CN116492350A

CN116492350A - Galanthamine combination composition and application thereof

Info

Publication number: CN116492350A
Application number: CN202310532915.6A
Authority: CN
Inventors: 熊鑫; 张强; 刘亚芳
Original assignee: Jiangxi Province Lushan Botanical Garden Chinese Academy Of Sciences
Current assignee: Jiangxi Province Lushan Botanical Garden Chinese Academy Of Sciences
Priority date: 2023-05-06
Filing date: 2023-05-06
Publication date: 2023-07-28

Abstract

The invention discloses a galanthamine combination composition and application thereof, and belongs to the technical field of natural medicines. The composition provided by the invention comprises EGCG and galanthamine, or polygonin and galanthamine, wherein the mass ratio of EGCG to galanthamine is selected from 80:0.2-100:0.1; the mass ratio of the polygonin to the galanthamine is selected from 150:0.2-200:0.1. The composition has obvious synergistic inhibition effect on acetylcholinesterase, so that the dosage of each component in the single administration can be effectively reduced, the toxic and side effects of the medicine can be reduced, the occurrence of organism drug resistance can be reduced, and the composition has a good medical application prospect in the treatment of Alzheimer's disease.

Description

Galanthamine combination composition and application thereof

Technical Field

The invention belongs to the technical field of natural medicines, and particularly relates to a galanthamine combination composition and application thereof.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease characterized by memory loss and cognitive decline and accompanied by irreversible neuronal loss, and is common to elderly people over 65 years of age. Acetylcholinesterase (AChE) is a key enzyme in biological nerve conduction, and catalyzes the hydrolysis of the neurotransmitter acetylcholine to choline and acetic acid, thereby blocking transmission of nerve signals. Therefore, the increase of AChE activity can lead to the reduction of the level of acetylcholine, which is a key cause of AD, so that the inhibition of AChE activity and the increase of the level of acetylcholine are important methods for treating AD. Currently, the acetylcholinesterase inhibitors (AChEI) approved by the United states food and drug administration for the treatment of AD are primarily Tacrine (Tacrine), donepezil (Donepezil), rivastigmine (Rivastigmine) and Galantamine (Galantamine). The three other medicines are synthetic medicines except galanthamine, and the synthetic medicines have certain treatment effects, but have the problems of low activity, low selectivity, large side effect and the like. Compared with the synthetic medicine, the galanthamine derived from natural plants can selectively inhibit AChE, has strong selectivity and long action time, but has the defects of weak enzyme inhibition and the like. Furthermore, the continuous use of a single drug also produces certain side effects and tolerability, which results in a lower therapeutic effect of galantamine in prolonged use.

Currently, new drugs for alzheimer's disease are developed at a slow rate. It is important to discuss the combination of active molecules without the availability of more and more optimal new drugs. The final objective of the combination of active molecules is to increase the effect, reduce the dosage, reduce the toxic side effects and avoid or delay the development of resistance. However, there are few reports of synergism between active molecules, especially with respect to the combination regimen of galantamine. Therefore, the development of the galanthamine combination scheme has important significance for improving human Alzheimer disease.

Disclosure of Invention

The invention provides a galanthamine composition, which consists of galanthamine and a compound A; wherein, the compound A is selected from EGCG or polygonin; when the compound A is EGCG, the mass ratio of EGCG to galanthamine is selected from 80:0.2-100:0.1; when the compound A is polygonin, the mass ratio of the polygonin to the galanthamine is selected from 150:0.2-200:0.1.

The galanthamine composition has an inhibitory effect on acetylcholinesterase, and based on the galanthamine composition, the invention provides application of the galanthamine composition in preparation of medicines with the acetylcholinesterase inhibitory effect.

Those skilled in the art know that acetylcholinesterase is closely related to Alzheimer's disease, based on which the invention provides application of the galantamine composition in preparation of medicines for treating Alzheimer's disease.

In the galanthamine composition, when the mass ratio of EGCG to galanthamine is 80:0.2-80:0.1, the EGCG and galanthamine have a synergistic effect on the aspect of acetylcholinesterase inhibition; when the mass ratio of the polygonin to the galanthamine is 150:0.2-150:0.1, the polygonin and the galanthamine have a synergistic effect on the aspect of acetylcholinesterase inhibition. Based on the above, in a specific embodiment, the galanthamine composition provided by the invention consists of EGCG and galanthamine according to the mass ratio of 80:0.2-80:0.1; or comprises knotgrass glycoside and galanthamine according to the mass ratio of 150:0.2-150:0.1.

In a specific embodiment, the galanthamine composition disclosed by the invention consists of EGCG and galanthamine according to the mass ratio of 80:0.1; or comprises herba Polygoni Avicularis glycoside and galanthamine at a mass ratio of 150:0.1.

According to the above, there is accordingly provided a medicament having a synergistic inhibitory effect on acetylcholinesterase, which comprises the galantamine composition described above. The medicine contains pharmaceutically acceptable carriers, solvents, diluents, excipients or other mediums, and can be prepared into corresponding dosage forms such as powder, granules, capsules, injections, oral liquid, tablets and the like according to different requirements.

The beneficial effects of the invention are as follows:

the composition can inhibit the activity of acetylcholinesterase, thereby achieving the effect of treating Alzheimer's disease. There is a synergistic effect between the ingredients in the composition. The combined medication of the components can greatly reduce the dosage of the components when the components are independently used, reduce the toxic and side effects of the medicine, and can obtain the synergistic treatment effect. Therefore, when the corresponding effect of treating the Alzheimer disease is achieved, the combined drug can effectively reduce the occurrence of drug resistance of organisms, and has better medical application prospect.

Drawings

FIG. 1 is a graph showing the Fa-CI trend of a combination of EGCG and galantamine (80:0.1) towards acetylcholinesterase;

FIG. 2 is a Fa-CI trend graph of a combination of Polygonum aviculare glycoside and galanthamine (150:0.1) versus acetylcholinesterase.

Detailed Description

EGCG with molecular formula of C ₂₂ H ₁₈ O ₁₁ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 458.38; CAS accession number: 989-51-5, the structural formula is:

herba Polygoni Avicularis glycoside (Avicelarin) with molecular formula of C ₂₀ H ₁₈ O ₁₁ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 434.35; CAS accession number: 572-30-5, structural formula:

galanthamine (Galanthamine) with molecular formula C ₁₇ H ₂₁ NO ₃ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 287.35; CAS accession number: 357-70-0, the structural formula is:

other terms used in the present invention, unless otherwise indicated, generally have meanings commonly understood by those of ordinary skill in the art. The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1

An in vitro inhibition assay of acetylcholinesterase activity was performed as follows:

the test subjects were dissolved in dimethyl sulfoxide (DMSO) to prepare a mother solution of 10 mg/mL. The mother liquor was diluted with PBS (DMSO content less than 5% after dilution) and a concentration gradient was set. From each gradient, 10. Mu.L was taken as the sample to be tested. To the sample to be measured, 40. Mu.L of PBS and 10. Mu.L of acetylcholinesterase (0.25U/mL) were added, after mixing, 20. Mu.L of the reaction substrate thiocholine (1.05 mM) was added, incubated at 37℃for 30 minutes, 30. Mu.L of sodium dodecyl sulfate (4%) was added to each well, and after developing with 100. Mu.L of 5,5-dithiobis (2-nitrobenzoic acid) (1.5 mM) solution, measurement was performed at a wavelength of 405nm in an microplate reader, and OD values were obtained. The inhibition rate of acetylcholinesterase activity under each gradient was calculated from the OD values, inhibition rate= [1- (OD) _{Sample of} -OD _{Sample blank} )/(OD _{Negative control} -OD _{Blank space} )]X 100%. Based on the inhibition ratio of acetylcholinesterase activity at each gradient, the semi-Inhibitory Concentration (IC) of the test subject was calculated ₅₀ Values), the calculation and statistics process uses SPSS 20.0.

In the above operation steps, specifically, each group is represented as a sample group: 10. Mu.L of sample to be tested +40. Mu.L of PBS +10. Mu.L of enzyme; sample blank: 10. Mu.L of sample to be tested+50. Mu.L of PBS; negative control group: 50. Mu.L of PBS+10. Mu.L of enzyme; blank group: 60 μL PBS.

The test subjects included EGCG, polygonin, galanthamine and compositions of EGCG and galanthamine, polygonin and galanthamine in different mass ratios.

The experimental instrument and the reagent are as follows: acetylcholinesterase (Type V-S ec3.1.1.7, C2888), 5-Dithiobis (2-nitrobenzoic acid) [5,5-Dithiobis (2-nitrobenzoic acid), D8130], substrate thioiodinated acetylcholine (Acetylthiocholine iodide, 01480) and galanthamine were purchased from Sigma; EGCG and polygonin (beijing solebone); millipore Simplicity Water purification System (Millipore, france), sodium phosphate buffer (pH 6.8,0.1 mol/L), microplate reader TECAN infinite M200 PRO (Teacan Group Ltd., swizer and).

Specifically, EGCG, polygonin and galanthamine are taken as test objects, the inhibition activity of each test object on acetylcholinesterase is tested by adopting the method, and the test objects are tested by adopting a half inhibition concentration IC ₅₀ The value represents.

The concentration gradient of each test object was set as follows: EGCG:100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL; polygonum aviculare glycoside: 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL; galanthamine: 0.4. Mu.g/mL, 0.2. Mu.g/mL, 0.1. Mu.g/mL, 0.05. Mu.g/mL.

The test results are shown in Table 1.

TABLE 1

Data were derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

Example 2

Using the combination of EGCG and galantamine as test subjects, the test subjects were tested for their inhibitory activity against acetylcholinesterase in the half inhibitory concentration IC by the method of example 1 ₅₀ The value represents.

Each test object is specifically: EGCG and galanthamine (80:0.2), EGCG and galanthamine (80:0.1) and EGCG and galanthamine (100:0.1) are all in mass ratio.

The concentration gradient of each test object was set as follows: EGCG and galanthamine (80:0.2): (80+0.2) μg/mL, (40+0.1) μg/mL, (20+0.05) μg/mL, (10+0.025) μg/mL; EGCG and galanthamine (80:0.1): (80+0.1) μg/mL, (40+0.05) μg/mL, (20+0.025) μg/mL, (10+0.0125) μg/mL; EGCG and galanthamine (100:0.1): (100+0.1) μg/mL, (50+0.05) μg/mL, (25+0.025) μg/mL, (12.5+0.0125) μg/mL.

Concentration gradient setting principle: taking EGCG and galantamine (80:0.1) as examples, the initial concentration of the composition was set to 80.1 μg/mL, i.e. the initial concentration of EGCG in the composition sample solution was 80 μg/mL and the initial concentration of galantamine was 0.1 μg/mL. The composition sample solutions were sequentially diluted 2-fold based on the initial concentration of 80.1. Mu.g/mL, followed by 80.1. Mu.g/mL, 40.05. Mu.g/mL, 20.025. Mu.g/mL, 10.0125. Mu.g/mL. Similarly, in a composition of EGCG and galanthamine (80:0.2), the initial concentration of the composition was set at 80.2 μg/mL, and a concentration gradient was formed by dilution 2-fold in sequence. Other compositions follow this principle as well.

The test results are shown in Table 2.

TABLE 2

As can be seen from tables 1 and 2: the combined use of EGCG and galantamine can obviously reduce the dosage of each component when being independently used under the condition of achieving similar inhibition rate, and improve the inhibition activity of acetylcholinesterase. Especially when the mass ratio of the two is 80:0.1, the effect is more obvious.

Example 3

The test subjects were tested for their inhibitory activity against acetylcholinesterase by the method of example 1 using a combination of polygonin and galanthamine at a half inhibitory concentration IC ₅₀ The value represents.

Each test object is specifically: the mass ratio of the polygonin to the galanthamine is 150:0.2, the polygonin to the galanthamine is 150:0.1 and the galanthamine is 200:0.1.

The concentration gradient of each test object was set as follows: polygonum aviculare glycoside and galanthamine (150:0.2): (150+0.2) μg/mL, (75+0.1) μg/mL, (37.5+0.05) μg/mL, (18.75+0.025) μg/mL; polygonum aviculare glycoside and galanthamine (150:0.1): (150+0.1) μg/mL, (75+0.05) μg/mL, (37.5+0.025) μg/mL, (18.75+0.0125) μg/mL; polygonin and galanthamine (200:0.1): (200+0.1) μg/mL, (100+0.05) μg/mL, (50+0.025) μg/mL, (25+0.0125) μg/mL.

Concentration gradient setting principle: taking polygonin and galanthamine (150:0.1) as examples, the initial concentration of the composition is set to 150.1 mug/mL, namely, the initial concentration of the polygonin in the composition sample solution is 150 mug/mL, and the initial concentration of the galanthamine is 0.1 mug/mL. The composition sample solutions were sequentially diluted 2-fold based on an initial concentration of 150.1. Mu.g/mL, followed by 150.1. Mu.g/mL, 75.05. Mu.g/mL, 37.525. Mu.g/mL, 18.7625. Mu.g/mL. Similarly, in the composition of polygonin and galanthamine (150:0.2), the initial concentration of the composition was set to 150.2. Mu.g/mL, and the concentration gradient was formed by dilution 2 times in sequence. Other compositions follow this principle as well.

The test results are shown in Table 3.

TABLE 3 Table 3

As can be seen from tables 1 and 3: the combined use of the polygonin and the galanthamine can obviously reduce the dosage of each component when being independently used under the condition of achieving similar inhibition rate, and improve the inhibition activity of acetylcholinesterase. Especially when the mass ratio of the two is 150:0.1, the effect is more obvious.

Determination of co-administration coefficient

The combination Coefficient (CI) of the compositions of example 2 and example 3 was measured, CI values were calculated according to the software CompuSyn, and the synergy between the drugs was evaluated.

The co-administration coefficient of the composition depends on the inhibition rate of the composition under different concentration gradients and the inhibition rate of each monomer compound in the composition under different concentration gradients, so that when the co-administration coefficient of the composition is measured, the concentration gradient setting needs to be carried out on each monomer compound in the composition to obtain the corresponding inhibition rate.

Taking EGCG and galantamine (80:0.1) as examples in example 2, the initial concentration of the composition was set to 80.1 μg/mL, the initial concentration of EGCG was 80 μg/mL, and the initial concentration of galantamine was 0.1 μg/mL; thus, EGCG was set to a gradient concentration of 80. Mu.g/mL, followed by 2-fold dilution at an initial concentration of 80. Mu.g/mL, followed by 80. Mu.g/mL, 40. Mu.g/mL, 20. Mu.g/mL, and 10. Mu.g/mL; the gradient concentration of galanthamine is set by 2 times dilution based on the initial concentration of 0.1 mug/mL, 0.05 mug/mL, 0.025 mug/mL and 0.0125 mug/mL.

The inhibition rates of EGCG, galanthamine, EGCG and galanthamine (80:0.1) at different concentration gradients were counted as follows:

the inhibition ratio of EGCG under the concentration gradient is 14.5+/-1.1%, 24.2+/-1.3%, 31.5+/-3.1%, 49.1+/-2.6% (from low concentration to high concentration), the inhibition ratio of galanthamine under the concentration gradient is 12.1+/-1.3%, 23.4+/-1.2%, 39.2+/-2.2%, 51.2+/-2.6% (from low concentration to high concentration), and the inhibition ratio of EGCG and galanthamine (80:0.1) under the concentration gradient is 32.2+/-2.1%, 48.4+/-1.6%, 65.8+/-3.3% and 82.1+/-5.3% (from low concentration to high concentration).

The above data were processed using CompuSyn software to obtain CI values for EGCG and galanthamine (80:0.1) in compositions of EGCG and galanthamine, as shown in Table 4. Thus, the Fa-CI trend of the combination of EGCG and galantamine (80:0.1) to inhibit acetylcholinesterase can be derived, as shown in FIG. 1.

Taking the example of the polygonum aviculare glycoside and galantamine (150:0.1) in the example 3, the initial concentration of the composition is set to be 150.1 mug/mL, the initial concentration of the polygonum aviculare glycoside is 150 mug/mL, and the initial concentration of the galantamine is 0.1 mug/mL; thus, the gradient concentration of the polygonin is set by sequentially carrying out 2 times dilution on the basis of the initial concentration of 150 mug/mL, and the concentration is sequentially 150 mug/mL, 75 mug/mL, 37.5 mug/mL and 18.75 mug/mL; the gradient concentration of galanthamine is set by 2 times dilution based on the initial concentration of 0.1 mug/mL, 0.05 mug/mL, 0.025 mug/mL and 0.0125 mug/mL.

The inhibition rates of polygonin, galanthamine, polygonin and galanthamine (150:0.1) under different concentration gradients were counted as follows:

the inhibition rate of the polygonin under the concentration gradient is 15.3+/-1.1%, 21.5+/-1.2%, 35.2+/-1.8%, 48.5+/-2.2% (from low concentration to high concentration), the inhibition rate of the galanthamine under the concentration gradient is 12.1+/-1.3%, 23.4+/-1.2%, 39.2+/-2.2%, 51.2+/-2.6% (from low concentration to high concentration), and the inhibition rate of the polygonin and the galanthamine (150:0.1) under the concentration gradient is 25.4+/-1.6%, 40.5+/-2.1%, 60.2+/-2.5% and 72.5+/-3.3% (from low concentration to high concentration).

The above data were processed using CompuSyn software to obtain CI values for the compositions of knotweed and galanthamine (150:0.1), as shown in Table 4. Thus, the Fa-CI trend of the composition of polygonin and galanthamine (150:0.1) to inhibit acetylcholinesterase can be obtained, as shown in FIG. 2.

Similarly, other compositions, including EGCG and galanthamine (80:0.2), EGCG and galanthamine (100:0.1), polygonin and galanthamine (150:0.2), and polygonin and galanthamine (200:0.1), concentration gradient settings of the individual monomer compounds, and calculation of CI values, etc., are all based on the above principles.

The measurement results are shown in Table 4:

TABLE 4 Table 4

As can be seen from table 4: (1) When EGCG and galantamine are combined, the combined drug coefficient CI is smaller than 1 when the mass ratio of EGCG to galantamine is 80:0.2, and the combination drug coefficient CI shows synergistic effect, which indicates that the EGCG and galantamine show synergistic effect when the mass ratio of EGCG to galantamine is 80:0.2, and the combined drug index mean (CI _avg ) 0.71. When the mass ratio of the two is 80:0.1, the GI ₅₀ 、GI ₇₅ And GI ₉₀ Are smaller than 0.60, which shows that the two components have strong synergistic effect when the mass ratio is 80:0.1, and the average value of the combined drug administration index (CI) _avg ) 0.45. When the mass ratio of the two is 100:0.1, the combined drug coefficient CI is larger than 1, and antagonism is shown. (2) When the mass ratio of the polygonin to the galanthamine is 150:0.2, the combination drug coefficient CI is smaller than 1, the synergy is shown, and the combination drug index average value (CI _avg ) 0.88. When the mass ratio of the two is 150:0.1, wherein, the GI ₇₅ And GI ₉₀ Are smaller than 0.75, which shows stronger synergistic effect when the mass ratio of the two is 150:0.1, and the average value of the combined drug index (CI) _avg ) 0.71. When the mass ratio of the two is 200:0.1, the combined drug coefficient CI is larger than 1, and antagonism is shown.

Comparative example

In addition to the above examples, the present invention provides other cases where no synergy with galanthamine is formed during the screening of the combination of compositions, in which case compound a involves kaempferol, quercetin, morin and phloretin. These case scenarios are only a part of the workload of the present invention at the time of creation. The following is shown:

first, IC of each monomer compound for acetylcholinesterase was tested according to the method in the above example ₅₀ Values, as shown in table 5 below:

TABLE 5

Then, the monomer compound IC is obtained in proximity to each ₅₀ Values the acetylcholinesterase inhibition rate was measured as shown in table 6:

TABLE 6

Compounds of formula (I)	Mass concentration (μg/mL)	Inhibition rate
			Kaempferol	200	48.1±1.8％
Quercetin	200	52.2±3.4％
			Mulberry pigment	20	54.2±2.8％
Phloretin	50	47.2±3.2％

The above-mentioned monomer compounds were combined with galanthamine, and the acetylcholinesterase inhibition ratios of the compositions were measured, as shown in the following table 7:

TABLE 7

As can be seen from table 7, each of the above monomer compounds, when combined with galanthamine, exhibited a composition inhibition directly lower than that of the individual compounds, and the composition actually exhibited antagonism, without synergy.

In addition, the invention also tests the inhibition rate of the acetylcholinesterase of EGCG and polygonin at the mass concentration ratio of 80:150, and the result shows that the inhibition rate of the composition is 43.3+/-2.1 percent, which is lower than the inhibition rate of the EGCG and the polygonin on the acetylcholinesterase alone (49.1+/-2.6 percent and 48.5+/-2.2 percent respectively), which shows that the antagonism exists between the EGCG and the polygonin.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A galanthamine composition, characterized by consisting of galanthamine and compound a; wherein, the compound A is selected from EGCG or polygonin; when the compound A is EGCG, the mass ratio of EGCG to galanthamine is selected from 80:0.2-100:0.1; when the compound A is polygonin, the mass ratio of the polygonin to the galanthamine is selected from 150:0.2-200:0.1.

2. Galanthamine composition according to claim 1, characterized in that the mass ratio of EGCG and galanthamine is 80:0.1; the mass ratio of the polygonin to the galanthamine is 150:0.1.

3. Use of a galanthamine composition according to claim 1 or 2 for the preparation of a medicament having a synergistic acetylcholinesterase inhibitory effect.

4. Use of a galanthamine composition according to claim 1 or 2 for the preparation of a medicament for the treatment of alzheimer's disease.

5. A medicament having synergistic inhibitory effect on acetylcholinesterase, characterized in that it contains a galantamine composition according to claim 1 or 2.

6. The medicament of claim 5, further comprising a pharmaceutically acceptable carrier, solvent, diluent, excipient, or other medium.

7. The medicament according to claim 5, wherein the dosage form of the medicament is selected from the group consisting of powders, granules, capsules, injections, oral liquids and tablets.