A longan pulp extract that inhibits acetylcholinesterase and helps prevent Alzheimer's disease, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a longan pulp extract for inhibiting acetylcholinesterase and preventing Alzheimer's disease, its preparation method, and its applications. This invention is the first to extract and isolate two longan pulp alkaloid compounds from longan pulp, and conduct activity studies on them. Experimental data show that these longan pulp alkaloid compounds inhibit acetylcholinesterase activity, preventing acetylcholinesterase from degrading acetylcholine and increasing the level of acetylcholine, which directly participates in cognitive impairment. This indicates that they can be used to prepare acetylcholinesterase inhibitors and Alzheimer's disease prevention and treatment drugs. The longan pulp extract provided by this invention mainly contains the above two compounds and has significant application value in the development of acetylcholinesterase inhibitors and Alzheimer's disease prevention and treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a longan pulp extract that inhibits acetylcholinesterase and helps prevent Alzheimer's disease, as well as its preparation method and application. Background Technology

[0002] Alzheimer's disease (AD) is a multifocal neurodegenerative disease and the leading cause of dementia in the elderly. Acetylcholine, a neurotransmitter, plays a crucial role in transmitting information between neurons. According to WHO data, there are currently about 50 million AD patients worldwide, and this number is projected to exceed 150 million by 2050. With the increasing aging population in my country, the number of AD patients in China not only ranks first in the world, but its growth rate is also the fastest globally. Therefore, developing drugs that can effectively alleviate the symptoms of AD is crucial for reducing the social burden caused by AD. Pharmaceutical companies worldwide have long been vying for the huge market for AD drugs. Pharmaceutical giants such as Pfizer, Merck, and Johnson & Johnson have attempted to develop various drugs to treat AD, but all have failed in the clinical trial stage.

[0003] Reports indicate that Alzheimer's patients often have low acetylcholine levels. This is because Alzheimer's disease damages the cells that produce and use acetylcholine, leading to impaired neuronal information transmission and weakened learning and memory functions. The cholinergic hypothesis posits that the pathophysiology of cognitive impairment associated with AD is due to the destruction or loss of cholinergic neurons, causing a decrease in acetylcholine levels, which in turn leads to a series of pathological features, including cognitive dysfunction. Inhibition of AChE leads to the accumulation of ACh, which in turn increases the stimulation of muscarinic substances, ultimately alleviating memory deficits caused by AD. Therefore, insufficient acetylcholine levels are considered a major factor in the development of Alzheimer's disease. Some drugs can increase acetylcholine levels by blocking the action of enzymes; these drugs are called acetylcholinesterase inhibitors or cholinesterase inhibitors (AChE). They can help alleviate symptoms related to thought processes, such as language, judgment, and memory.

[0004] Chinese patent application CN103533948A discloses a composition for the prevention or treatment of neurological diseases containing extracts of longan pulp, salvia miltiorrhiza, and gastrodia elata as active ingredients. This composition inhibits the activity of acetylcholinesterase. However, longan pulp, salvia miltiorrhiza, and gastrodia elata extracts contain multiple active ingredients, making it impossible to identify which ingredient inhibits acetylcholinesterase activity. Furthermore, scholars such as Ran Yubing (Ran Yubing, Liu Lei, Zhang Ruifen, et al. Longan pulp extract improves learning and memory function in SAMP8 mice [J]. Modern Food Science and Technology, 2017, 33(06):1-8. DOI:10.13982 / j.mfst.1673-9078.2017.6.001.) disclosed that both the petroleum ether phase and aqueous phase of the longan pulp alcohol extract effectively improve the learning and memory function of SAMP8 mice, a model of Alzheimer's disease. However, the chemical composition of the petroleum ether phase and aqueous phase of the longan pulp alcohol extract remains unclear. The aforementioned patents and journal articles all disclose studies on the efficacy of traditional Chinese medicine extracts as a whole, failing to confirm the specific active ingredients contained within or the specific efficacy of the individual compounds. Traditional Chinese medicine extracts are mixtures of various components from medicinal materials after extraction and concentration. Some of these components may have medicinal effects, while others may have no medicinal effect or even have adverse effects on the human body. Therefore, it is essential to conduct in-depth research on the components of traditional Chinese medicine extracts to better understand the mechanisms of action of traditional Chinese medicine, improve its efficacy and safety, and promote the modernization and internationalization of traditional Chinese medicine. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings and deficiencies of the existing technology, which often studies longan pulp extract as a whole and lacks in-depth research on the enrichment or purification of specific effective monomeric compounds and their pharmacological effects. The present invention provides a longan pulp extract that inhibits acetylcholinesterase and prevents Alzheimer's disease.

[0006] Another object of the present invention is to provide the use of the longan pulp extract in the preparation of acetylcholinesterase inhibitors or Alzheimer's disease prevention and treatment drugs.

[0007] Another objective of this invention is to provide an acetylcholinesterase inhibitor or an Alzheimer's disease prevention and treatment drug, with the longan pulp extract as the active ingredient.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention protects a longan pulp extract that inhibits acetylcholinesterase and prevents Alzheimer's disease, the extract mainly containing compound 1 and / or compound 2:

[0010]

[0011] The above-mentioned longan pulp extract was prepared by method one and / or method two:

[0012] Method 1: The dried extract of longan pulp obtained by alcohol extraction and precipitation was subjected to silica gel column chromatography: elution was performed using an ethyl acetate-methanol solution with a volume ratio of (1-2):1 (preferably a 1:1 ethyl acetate-methanol solution, and / or a 2:1 ethyl acetate-methanol solution) to obtain fraction A; fraction A was subjected to silica gel column chromatography, eluted with ethyl acetate to obtain fraction A-1; fraction A-1 was then separated and purified by medium-pressure preparative chromatography, eluted with a water-methanol solution with a volume ratio of 20:80, and the eluent was dried to obtain the longan pulp extract containing compound 1.

[0013] Method 2: The dried extract of longan pulp prepared by alcohol extraction and precipitation was subjected to silica gel column chromatography: using ethyl acetate as the eluent, fraction B was obtained; fraction B was separated and purified by semi-preparative liquid chromatography, using a water-methanol solution with a volume ratio of 8:92 as the eluent, and the eluent was dried to obtain the longan pulp extract containing compound 2.

[0014] The longan pulp extract obtained by combining Method 1 and Method 2 mainly contains compounds 1 and 2.

[0015] Preferably, in Method 1, the eluent ethyl acetate-methanol solution is a 1:1 ethyl acetate-methanol solution and / or a 2:1 ethyl acetate-methanol solution.

[0016] Furthermore, in methods one and two above, the preparation method of the dry extract made from longan pulp through alcohol extraction and alcohol precipitation includes the following steps:

[0017] S1. Take longan pulp, add alcohol solvent, heat to reflux, cool, shake well, filter, and concentrate the filtrate under reduced pressure to obtain crude extract.

[0018] S2. Add 60-95 vol% ethanol to the crude extract obtained in step S1 for alcohol precipitation, filter, concentrate the filtrate under reduced pressure and then dry to obtain lyophilized extract.

[0019] Furthermore, the silicone column in Method 1 is a glass column, and the mesh size of the silicone filling is 80-100 mesh.

[0020] Furthermore, in Method 2, the silicone column is a glass column, and the mesh size of the silicone filling is 200-300 mesh.

[0021] Furthermore, the chromatographic column used in the medium-pressure preparative chromatography is a carbon-18 column.

[0022] Furthermore, the chromatographic column used in the medium-pressure preparative chromatography has a specification of 36mm*460mm and a particle size of 40-63um.

[0023] Furthermore, the chromatographic column used in the semi-preparative liquid chromatography is a C18 reversed-phase column, preferably an Agilent 10Prep-C column. 18 The dimensions are 30mm*150mm.

[0024] More specifically, as one possible implementation, the above-mentioned longan pulp extract is obtained by a method comprising the following steps:

[0025] S1. Take longan pulp, add alcohol solvent, heat to reflux, cool, shake well, filter, and concentrate the filtrate under reduced pressure to obtain crude extract.

[0026] S2. Add 60-95 vol% ethanol to the crude extract obtained in step S1 for alcohol precipitation, filter, concentrate the filtrate under reduced pressure and then dry to obtain lyophilized extract.

[0027] S3. The lyophilized extract obtained in step S2 was subjected to silica gel column chromatography with gradient elution using ethyl acetate-methanol solution as the eluent. The fractions with the same volume ratio were combined by TLC (thin-layer chromatography) and HPLC (High Performance Liquid Chromatography). The fractions with the same volume ratio of ethyl acetate-methanol of 2:1 and 1:1 were collected, dried, and obtained fraction A. The fraction with the same volume ratio of ethyl acetate-methanol of 1:0 was collected, dried, and obtained fraction B.

[0028] S4. The fraction A obtained in step S3 is subjected to silica gel column chromatography, using an ethyl acetate-methanol solution with a volume ratio of 1:0 as the eluent, and dried to obtain fraction A-1.

[0029] S5. The fraction A-1 obtained in step S4 is separated and purified by medium-pressure preparative chromatography, using a water-methanol solution with a volume ratio of 20:80 as the eluent, and the fraction is collected and dried.

[0030] S6. Separate and purify fraction B obtained in step S3 by semi-preparative liquid chromatography, using a water-methanol solution with a volume ratio of 8:92 as the eluent, collect the fraction, and dry it.

[0031] The products obtained from S7, S5, S6, or a combination of S5 and S6, are longan pulp extracts that have the activity of inhibiting acetylcholinesterase and preventing Alzheimer's disease.

[0032] Specifically, the TLC conditions were as follows: the developing solvent was ethyl acetate and methanol, the sample volume was 3-5 μL, and the colorimetric reagents were iodine fumigation and sulfuric acid ethanol. TLC was used to assess the purity of the compound by observing the shape and color uniformity of the sample spots.

[0033] The HPLC detection conditions were as follows: Column: Agilent 10 Prep-C 18 The HPLC apparatus was 30mm x 150mm, with methanol-water as the mobile phase, a flow rate of 4ml / min, and a detection wavelength of 254nm. The purity of the compound was evaluated by HPLC using spectra.

[0034] Specifically, the product obtained in step S5 is compound 1, and the product obtained in step S6 is compound 2.

[0035] Through extensive experiments, the inventors extracted for the first time the two longan pulp alkaloids that have inhibitory activity against acetylcholinesterase from longan pulp. These alkaloids can prevent acetylcholinesterase from degrading acetylcholine and increase the level of acetylcholine, which is directly involved in cognitive impairment. This indicates that the two longan pulp alkaloids can be used to prepare acetylcholinesterase inhibitors and can be developed into drugs for the prevention and treatment of Alzheimer's disease.

[0036] Structural comparison search revealed that the structures of compounds 1 and 2 were disclosed in existing technologies (Compound 1: Chen Dongjie. Analysis and Origin Identification of Amide and Phenylpropanoid Components of Chinese Herbal Medicine Lycium barbarum [D]. Nanjing University of Chinese Medicine, 2021.; Compound 2: Jung HJ, Jung HA, Kang SS, et al. Inhibitory activity of Aralia continentalis roots on protein tyrosine phosphatase 1B and rat lens aldose reductase. [J]. Archives of pharmacal research, 2012, 35(10).), but their activities were not disclosed.

[0037] Therefore, this invention claims protection for the two compounds mentioned above, extracts rich in the compounds, and their use in the preparation of acetylcholinesterase inhibitors and Alzheimer's disease prevention and treatment drugs.

[0038] According to the present invention, extracting an extract from longan pulp that can inhibit acetylcholinesterase and prevent Alzheimer's disease requires maximizing the enrichment of compound 1 and / or compound 2. The aforementioned longan pulp extract is an extract enriched with compound 1 and / or compound 2.

[0039] In the above-mentioned method for extracting longan pulp extract, preferably, in step S1, the alcohol solvent is any one or more of ethanol, methanol, isopropanol, and n-butanol.

[0040] More preferably, when the alcohol solvent is ethanol, the concentration of ethanol used is preferably 60-95 vol%.

[0041] Preferably, in step S1, the mass-to-volume ratio of the longan pulp to the alcohol solvent is 1:8-10 (kg / L).

[0042] Preferably, in step S1, the temperature of the heating reflux is 50–80°C.

[0043] Preferably, in step S1, the heating reflux time is 0.5 to 1 hour.

[0044] Preferably, in step S1, the relative density of the crude extract is 0.9 to 1.2.

[0045] Preferably, in steps S1 and S2, the temperature of the vacuum concentration is 50–65°C.

[0046] Preferably, in step S2, the drying is freeze-drying.

[0047] Preferably, in step S2, the addition of 60-95 vol% ethanol for precipitation is performed 2-5 times.

[0048] Specifically, in step S3, the specific operation of performing silica gel column chromatography is as follows: the obtained lyophilized extract is thoroughly mixed and ground with silica gel (80-100 mesh) at a mass ratio of 1:2-4, dry-packed into a column, dry-loaded, and subjected to silica gel column chromatography.

[0049] Specifically, in step S4, the specific operation of performing silica gel column chromatography is as follows: the fraction A obtained in step 3 is thoroughly mixed and ground with silica gel (200-300 mesh) at a mass ratio of 1:1, dry-packed into a column, dry-loaded, and subjected to silica gel column chromatography.

[0050] This invention also protects the use of the longan pulp extract in the preparation of acetylcholinesterase inhibitors or Alzheimer's disease prevention and treatment drugs.

[0051] This invention also protects an acetylcholinesterase inhibitor or an Alzheimer's disease prevention and treatment drug, with the longan pulp extract as the active ingredient.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] This invention is the first to extract and isolate two longan pulp alkaloids that inhibit acetylcholinesterase activity from longan pulp. Activity studies were conducted, and experimental data showed that these longan pulp alkaloids exhibit acetylcholinesterase activity, preventing the degradation of acetylcholine by acetylcholinesterase and increasing the level of acetylcholine, which directly participates in cognitive impairment. This indicates that they can be used to prepare acetylcholinesterase inhibitors and Alzheimer's disease prevention and treatment drugs.

[0054] Based on this, the longan pulp extract provided by the present invention has a simpler composition and higher purity, mainly containing compound 1 and / or compound 2, and has important application value in the development of acetylcholinesterase inhibitors and Alzheimer's disease prevention and treatment drugs. Attached Figure Description

[0055] Figure 1 This is the hydrogen spectrum of compound 1.

[0056] Figure 2 This is the carbon spectrum of compound 1.

[0057] Figure 3 This is the secondary mass spectrum of compound 1.

[0058] Figure 4 This is the ultraviolet spectrum of compound 1.

[0059] Figure 5 This is the hydrogen spectrum of compound 2.

[0060] Figure 6 This is the carbon spectrum of compound 2.

[0061] Figure 7 This is the secondary mass spectrum of compound 2.

[0062] Figure 8 This is the ultraviolet spectrum of compound 2.

[0063] Figure 9 This is the hydrogen spectrum of compound 3.

[0064] Figure 10 This is the carbon spectrum of compound 3.

[0065] Figure 11 This is a statistical graph showing the inhibitory effects of compounds 1–3 on acetylcholinesterase activity. Detailed Implementation

[0066] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0067] The packing material for the silica gel glass column below can be column chromatography silica gel purchased from Qingdao Ocean Chemical Co., Ltd.

[0068] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0069] Through extensive experimental research, our team has compared the effects of active ingredients extracted from longan pulp on acetylcholinesterase activity. The results show that compounds 1 and 2 significantly inhibit acetylcholinesterase activity. Therefore, this invention aims to develop an extract rich in compounds 1 and 2 from longan pulp for use in the development of acetylcholinesterase inhibitors and Alzheimer's disease prevention drugs. The following examples illustrate the extraction process of the extract and the effect of the extract product on acetylcholinesterase activity.

[0070] Example 1: Study on the extraction process of active ingredients from longan pulp

[0071] 1. Extraction process of active ingredients from longan pulp

[0072] S1. Take 1 kg of longan pulp slices and place them in a stoppered conical flask. Add 8 L of 80 vol% ethanol at a mass-to-volume ratio of 1:8 (kg / L). Heat and reflux at 65°C for 1 h. Cool, shake well, filter, and concentrate the filtrate under reduced pressure at 60°C to obtain a crude extract with a relative density of 1.1.

[0073] S2. The crude extract obtained in step S1 is added to 80 vol% ethanol for precipitation twice, filtered, and the filtrate is concentrated under reduced pressure at 60°C and then freeze-dried to obtain the freeze-dried extract.

[0074] S3. The lyophilized extract obtained in step S3 is thoroughly mixed and ground with silica gel (80-100 mesh) at a mass ratio of 1:2. The mixture is then dry-packed into a column and loaded with the sample using a silica gel glass column chromatography process. A gradient elution is performed using ethyl acetate-methanol solution as the eluent, with volume ratios of 1:0, 50:1, 10:1, 5:1, 2:1, 1:1, and 1:2. The fractions with ethyl acetate-methanol volume ratios of 2:1 and 1:1 are collected, combined, and dried to obtain fraction A. The fraction with an ethyl acetate-methanol volume ratio of 1:0 is collected and dried to obtain fraction B. Other fractions do not contain the target compound or contain only a small amount; therefore, these fractions are discarded.

[0075] S4. The fraction A obtained in step S3 is thoroughly mixed and ground with silica gel (200-300 mesh) at a mass ratio of 1:1. The mixture is then dry-packed and loaded onto a silica gel glass column for chromatography. Eluent is ethyl acetate-methanol solution as the eluent, and gradient elution is performed sequentially at ethyl acetate-methanol volume ratios of 1:0, 5:1, 2:1, 1:1, 1:2, and 1:5. The elution is then analyzed by TLC and HPLC (column: Agilent 10 Prep-C).18 Using a 30mm*150mm sample, with methanol-water as the mobile phase, a flow rate of 4ml / min, and a detection wavelength of 254nm, the same fractions were detected and combined. The fraction with an ethyl acetate-methanol volume ratio of 1:0 was collected, dried, and obtained fraction A-1. The fractions with ethyl acetate-methanol volume ratios of 5:1 and 2:1 were collected, combined, and dried to obtain fraction A-2.

[0076] S5. The fraction A-1 obtained in step S4 was separated and purified by medium-pressure preparative chromatography (C18 column, specifications: 36mm*460mm, particle size: 40-63um, flow rate: 20ml / min, detection wavelength: 210nm). The fraction was eluted with water-methanol solution at water-methanol volume ratios of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100. The fraction with a water-methanol volume ratio of 20:80 was collected and dried to obtain compound 1 (structural formula and identification data are shown below).

[0077] S6. The fraction B obtained in step S3 is subjected to semi-preparative liquid chromatography (column: Agilent 10Prep-C). 18 The sample was separated and purified using a 30mm*150mm membrane, a flow rate of 4ml / min, and a detection wavelength of 254nm. The eluent was a water-methanol solution with a volume ratio of 8:92. The fractions were collected, dried, and obtained as compound 2 (structural formula and identification data are shown below).

[0078] In addition, fraction A-2 obtained in step S4 was subjected to semi-preparative liquid chromatography (column: Agilent 10Prep-C). 18 The sample was separated and purified using a 30mm*150mm membrane, a flow rate of 4ml / min, and a detection wavelength of 254nm. The eluent was a water-methanol solution with a volume ratio of 90:10. The fractions were collected, dried, and obtained as compound 3 (structural formula and identification data are shown below).

[0079] 2. Structure and identification of compounds 1-3

[0080] The structures of the obtained compounds 1 to 3 are shown below:

[0081]

[0082] The structural identification data of compounds 1-3 are shown below:

[0083] Compound 1: White powder, UV (MeOH) λmax 254 nm, HR-ESI-MS m / z 180.0655 [M+H] + (Theoretical value 179.06), molecular formula C9H9NO3. 1H NMR (600MHz, CDCl3) δ9.52 (s, 1H), 6.96 (d, J = 4.0Hz, 2H), 6.19 (d, J = 3.9Hz, 2H), 5.83 ( q,J=7.3Hz,1H),5.46(d,J=14.9Hz,1H),5.37(d,J=14.9Hz,1H),1.71(d,J=2.3Hz,3H); 13 C10 NMR (151MHz, CDCl3) δ 179.44 (C-11), 168.38 (C-1), 131.04 (C-7), 130.53 (C-10), 124.74 (C-8), 106.75 (C-9), 63.51 (C-2), 53.99 (C-13), 19.31 (C-3). The above data are basically consistent with compound 9 in the literature (Chen Dongjie. Analysis and Identification of Amide and Phenylpropanoid Components in Lycium barbarum [D]. Nanjing University of Traditional Chinese Medicine, 2021.), identifying this compound as 2-(5-Hydroxymethyl-2-formylpyrrol-1-yl)propionic Acid Lactone.

[0084] Compound 2: White powder, UV (MeOH) λmax: 254 nm, HR-ESI-MS m / z: 224.0927 [MH]-, molecular formula C 11 H 15 NO4. 1 H NMR(600MHz,DMSO-d6)δ9.49(s,1H),7.00(d,J=4.0Hz,1H),6.28(d,J=3.9Hz,1H),4.44(s, 2H), 4.25 (d, J = 165.5Hz, 2H), 3.27 (s, 3H), 2.21 (t, J = 7.3Hz, 2H), 1.85 (d, J = 344.5Hz, 2H); 13C NMR(151MHz,DMSO)δ179.48(CHO),173.88(COOH),139.02(C-5),132.00(C-2),123.69(C -3),111.33(C-4),64.62(C-6),57.42(OCH3),44.26(C-1′),30.66(C-3′),26.34(C-2′). The above data are basically consistent with the control of compound 12 in the literature (Jung HJ, Jung HA, Kang SS, et al. Inhibitory activity of Aralia continentalis roots on protein tyrosinephosphatase 1B and rat lens aldose reductase.[J]. Archives of pharmacalresearch,2012,35(10).), and the compound was identified as 4-[formy-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoic acid.

[0085] Compound 3: Brownish-yellow oily substance. ESI-MS m / z 124 [M-H]−; 1H-NMR (DMSO-d6, 500MHz) δ: 9.40 (1H, s, H−6), 6.92 (1H, d, J=3.5Hz, H−3), 6.18 (1H, d, J=3.5Hz, H−4), 4.46 (2H, s, H−7). 13C-NMR (DMSO-d6, 125MHz) δ: 178.6 (C−6), 1142.5

[0086] (C-2), 1132.1 (C-5), 1120.9 (C-3), 1108.8 (C-4), 556.1 (C-7). The above data are basically consistent with compound 1 in the literature (Health, Li Meng, Zheng Xiaoke et al. Study on chemical constituents of Rehmannia glutinosa [J]. Chinese Pharmaceutical Journal, 2014, 49(17):1496-1502.), and the compound is identified as 5-hydroxymethyl-pyrrole-2-carbaldehyde.

[0087] The proton, carbon, secondary mass, and ultraviolet spectra of compound 1 are as follows: Figures 1-4 As shown. Combining these spectral data, the extracted compound 1 can be further identified as 2-(5-Hydroxymethyl-2-formylpyrrol-1-yl)propionic Acid Lactone.

[0088] The proton, carbon, secondary mass, and ultraviolet spectra of compound 2 are as follows: Figures 5-8 As shown. Combining these spectral data, the extracted compound 2 can be further identified as 4-[formy-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoic acid.

[0089] The proton and carbon spectra of compound 3 are as follows: Figures 9-10 As shown in the figure. Combining these spectral data, the extracted compound 3 can be further identified as 5-hydroxymethyl-pyrrole-2-carbaldehyde.

[0090] Example 2: Determination of the acetylcholinesterase inhibitory activity of compounds 1-3 (extracts of the main constituent compounds) obtained from Example 1.

[0091] This method employs a modified Ellman colorimetric assay. The basic principle is based on acetylthiocholine iodide (ATCI) as a substrate. Upon addition of AChE (acetylcholinesterase), ATCI hydrolyzes under the action of AChE to generate thiocholine. This thiocholine then reacts with DTNB to produce 2-nitrobenzoic acid-5-mercaptothiocholine and 2-nitro-5-mercaptobenzoic acid (TNB). The absorbance of the yellow ion (TNB) can be detected at 405 nm. In determining the inhibitory activity of the sample against AChE, if the sample inhibits AChE, the amount of ATCI participating in the reaction decreases, leading to a reduction in the product (thiocholine). The amount of product reacting with DTNB also decreases accordingly, ultimately resulting in less TNB formation, thus lowering the absorbance value at 405 nm. Conversely, increased absorbance at 405 nm leads to an increased absorbance value.

[0092] Using the above method and determination principle, the enzyme inhibitory activity of compounds 1-3 obtained in Example 1 was determined.

[0093] The specific operating steps are as follows: Divide this experiment into a blank group, a sample group, and a model group. Perform three parallel experiments in each group. Add samples according to Table 1, keeping the total volume of each tube constant.

[0094] Table 1

[0095]

[0096] The AChE enzyme inhibition rate of the sample groups was calculated using formula (2.1):

[0097] Sample group:

[0098] Inhibition rate = (Model A - Sample A) / (Model A - Blank A) × 100% (2.1)

[0099] The enzyme inhibition test results of compounds 1-3 are as follows: Figure 11 As shown, the results indicate that compounds 1 and 2 inhibit the activity of acetylcholinesterase, preventing the degradation of acetylcholine by acetylcholinesterase and increasing the level of acetylcholine, which is directly involved in cognitive impairment. This suggests that compounds 1 and 2 can be used to prepare acetylcholinesterase inhibitors and could be further developed into drugs for the prevention and / or treatment of Alzheimer's disease. Compound 3, however, showed no inhibitory effect on acetylcholinesterase activity, indicating that not all alkaloid compounds extracted and isolated from longan pulp inhibit acetylcholinesterase activity.

[0100] Therefore, to extract an extract from longan pulp that can inhibit acetylcholinesterase and prevent Alzheimer's disease, it is necessary to enrich compound 1 and / or compound 2 as much as possible.

[0101] Based on the above research, the present invention also provides a longan pulp extract with activity of inhibiting acetylcholinesterase and preventing Alzheimer's disease, as specifically shown in Examples 3 to 7 below.

[0102] Example 3: Longan pulp extract with activity in inhibiting acetylcholinesterase and preventing Alzheimer's disease

[0103] The extraction method is as follows:

[0104] S1. Take 1 kg of longan pulp slices and place them in a stoppered conical flask. Add 8 L of 80 vol% ethanol at a mass-to-volume ratio of 1:8 (kg / L). Heat and reflux at 65°C for 1 h. Cool, shake well, filter, and concentrate the filtrate under reduced pressure at 60°C to obtain a crude extract with a relative density of 1.1.

[0105] S2. The crude extract obtained in step S1 is added to 80 vol% ethanol for precipitation twice, filtered, and the filtrate is concentrated under reduced pressure at 60°C and then freeze-dried to obtain the freeze-dried extract.

[0106] S3. The lyophilized extract obtained in step S3 is thoroughly mixed and ground with silica gel (80-100 mesh) at a mass ratio of 1:2. The mixture is then packed into a dry column and loaded onto the column using a dry method. Silica gel glass column chromatography is performed using ethyl acetate-methanol solution as the eluent, with gradient elution at volume ratios of 1:0, 2:1, and 1:1. The elution is then analyzed by TLC and HPLC (column: Agilent 10Prep-C). 18 Using a 30mm*150mm membrane, with methanol-water as the mobile phase, a flow rate of 4ml / min, and a detection wavelength of 254nm, the same fractions were detected and combined. Fractions with an ethyl acetate-methanol volume ratio of 2:1 and 1:1 were collected, combined, and dried to obtain fraction A. Fraction with an ethyl acetate-methanol volume ratio of 1:0 was collected, dried, and obtained fraction B.

[0107] S4. Mix and grind fraction A obtained in step S3 with silica gel (200-300 mesh) at a mass ratio of 1:1, dry pack the sample into a column, dry load the sample, and perform silica gel column chromatography. Use ethyl acetate-methanol solution at a volume ratio of 1:0 as the eluent for elution, dry the sample, and obtain fraction A-1.

[0108] S5. The fraction A-1 obtained in step S4 is separated and purified by medium-pressure preparative chromatography (C18 column, specifications are 36mm*460mm, particle size is 40-63um, flow rate is 20ml / min, detection wavelength is 210nm). The fraction is eluted with a water-methanol solution with a volume ratio of 20:80, the fraction is collected and dried.

[0109] S6. The fraction B obtained in step S3 is subjected to semi-preparative liquid chromatography (column: Agilent 10Prep-C). 18 Separation and purification were performed using a 30mm*150mm sample, a flow rate of 4ml / min, and a detection wavelength of 254nm. Elution was carried out using a water-methanol solution with a volume ratio of 8:92. The fractions were collected and dried.

[0110] The products obtained from S7, S5, S6, or a combination of S5 and S6, are longan pulp extracts that have the activity of inhibiting acetylcholinesterase and preventing Alzheimer's disease.

[0111] The above extraction method yielded a longan pulp extract enriched with compound 1 and / or compound 2, which was able to inhibit the activity of acetylcholinesterase.

[0112] Example 4: Longan pulp extract with activity in inhibiting acetylcholinesterase and preventing Alzheimer's disease

[0113] The extraction method is as follows:

[0114] S1. Take 1 kg of longan pulp slices and place them in a stoppered conical flask. Add 10 L of 90 vol% ethanol at a mass-to-volume ratio of 1:10 (kg / L). Heat under reflux at 80°C for 0.8 h, cool, shake well, filter, and concentrate the filtrate to obtain a crude extract with a relative density of 1.2.

[0115] S2. The crude extract obtained in step S1 is added to 80 vol% ethanol for precipitation twice, filtered, and the filtrate is concentrated under reduced pressure at 50°C and then freeze-dried to obtain the freeze-dried extract.

[0116] S3. The lyophilized extract obtained in step S3 is thoroughly mixed and ground with silica gel (80-100 mesh) at a mass ratio of 1:2. The mixture is then packed into a dry column and loaded onto the column using a dry method. Silica gel glass column chromatography is performed using ethyl acetate-methanol solution as the eluent, with gradient elution at volume ratios of 1:0, 2:1, and 1:1. The elution is then analyzed by TLC and HPLC (column: Agilent 10Prep-C). 18 Using a 30mm*150mm filter, with methanol-water as the mobile phase, a flow rate of 4ml / min, and a detection wavelength of 254nm, the same fractions were detected and combined. Fractions with an ethyl acetate-methanol volume ratio of 2:1 and 1:1 were collected, combined, and dried to obtain fraction A. Fraction with an ethyl acetate-methanol volume ratio of 1:0 was collected, dried, and obtained fraction B.

[0117] S4. Mix and grind fraction A obtained in step S3 with silica gel (200-300 mesh) at a mass ratio of 1:1, dry pack the column, dry load the sample, and perform silica gel glass column chromatography. Elute with ethyl acetate solution to obtain fraction A-1.

[0118] S5. The fraction A-1 obtained in step S4 is separated and purified by medium-pressure preparative chromatography (C18 column, specifications are 36mm*460mm, particle size is 40-63um, flow rate is 20ml / min, detection wavelength is 210nm). The fraction is eluted with a water-methanol solution with a volume ratio of 20:80, the fraction is collected and dried.

[0119] S6. The fraction B obtained in step S3 is subjected to semi-preparative liquid chromatography (column: Agilent 10Prep-C). 18 Separation and purification were performed using a 30mm*150mm sample, a flow rate of 4ml / min, and a detection wavelength of 254nm. Elution was carried out using a water-methanol solution with a volume ratio of 8:92. The fractions were collected and dried.

[0120] The products obtained from S7, S5, S6, or a combination of S5 and S6, are longan pulp extracts that have the activity of inhibiting acetylcholinesterase and preventing Alzheimer's disease.

[0121] The above extraction method yielded a longan pulp extract enriched with compound 1 and / or compound 2, which was able to inhibit the activity of acetylcholinesterase.

[0122] Example 5: Longan pulp extract with activity in inhibiting acetylcholinesterase and preventing Alzheimer's disease

[0123] The extraction method is as follows:

[0124] S1. Take 1 kg of longan pulp slices, place them in a stoppered conical flask, add 9 L of methanol at a mass-to-volume ratio of 1:9 (kg / L), heat and reflux at 55℃ for 1 h, cool, shake well, filter, and concentrate the filtrate to obtain a crude extract with a relative density of 0.95.

[0125] S2. The crude extract obtained in step S1 is added to 60 vol% ethanol for precipitation twice, filtered, and the filtrate is concentrated under reduced pressure at 55°C and then freeze-dried to obtain the freeze-dried extract.

[0126] S3. The lyophilized extract obtained in step S3 is thoroughly mixed and ground with silica gel (80-100 mesh) at a mass ratio of 1:2. The mixture is then packed into a dry column and loaded onto the column using a dry method. Silica gel glass column chromatography is performed using ethyl acetate-methanol solution as the eluent, with gradient elution at volume ratios of 1:0, 2:1, and 1:1. The elution is then analyzed by TLC and HPLC (column: Agilent 10Prep-C). 18 Using a 30mm*150mm filter, with methanol-water as the mobile phase, a flow rate of 4ml / min, and a detection wavelength of 254nm, the same fractions were detected and combined. Fractions with an ethyl acetate-methanol volume ratio of 2:1 and 1:1 were collected, combined, and dried to obtain fraction A. Fraction with an ethyl acetate-methanol volume ratio of 1:0 was collected, dried, and obtained fraction B.

[0127] S4. Mix and grind fraction A obtained in step S3 with silica gel (200-300 mesh) at a mass ratio of 1:1, dry pack the column, dry load the sample, and perform silica gel glass column chromatography. Elute with ethyl acetate solution to obtain fraction A-1.

[0128] S5. The fraction A-1 obtained in step S4 is separated and purified by medium-pressure preparative chromatography (C18 column, specifications are 36mm*460mm, particle size is 40-63um, flow rate is 20ml / min, detection wavelength is 210nm). The fraction is eluted with a water-methanol solution with a volume ratio of 20:80, the fraction is collected and dried.

[0129] S6. The fraction B obtained in step S3 is subjected to semi-preparative liquid chromatography (column: Agilent 10Prep-C). 18 Separation and purification were performed using a 30mm*150mm sample, a flow rate of 4ml / min, and a detection wavelength of 254nm. Elution was carried out using a water-methanol solution with a volume ratio of 8:92. The fractions were collected and dried.

[0130] The products obtained from S7, S5, S6, or a combination of S5 and S6, are longan pulp extracts that have the activity of inhibiting acetylcholinesterase and preventing Alzheimer's disease.

[0131] The above extraction method yielded a longan pulp extract enriched with compound 1 and / or compound 2, which was able to inhibit the activity of acetylcholinesterase.

[0132] Example 6: Longan pulp extract with inhibitory activity against acetylcholinesterase and activity in preventing and treating Alzheimer's disease

[0133] The difference from Example 3 is that in step S1, 80 vol% ethanol is replaced with isopropanol.

[0134] For other steps and parameters, refer to Example 3.

[0135] The above extraction method yielded a longan pulp extract enriched with compound 1 and / or compound 2, which was able to inhibit the activity of acetylcholinesterase.

[0136] Example 7: Longan pulp extract with inhibitory activity against acetylcholinesterase and activity in preventing and treating Alzheimer's disease

[0137] The difference from Example 3 is that in step S1, 80 vol% ethanol is replaced with n-butanol.

[0138] For other steps and parameters, refer to Example 3.

[0139] The above extraction method yielded a longan pulp extract enriched with compound 1 and / or compound 2, which was able to inhibit the activity of acetylcholinesterase.

[0140] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a longan pulp extract containing compound 1 as the main component, characterized in that, The process includes the following steps: Longan pulp is extracted and precipitated with alcohol to obtain a dry extract, which is then subjected to silica gel column chromatography: elution is performed using an ethyl acetate-methanol solution with a volume ratio of (1-2):1) to obtain fraction A; fraction A is further subjected to silica gel column chromatography, eluted with ethyl acetate to obtain fraction A-1; fraction A-1 is then separated and purified by medium-pressure preparative chromatography, eluted with a water-methanol solution with a volume ratio of 20:80, and the eluent is dried to obtain the longan pulp extract containing compound 1. The structure of compound 1 is shown below: ； The method for preparing the dry extract of longan pulp by alcohol extraction and precipitation includes the following steps: S1. Take longan pulp, add alcohol solvent, heat to reflux, cool, shake well, filter, and concentrate the filtrate under reduced pressure to obtain crude extract. S2. Add 60-95 vol% ethanol to the crude extract obtained in step S1 for alcohol precipitation, filter, take the filtrate, concentrate under reduced pressure and dry to obtain lyophilized extract. The column used in the medium-pressure preparative chromatography is a carbon-18 column.

2. The preparation method according to claim 1, characterized in that, The eluent ethyl acetate-methanol solution is a 1:1 ethyl acetate-methanol solution and / or a 2:1 ethyl acetate-methanol solution.

3. The preparation method according to claim 1, characterized in that, In step S1, the alcohol solvent is any one or more of ethanol, methanol, isopropanol, and n-butanol.

4. The preparation method according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of longan pulp to alcohol solvent is 1:8~10 (kg / L).

5. The preparation method according to claim 1, characterized in that, In step S1, the temperature of the heating reflux is 50~80℃.

6. The preparation method according to claim 1, characterized in that, In step S1, the relative density of the crude extract is 0.9~1.

2.

7. A method for preparing a longan pulp extract containing compound 2 as the main component, characterized in that, Includes the following steps: The dried extract of longan pulp, prepared by alcohol extraction and precipitation, was subjected to silica gel column chromatography: using ethyl acetate as the eluent, fraction B was obtained; fraction B was separated and purified by semi-preparative liquid chromatography, using a water-methanol solution with a volume ratio of 8:92 as the eluent, and the eluent was dried to obtain the longan pulp extract containing compound 2 as the main component. The structure of compound 2 is shown below: ； The method for preparing the dry extract of longan pulp by alcohol extraction and precipitation includes the following steps: S1. Take longan pulp, add alcohol solvent, heat to reflux, cool, shake well, filter, and concentrate the filtrate under reduced pressure to obtain crude extract. S2. Add 60-95 vol% ethanol to the crude extract obtained in step S1 for alcohol precipitation, filter, take the filtrate, concentrate under reduced pressure and dry to obtain lyophilized extract. The column used in the semi-preparative liquid chromatography is a C18 reversed-phase column.

8. The preparation method according to claim 7, characterized in that, In step S1, the alcohol solvent is any one or more of ethanol, methanol, isopropanol, and n-butanol.

9. The preparation method according to claim 7, characterized in that, In step S1, the mass-to-volume ratio of longan pulp to alcohol solvent is 1:8~10 (kg / L).

10. The preparation method according to claim 7, characterized in that, In step S1, the temperature of the heating reflux is 50~80 ℃.

11. The preparation method according to claim 7, characterized in that, In step S1, the relative density of the crude extract is 0.9~1.

2.

12. A method for preparing a longan pulp extract mainly containing compound 1 and compound 2, characterized in that, The process includes the following steps: combining the longan pulp extract containing compound 1 obtained by the preparation method according to any one of claims 1 to 6 and the longan pulp extract containing compound 2 obtained by the preparation method according to any one of claims 7 to 11, and the result is the longan pulp extract containing compound 1 and compound 2. The structures of compound 1 and compound 2 are shown below: 。

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