Application of extracts from Nasturtium officinale in the preparation of acetylcholinesterase inhibitors

Abstract

The present invention relates to the technical field of plant extracts, and in particular to the use of a ground nasturtium extract in the preparation of an acetylcholinesterase inhibitor. Studies have shown that the ethanol extract of the ground nasturtium and its flower buds (i.e., the ground nasturtium crude extract), the refined extract (RC1-RC6) and the C1-C6 compounds of the present invention have a strong inhibitory effect on acetylcholinesterase, and have good application prospects in the development of drugs for the prevention and treatment of dementia and Alzheimer's disease.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a nephrite extract in the preparation of acetylcholinesterase inhibitors. Background Art

[0002] As the global population ages, the number of patients with Alzheimer's disease (AD) increases year by year. AD is characterized by cognitive decline, which seriously affects the quality of life of patients and places a heavy burden on their families and society. Demand for other neurodegenerative diseases: In addition to AD, other neurodegenerative diseases such as Parkinson's disease are also related to abnormal function of the acetylcholine system, and there is also a demand for acetylcholinesterase inhibitors.

[0003] Acetylcholinesterase plays a key role in neuromuscular junctions and cholinergic synapses. Its structure is relatively clear, including the catalytic active site CAS region, the active pocket entrance PAS region, etc., which provides a clear target for drug design for neurodegenerative diseases such as AD and Parkinson's disease.

[0004] Although existing drugs such as tacrine can improve cognitive function, they have limited therapeutic effects and large side effects. For example, tacrine is hepatotoxic, which limits its clinical application. The development of new, highly effective and low-toxic acetylcholinesterase inhibitors has become a top priority. Natural products are generally considered to be healthier products that are "greener" and closer to nature. Therefore, developing a natural product with acetylcholinesterase inhibitory activity and screening out compounds with better effects from natural products is of great significance and has good prospects. Summary of the invention

[0005] In view of this, the present invention provides an application of a Herba Lycopodii Extract in preparing an antioxidant-reducing product.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] Acetylcholinesterase (AChE) is a key enzyme involved in neural function and signal transmission in the brain. However, during the aging process, excessive AChE activity often leads to rapid and progressive depletion of acetylcholine (ACh), the main neurotransmitter. A deficiency of ACh leads to reduced neurotransmission and the development of diseases such as dementia and Alzheimer's disease (AD). Therefore, an effective clinical treatment for AD is to use compounds that inhibit AChE activity to increase the level of AChE in the brain and restore normal neural function in the brain.

[0008] Studies have shown that the ethanol extracts of the ground nasturtium and flower buds described in the present invention (including the ground nasturtium crude extract and refined extracts RC1~RC6) have a strong inhibitory effect on acetylcholinesterase, can be used to prepare acetylcholinesterase inhibitors, and have good application prospects in the development of drugs for preventing and treating dementia and Alzheimer's disease.

[0009] Based on the above results, the present invention provides the use of the extract of the Herba Lycoris Radiatae in the preparation of the following products:

[0010] (1) Acetylcholinesterase inhibitors;

[0011] (2) Drugs for the prevention and treatment of neurodegenerative diseases;

[0012] The nasturtium schreberi extract is an ethanol extract of nasturtium schreberi and its flower buds;

[0013] Such neurodegenerative diseases include dementia and Alzheimer's disease.

[0014] In the present invention, the Herba Lycopodii extract comprises at least one active ingredient of the following C1-C6 compounds:

[0015] Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0016] Compound C2: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0017] C3 compound: mumeose K;

[0018] C4 compound: carotene linoleate;

[0019] C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid);

[0020] C6 compound: 2-linolenoyl-rac-glycerol.

[0021] Among them, in C1~C6 compounds, C is the abbreviation of compound, and C1~C6 compounds are compound1~compound6.

[0022] In some embodiments, the extract of the Herba Lycoris Radiatae is an ethanol extract of the flowers and buds of the Herba Lycoris Radiatae, which is named herein as a crude Herba Lycoris Radiatae extract, and the preparation method thereof comprises:

[0023] The flowers and buds of the Herba Lycoris Radiatae were extracted by cold soaking with 75% ethanol, and then concentrated to dryness under reduced pressure to obtain a crude Herba Lycoris Radiatae extract.

[0024] In some specific embodiments, the preparation method of the crude extract of the Herba Lycopodii includes:

[0025] The flowers and buds of the ground jellyfish were taken, and cold-immersed and extracted three times with 3 times the volume of 75% ethanol aqueous solution, each time for 24 hours, and the extract was concentrated to dryness to obtain a crude extract of the ground jellyfish.

[0026] In other embodiments, the extract of the ground jellyfish is ground jellyfish refined extract RC1~RC6, RC is the abbreviation of Refined component, and its preparation method includes: taking the flowers and buds of the ground jellyfish, cold-immersing and extracting with 75% ethanol aqueous solution, and concentrating under reduced pressure to dryness to obtain a ground jellyfish crude extract; applying the obtained ground jellyfish crude extract to an MCI column, and eluting with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution and 100% methanol solution in sequence to obtain ground jellyfish refined extract RC1~RC6 respectively. In a specific embodiment, the preparation method of the ground jellyfish crude extract includes: taking the flowers and buds of the ground jellyfish, cold-immersing and extracting with 3 times the volume of 75% ethanol aqueous solution for 3 times, each time for 24 hours, concentrating the extract to dryness, and obtaining a ground jellyfish crude extract.

[0027] In the present invention, in the elution, each gradient elution has 3 column volumes.

[0028] Specifically, RC1 is the fraction eluted with water after MCI chromatography separation;

[0029] RC2 is the fraction eluted with 20% methanol in water after MCI chromatography separation;

[0030] RC3 is the fraction eluted with 40% methanol in water after MCI chromatography separation;

[0031] RC4 is the fraction eluted with 60% methanol in water after MCI chromatography separation;

[0032] RC5 is the fraction eluted with 80% methanol in water after MCI chromatography separation;

[0033] RC6 is the fraction eluted with 100% methanol after MCI chromatography separation.

[0034] In the present invention, the C1-C6 compounds are separated from the RC2, RC3, RC4, RC5, and RC6, and the specific separation method is as follows:

[0035] The extract RC2 was loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9:1-8:2), petroleum ether-isopropanol (volume ratio 8:2-7:3) and ethyl acetate:methanol 9.8:0.2 to separate C4.

[0036] The extract RC4 was loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9.8:0.2-8:2), dichloromethane-isopropanol (volume ratio 9.8:0.2-9:1), and petroleum ether-isopropanol (volume ratio 8:2-6:4); the extract was loaded onto a Sephdex column and eluted with methanol to separate C3.

[0037] The refined extract RC5 was loaded onto a silica gel column and eluted with dichloromethane-acetone (volume ratio 9:1-8:2), dichloromethane-methanol (volume ratio 9:1), and petroleum ether-acetone (volume ratio 8:2-7:3) to separate C1.

[0038] The refined extract RC6 was loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9.8:0.2-9:1), dichloromethane-acetone, petroleum ether-acetone (volume ratio 9:1-7:3), petroleum ether-ethyl acetate (volume ratio 9:1-8:2), and loaded onto a Sephdex column and eluted with dichloromethane-methanol (volume ratio 1:1). After multiple separations, refined compounds C2, C5, and C6 were obtained.

[0039] Specifically, the name of C1 compound is 1,2',3',4',6'-O-pentaacetyl-3-O-trans-p-coumaroylsucrose, CAS No: 1392307-46-8, i.e. Mumeose D, Chinese name: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumaroylsucrose, structural formula: C 31 H 38 O 18 , the specific structure is as follows:

[0040] .

[0041] Compound C2: 2', 3', 4', 6'-tetra-O-acetyl-3-O-(E)-p-coumaroyl sucrose, CAS No: 1603815-30-0, Chinese name: 2', 3', 4', 6'-tetra-O-acetyl-3-O-(E)-p-coumaroyl sucrose, structural formula: C 25 H 32 O 15 , the specific structure is as follows:

[0042] .

[0043] Compound C3: mumeose K, CAS No: 2132384-01-9, structural formula: C 25 H 32 O 15 , the specific structure is as follows:

[0044] .

[0045] Compound C4: daucosterol linoleate, CAS No: 79380-30-6,

[0046] Chinese name: Carotene linoleate, 3-O-(6'-O-linoleyl-β-D-glucose)-β-sitosterol; structural formula: C 53 H 90 O7, the specific structure is as follows:

[0047] .

[0048] Compound C5: Glycerol 1-eicosanoate 3-(4E,7E,10E-hexadecatrienoate), CAS No: 1415934-71-2, Name: Glycerol-1-eicosanoate-3-(4E,7E,10E-hexadecatrienoate), Structural formula: C 39 H 70 O5, the specific structure is as follows:

[0049] .

[0050] Compound C6: 1,3-dihydroxylpropyl-(9Z,12Z)-octadeca-9,12-dienate, CAS No: 3443-82-1;

[0051] Chinese name: benfuranolol, 2-linolenoyl-rac-glycerol;

[0052] Structural formula: C 21 H 38 O4, the specific structure is as follows:

[0053] .

[0054] In the present invention, the effective concentration of the Herba Lycoris Radiatae Extract (Herba Lycoris Radiatae Crude Extract or Refined Extract RC1~RC6) in the product is 1~100μg / ml, specifically 1μg / ml, 10μg / ml, 20μg / ml, 30μg / ml, 40μg / ml, 50μg / ml, 60μg / ml, 70μg / ml, 80μg / ml, 90μg / ml, 100μg / ml.

[0055] Studies have shown that the C1-C6 compounds of the present invention have a strong inhibitory effect on acetylcholinesterase, among which the C5 compound has the best inhibitory effect. When the concentration is 100ug / ml, the inhibition rate of acetylcholinesterase is as high as 92%. It can be used to prepare acetylcholinesterase inhibitors and has great potential in the development of drugs for the prevention and treatment of dementia and Alzheimer's disease.

[0056] The prior art has generally confirmed that the lack of the neurotransmitter acetylcholine in the brain is the key cause of Alzheimer's disease. Acetylcholinesterase inhibitors are a type of cholinergic enhancer and are currently the only class of drugs that are clearly used for Alzheimer's disease. Currently, there are a variety of acetylcholinesterase inhibitors, such as huperzine A, tacrine, donepezil, etc., which are used as the main drugs for cholinergic compensation therapy to treat senile dementia, such as Alzheimer's disease. Its mechanism of action is to inhibit the activity of intersynaptic acetylcholinesterase, delay the rate of hydrolysis of released acetylcholine, increase the level of acetylcholine, thereby improving cognitive function and achieving the purpose of treatment. Therefore, the mechanism of "inhibiting brain acetylcholinesterase activity" corresponds to the treatment of senile dementia (such as Alzheimer's disease), that is, drugs that inhibit acetylcholinesterase activity have a therapeutic effect on senile dementia (such as Alzheimer's disease).

[0057] Experiments show that the Herba Lysimachiae extract, refined extract and C1-C6 compounds of the present invention have a strong inhibitory effect on acetylcholinesterase, and can be used as acetylcholinesterase inhibitors to prevent and treat dementia and Alzheimer's disease.

[0058] Based on the above results, the present invention also provides the use of one or a combination of two or more of the C1-C6 compounds in the preparation of any of the following products (1)-(2):

[0059] (1) Acetylcholinesterase inhibitors;

[0060] (2) Drugs for the prevention and treatment of neurodegenerative diseases;

[0061] Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0062] Compound C2: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0063] C3 compound: mumeose K;

[0064] C4 compound: carotene linoleate;

[0065] C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid);

[0066] C6 compound: 2-linolenoyl-rac-glycerol;

[0067] The neurodegenerative disease includes dementia and / or Alzheimer's disease.

[0068] In the present invention, the prevention and treatment includes inhibiting the activity of acetylcholinesterase.

[0069] In the present invention, the effective concentration of the C1~C6 compounds is independently selected from 1~100μg / ml, specifically 1μg / ml, 10μg / ml, 20μg / ml, 30μg / ml, 40μg / ml, 50μg / ml, 60μg / ml, 70μg / ml, 80μg / ml, 90μg / ml, 100μg / ml.

[0070] The present invention also provides an acetylcholinesterase inhibitor, comprising at least one of the following C1-C6 compounds or a Herba Lycopodii extract, and auxiliary materials:

[0071] Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0072] Compound C2: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0073] C3 compound: mumeose K;

[0074] C4 compound: carotene linoleate;

[0075] C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid);

[0076] C6 compound: 2-linolenoyl-rac-glycerol;

[0077] The extract of the ground nasturtium is an ethanol extract of the ground nasturtium and its flower buds, which contains at least one of the C1-C6 compounds;

[0078] The C1-C6 compounds are extracted and separated from the extract of the Herba Lycoris Radiatae.

[0079] The present invention also provides a drug for preventing and treating neurodegenerative diseases, comprising one of the following C1-C6 compounds or a Herba Lycopodii extract, and auxiliary materials:

[0080] Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0081] Compound C2: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumarylsucrose;

[0082] C3 compound: mumeose K;

[0083] C4 compound: carotene linoleate;

[0084] C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid);

[0085] C6 compound: 2-linolenoyl-rac-glycerol;

[0086] The nephrite extract contains at least one of C1-C6 compounds;

[0087] The nasturtium schreberi extract is an ethanol extract of nasturtium schreberi and its flower buds;

[0088] Such neurodegenerative diseases include dementia and Alzheimer's disease.

[0089] The dosage forms of the drug of the present invention include tablets, capsules, granules, liquid preparations, pills or powders, etc., and any common dosage forms in the art can be used.

[0090] The present invention also provides a method for preventing and treating neurodegenerative diseases, comprising: administering at least one of the Herba Lycoris Radiatae Extract or C1-C6 Compounds of the present invention. The Herba Lycoris Radiatae Extract and C1-C6 Compounds are as described above. The neurodegenerative diseases include dementia and Alzheimer's disease.

[0091] Studies have shown that the crude ethanol extract of the ground nasturtium and flower buds (i.e., the ground nasturtium crude extract), refined extracts (RC1~RC6) and C1~C6 compounds of the present invention all have strong inhibitory activity on acetylcholinesterase, especially the inhibitory effect of the ground nasturtium crude extract and C5 compound is significantly better than that of other refined extracts and other compounds, showing great potential in the development of drugs for preventing and treating dementia and Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 It indicates the acetylcholinesterase inhibition rate of crude and refined extracts of Herba Lycoris Radiatae;

[0093] Figure 2 It represents the acetylcholinesterase inhibition rate of crude extract and refined compound of Herba Lycoris Radiatae. DETAILED DESCRIPTION

[0094] The present invention provides the use of Herba Lycopodii Extract in the preparation of acetylcholinesterase inhibitors. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0095] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0096] In a specific embodiment of the present invention, the crude extract of Herba Lycoris Radiatae is prepared by the following method:

[0097] The flowers and buds of the ground jellyfish were taken, and cold-immersed and extracted three times with 3 times the volume of 75% ethanol aqueous solution, each time for 24 hours, and the extract was concentrated to dryness to obtain a crude extract of the ground jellyfish.

[0098] The prepared Nasturtium schrenkiana crude extract was made into a sample solution of required concentration and then the acetylcholinesterase inhibition rate was determined.

[0099] In a specific embodiment of the present invention, the preparation method of the extracts RC1 to RC6 of the Herba Lysimachiae comprises:

[0100] Take the flowers and buds of the ground jellyfish, use 3 times the volume of 75% ethanol aqueous solution to cold-immerse and extract 3 times, each time for 24 hours, and concentrate the extract to dryness to obtain the ground jellyfish crude extract;

[0101] The crude extract of the Herba Lycoris Radiatae was loaded onto an MCI column and eluted with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution and 100% methanol solution in sequence to obtain Herba Lycoris Radiatae refined extracts RC1 to RC6, respectively.

[0102] In the elution, each gradient elution was performed for 3 column volumes.

[0103] Specifically, RC1 is the fraction eluted with water after MCI chromatography separation;

[0104] RC2 is the fraction eluted with 20% methanol in water after MCI chromatography separation;

[0105] RC3 is the fraction eluted with 40% methanol in water after MCI chromatography separation;

[0106] RC4 is the fraction eluted with 60% methanol in water after MCI chromatography separation;

[0107] RC5 is the fraction eluted with 80% methanol in water after MCI chromatography separation;

[0108] RC6 is the fraction eluted with 100% methanol after MCI chromatography separation.

[0109] The prepared nephrolepis schreberi extracts RC1 to RC6 were prepared into sample solutions of required concentrations and then the acetylcholinesterase inhibition rate was determined.

[0110] The present invention will be further described below in conjunction with embodiments:

[0111] Example 1 Acetylcholinesterase inhibition rate experimental determination of anti-acetylcholinesterase effect

[0112] (I) Determination of sample acetylcholinesterase inhibition rate

[0113] 1. Solution preparation:

[0114] (1) Phosphate buffer (pH 7.5)

[0115] (2) 3 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) solution: Accurately weigh an appropriate amount of DTNB powder and prepare a 0.1 M stock solution with DMSO. When required for the experiment, prepare a solution of the corresponding concentration with phosphate buffer.

[0116] (3) 0.4 U / mL acetylcholinesterase solution: Accurately weigh acetylcholinesterase and dissolve it in phosphate buffer to prepare a 0.4 U / mL enzyme solution.

[0117] (4) 15 mM ATCI solution: Accurately weigh an appropriate amount of ATCI powder and prepare a solution with phosphate buffer.

[0118] (5) Galantamine: Accurately weigh an appropriate amount of galantamine powder and prepare a 3 mg / mL stock solution with DMSO. When required for the experiment, use phosphate buffer to prepare a secondary stock solution of corresponding concentration.

[0119] (6) Samples: Prepare the following samples as described above: crude extract of Herba Lysimachiae, refined extracts RC1 to RC6, and compounds C1 to C6. Accurately weigh appropriate amounts of samples and prepare sample solutions of corresponding concentrations using phosphate buffer as required for the experiment.

[0120] 2. Prepare a 96-well plate, add 75 μL of 3 mM-DTNB solution, 15 μL of 15 mM-ATCI solution and 50 μL of sample solution, and incubate at 37°C for 10 min.

[0121] 3. Add 10 μL of 0.4 U / mL acetylcholinesterase to start the reaction and incubate at 37°C for 5 min.

[0122] 4. Measure the absorbance (A) value at 405 nm.

[0123] 5. The sample group was As, with buffer replacing the sample as the blank control (Ab), phosphate buffer replacing acetylcholine as the negative control (Ac), and galantamine replacing the sample as the positive control.

[0124] 6. Acetylcholinesterase inhibition rate (%) = [Ab−(As−Ac)] / Ab× 100%.

[0125] (II) Anti-acetylcholinesterase effects of crude and refined extracts of Nymphaeaceae

[0126] Table 1 Acetylcholinesterase inhibition rate of crude and refined extracts of Herba Lycopodii

[0127]

[0128]

[0129] Conclusion: The three concentration gradients of galantamine showed good inhibition rate of acetylcholinesterase activity, indicating that the experimental system was successful. The crude extract of Hedyssa sutchuenensis and the six extracts of Hedyssa sutchuenensis all showed good anti-acetylcholinesterase activity and had strong anti-acetylcholinesterase efficacy.

[0130] 3. Anti-acetylcholinesterase effects of crude and refined extracts of Herba Lycopodii

[0131] Table 2 Acetylcholinesterase inhibition rate of crude and refined compounds of Herba Lycopodii

[0132]

[0133]

[0134] Summary: The three concentration gradients of galantamine showed good inhibition rates of acetylcholinesterase activity, indicating that the experimental system was successful. The crude extract of Herba Lycoris and the six Herba Lycoris extract compounds all showed good anti-acetylcholinesterase activity (especially C5, whose acetylcholinesterase inhibition rate reached 92% at a concentration of 100 μg / ml), and had extremely strong anti-acetylcholinesterase efficacy.

[0135] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of compound C5 in the preparation of any of the following products (a) to (b): (a) acetylcholinesterase inhibitors; (b) drugs for the prevention and treatment of neurodegenerative diseases; The C5 compound is glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid), and its structural formula is as follows: ； The neurodegenerative disease is Alzheimer's disease.

2. The use according to claim 1, characterized in that: The control is carried out by inhibiting the activity of acetylcholinesterase.

3. The use according to claim 1, characterized in that: The effective concentration of the C5 compound is selected from 1 to 100 μg / ml.

4. The use according to any one of claims 1 to 3, characterized in that: The C5 compound is extracted and separated from the ground jellyfish.

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