WO2016127399A1 - Use of chlorogenic acid in preparing drug for treating amyotrophic lateral sclerosis - Google Patents

Abstract

The present invention provides use of chlorogenic acid in preparing a drug for treating amyotrophic lateral sclerosis. The chlorogenic acid is beneficial to alleviating disease symptoms of amyotrophic lateral sclerosis (ALS), beneficial to biologically synthesizing neurotrophic factors and beneficial to repairing and protecting neurons.

Description

Use of chlorogenic acid in the preparation of a medicament for treating amyotrophic lateral sclerosis Technical field

The present invention relates to the use of chlorogenic acid for the preparation of a medicament for the treatment of amyotrophic lateral sclerosis.

Background technique

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), commonly known as "frozen disease", is a serious neurodegenerative disease with cerebral cortex, brainstem, and anterior horn of the spinal cord. Neuronal loss is characterized by muscle weakness, muscle atrophy, fasciculation, and pyramidal tract signs. The disease is a fatal motor neuron degenerative disease with hand muscle weakness and atrophy as the first symptom. The average survival time of the patient is about 4 years.

The pathogenesis of amyotrophic lateral sclerosis (ALS) is very complicated, and its clinical treatment is still at the stage of the right medicine, and there are few drugs for the treatment of the disease. Riluzole is commonly used. ). Previous studies have found that neurotrophic factors have good control, repair and treatment effects on the course of amyotrophic lateral sclerosis (ALS), and the amount of neurotrophic factors synthesized by the body itself is very limited. The effect of neurotrophic factors on the disease is even more drastic. In recent years, studies have found that erythropoietin (EPO) can effectively promote the synthesis of neurotrophic factors in the body. Unlike foreign-introduced neurotrophic factors, it promotes EPO production to up-regulate neurotrophic factors in the body. Gentle and safe.

Chlorogenic acid CGA, also known as caffeic acid, is a phenolic acid consisting of caffeic acid CA and quinic acid QA. Its chemical name is 3-o-caffeoyl quinine. Acid (3-o-caffeoylquinic acid CGA). Chlorogenic acid is a phenylpropanoid synthesized by plants in the process of aerobic respiration through the pentose phosphate pathway intermediate. Chlorogenic acid has been used in many fields such as food, health care products, cosmetics and pharmaceuticals. Because it is widely found in a variety of common vegetables and fruits, it has a variety of biological activities, such as: cardiovascular protection, anti-oxidation, anti-ultraviolet and anti-radiation effects, anti-mutagenic and anti-cancer effects, antibacterial effects, anti- Viral action, lipid-lowering and hypoglycemic effects, immunomodulatory effects, etc. It has a wide range of applications in the fields of pharmaceutical, chemical and food.

Summary of the invention

The technical solution of the present invention is to provide the use of chlorogenic acid for the preparation of a medicament for treating amyotrophic lateral sclerosis (ALS).

The present invention provides the use of chlorogenic acid for the preparation of a medicament for the treatment of amyotrophic lateral sclerosis (ALS).

Among them, the drug is a drug that promotes the production of erythropoietin (EPO). .

Among them, the drug is a drug that promotes the production of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB protein).

Among them, the drug is a drug that increases the number of spinal motor neurons and protects neuronal cells.

Wherein, the medicament is prepared by adding chlorogenic acid as an active ingredient, adding a pharmaceutically acceptable adjuvant or an auxiliary component.

Among them, the preparation is an oral preparation and an injection preparation.

Wherein, the preparation is used in a dose of 1-100 mg/kg.

The test of the present invention shows that chlorogenic acid has a therapeutic effect on amyotrophic lateral sclerosis (ALS). The results showed that chlorogenic acid can effectively improve the behavioral characteristics of mice with amyotrophic lateral sclerosis (ALS) and reduce the onset time. Chlorogenic acid can effectively increase the erythropoietin content in the serum of mice with amyotrophic lateral sclerosis (ALS) model. Chlorogenic acid can effectively promote brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) production in mice with amyotrophic lateral sclerosis (ALS) model, significantly increased The number of spinal motor neurons. In summary, chlorogenic acid is beneficial for improving the symptoms of amyotrophic lateral sclerosis (ALS), beneficial to the biosynthesis of neurotrophic factors, and beneficial for repairing and protecting neuronal cells.

DRAWINGS

Figure 1 Results of neurobehavioral investigation in mice

Figure 2 Number of spinal motor neurons in each group of mice (staining results of spinal motor neuron cells in each group; (b) Cell count analysis results)

Figure 3 overall access map

detailed description

Example 1: Preparation of lyophilized powder injection using chlorogenic acid

1. Extraction of chlorogenic acid:

The chlorogenic acid raw material used in the present embodiment is obtained by extracting and purifying from honeysuckle, and the purity is

99.11%.

2. Preparation of chlorogenic acid freeze-dried powder injection

2.1 Prescription:

The above prescription was completely dissolved in water for injection, filtered, and then finely filtered with a 0.22 μm sterile microfiltration membrane to adjust the pH. After the conventional operation of the sterile powder injection, 1000 ml of 2 ml powder injections were prepared. Contains 30mg of chlorogenic acid.

Example 2: Preparation of pills with chlorogenic acid

1. Extraction of chlorogenic acid

The chlorogenic acid used in the present example was extracted and purified from Eucommia ulmoides leaves, and the purity was 99.26%.

2. Preparation of chlorogenic acid pills

2.1 prescription

2.2. Method of production:

Take appropriate amount of povidone K30, prepare solution with absolute ethanol, then take the prescribed amount of chlorogenic acid and powdered sugar powder and mix them evenly by equal dilution method. Add the dextrin powder in ethanol solution and stir well. To obtain soft materials, 1000 tablets of chlorogenic acid pills were prepared by the method of sputum pill, and each pill contained 1 mg of chlorogenic acid.

Example 3: Preparation of oral solution with chlorogenic acid

1. Extraction of chlorogenic acid

The chlorogenic acid used in the present example was extracted and purified from the burdock leaves, and the purity was 99.34%.

1. Preparation of chlorogenic acid oral solution

2.1 prescription

2.2 Method

Take the prescribed amount of chlorogenic acid and sodium bisulfite, dissolve 100L of water for injection, follow the conventional preparation process of oral liquid, filter, and aseptically fill into 1000 oral liquids, each oral solution is 100mL, Contains 200mg of chlorogenic acid.

Example 4: Preparation of tablets with chlorogenic acid

1. Extraction of chlorogenic acid:

The chlorogenic acid used in the present example was extracted and purified from Eucommia ulmoides leaves, and the purity was 98.47%.

2. Preparation of chlorogenic acid tablets

2.1 Prescription:

2.2 Method of production:

In this embodiment, a chlorogenic acid tablet is prepared by a wet granule tableting method. (1) Prepare an aqueous solution of hypromellose according to the prescription; (2) mix the prescribed amount of chlorogenic acid, powdered sugar and lactose, add the hypromellose aqueous solution, stir well and make it soft. (3) the prepared soft material according to the conventional wet granulation operation procedure, sieving, drying and granulating to obtain granules of uniform size; (4) mixing the prepared granules with magnesium stearate uniformly After compression, a total of 1000 tablets were prepared, each containing 100 mg of chlorogenic acid.

Example 5: Preparation of capsules with chlorogenic acid

1. Extraction of chlorogenic acid:

The chlorogenic acid used in the present example was obtained by extraction and purification from Eucommia ulmoides leaves, and the purity was 99.35%.

2. Preparation of chlorogenic acid capsules:

2.1 Prescription:

2.2 Method of production:

Take the prescribed amount of chlorogenic acid and starch, mix well, add 80% ethanol solution to make soft material, dry, and then prepare 2000 capsules according to the conventional preparation process of capsules, each capsule contains 50mg of chlorogenic acid.

Example 6: Preparation of granules with chlorogenic acid

1. Extraction of chlorogenic acid

The chlorogenic acid used in the present example was extracted and purified from Eucommia ulmoides leaves, and the purity was 98.92%.

2. Preparation of chlorogenic acid granules

2.1 Prescription:

2.2 Method of production:

Povidone K30 was taken and water for injection was added to prepare a solution. After the prescription amount of chlorogenic acid, mannitol and sucrose were uniformly mixed, the povidone K30 solution was added to prepare a soft material. According to the conventional preparation process of the granules, the granules are obtained after sieving, drying and granulating the soft materials. The granules were dispensed under aseptic conditions to prepare 400 bags of granules, each bag containing 500 mg of chlorogenic acid.

Example 7: Preparation of powder with chlorogenic acid

1. Extraction of chlorogenic acid:

The chlorogenic acid raw material used in the present embodiment is obtained by extracting and purifying from the burdock leaves, and the purity is 99.82%.

2. Preparation of chlorogenic acid powder:

2.1 prescription

Chlorogenic acid 1000g with a purity of 99.82%

2.2 Method

After sifting the prescription amount of chlorogenic acid, according to the conventional preparation process of the powder, it is aseptically packaged into 1000 bottles/bag of powder, and each bottle/bag powder contains 1000 mg of chlorogenic acid.

The beneficial effects of the present invention are demonstrated by specific pharmacodynamic tests below.

Test Example 1. Pharmacological investigation of chlorogenic acid for the treatment of amyotrophic lateral sclerosis (ALS) and examination of various biochemical indicators.

Experimental material

1.1 Experimental reagents and instruments

Chlorogenic acid, riluzole, Rotarod tester, hemoglobin gene (EPO) ELISA test kit, brain-derived neurotrophic factor (BDNF) ELISA test kit, BDNF receptor tyrosine receptor kinase B ( TrkB protein) ELISA kit, cresyl violet, EDTA-2Na, high power microscope.

1.2 Animal models and grouping

Thirty hSOD1-G93A transgenic mice were purchased (this mouse is an internationally recognized animal model of ALS pathogenesis and preclinical drug pressure). ALS model mice were randomly divided into three groups, named ALS control group (n=10), positive drug riluzole treatment group (Riluzole group, n=10) and chlorogenic acid treatment group (LYS group n= 10) Wild type BALB/C mice were used as a normal control group (NC group, n=10). The specific grouping and dosage and mode of administration are shown in Table 1:

Table 1 Experimental grouping and dosing schedule

2. Experimental content

2.1 Evaluation of neuromotor function in mice

The motor strength and coordination function of each group of mice were evaluated using the Rotarod test. The Rotarod speed is set to 5, 15r/min. The time that each mouse stayed on the rotating rod was automatically recorded. If the mouse stayed for 7 minutes, the test could be ended and scored 7 minutes. The mice were evaluated for behavioral behavior every day, 3 times at each rotation speed, and the average time was taken for 28 days, and the average turning time of the mice was determined by taking one week as a small unit. The first day when the mouse could not stay on the 15r/min rod for 7 minutes was used as the onset of the onset.

2.2 Determination of serum erythropoietin (EPO) in mice

Erythropoietin (EPO) is a sialic acid-containing acidic glycoprotein that acts on endogenous hormones that promote the proliferation, differentiation, and eventual maturation of erythroid progenitor cells by bone marrow hematopoietic cells. EPO is a multifunctional protective factor with anti-inflammatory and immunomodulatory effects, which promotes the production of nerve growth factor and has neuroprotective effects.

During the 4 weeks of the experiment, each group of mice was bled by eyelid blood collection every weekend (experiment 7th, 14th, 21st, and 28th days), and the collected blood was placed on the blood. The tube was immersed in EDTA-2Na, centrifuged at 5000 r/min for 10 min, and the supernatant serum was taken. The EPO content in the serum was measured using an ELISA kit for mouse erythropoietin.

2.3 Detection of expression of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB protein) in mouse hippocampus

A neuropeptide is a type of polypeptide or protein molecule that promotes the survival, growth, and differentiation of nerve cells. It organizes the death of neurons after injury and promotes the repair of neurons. Is reflecting the brain and ridge An important indicator of myeloid repair.

BDNF is the most representative member of the neurotrophic factor family and is widely distributed in the nervous system, with the hippocampus being the most abundant. BDNF neuroprotection is achieved primarily by binding to Trk. BDNF helps the body maintain normal neuronal physiological functions, and plays an important role in reducing the apoptosis of damaged neurons and promoting neuronal repair. In this embodiment, the hippocampal tissue samples of each group of mice were centrifuged at 12,000 r/min for 10 min, and the supernatant was collected and stored in a refrigerator at -20 ° C for use. The BDNF and TrkB of hippocampus were determined by double antibody boost ELISA. Protein level.

2.4 mouse spinal cord motor neuron cell count

The 2-3 horizontal spinal cords of the mice in each group were taken, and 10 slices were taken and stained with cresyl violet. The anterior horn motor neurons were counted and the average value was taken as the number of motor neurons in each slice of each mouse. .

3. Experimental results

3.1 Evaluation of neuromotor function in mice

The evaluation of neuromotor function in mice was mainly tested by the Rotarod test rod method. The experimental results of the test showed that compared with the control group, the average time of the rotation of the mice in the ALS model group was significantly decreased during the 4 weeks (28 days) of the experimental test. In the Riluzole treatment group and the LYS treatment group, the average turning time also decreased, but the rate of decline was relatively slow, and the daily activities of the mice were more healthy than those of the ALS group. More good. Observations on motor function and behavior in mice revealed no significant differences between the Riluzole and LYS treatment groups. The overall trend of mouse turn time is shown in Figure 1. In addition, the first day when the mice could not stay on the 15r/min rod for 7 minutes was used as the onset of the onset, and the average onset time of each group of mice was counted as shown in Table 2. In conclusion, chlorogenic acid can delay the loss of neuromotor function in mice to a certain extent, and the decreasing trend of the turning time is slower than that in other groups. The number of days in mice in the chlorogenic acid treatment group is higher than that in the ALS control group. There is a certain degree of reduction.

Table 2. Incidence time of each group of mice (days)

3.2 Determination of erythropoietin (EPO) in mouse serum

The results of detection of erythropoietin in mouse serum in the four weeks of the experiment showed: Compared with the normal group of mice, the serum EPO content of the ALS model group increased slightly, but the increase was not significant. The EPO content of the two groups showed no statistical difference in the four weeks (p>0.05). The serum EPO levels of the mice in the Riluzole and LYS treatment groups increased to some extent. Among them, the serum EPO content of the mice in the LYS treatment group increased significantly, and the weekly data values were significantly higher than those of the other three groups. Sexual differences (p <0.05). Compared with the NC group and the ALS group, the serum EPO content of the positive drug Riluzole treatment group was significantly different (P<0.05), and the increase of EPO was lower than that of the LYS treatment group. There was a significant difference (p<0.05), and the specific experimental results are shown in Table 3:

Table 3. Mouse serum EPO content (

) U/L

Compared with NC group *p<0.05 compared with ALS group#p<0.05

△p<0.05 compared with the Riluzole group

3.3 Detection of expression of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB protein) in mouse hippocampus

The detection results of BDNF protein and TrkB protein showed that the expression levels of these two proteins increased slightly in mice of ALS group, suggesting that the body accelerates the production of neurotrophic factors in order to repair neurons. The expression level of TrkB protein was significantly different from that of NC group (p<0.05). The expression of BDNF and TrkB protein in Riluzole and LYS treatment groups increased significantly. Among the mice in the LYS treatment group, the expression of BDNF and TrkB protein increased more than other groups, and the other three groups were smaller. The data of the two protein expression levels of the mice were compared and showed significant differences (p<0.05). The results showed that chlorogenic acid can effectively promote the expression of BDNF and TrkB proteins. The specific experimental data is shown in Table 4:

Table 4. Expression levels of BDNF and TrkB proteins in each group of mice (±SD, pg/mL)

Grouping	BDNF protein	TrkB protein
			NC group	3.42±0.48	0.33±0.08#
ALS group	4.22±0.57	0.48±0.06*
			Riluzole group	5.02±0.73*	0.54±0.07*
LYS group	7.01±0.48*#△	0.68±0.05*#△

Compared with NC group *p<0.05 compared with ALS group#p<0.05

△p<0.05 compared with the Riluzole group

3.4 mouse spinal motor neuron cell count

Counting the spinal motor neurons of each group of mice revealed that the number of motor neuron cells in the LYS and Riluzole-treated mice was significantly higher than that in the ALS group, and the results after statistics showed that green The original acid and Riluzole can increase the number of spinal motor neurons in mice to a greater extent, and there is a significant difference between the two groups (p<0.05). Chlorogenic acid can effectively increase the number of spinal motor neurons and protect spinal cord motor neurons. The results of spinal motor neuron cell staining are shown in Figure 2a, and the statistical results are shown in Figure 2b.

4 statistical processing

Experimental data in the form of continuous variables

It is indicated that the comparison between the two sets of data uses a two-sample t test. Using SPSS13.0 statistical software, P <0.05 was considered statistically significant.

5 Conclusion

In this study, an internationally recognized hSOD-G93A transgenic mouse that studied amyotrophic lateral sclerosis (ALS) was used as a model mouse to investigate the therapeutic effect of chlorogenic acid on ALS. The effects of chlorogenic acid on ALS were systematically studied from the aspects of pharmacodynamics and biochemical indicators. The results showed that chlorogenic acid can effectively improve the behavioral characteristics of mice with amyotrophic lateral sclerosis (ALS) model. Reduce the onset time. Chlorogenic acid can effectively increase the erythropoietin content in the serum of mice with amyotrophic lateral sclerosis (ALS) model and effectively promote brain-derived neurotrophic factor (BDNF) and its receptor tyrosine The production of somatic kinase B (TrkB protein) significantly increased the number of spinal motor neurons (see Figure 3). It provides a theoretical basis for the development of chlorogenic acid as a drug for the treatment of amyotrophic lateral sclerosis (ALS).

Claims (7)

1. Use of chlorogenic acid for the preparation of a medicament for the treatment of amyotrophic lateral sclerosis.
2. The use according to claim 1, characterized in that the drug is a drug which promotes the production of erythropoietin (EPO).
3. The use according to claim 1, characterized in that the drug is a drug which promotes the production of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB protein).
4. The use according to claim 1, characterized in that the drug is a drug which increases the number of spinal motor neurons and protects neuronal cells.
5. The use according to any one of claims 1 to 4, characterized in that the medicament is a preparation prepared by adding chlorogenic acid as an active ingredient and adding a pharmaceutically acceptable adjuvant or auxiliary ingredient.
6. The use according to claim 5, characterized in that the pharmaceutical preparation is an oral preparation or an injection preparation.
7. The use according to claim 6, wherein the preparation contains 1-10 mg of chlorogenic acid per unit preparation; and the clinical use dose of the preparation is 1-100 mg/kg.

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