Application of boxthorn leaf total alkaloids in preparation of acetylcholin esterase inhibitor

Abstract

The invention provides application of boxthorn leaf total alkaloids in preparation of an acetylcholin esterase inhibitor, and belongs to the technical field of traditional Chinese medicines. The invention proposes that the boxthorn leaf total alkaloid has an acetylcholin esterase inhibiting effect for the first time, and the boxthorn leaf total alkaloid can be developed into an acetylcholin esterase inhibitor for preventing and treating diseases related to abnormal rise of acetylcholin esterase. Meanwhile, compared with an existing boxthorn leaf total alkaloid preparation method in the field, the boxthorn leaf total alkaloid preparation method provided by the invention is simpler, and the boxthorn leaf total alkaloid can be obtained without extracting by using organic solvents such as ethyl acetate and the like which are mutually soluble with water.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to the application of total alkaloids from wolfberry leaves in the preparation of acetylcholinesterase inhibitors. Background Art

[0002] Acetylcholinesterase (AChE) is a key hydrolytic enzyme in the cholinergic system, which is widely distributed in the central nervous system, neuromuscular junctions, and non-neural tissues (such as red blood cells, retina, etc.). It catalyzes the decomposition of the neurotransmitter acetylcholine (ACh) into choline and acetic acid, thereby terminating the signal transmission effect of ACh on the postsynaptic membrane. Abnormal functions of AChE are closely related to the occurrence and development of various diseases: in Alzheimer's Disease (AD), abnormal elevation of AChE activity leads to the degeneration of central cholinergic neurons, and a significant decrease in the level of ACh in the synaptic cleft, resulting in cognitive dysfunction and memory decline; in Myasthenia Gravis (MG), ACh at the neuromuscular junction is excessively decomposed, weakening the muscle contraction signal conduction and exacerbating the myasthenia symptoms; in Glaucoma, AChE may affect intraocular pressure and retinal blood flow by regulating the cholinergic pathway, accelerating the apoptosis of ganglion cells in the optic nerve. Therefore, inhibiting AChE activity to restore the function of the cholinergic system has become an important therapeutic target for improving the above diseases.

[0003] Currently, the AChE inhibitors used clinically are mainly chemically synthesized drugs. Although they can quickly inhibit AChE activity and relieve symptoms, their long-term application has significant limitations: one is insufficient selectivity. For example, the highly efficient inhibition of central AChE by donepezil can improve the cognitive function of AD patients, but the inhibition of peripheral tissue AChE is likely to cause side effects such as nausea, diarrhea, and arrhythmia; the other is low bioavailability, easy to develop drug resistance, and unable to intervene in the disease process through multi-target mechanisms (such as β-amyloid deposition in AD).

[0004] In recent years, natural extracts and their active components (such as polysaccharides, total alkaloids, total flavonoids, etc.) have become a research hotspot for new AChE inhibitors due to their multi-component synergistic effects, low toxicity, and sustainable sources. Wolfberry leaves, as a traditional resource for both medicine and food, are rich in active ingredients such as alkaloids, flavonoids, polysaccharides, and polyphenols. Studies have shown that wolfberry leaf alkaloids have various pharmacological activities such as antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective effects, but their regulatory effects on the cholinergic system have not been taken seriously for a long time. Particularly crucial is that total alkaloids from wolfberry leaves, as a class of multi-component alkaloid complexes, may intervene in AChE activity and disease-related pathways through multi-target synergistic effects, but their specific inhibitory effects have not been reported in the literature.

[0005] The present invention first reveals the highly efficient inhibitory effect of total alkaloids from wolfberry leaves on AChE. Compared with chemically synthesized inhibitors, total alkaloids from wolfberry leaves are widely sourced and have a simple extraction process. Compared with the previously authorized invention patent of the research group (a preparation method and application of an alkaloid extract from wolfberry leaves, application number 202210611065.4, which uses ethyl acetate for extraction before purifying the wolfberry leaf extract with a cation exchange resin, with a cumbersome process and possible residual toxic reagent residues), the preparation method of total alkaloids from wolfberry leaves is further simplified, avoiding the use of organic solvents such as ethyl acetate and having higher safety. The proposal of the present invention not only provides innovative candidate components for the development of new AChE inhibitors, but also opens up a new strategy for treating neurodegenerative diseases, neuromuscular diseases, and ophthalmic diseases using traditional medicinal resources, with significant clinical application value and social and economic benefits. Summary of the Invention

[0006] Through previous in vitro enzyme activity inhibition experiments, the present invention found that total alkaloids from wolfberry leaves can significantly inhibit the activity of acetylcholinesterase.

[0007] Therefore, the object of the present invention is to provide the application of total alkaloids from wolfberry leaves in the preparation of acetylcholinesterase inhibitors.

[0008] The second object of the present invention is to provide a preparation method of total alkaloids from wolfberry leaves.

[0009] The third object of the present invention is to provide the use of total alkaloids from wolfberry leaves in the preparation of drugs for treating diseases related to abnormal elevation of acetylcholinesterase.

[0010] To achieve the above objects, the present invention adopts the following technical solutions: The application of total alkaloids from wolfberry leaves in the preparation of acetylcholinesterase inhibitors, wherein the wolfberry leaves are from a new wolfberry variety selected by interspecific hybridization between wild wolfberry and Lycium barbarum L. of the genus Lycium in Solanaceae, and then through artificial domestication and cultivation into the bud and leaf of a fruitless wolfberry that does not flower or bear fruit. Lycium L. Ningxia wolfberry Lycium barbarum L. The bud and leaf of the fruitless wolfberry that does not flower or bear fruit after artificial domestication and cultivation.

[0011] Furthermore, the total alkaloids from Lycium barbarum leaves are prepared by the following method: using dried Lycium barbarum leaves as raw materials, adding an ethanol aqueous solution for extraction, filtering and combining the filtrates, concentrating to an appropriate concentration, and then purifying with a cation exchange resin. First, wash with absolute ethanol to remove impurities, and then elute with an appropriate concentration of NaCl-ethanol aqueous solution. Collect the eluate and concentrate it. Then, use macroporous adsorption resin or nanofiltration membrane to remove salts. After desalting, concentrate the solution and evaporate to dryness to obtain the total alkaloids from Lycium barbarum leaves. Among them, the volume concentration of ethanol in the ethanol aqueous solution is 0-100%, preferably 60% ethanol aqueous solution; the ratio of dried Lycium barbarum leaves to the ethanol aqueous solution is mass (g): volume (mL) = 1:5-50, preferably 1:20; the extraction method is heating reflux extraction or ultrasonic extraction, and reflux extraction is preferred; the extraction times are 1-3 times, and the extraction time is 0.5-3 h; the concentration of the NaCl-ethanol aqueous solution is 2%-8% NaCl-55%-80% ethanol aqueous solution, preferably 8% NaCl-55% ethanol aqueous solution.

[0012] Use of the above-mentioned total alkaloids from Lycium barbarum leaves in the preparation of a medicament for treating diseases related to abnormally elevated acetylcholinesterase.

[0013] The diseases related to abnormally elevated acetylcholinesterase are Alzheimer's disease, myasthenia gravis or glaucoma.

[0014] Advantages of the present invention: By using in vitro enzyme activity test experiments, the present invention first discovers that the total alkaloids from Lycium barbarum leaves have acetylcholinesterase inhibitory activity and can be used for the treatment of diseases with abnormally elevated acetylcholinesterase, providing a new use for the total alkaloids from Lycium barbarum leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0016] Figure 1 For the half-inhibitory concentration IC of acetylcholinesterase by the total alkaloids from Lycium barbarum leaves 50 Test result graph. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] The present invention will be described in detail below with reference to the drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0018] The wolfberry leaves used in the present invention are derived from the fresh bud leaves of the fruitless wolfberry obtained from the interspecific hybridization of wild wolfberry and Lycium barbarum L. of the Solanaceae family, and are the wolfberry leaves obtained after drying. Lycium L. barbarum Lycium barbarum The wolfberry leaves used in the present invention are derived from the fresh bud leaves of the fruitless wolfberry obtained from the interspecific hybridization of wild wolfberry and Lycium barbarum L. of the Solanaceae family, and are the wolfberry leaves obtained after drying. Example 1

[0019] Take dried wolfberry leaves (2 kg), add an aqueous solution of absolute ethanol (10 L), with a material-liquid ratio of (mass g: volume mL = 1:5), soak for 0.5 h, heat under reflux for extraction, extract once, 3 h each time, filter while it is hot, combine the filtrates, and evaporate the filtrates under reduced pressure to dryness to obtain an extract (1079 g). The extract is dissolved in absolute ethanol to prepare a sample loading solution of 25 mg / mL, and the sample loading solution is purified using 001X7 cation exchange resin. The specific steps are as follows: Take 3.5 BV (column volume) of the sample loading solution and add it to the 001X7 resin column, adsorb at a speed of 2 BV / h, then add 5 BV of absolute ethanol to the resin column and wash and remove impurities at a speed of 4 BV / h, and finally use 5 BV of 8% NaCl - 60% ethanol solution to elute at a flow rate of 2 BV / h. Take the eluate, rotary evaporate and concentrate it to dryness, add an appropriate amount of water to dissolve it into a concentrated solution, load it onto D101 macroporous adsorption resin, wash and remove salts with water, and then elute with ethanol. Collect the eluate, concentrate and evaporate it to dryness to obtain the total alkaloids of wolfberry leaves. Example 2

[0020] Take dried wolfberry leaves (50 g), add an aqueous solution (2.5 L), with a material-liquid ratio of (mass g: volume mL = 1:50), soak for 1 h, decoct for extraction, extract 3 times, 0.5 h each time, filter while it is hot, combine the filtrates, and evaporate the filtrates under reduced pressure to dryness to obtain an extract (18 g). The extract is dissolved in water to prepare a sample loading solution of 25 mg / mL, and the sample loading solution is purified using 001X7 cation exchange resin. The specific steps are as follows: Take 3.5 BV (column volume) of the sample loading solution and add it to the 001X7 resin column, adsorb at a speed of 2 BV / h, then add 5 BV of absolute ethanol to the resin column and wash and remove impurities at a speed of 4 BV / h, and finally use 5 BV of 2% NaCl - 80% ethanol solution to elute at a flow rate of 2 BV / h. Take the eluate, rotary evaporate and concentrate it to dryness, add an appropriate amount of water to dissolve it into a concentrated solution, load it onto D101 macroporous adsorption resin, wash and remove salts with water, and then elute with ethanol. Collect the eluate, concentrate and evaporate it to dryness to obtain the total alkaloids of wolfberry leaves. Example 3

[0021] Take dry wolfberry leaves (2 kg), add 60% ethanol aqueous solution (20 L), with a solid-liquid ratio (mass g: volume mL = 1:10), soak for 1 h, heat under reflux for extraction, extract twice, 1 h each time, filter while it is hot, combine the filtrates, and evaporate the filtrates to dryness under reduced pressure to obtain an extract (961 g). The extract is dissolved in 60% ethanol to prepare a sample solution with a concentration of 25 mg / mL. The sample solution is purified using 001X7 cation exchange resin. The specific steps are as follows: Take 3.5 BV (column volume) of the sample solution and add it to the 001X7 resin column, adsorb at a rate of 2 BV / h, then add 5 BV of absolute ethanol to the resin column and wash to remove impurities at a rate of 4 BV / h. Finally, use 5 BV of 8% NaCl - 55% ethanol solution to elute at a flow rate of 2 BV / h. Take the eluate, remove salt with a nanofiltration membrane, and then concentrate and evaporate to dryness to obtain the total alkaloids from wolfberry leaves. Example 4

[0022] Inhibitory effect of total alkaloids from wolfberry leaves on acetylcholinesterase (IC 50 ). Prepare a series of sample solutions of total alkaloids from wolfberry leaves with concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 1000 μg / mL respectively. Take 200 μL of each, and add 400 μL of acetylcholinesterase solution with a concentration of 1 mg / mL respectively. After mixing, react in the dark at room temperature for 30 minutes. Then add 400 μL of acetylcholine solution with a concentration of 500 μg / mL respectively. After mixing, react in the dark for 30 minutes. Subsequently, add 200 μL of choline oxidase with a concentration of 50 μg / mL, 400 μL of 3,3',5,5'-tetramethylbenzidine with a concentration of 1 mg / mL, and 200 μL of horseradish peroxidase with a concentration of 5 μg / mL. After reacting for 30 minutes, add 200 μL of 3,3',5,5'-tetramethylbenzidine color development termination solution. Finally, measure the absorbance values of each solution at a wavelength of 450 nm, and calculate the IC 50 value of the total alkaloids inhibiting acetylcholinesterase. The results are shown in Figure 1 . The results show that the IC 50 value of the total alkaloids from wolfberry leaves is 14.4 μg / mL. The above shows that the total alkaloids from wolfberry leaves have a good effect on inhibiting the activity of acetylcholinesterase. Therefore, the total alkaloids from wolfberry leaves can be used to prepare drugs for treating diseases with abnormally elevated acetylcholinesterase, and the diseases related to abnormally elevated acetylcholinesterase are Alzheimer's disease, myasthenia gravis, or glaucoma.

[0023] As described above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. Application of total alkaloids from wolfberry leaves in the preparation of acetylcholinesterase inhibitors.

2. The use according to claim 1, characterized in that: The wolfberry leaf is derived from wild wolfberry and the genus Lycium of the Solanaceae family. Lycium L. Ningxia wolfberry Lycium barbarum L. A new variety of wolfberry bred through interspecific hybridization, which was later artificially domesticated and cultivated into the buds and leaves of fruitless wolfberry that does not bloom or bear fruit.

3. The use according to claim 1, characterized in that: The total alkaloids from wolfberry leaves are prepared by the following method: using dried wolfberry leaves as raw materials, adding ethanol aqueous solution to extract, filtering and combining the filtrate, concentrating to a suitable concentration, purifying by using a cation exchange resin, first washing with anhydrous ethanol to remove impurities, then eluting with a NaCl-ethanol aqueous solution of a suitable concentration, collecting and concentrating the eluate, and then desalting with a macroporous adsorption resin or a nanofiltration membrane. After desalting, the solution is concentrated and evaporated to dryness to obtain the total alkaloids from wolfberry leaves.

4. The method for preparing total alkaloids from wolfberry leaves according to claim 3, characterized in that: The volume concentration of ethanol in the ethanol aqueous solution is 0-100%, preferably 60% ethanol aqueous solution; the ratio of the dried wolfberry leaves to the ethanol aqueous solution is mass (g): volume (mL) = 1:5-50, preferably 1:20; the extraction method is heating reflux extraction or ultrasonic extraction, preferably reflux extraction; the number of extractions is 1-3 times, and the extraction time is 0.5-3 h; the concentration of the NaCl-ethanol aqueous solution is 2%~8%NaCl-55%~80% ethanol aqueous solution, preferably 8%NaCl-55% ethanol aqueous solution.

5. The use according to claim 1, characterized in that: Use of total alkaloids from wolfberry leaves with acetylcholinesterase inhibitory effect in preparing medicines for treating diseases related to abnormally elevated acetylcholinesterase.

6. The use according to claim 5, characterized in that The disease associated with abnormal increase of acetylcholinesterase is Alzheimer's disease, myasthenia gravis or glaucoma.

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