and add 12.5 mL of anhydrous methanol, and stir until the NaOH dissolves;

[0043] (2) Place 100 mg of ilicicolin H in a round-bottom flask and add 1 mL of NaOH methanol solution;

[0044] (3) Place the round-bottom flask in an oil bath and reflux at 60 °C overnight;

[0045] (4) After monitoring the reaction to completion by TLC, cool the reaction solution to room temperature;

[0046] (5) Slowly add dilute hydrochloric acid dropwise to the reaction solution to adjust the pH to 2.5, and the solution gradually becomes turbid and forms a rice paste;

[0047] (6) Filter the reaction solution through a Buchner funnel to obtain the solid, and dry it to obtain ilicic acid.

[0048] Example 2

[0049] This example provides a method for preparing ilicic acid, and its synthetic route is as follows:

[0050]

[0051] The preparation method is as follows:

[0052] (1) Weigh 1 g of NaOH in a beaker and add 25 mL of anhydrous methanol, and stir until the NaOH dissolves;

[0053] (2) Place 100 mg of ilicicolin H in a round-bottom flask and add 2 mL of NaOH methanol solution;

[0054] (3) Place the round-bottom flask in an oil bath and reflux at 60 °C overnight;

[0055] (4) After monitoring the reaction to completion by TLC, cool the reaction solution to room temperature;

[0056] (5) Slowly add dilute hydrochloric acid dropwise to the reaction solution to adjust the pH to 2, and the solution gradually becomes turbid and forms a rice paste;

[0057] (6) Filter the reaction solution through a Buchner funnel to obtain the solid, and dry it to obtain ilicic acid.

[0058] Example 3

[0059] This example provides a method for preparing sodium ilicicate, and its synthetic route is as follows:

[0060]

[0061] The preparation method is:

[0062] (1) Weigh 40 mg of NaOH and 50 mg of ilicic acid and place them in a round-bottom flask;

[0063] (2) Add 5 mL of absolute ethanol as the solvent.

[0064] (3) Place the round-bottom flask in an oil bath and reflux at 100 °C for 30 min.

[0065] (4) After 30 min, cool the reaction solution to room temperature.

[0066] (5) Slowly add chloroform dropwise to the reaction solution, and the solution will gradually become turbid.

[0067] (6) Cool and crystallize in the refrigerator for 24 h, and filter with suction and wash 2 - 3 times with chloroform.

[0068] Effect experiment

[0069] Determination of the solubility of ilicic acid and ilicic acid sodium salt

[0070] Experimental procedure:

[0071] (1) Grind ilicic acid and ilicic acid sodium salt separately through a 75-mesh sieve. Weigh 50 mg of the sieved ilicic acid and ilicic acid sodium salt powders respectively, place them in 2 50-ml volumetric flasks, and add fresh distilled water to the scale.

[0072] (2) At room temperature (23 - 25 °C), shake 60 times every 5 min. After 1 h, filter naturally with quantitative filter paper.

[0073] (3) Gradually dilute the solution. Add 5 mL of the solution to a 50-ml volumetric flask, add distilled water to the scale to obtain a 10-fold diluted solution. Then add 5 mL of the 10-fold diluted solution to a 50-ml volumetric flask, add distilled water to the scale to obtain a 100-fold diluted solution, and so on, to prepare a series of solutions with different concentrations.

[0074] (4) Take another 200 mg of ilicic acid and ilicic acid sodium salt respectively, place them in 2 50-ml beakers, add 10 ml of fresh distilled water, shake 60 times every 5 min at room temperature (23 - 25 °C). After 1 h, filter naturally with quantitative filter paper to obtain the saturated solutions of ilicic acid and ilicic acid sodium salt.

[0075] (5) Preheat the ultraviolet spectrophotometer for 30 min. After rinsing the cuvette with distilled water, adjust the absorbance to 0 to eliminate the influence of background absorbance, measure the absorbance of each solution, and record it.

[0076] (6) Import the data into an Excel spreadsheet, plot a scatter diagram, and obtain the linear regression equation.

[0077] Experimental results:

[0078] 1. Ilex pedunculosa acid is a poorly soluble substance. 1 mg dissolves in 500 ml of water, and its ultraviolet absorbance is similar to that of water, so no subsequent experiments were conducted.

[0079] 2. Using an ultraviolet spectrophotometer, the full ultraviolet wavelength of sodium Ilex pedunculosa was measured (200 nm - 600 nm), and the maximum absorption wavelength of sodium Ilex pedunculosa was found to be 200 nm, and the minimum absorption wavelength was 590 nm.

[0080] 3. At 200 nm, the fluorescence intensities of sodium Ilex pedunculosa at different concentrations were measured. And Table 1 was plotted and Figure 1 ;

[0081] Table 1

[0082] Concentration (mg / ml) Fluorescence intensity 1 1.502 0.1 0.32 0.01 0.146 0.001 0.086 0.0001 0.076 0.00001 0.126 0.000001 0.053

[0083] The linear regression equation was: y = 1.4022x + 0.1073, r = 0.9967. The absorbance of the supersaturated solution of sodium Ilex pedunculosa was 1.748. Substituting it into the formula, the saturation concentration was obtained as 1.17 mg / ml. According to the solubility classification in the Chinese Pharmacopoeia, it belongs to slightly soluble. The molecular weight of sodium Ilex pedunculosa is: 510.7.

[0084] Verify the protective effect of sodium Ilex pedunculosa on myocardial ischemia induced by coronary artery ligation in rats

[0085] 1 Experimental animals

[0086] 88 male SD rats, weighing 180 - 200 g. Purchased from Changchun Yisi Experimental Animal Technology Co., Ltd., license number: SCXK(Ji)2023 - 0002, certificate number: 01021730353814038, 01021730777149469.

[0087] 2 Drugs and reagents

[0088] Sodium Ilex pedunculosa, prepared in Example 2.

[0089] Diao Xin Xue Kang Capsules, produced by Chengdu Diao Pharmaceutical Group Co., Ltd., batch number: 2303052.

[0090] 3 Instrumentation

[0091] CS - 600B fully automatic biochemical analyzer, produced by Changchun Dirui Industry Co., Ltd., China.

[0092] PowerLab / 8SP type multi - channel physiological recorder, produced by ADInstruments company.

[0093] 4 Experimental methods

[0094] 4.1 Model establishment

[0095] The rats were randomly divided into a blank group, a model group, a Dioscoreae Hypoglaucae Radix et Rhizoma group at 60 mg / kg, and three dosage groups of sodium ilicifoliaate at 47.08 mg / kg (14 rats), 23.54 mg / kg, and 11.77 mg / kg (13 rats). Medication was administered continuously for 5 days. After the medication on the 4th day, the rats were anesthetized by inhaling isoflurane. After detecting the electrocardiogram in lead II, an incision was made along the left edge of the sternum on the chest wall skin of the rats. A suture was passed through the incision, and after bluntly separating the pectoralis major muscle, the chest wall was punctured between the 4th and 5th intercostal spaces with a hemostatic forceps. Immediately, the chest wall opening was expanded with a vascular retractor, the heart was extruded, a No. 4 suture was inserted at the lower edge of the left auricle and exited at the pulmonary artery conus, the left anterior descending coronary artery was ligated, and the heart was quickly placed back into the thoracic cavity. The chest wall was closed, the suture of the skin incision was tightened, and the thoracic cavity was compressed to expel air. On the 5th day, the following detections were carried out after the last dose was administered to the surviving animals.

[0096] 4.2 Index detection

[0097] 4.2.1 Effect on the heart rate of animals

[0098] One hour after the last dose, the electrocardiogram was detected after anesthesia, and the heart rate and ST height were statistically analyzed.

[0099] 4.2.2 Myocardial enzyme examination

[0100] After electrocardiogram detection, blood was collected from the abdominal aorta, centrifuged to obtain serum, and the contents of lactate dehydrogenase (LDH), creatine kinase (CK), and creatine kinase isoenzyme (CK-MB) in the serum were detected.

[0101] 4.2.3 Heart index

[0102] The heart was taken and weighed, and the heart index was calculated. The calculation formula is: Heart index = Heart weight ÷ Body weight × 100%

[0103] 5 Statistical method

[0104] The data were compared between groups using the intergroup T-test method.

[0105] 6 Experimental results

[0106] 6.1 Effect on the heart index

[0107] Compared with the blank group, the heart index of the model group increased significantly. Compared with the model group, the high, medium, and low dosage groups of sodium ilicifoliaate could all reduce the heart index. The results are shown in Table 2.

[0108] Table 2 Effect on the heart index

[0109]

[0110] Note: Compared with the model group, *p < 0.05, **p < 0.01, the same applies to the following tables.

[0111] 6.2 Effects on electrocardiogram heart rate and ST segment height

[0112] Compared with the blank group, the heart rate of the model group increased significantly, and the height of the ST segment decreased. Compared with the model group, the heart rate of the high-dose Illicium longipedunculatum sodium group decreased significantly, and the height of the ST segment increased significantly; the heart rate of the medium-dose Illicium longipedunculatum sodium group decreased significantly. The results are shown in Table 3.

[0113] Table 3 Effects on electrocardiogram heart rate and ST segment height

[0114]

[0115]

[0116] 6.3 Effects on serum myocardial enzymes

[0117] Compared with the blank group, the contents of LDH, CK, and CK-MB in the serum of the model group increased significantly. Compared with the model group, the contents of LDH, CK, and CK-MB in the high-dose and medium-dose Illicium longipedunculatum sodium groups decreased significantly; the content of LDH in the low-dose Illicium longipedunculatum sodium group decreased significantly. The results are shown in Table 4.

[0118] Table 4 Effects on serum myocardial enzymes

[0119]

[0120] 7 Result analysis

[0121] Illicium longipedunculatum sodium can reduce the content of myocardial enzymes in the serum, improve the electrocardiogram manifestations caused by myocardial ischemia, and reduce the increase in cardiac index caused by myocardial ischemia for myocardial ischemia induced by coronary artery ligation in rats. Therefore, Illicium longipedunculatum sodium has a protective effect on myocardial ischemia induced by coronary artery ligation in rats.

[0122] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and reference can be made to the description in the method part for relevant parts.

[0123] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing long-pedunculated holly acid, characterized in that: Its synthetic route is as follows: The preparation method is as follows: (1) Weigh NaOH in a beaker and add anhydrous methanol. Stir until the NaOH is dissolved. (2) Place ferric holly glycoside in a round-bottom flask and add NaOH methanol solution; (3) Place the round-bottom flask in an oil bath and reflux, and react overnight; (4) After the reaction is complete as monitored by TLC, the reaction solution is cooled to room temperature; (5) The pH value is adjusted, and the reaction solution gradually becomes turbid and becomes rice paste-like. The reaction solution is filtered through a Buchner funnel to obtain the solid, which is dried to obtain holly acid.

2. The method for preparing long-pedunculated holly acid according to claim 1, characterized in that: The mass volume ratio of NaOH to anhydrous ethanol in step (1) is 1 g: (12.5-25) mL.

3. The method for preparing holly acid according to claim 1, characterized in that: The mass volume ratio of ferric holly glycoside to NaOH methanol solution in step (2) is 100 mg: (1-2) mL.

4. The method for preparing holly acid according to claim 1, characterized in that: The reflux temperature in step (3) is 60°C.

5. The method for preparing holly acid according to claim 1, characterized in that: The pH adjustment in step (5) is to add dilute hydrochloric acid dropwise into the reaction solution to adjust the pH to 1.5-2.

5.

6. A method for preparing sodium salt of holly acid, characterized in that: The synthetic route is as follows: The preparation method comprises: 1) Weigh NaOH and holly acid and place them in a round-bottom flask; 2) adding anhydrous ethanol as a solvent; 3) Place the round-bottom flask in an oil bath, and cool the reaction solution to room temperature after reflux reaction; 5) Add chloroform dropwise into the reaction solution until the solution becomes gradually turbid; 6) Place the turbid reaction solution in a refrigerator to cool and crystallize, and filter and rinse with chloroform 2-3 times.

7. The method for preparing sodium salt of hollycenic acid according to claim 6, characterized in that: The mass ratio of NaOH to holly acid in step 1) is 4:

5.

8. The method for preparing sodium salt of holly acid according to claim 6, characterized in that: The reflux in step 3) is reflux at 100° C. for 30 min.

9. The method for preparing sodium salt of holly acid according to claim 6, characterized in that: The cooling crystallization time in step 6) is 24h.

10. Use of sodium salt of ilexine acid obtained by the method for preparing sodium salt of ilexine acid as claimed in any one of claims 6 to 9 in drugs for treating coronary heart disease and myocardial infarction caused by myocardial ischemia.

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