Alkaloid with acetylcholin esterase inhibitory activity and preparation method thereof

Abstract

The invention provides alkaloid with acetylcholin esterase inhibitory activity and a preparation method of the alkaloid. The structural formula of the alkaloid compound 6, 8-dihydroxy-1-oxo-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid is shown as a formula (I), and the alkaloid compound 6, 8-dihydroxy-1-oxo-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid is separated from dry products or fresh products of fruits, stems or leaves of carambola. In-vitro pharmacological experiments prove that the compound 6, 8-dyhydroxyl-1-oxo-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid has good acetylcholin esterase capability, and the half inhibitory concentration of acetylcholin esterase is 304.4 + / -7.3 mu M, which indicates that the compound 6, 8-dyhydroxyl-1-oxo-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid has good acetylcholin esterase inhibitory activity. The alkaloid compound 6, 8-dyhydroxyl-1-oxo-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid is expected to be used as a lead compound to develop a novel cholinesterase resisting drug. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicines, and specifically relates to a new alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and a preparation method thereof and application thereof in the preparation of anticholinesterase drugs. Background Art

[0002] Acetylcholine is the most important neurotransmitter involved in learning and memory. Improving the level of acetylcholine in the brain can effectively improve the cognitive and learning and memory abilities of patients with Alzheimer's disease. Acetylcholinesterase is a key enzyme in biological nerve conduction, which can degrade acetylcholine and terminate the excitatory effect of neurotransmitters on the postsynaptic membrane. Acetylcholinesterase inhibitors are substances that can reversibly inhibit acetylcholinesterase. They can accumulate acetylcholine at synapses and increase its content, ensuring the normal transmission of nerve signals in the body, thereby improving learning and memory functions. It is currently the most widely used Alzheimer's disease treatment drug in clinical practice. Currently, the acetylcholinesterase inhibitors on the market mainly include tacrine, rivastigmine, donepezil and galanthamine. Although the above drugs have clear efficacy, most of them have shortcomings such as short half-life or more serious peripheral cholinergic system side effects, which are not conducive to long-term use by patients. Natural products have always been an important source for drug development. Finding acetylcholinesterase inhibitors from nature is of great significance for the development of new anticholinesterase drugs.

[0003] Carambola (Averrhoa carambola Linn) is a plant of the genus Averrhoa in the family Oxalidaceae. The fruit of carambola has both edible and medicinal value. Previous studies have shown that carambola is not only rich in nutrients, but also contains a wealth of active substances such as dihydrochalcone, norsesquiterpenes, anthocyanins, and alkaloids. So far, there has been no research report on the 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid component in carambola with acetylcholinesterase inhibitory activity. Summary of the invention

[0004] The purpose of the present invention is to provide a new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and to provide a preparation method thereof, as well as to provide an application thereof in the preparation of an anticholinesterase drug.

[0005] The novel alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or a pharmaceutically acceptable salt thereof, or an esterified derivative thereof, of the present invention has a structure as shown in formula (I):

[0006]

[0007] The new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid of the present invention is separated and obtained by the inventors for the first time from dry or fresh products of various tissue parts of carambola (Averrhoa carambola Linn), and the specific parts may be peel and flesh.

[0008] The new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid provided by the present invention has been confirmed by in vitro pharmacological experiments to be a good acetylcholinesterase inhibitor with a half inhibition concentration of 304.4 μM. The new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or its pharmaceutically acceptable salt, or its esterified derivative can be used to prepare anticholinesterase drugs.

[0009] The new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or its pharmaceutical salt, or its esterified derivative can be combined with an excipient or carrier permitted by a preparation or medicine to prepare a medicine or pharmaceutical composition having acetylcholinesterase inhibitory activity and being used to relieve oxidative stress. The medicine or pharmaceutical composition can be in the form of wettable powder, tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol, etc.; it can also be in the form of controlled release or sustained release or nano-preparation known in the modern pharmaceutical industry.

[0010] Therefore, the present invention also provides an anticholinesterase sugar drug, which comprises the above-mentioned new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or a pharmaceutically acceptable salt thereof, or an esterified derivative thereof, as an active ingredient.

[0011] The anticholinesterase drug also includes excipients or carriers permitted by the preparation or medicine, such as wettable powder, tablets, granules, capsules, oral liquid, pills, injections, aerosols and the like; controlled release or sustained release dosage forms or nano preparations known in the modern pharmaceutical industry may also be used.

[0012] The present invention also provides a method for preparing a new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, which is isolated from the carambola (Averrhoa carambola Linn) plant, including the dried or fresh products of carambola fruit or leaves. Preferably, it is isolated from the dried or fresh products of various tissue parts of the carambola fruit, and the specific parts can be the peel and / or the flesh.

[0013] The preparation method of the new alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid of the present invention preferably comprises the following specific steps: homogenizing carambola with a volume fraction of 95% ethanol aqueous solution and then leaching for 48 hours, collecting the extract, and removing the organic solvent by decompression concentration to obtain an extract. The extract is subjected to normal phase silica gel column chromatography, gradient eluted with chloroform / methanol from a volume ratio of 100:0 to 60:40, and component F9 eluted with a volume ratio of chloroform / methanol of 80:20 is collected. Component F9 is subjected to ODS-A medium pressure column chromatography, gradient eluted with methanol / water from a volume ratio of 20:80 to 80:20, and component F9-4 eluted with a volume ratio of methanol / water of 30:70 is collected. Flow fraction F9-4 is separated and purified by high performance liquid phase to obtain 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

[0014] The invention extracts and separates acetylcholinesterase inhibitors from carambola fruit, and the preparation process conditions are controllable, the extraction is convenient, the product is environmentally friendly and has potential good economic benefits. The obtained new alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is stable and easy to store. The product has good acetylcholinesterase inhibition activity and is expected to be used as a lead compound to develop a new type of anticholinesterase drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 1 HNMR spectrum;

[0016] Figure 2 It is the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 13 C NMR spectrum;

[0017] Figure 3 It is the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 1 H -1 HCOSY spectrum;

[0018] Figure 4 is the HSQC spectrum of the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0019] Figure 5 is the HMBC spectrum of the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0020] Figure 6It is the HR-EI-MS spectrum of compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. DETAILED DESCRIPTION

[0021] The following examples are further explanations of the present invention, but are not limitations of the present invention. Simple improvements made to the present invention based on the essence of the present invention all fall within the scope of protection claimed by the present invention.

[0022] Example 1: Preparation of alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

[0023] 1.1 Plant origin and identification

[0024] Plant material for extraction: Fruit samples of carambola (Averrhoa carambola Linn) were purchased from Guangzhou Tianpingjia Fruit Wholesale Market.

[0025] 1.2 Extraction and separation

[0026] The carambola was homogenized with a 95% by volume ethanol aqueous solution and then soaked for 48 hours, the extract was collected, and the extract was concentrated under reduced pressure to remove the organic solvent to obtain an extract concentrate. The extract was chromatographed on a normal phase silica gel column, eluted with chloroform / methanol in a gradient ratio of 100:0 to 60:40, and fraction F9 eluted with a chloroform / methanol volume ratio of 80:20 was collected. Component F9 was chromatographed on an ODS-A medium pressure column, eluted with methanol / water in a gradient ratio of 20:80 to 80:20, and fraction F9-4 eluted with a methanol / water volume ratio of 30:70 was collected. Fraction F9-4 used 17% acetonitrile aqueous solution (v / v) as the mobile phase at a flow rate of 6 mL / min, and was further chromatographically separated and purified by preparative HPLC using a SunFire C18 OBD Prep column (5 μm, 19×250 mm) to obtain compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (14 mg, tR 51 min).

[0027] 1.3 Structural identification of new alkaloid compounds

[0028] Figure 1 It is the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 1 HNMR spectrum; Figure 2 It is the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 13 C NMR spectrum; Figure 3 It is the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 1 H-1 H COSY spectrum; Figure 4 is the HSQC spectrum of the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Figure 5 is the HMBC spectrum of the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Figure 6 It is the HR-EI-MS spectrum of the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, colorless oil; ESI-MS m / z 224[M+H] + , 246[M+Na] + ; 1 H(500MHz)and 13 The C (125MHz) NMR data are shown in Table 1.

[0029] Table 1. NMR data of compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (in D2O)

[0030]

[0031]

[0032] Based on the comprehensive analysis of the above mass spectrometry and one-dimensional and two-dimensional NMR spectroscopic data, the structural formula of the compound is deduced as shown in formula (I), and is named 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid:

[0033]

[0034] Example 2: Assay for the acetylcholinesterase inhibitor activity of the alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

[0035] 1. Evaluation of Acetylcholinesterase Inhibitory Activity

[0036] Using thioiodoacetylcholine as substrate and 5,5-dithiobis(2-nitrobenzoic acid) as colorimetric reagent, the inhibitory activity of the compound on the enzyme was calculated based on the absorbance value of each well after the reaction.

[0037] a) 1 μg / mL acetylcholinesterase solution, 1.05 mM iodothioacetylcholine solution, and 1.5 mM 5,5-dithiobis(2-nitrobenzoic acid) solution were prepared using PBS (pH=7.4).

[0038] b) The compound and the positive control were diluted with DMSO in equal proportions to prepare the test solutions with initial concentrations of 10, 5, 2.5, 1.25, 0.625, and 0.3125 mM, respectively.

[0039] c) Add the sample according to the following reaction system (DMSO concentration does not exceed 5% of the whole system):

[0040] Sample group: 10μL sample + 40μL PBS + 10μL enzyme

[0041] Sample blank group: 10 μL sample + 50 μL PBS

[0042] Negative group: 10μL DMSO + 40μL PBS + 10μL enzyme

[0043] Negative blank group: 10μL DMSO + 50μL PBS

[0044] After the sample is added and mixed, 20 μL of the reaction substrate iodothioacetylcholine (1.05 mM) is added to each well. After reacting in a 37°C constant temperature incubator for 30 minutes, 30 μL of sodium dodecyl sulfate solution (4%) is added to each sample well to terminate the reaction. Then 100 μL of 5,5-dithiobis(2-nitrobenzoic acid) solution is added to each well for color development and the absorbance value of each well at 405 nm is measured using an ELISA reader. Each sample is repeated at least 3 times, and the inhibition rate is calculated as follows: Inhibition rate (%) = [1-(A 样品 –A 样品空白 ) / (A 阴性 –A 阴性空白 )]×100

[0045] SPSS 19.0 statistical analysis software was used to calculate the half inhibitory concentration IC 50 value.

[0046] 2. Experimental data see Table 2:

[0047] Table 2. Acetylcholinesterase inhibitory activity of compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

[0048]

[0049] 4. Experimental conclusion:

[0050] This experiment shows that the alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid has good acetylcholinesterase inhibitory activity and is expected to be developed for the preparation of anticholinesterase drugs, with broad application potential and promising prospects.

[0051] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or its pharmaceutically acceptable salt, or its esterified derivative, whose structure is shown in formula (I):

2. A method for preparing the alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid according to claim 1, characterized in that: The 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is separated from carambola.

3. The preparation method according to claim 2, characterized in that: The specific steps are: a. Extract preparation: The carambola was homogenized with a 95% ethanol aqueous solution and then extracted for 48 hours. The extract was collected and concentrated under reduced pressure to remove the organic solvent to obtain an extract concentrate. b. Separation and purification: The extract was chromatographed on a normal phase silica gel column, with chloroform / methanol gradient elution from a volume ratio of 100:0 to 60:40, and the fraction F9 eluted with a chloroform / methanol volume ratio of 80:20 was collected. The fraction F9 was chromatographed on an ODS-A medium pressure column, with methanol / water gradient elution from a volume ratio of 20:80 to 80:20, and the fraction F9-4 eluted with a methanol / water volume ratio of 30:70 was collected. The fraction F9-4 was separated and purified by high performance liquid phase to obtain 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

4. The preparation method according to claim 2 or 3, characterized in that: The carambola comprises the dried or fresh carambola fruit or leaf.

5. The preparation method according to claim 4, characterized in that: The carambola is a carambola fruit, including peel and / or pulp.

6. Use of the alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or a pharmaceutically acceptable salt thereof, or an esterified derivative thereof as claimed in claim 1 in the preparation of an anticholinesterase drug.

7. An anticholinesterase drug, characterized in that: A composition comprising an effective amount of 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid according to claim 1, or a pharmaceutically acceptable salt thereof, or an esterified derivative thereof as an active ingredient.

8. The anticholinesterase drug according to claim 7, characterized in that: It also includes excipients or carriers that are acceptable for formulation or medicine.

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