Application of boxthorn leaf extract in preparation of acetylcholin esterase inhibitor

Abstract

The invention discloses an application of a boxthorn leaf extract in preparation of an acetylcholin esterase inhibitor, and belongs to the technical field of traditional Chinese medicines. An in-vitro enzyme activity test experiment proves that the boxthorn leaf extract has an acetylcholin esterase inhibiting effect, can be developed into an acetylcholin esterase inhibitor and is used for treating diseases related to abnormal rise of acetylcholin esterase.

Description

Technical Field

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

[0002] Acetylcholinesterase (AChE) is a key enzyme widely distributed in the nervous system, neuromuscular junctions, and non-neural tissues (such as red blood cells and retina). Its main function is to catalyze the hydrolysis of the neurotransmitter acetylcholine (ACh) into choline and acetic acid, thereby terminating the signal transmission of ACh to the postsynaptic membrane. Abnormal AChE activity is closely related to various diseases: in Alzheimer's Disease (AD), elevated AChE activity leads to the degeneration of central cholinergic neurons, a significant reduction in the level of ACh in the synaptic cleft, and the onset of cognitive impairment and memory decline; in Myasthenia Gravis (MG), ACh at the neuromuscular junction is excessively decomposed, hindering the conduction of muscle contraction signals and resulting in myasthenia symptoms; in Glaucoma, AChE may affect intraocular pressure and optic nerve blood flow by regulating the cholinergic pathway, exacerbating the damage of retinal ganglion cells. Therefore, inhibiting AChE activity to restore the function of the cholinergic system has become an important treatment strategy for improving the above diseases.

[0003] Currently, the AChE inhibitors used clinically are mainly chemically synthesized drugs (such as donepezil, etc.). Although they can rapidly inhibit AChE activity and temporarily relieve symptoms, their long-term use has significant drawbacks, such as being prone to side effects such as nausea, diarrhea, and arrhythmia, poor patient tolerance, low bioavailability, easy development of drug resistance, and the inability to intervene in the disease process through multi-target mechanisms. For example, the high selectivity of donepezil for central AChE can improve the cognitive function of AD patients, but the inhibition of peripheral tissue AChE may exacerbate gastrointestinal adverse reactions and cannot reverse neurodegenerative lesions.

[0004] In recent years, natural extracts have gradually become a research hotspot for new AChE inhibitors due to their multi-component synergistic effects, low toxicity, and sustainable sources. Ginkgo biloba extract can synergistically protect nerve function through antioxidant, anti-inflammatory, and other pathways. However, existing natural source inhibitors still face many challenges, including low natural content of some active ingredients and insufficient extraction efficiency. The action targets of single compounds are limited, making it difficult to cope with the complex pathological networks of diseases such as AD and MG. In addition, the action mechanisms of most natural extracts have not been fully elucidated, restricting their clinical transformation potential.

[0005] Wolfberry leaves, named "Tianjingcao" in "Compendium of Materia Medica", are recorded in "Japanese Pharmacopoeia of Flowering Plants" as having effects such as "relieving vexation and enhancing will, and treating the five types of overstrain and the seven types of impairment". As a traditional resource for both medicine and food, wolfberry leaves are rich in active ingredients such as polysaccharides, flavonoids, alkaloids, and polyphenols. Existing research has confirmed that they have antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective effects. However, there is very little research on the regulatory effect of wolfberry leaf extracts on the cholinergic system at present, and whether it has AChE inhibitory activity has not been systematically explored. The wolfberry leaves described in this invention are derived from wild wolfberries and the Lycium Lycium barbarum L. Lycium barbarum new wolfberry variety bred through interspecific hybridization between Lycium barbarum L. and then artificially domesticated and cultivated into the fresh bud leaves of fruitless wolfberry that do not flower or bear fruit, also known as fruitless wolfberry leaves, fruitless wolfberry leaf buds, wolfberry bud tea, fruitless wolfberry bud tea, etc. This invention first discovers that wolfberry leaf extracts have a significant inhibitory effect on AChE, providing an experimental basis for its treatment of diseases with abnormally elevated acetylcholinesterase. Compared with chemically synthesized inhibitors, wolfberry leaf extracts are widely sourced, low-cost, and highly safe, providing a new strategy for the development of natural-source AChE inhibitors with both high efficiency and wide applicability. Summary of the Invention

[0006] Through preliminary in vitro enzyme activity inhibition experiments, the present invention discovers that wolfberry leaf extracts can significantly inhibit the activity of acetylcholinesterase.

[0007] Therefore, the purpose of the present invention is to provide the application of wolfberry leaf extracts in the preparation of acetylcholinesterase inhibitors.

[0008] The second purpose of the present invention is to provide the use of wolfberry leaf extracts in the preparation of drugs for treating diseases related to abnormally elevated acetylcholinesterase.

[0009] To achieve the above purposes, the present invention adopts the following technical solutions: The application of wolfberry leaf extracts in the preparation of acetylcholinesterase inhibitors, wherein the wolfberry leaves are derived from wild wolfberries and the Lycium Lycium barbarum L. Lycium barbarum new wolfberry variety bred through interspecific hybridization between Lycium barbarum L. and then artificially domesticated and cultivated into the bud leaves of fruitless wolfberry that do not flower or bear fruit.

[0010] Further, the wolfberry leaf extract is prepared by the following method: using dried wolfberry leaves as raw materials, adding an ethanol aqueous solution for soaking, extraction, filtering, combining the filtrates, and concentrating to obtain the wolfberry leaf extract. Among them, the volume concentration of ethanol in the ethanol aqueous solution is 0-100%, preferably 60% ethanol aqueous solution; the ratio of dried wolfberry leaves to the ethanol aqueous solution is mass (g): volume (mL) = 1:5-50, preferably 1:20; the soaking time is 0.5-1 h; the extraction method is heating reflux extraction or ultrasonic extraction, the extraction times are 1-3 times, and the extraction time is 0.5-3 h.

[0011] Use of the above wolfberry leaf extract in the preparation of a medicament for treating diseases related to abnormal elevation of acetylcholinesterase.

[0012] The diseases related to abnormal elevation of acetylcholinesterase are Alzheimer's disease, myasthenia gravis or glaucoma.

[0013] Advantages of the present invention: The present invention adopts an in vitro enzyme activity test experiment and for the first time discovers that the wolfberry leaf extract has acetylcholinesterase inhibitory activity and can be used for the treatment of diseases with abnormal elevation of acetylcholinesterase, providing a new use for the wolfberry leaf extract.

[0014] 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 the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0015] Figure 1 For the half inhibitory concentration IC of acetylcholinesterase by wolfberry leaf extract 50 Test result graph. Detailed implementation manners

[0016] 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.

[0017] The wolfberry leaves used in the present invention are obtained from the fresh bud leaves of the fruitless wolfberry obtained by interspecific hybridization between wild wolfberry and Lycium barbarum L. of the genus Lycium in Solanaceae Lycium L. barbarum Lycium barbarum L. species, and are the wolfberry leaves obtained after drying. Example 1

[0018] Take dry wolfberry leaves (2 kg), add anhydrous ethanol aqueous solution (10 L), the material-liquid ratio (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 to dryness under reduced pressure to obtain the extract (1079 g), thus obtaining the wolfberry leaf extract. Example 2

[0019] Take dry wolfberry leaves (50 g), add aqueous solution (2.5 L), the material-liquid ratio (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 to dryness under reduced pressure to obtain the extract (18 g), thus obtaining the wolfberry leaf extract. Example 3

[0020] Take dry wolfberry leaves (2 kg), add 60% ethanol aqueous solution (20 L), the material-liquid ratio (mass g: volume mL = 1:10), soak for 1 h, heat under reflux for extraction, extract 2 times, 1 h each time, filter while it is hot, combine the filtrates, and evaporate the filtrates to dryness under reduced pressure to obtain the extract (961 g), thus obtaining the wolfberry leaf extract. Example 4

[0021] Inhibitory effect of wolfberry leaf extract on acetylcholinesterase (IC 50 ). Prepare a series of wolfberry leaf extract sample solutions 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, 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 for 30 minutes in the dark at room temperature. Then add 400 μL of acetylcholine solution with a concentration of 500 μg / mL respectively, and react for 30 minutes in the dark after mixing. Subsequently, add 200 μL of choline oxidase at 50 μg / mL, 400 μL of 3,3',5,5'-tetramethylbenzidine at 1 mg / mL and 200 μL of horseradish peroxidase at 5 μg / mL respectively. After reacting for 30 minutes in the dark, add 200 μL of 3,3',5,5'-tetramethylbenzidine color development termination solution, and detect the absorbance values of each solution at a wavelength of 450 nm, and calculate the IC 50 value of the wolfberry leaf extract inhibiting acetylcholinesterase. The results are shown in Figure 1 as follows. The results show that the IC 50The value is 43.63 μg / mL. The above indicates that the wolfberry leaf extract has a good effect on inhibiting acetylcholinesterase activity. Therefore, the wolfberry leaf extract can be used to prepare a drug for treating diseases with abnormally elevated acetylcholinesterase, and the diseases related to abnormally elevated acetylcholinesterase are Alzheimer's disease, myasthenia gravis or glaucoma.

[0022] As mentioned above, it is only a preferred specific embodiment 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 and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered within the protection scope of the present invention.

Claims

1. Application of wolfberry leaf extract 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 wolfberry leaf extract is prepared by the following method: using dried wolfberry leaves as raw materials, adding ethanol aqueous solution to soak, extracting, filtering, combining filtrates, and concentrating to obtain the wolfberry leaf extract.

4. The method for preparing the wolfberry leaf extract 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 soaking time is 0.5-1 h; the extraction method is heating reflux extraction or ultrasonic extraction, the number of extractions is 1-3 times, and the extraction time is 0.5-3 h.

5. The use according to claim 1, characterized in that: Use of a wolfberry leaf extract with acetylcholinesterase inhibitory effect in preparing a medicine 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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