A combined drug of astragaloside IV, chlorogenic acid and scutellarin for treating chronic cerebral ischemia

Abstract

The present invention belongs to the field of chemical medicine, and particularly relates to a combined drug of astragaloside IV, chlorogenic acid and scutellarin for treating chronic cerebral ischemia. The present invention provides the use of the combined use of astragaloside IV, chlorogenic acid and scutellarin in the preparation of a drug for preventing and / or treating chronic cerebral ischemia. In addition, calycosin can also be used as an additional component to be combined with the above three components to prepare a drug for preventing and / or treating chronic cerebral ischemia. The drug of the present invention can have synergistic effects, is used for treating chronic cerebral ischemia, and produces good therapeutic effects, providing a new option for the clinical treatment of chronic cerebral ischemia and having good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine, and particularly relates to a combined drug of astragaloside IV, chlorogenic acid and breviscapine for treating chronic cerebral ischemia. Background Art

[0002] Chronic cerebral ischemia (slow cerebral ischemia) refers to a state of reduced blood supply at the overall level of the brain. It is a common and frequently-occurring disease in the middle-aged and elderly, and is also an important link in the occurrence and development of ischemic stroke. According to statistics, more than half of people over 80 years old have symptoms of chronic cerebral hypoperfusion to varying degrees, and 2 / 3 of the population over 60 years old have a history of chronic cerebral hypoperfusion.

[0003] Under the guidance of the treatment principle of replenishing qi and activating blood circulation in traditional Chinese medicine clinical practice, astragalus injection and breviscapine injection are combined to treat cerebral ischemia diseases, and the effect is good, and the combined use effect is better than that of either preparation used alone or conventional western medicine treatment (the optimal ratio of astragalus injection combined with breviscapine injection for anti-rat cerebral ischemia-reperfusion injury was optimized by the baseline equal ratio increase and decrease design method, "China Pharmacy", the 14th issue in 2019).

[0004] Astragalus injection is a sterilized aqueous solution made mainly of astragalus, and breviscapine is a sterilized aqueous solution made by extracting phenolic acid components. The chemical components contained in these two injections are complex. This makes the material basis of their combined treatment of cerebral ischemia diseases unclear. Therefore, although astragalus injection and breviscapine injection can currently be combined to treat cerebral ischemia, after clarifying the material basis, directly combining the active substances is expected to further improve its curative effect.

[0005] The inducements and causes of slow cerebral ischemia diseases are complex, so more diverse and effective treatment methods are needed clinically. In addition, people still hope to continue to improve the treatment effect of drugs on slow cerebral ischemia diseases. Therefore, it is necessary to continue to develop new drugs for treating slow cerebral ischemia to provide more new choices for clinical treatment. Summary of the Invention

[0006] Aiming at the defects in the prior art, the present invention provides a combined drug of astragaloside IV, chlorogenic acid and breviscapine for treating chronic cerebral ischemia, aiming to provide a new drug composition and combined medication method that can be used to prevent or treat chronic cerebral ischemia, and provide a new choice for clinical treatment.

[0007] Use of the combination of astragaloside IV, chlorogenic acid and breviscapine, or the combination of astragaloside IV, chlorogenic acid, breviscapine and calycosin in the preparation of a drug for preventing and / or treating slow cerebral ischemia.

[0008] Preferably, the dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is 2.5-10:25-100:25-70 in molar ratio;

[0009] Or, the dosage ratio of astragaloside IV, chlorogenic acid, scutellarin and calycosin is 2.5-10:25-100:25-70:3.95-31.6 in molar ratio.

[0010] Preferably, the dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is 10:75:40 in molar ratio;

[0011] Or, the dosage ratio of astragaloside IV, chlorogenic acid, scutellarin and calycosin is 10:75:40:15.8 in molar ratio.

[0012] The present invention also provides a pharmaceutical composition for preventing and / or treating chronic cerebral ischemia, which is made of a raw material composition including astragaloside IV, chlorogenic acid and scutellarin or their pharmaceutically acceptable salts as active ingredients, plus pharmaceutically acceptable excipients or auxiliary components.

[0013] Preferably, the dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is 2.5-10:25-100:25-70 in molar ratio.

[0014] Preferably, the dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is 10:75:40 in molar ratio.

[0015] Preferably, the dosage form of the pharmaceutical composition is a solid preparation or a liquid preparation. The solid preparation is a pill, powder, tablet or granule, and the liquid preparation is an oral liquid.

[0016] Preferably, the dosage form of the pharmaceutical composition is a liquid preparation. The concentration of astragaloside IV is 2.5-10 μmol / L, the concentration of chlorogenic acid is 25-100 μmol / L, and the concentration of scutellarin is 25-70 μmol / L.

[0017] Preferably, the concentration of astragaloside IV is 10 μmol / L, the concentration of chlorogenic acid is 75 μmol / L, and the concentration of scutellarin is 40 μmol / L.

[0018] Preferably, the raw material composition further includes calycosin, and the dosage ratio of astragaloside IV, chlorogenic acid, scutellarin and calycosin is 2.5-10:25-100:25-70:3.95-31.6 in molar ratio.

[0019] In the present invention, the term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or the salt formed is generally chemically or physically compatible with the other components constituting a pharmaceutical dosage form and is physiologically compatible with the receptor.

[0020] The present invention provides a novel combination drug and its pharmaceutical composition. The combination drug of the present invention can effectively improve the survival rate of PC12 cells damaged by cobalt chloride and can improve the bilateral cerebral blood flow in mice with chronic cerebral ischemia in animal experiments. Therefore, the combination drug of the present invention can be used to prepare a drug for treating chronic cerebral ischemia, producing a good therapeutic effect, and providing a new option for the clinical treatment of chronic cerebral ischemia.

[0021] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution, or change can be made.

[0022] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0023] Figure 1 It is the cerebral blood flow map for Experimental Example 3. Detailed Description of the Invention

[0024] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0025] Example 1 Oral Liquid for Treating Chronic Cerebral Ischemia

[0026] This example provides an oral liquid with astragaloside IV, chlorogenic acid, and scutellarin as active ingredients, and their concentrations are: astragaloside IV 10 μmol / L, chlorogenic acid 75 μmol / L, and scutellarin 40 μmol / L.

[0027] Example 2 Oral Liquid for Treating Chronic Cerebral Ischemia

[0028] This example provides an oral liquid with astragaloside IV, chlorogenic acid, and scutellarin as active ingredients, and their concentrations are: astragaloside IV 2.5 μmol / L, chlorogenic acid 25 μmol / L, and scutellarin 25 μmol / L.

[0029] Example 3 Oral Liquid for Treating Chronic Cerebral Ischemia

[0030] This example provides an oral liquid with astragaloside IV, chlorogenic acid, and scutellarin as active ingredients, and their concentrations are: astragaloside IV 5 μmol / L, chlorogenic acid 100 μmol / L, and scutellarin 70 μmol / L.

[0031] Powder for treating chronic cerebral ischemia in Example 4

[0032] This example provides a powder with astragaloside IV, chlorogenic acid, and scutellarin as active ingredients, which includes: astragaloside IV 2.5 g, chlorogenic acid 25 g, and scutellarin 25 g.

[0033] Granules for treating chronic cerebral ischemia in Example 5

[0034] This example provides granules with astragaloside IV, chlorogenic acid, and scutellarin as active ingredients, which includes: astragaloside IV 5 g, chlorogenic acid 100 g, and scutellarin 70 g.

[0035] Oral liquid for treating chronic cerebral ischemia in Example 6

[0036] This example provides an oral liquid with astragaloside IV, chlorogenic acid, scutellarin, and calycosin as active ingredients, and their concentrations are: astragaloside IV 10 μmol / L, chlorogenic acid 75 μmol / L, scutellarin 40 μmol / L, and calycosin 15.8 μmol / L.

[0037] Oral liquid for treating chronic cerebral ischemia in Example 7

[0038] This example provides an oral liquid with astragaloside IV, chlorogenic acid, scutellarin, and calycosin as active ingredients, and their concentrations are: astragaloside IV 2.5 μmol / L, chlorogenic acid 25 μmol / L, scutellarin 25 μmol / L, and calycosin 3.95 μmol / L.

[0039] Oral liquid for treating chronic cerebral ischemia in Example 8

[0040] This example provides an oral liquid with astragaloside IV, chlorogenic acid, scutellarin, and calycosin as active ingredients, and their concentrations are: astragaloside IV 5 μmol / L, chlorogenic acid 100 μmol / L, scutellarin 70 μmol / L, and calycosin 31.6 μmol / L.

[0041] Tablet for treating chronic cerebral ischemia in Example 9

[0042] This example provides a tablet with astragaloside IV, chlorogenic acid, scutellarin, and calycosin as active ingredients, which includes: astragaloside IV 10 mg, chlorogenic acid 75 mg, scutellarin 40 mg, and calycosin 15.8 mg.

[0043] The technical solution of the present invention is further illustrated by experiments below.

[0044] Experimental Example 1 Evaluation of the in vitro efficacy of astragaloside IV, chlorogenic acid and scutellarin combined with other drugs in the treatment of chronic cerebral ischemia

[0045] 1 Materials

[0046] 1.1 Cell lines

[0047] Rat adrenal pheochromocytoma cells PC12. PC12 cells are a commonly used neural cell line. By studying the effect of drugs on PC12 cells damaged by sugar-free culture and cobalt chloride, we can understand the effect of the drug on neural cells damaged by glucose and hypoxia, and thus infer its efficacy in treating chronic cerebral ischemia.

[0048] 1.2 Drugs and reagents

[0049] Astragaloside IV standard, chlorogenic acid standard, and scutellarin standard were purchased from Chongqing Gaoren Biotechnology Co., Ltd. (Batch No. 84687-43-4, 327-97-9, and 27740-01-8), cobalt chloride (Sigma, Batch No. 7791-13-1), DMEM high-glucose medium (Hyclon), DMEM sugar-free medium (BI), fetal bovine serum (Hangzhou Sijiqing Bioengineering Research Institute); dimethyl sulfoxide (BioFroxx), trypsin (HyClone), and MTT (Biofroxx).

[0050] 1.3 Main instruments

[0051] 1300 series Class II biological safety cabinet (Thermo Fisher Scientific (Shanghai) Instrument Co., Ltd.), low-speed centrifuge (Anhui Jiawen Instrument Equipment Co., Ltd.), ordinary inverted microscope (Motic), electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), micro-oscillator (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.), microplate reader (Victor Nivo), flow cytometer (ACEA Bioscience Inc), pipette (Thermo Fisher Scientific Instrument Co., Ltd.), carbon dioxide incubator (Thermo Fisher Scientific Instrument Co., Ltd.).

[0052] 2. Methods and Results

[0053] 2.1 Preparation of the proportion of each component

[0054] Determine the effective concentration ranges of the three active ingredients, astragaloside IV, chlorogenic acid, and scutellarin, on cobalt chloride-induced damaged PC12 cells. The concentration of astragaloside IV in the combination is 2.5 - 10 μmol / L, the concentration of chlorogenic acid is 25 - 100 μmol / L, and the concentration of scutellarin is 25 - 70 μmol / L. Combine astragalus, chlorogenic acid, and scutellarin, and the combination ratios of each experimental group are shown in Table 1.

[0055] Table 1 Combination Table of Astragaloside IV, Chlorogenic Acid, and Scutellarin

[0056]

[0057] 2.2 Effects of Each Combination on the Viability of Cobalt Chloride-Induced Damaged PC12 Cells

[0058] Take PC12 cells in the logarithmic growth phase, digest and centrifuge (800 rpm, 5 min), and dilute them with complete medium to a cell suspension of 1×10 5 cells / mL. Inoculate 100 μL / well into a 96-well plate and culture for 24 h. Divide the cells into: normal group, model group, and 6 combination drug administration groups (drug concentrations are shown in Table 1). The normal control group is given 120 μL of complete medium, and the rest of the groups are first added with 0.4 mmol / L cobalt chloride prepared with sugar-free DMEM medium, 100 μL / well. Then, each drug administration group is added with 20 μL of the drug solution (drug concentrations are shown in Table 1), and the model group is added with 20 μL of sugar-free medium. Each group is set with 6 replicate wells and incubated in an incubator at 37 °C and 5% CO2 for 24 h. Discard the supernatant, add 100 μL of serum-free DMEM high-glucose medium and 20 μL of MTT to each well, incubate in the dark for 4 h, then discard the supernatant, add 150 μL of DMSO to each well, shake for 10 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of each well at 490 nm. The experiment is repeated 3 times to calculate the cell survival rate.

[0059] 2.3 Results of the Effects of Each Combination on Cell Survival Rate

[0060] Perform statistical analysis on the cell survival rate. As shown in Table 2, compared with the model group, the survival rates of each combination have statistical differences (P < 0.01). Among them, combination 1 increases the cell survival rate relatively the most and is the closest to the optimal combination obtained from the fitting equation. Determine it as the optimal combination. The corresponding monomer concentrations are astragaloside IV 10 μmol / L, chlorogenic acid 75 μmol / L, and scutellarin 40 μmol / L.

[0061] Table 2 Effects of Each Combination on Cell Survival Rate (x±s, n = 6)

[0062]

[0063] Note: Compared with the model group, *P<0.05, **P<0.01;

[0064] 2.4 Verification of the effect of combined drug use

[0065] To prove that astragaloside IV, chlorogenic acid, and scutellarin have a synergistic effect. Experimental data on the effect of single-component drug use of astragaloside IV, chlorogenic acid, and scutellarin on cell viability were further provided. Among them, the dose of single-component drug use was the same as the total dose of the three drugs in the above combination 1, that is, the concentration of single-component drug use was 125 μmol / L. Other unspecified experimental steps and conditions were the same as those in section 2.2.

[0066] The results are shown in Table 3. It can be seen that under the condition of the same total drug dose, the effect of combination 1 (optimal combination) in improving cell viability is better than that of any single-component drug experimental group. This indicates that in combination 1, the three drugs have a synergistic effect, thus having a better effect in improving cell viability.

[0067] Table 3 Effects of the same-dose combination and monomer components on cell viability (x±s, n = 6

[0068]

[0069]

[0070] Note: Compared with the model group, *P<0.05, **P<0.01; compared with the optimal combination group, # P<0.05

[0071] Experimental Example 2 In vitro pharmacodynamic evaluation of the combined use of astragaloside IV, chlorogenic acid, scutellarin, and calycosin for the treatment of chronic cerebral ischemia

[0072] This example was experimented in the same way as Example 1, except that the liquid medicine was prepared by adding calycosin on the basis of combination 1 in Example 1. The concentration of calycosin in the liquid medicine of each experimental group and the results of cell viability are shown in Table 4.

[0073] Table 4 Effects of adding calycosin to the optimal combination drug on cell viability ( n = 6)

[0074]

[0075] Note: Compared with the model group, *P<0.05, **P<0.01;

[0076] The results showed that when formononetin at various concentrations was added to the combination, especially at a concentration of 15.8 μmol / L, the improvement in survival rate was more obvious. However, whether the concentration of formononetin was further decreased or increased, it would lead to a significant decrease in cell survival rate. Therefore, when formononetin was used in combination with Combination 1, the dosage of each component had a significant impact on the therapeutic effect of the pharmaceutical composition. The optimal dosage ratio was: formononetin 15.8 μmol / L, astragaloside IV 10 μmol / L, chlorogenic acid 75 μmol / L, and scutellarin 40 μmol / L.

[0077] Experimental Example 3 Pharmacodynamic Evaluation of the Composition of Astragaloside IV, Chlorogenic Acid, and Scutellarin on Chronic Cerebral Ischemia Mice

[0078] 1 Experimental Equipment and Methods

[0079] 1.1 Experimental Animals

[0080] Kunming mice, provided by Chengdu Dashuo Experimental Animal Co., Ltd.

[0081] 1.2 Instruments, Equipment and Reagents

[0082]

[0083] 2 Methods

[0084] 2.1 Establishment of Chronic Cerebral Ischemia Mouse Model

[0085] The right common carotid artery ligation method was used to establish a chronic cerebral ischemia mouse model. The mice were fasted for 12 h before surgery and weighed. The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate, fixed in the supine position, and the neck was shaved and disinfected. A small incision of about 1 - 2 cm was made along the midline of the neck with scissors, the fascia and muscles were separated, and the left common carotid artery was found. Then, the distal and proximal ends were ligated with 0 - 4 silk threads, and the right common carotid artery was cut in the middle. Finally, the neck incision was sutured with surgical thread, disinfected with iodophor, and the sham operation group only separated the common carotid artery without ligation. Penicillin sodium was intramuscularly injected continuously for 3 days after surgery to prevent infection.

[0086] 2.2 Grouping and Administration

[0087] The mice were randomly divided into 5 groups, namely the sham operation group, the model group, the positive group (Naoluotong), the high-dose combination group (astragaloside IV-chlorogenic acid-scutellarin combination), and the low-dose combination group (astragaloside IV-chlorogenic acid-scutellarin combination), with 10 mice in each group. The dosage of the positive drug group was 97.5 mg / kg, the high-dose combination group (astragaloside IV 28 mg / kg, chlorogenic acid 84 mg / kg, scutellarin 56 mg / kg), the low-dose combination group (astragaloside IV 14 mg / kg, chlorogenic acid 42 mg / kg, scutellarin 28 mg / kg), and the sham operation group and the model group were given an equal amount of normal saline. The drugs were dissolved in sodium carboxymethylcellulose and administered continuously by gavage for 30 days. The dosage of the combination group was derived from cell experiments and references: in cell experiments, the concentration ratio (mg / ml) of astragaloside IV:chlorogenic acid:scutellarin = 1:3:2. The rat dosage of astragaloside IV in the references was 20 mg / kg, and the mouse dosage was converted according to 1.4 times that of rats.

[0088] 2.3 Cerebral blood flow measurement

[0089] The cerebral blood flow on both sides was measured after surgery and 30 days after drug administration. Under the anesthesia of the mice, the scalp was cut open along the midline of the mouse brain with scissors, about 2 cm in length. The scalp was separated with forceps to expose the skull, and the skull was kept moist with normal saline and placed on a laser speckle imager to measure the bilateral cerebral blood flow, take pictures and record, and calculate the difference in cerebral blood flow on both sides. Subsequently, the wound was sutured with surgical thread and disinfected.

[0090] Cerebral blood flow difference on both sides = left cerebral blood flow - right cerebral blood flow

[0091] Right blood flow recovery rate = (cerebral blood flow difference on both sides after surgery - cerebral blood flow difference on both sides 30 days after drug administration) / cerebral blood flow difference on both sides after surgery

[0092] 2.4 Statistical analysis

[0093] The experimental results were statistically analyzed using SPSS 19.0 software. The data were expressed as mean ± standard deviation (X-±S), and one-way analysis of variance was used for multiple groups of data. P < 0.05 indicated statistical significance.

[0094] 3 Results

[0095] 3.1 Cerebral blood flow on both sides of chronic cerebral ischemia mice

[0096] As can be seen from Table 5, the bilateral cerebral blood flow of mice was measured immediately after the operation. Compared with the sham operation group, the right cerebral blood flow in the model group was significantly decreased after the operation, and there was a statistically significant difference in bilateral blood flow analysis (P < 0.01). This indicates that the model of chronic cerebral ischemia in mice was successfully established. Compared with the model group, there was no statistically significant difference in the blood flow difference between the two sides in each drug administration group (P > 0.05). Thirty days after the operation (i.e., 30 days of drug administration), compared with the model group, the blood flow difference between the two sides in each drug administration group became smaller, with statistical significance (p < 0.05 or 0.01), and the blood flow recovery rate in each drug administration group was higher than that in the model group (56.93%). From the cerebral blood flow Figure 1 it can be seen that there was no difference in the cerebral blood flow between the left and right sides in the sham operation group; the left cerebral blood flow in the model group was significantly less than that on the right; compared with the model group, the right cerebral blood flow in the high- and low-dose combination groups of mice increased significantly, indicating that the combined drug can significantly promote the recovery of right cerebral blood flow in chronic cerebral ischemia mice.

[0097] Table 5 Bilateral cerebral blood flow difference in mice (x±s, n = 10)

[0098]

[0099] Note: Compared with the model, * P < 0.05, ** P < 0.01

[0100] As can be seen from the above embodiments, the combined drug of the present invention can effectively improve the survival rate of PC12 cells damaged by cobalt chloride and can improve the bilateral cerebral blood flow in chronic cerebral ischemia mice in animal experiments. Therefore, it can be used to prepare drugs for preventing and / or treating chronic cerebral ischemia, and has broad application prospects.

Claims

1. Use of astragaloside IV, chlorogenic acid and breviscapine in combination for preparing a medicament for preventing and / or treating chronic cerebral ischemia, characterized in that: The dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is a molar ratio of 2.5 - 10:25 - 100:25 - 70.

2. The use according to claim 1, characterized in that: The dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is a molar ratio of 10:75:

40.

3. Use of astragaloside IV, chlorogenic acid, scutellarin and calycosin in the preparation of a medicament for preventing and / or treating chronic cerebral ischemia, characterized in that: the dosage ratio of astragaloside IV, chlorogenic acid, scutellarin and calycosin is a molar ratio of 2.5 - 10:25 - 100:25 - 70:3.95 - 31.

6.

4. The use according to claim 3, characterized in that: The dosage ratio of astragaloside IV, chlorogenic acid, scutellarin and calycosin is a molar ratio of 10:75:40:15.

8.

5. A pharmaceutical composition for preventing and / or treating chronic cerebral ischemia, characterized in that: It is prepared from a raw material composition of astragaloside IV, chlorogenic acid and scutellarin or their pharmaceutically acceptable salts as active ingredients, plus pharmaceutically acceptable excipients or auxiliary components, and the dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is a molar ratio of 2.5 - 10:25 - 100:25 - 70.

6. The pharmaceutical composition according to claim 5, characterized in that: The dosage ratio of astragaloside IV, chlorogenic acid and scutellarin is a molar ratio of 10:75:

40.

7. The pharmaceutical composition according to claim 5, wherein: The dosage form of the pharmaceutical composition is a solid preparation or a liquid preparation. The solid preparation is a pill, powder, tablet or granule, and the liquid preparation is an oral liquid.

8. The pharmaceutical composition according to claim 7, wherein: The dosage form of the pharmaceutical composition is a liquid preparation. The concentration of astragaloside IV is 2.5 - 10 μmol / L, the concentration of chlorogenic acid is 25 - 100 μmol / L, and the concentration of scutellarin is 25 - 70 μmol / L.

9. The pharmaceutical composition according to claim 8, characterized in that: The concentration of astragaloside IV is 10 μmol / L, the concentration of chlorogenic acid is 75 μmol / L, and the concentration of scutellarin is 40 μmol / L.

10. A pharmaceutical composition for preventing and / or treating chronic cerebral ischemia, characterized in that: It is prepared from a raw material composition of astragaloside IV, chlorogenic acid, scutellarin and calycosin or their pharmaceutically acceptable salts as active ingredients, plus pharmaceutically acceptable excipients or auxiliary components, and the dosage ratio of astragaloside IV, chlorogenic acid, scutellarin and calycosin is a molar ratio of 2.5 - 10:25 - 100:25 - 70:3.95 - 31.6.

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