An alkaloid with acetylcholinesterase inhibitory activity and its preparation method

Abstract

This invention provides an alkaloid with acetylcholinesterase inhibitory activity and a method for its preparation. The alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid of this invention has the structural formula shown in Formula (I), and is isolated from the dried or fresh fruit, stem, or leaves of star fruit. In vitro pharmacological experiments have confirmed that the compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid exhibits good acetylcholinesterase inhibitory activity, with a half-maximal inhibitory concentration (IC50) of 304.4 ± 7.3 μM, indicating its excellent acetylcholinesterase inhibitory activity. The alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid shows promise as a lead compound for the development of novel anticholinesterase drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to a novel alkaloid compound, 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, its preparation method, and its application in the preparation of anticholinesterase drugs. Background Technology

[0002] Acetylcholine is the most important neurotransmitter involved in learning and memory. Increasing acetylcholine levels in the brain can effectively improve cognitive and learning / memory abilities in Alzheimer's patients. Acetylcholinesterase is a key enzyme in biological neurotransmission that degrades acetylcholine, terminating the excitatory effect of neurotransmitters on the postsynaptic membrane. Acetylcholinesterase inhibitors are substances that reversibly inhibit acetylcholinesterase, causing acetylcholine to accumulate at the synapse, increasing its concentration, and ensuring the normal transmission of nerve signals in the body, thereby improving functions such as learning and memory. They are currently the most widely used drugs for the clinical treatment of Alzheimer's disease. Currently, the main acetylcholinesterase inhibitors on the market include tacrine, rivastigmine, donepezil, and galanthamine. Although these drugs have clear efficacy, most of them have disadvantages such as short half-lives or relatively severe 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, and finding acetylcholinesterase inhibitors from nature is of great significance for developing novel anticholinesterase drugs.

[0003] Star fruit (Averrhoa carambola Linn.) is a plant belonging to the genus Averrhoa in the Oxalidaceae family. Its fruit has both edible and medicinal value. Previous studies have shown that, in addition to being rich in nutrients, star fruit also contains abundant dihydrochalcone, norosesquiterpenes, anthocyanins, alkaloids, and other active substances. Currently, there are no research reports on the 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid component in star fruit that exhibits acetylcholinesterase inhibitory activity. Summary of the Invention

[0004] The purpose of this invention is to provide a novel alkaloid component, 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, a method for its preparation, and its application in the preparation of anticholinesterase drugs.

[0005] The novel alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or its pharmaceutical salt, or its esterified derivative, described in this invention, has the structure represented by formula (Ⅰ):

[0006]

[0007] The novel alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid described in this invention was first isolated by the inventors from dried or fresh samples of various tissues and parts of star fruit (Averrhoa carambola Linn), specifically the peel and pulp.

[0008] The novel alkaloid 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid provided by this invention has been confirmed by in vitro pharmacological experiments to be a good inhibitor of acetylcholinesterase, with a half-maximal inhibitory concentration (IC50) of 304.4 μM. The novel alkaloid 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, its pharmaceutical salt, or its esterified derivatives can be used to prepare anticholinesterase drugs.

[0009] The novel alkaloid component of this invention, 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 excipients or carriers permitted in formulations or pharmaceuticals to prepare drugs or pharmaceutical compositions with acetylcholinesterase inhibitory activity that can be used to alleviate oxidative stress. This drug or pharmaceutical composition can be in dosage forms such as wettable powders, tablets, granules, capsules, oral liquids, pellets, injections, and aerosols; it can also be in controlled-release or sustained-release dosage forms or nanoformulations known in modern pharmaceutical industry.

[0010] Therefore, the present invention also provides an anticholinesterase sugar drug comprising the above-mentioned novel alkaloid component 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, or its pharmaceutical salt, or its esterified derivative, as an active ingredient.

[0011] The anticholinesterase drugs also include excipients or carriers permitted by the formulation or drug, such as wettable powders, tablets, granules, capsules, oral liquids, pellets, injections, aerosols, etc.; and can also adopt controlled-release or sustained-release dosage forms or nano-formulations known in the modern pharmaceutical industry.

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

[0013] The preferred steps for preparing the novel alkaloid 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid of this invention are as follows: Star fruit is homogenized in a 95% (v / v) aqueous ethanol solution and extracted for 48 hours. The extract is collected, concentrated under reduced pressure to remove the organic solvent, and then the extract is obtained as a paste. The extract is subjected to normal-phase silica gel column chromatography, eluted with a chloroform / methanol gradient from 100:0 to 60:40 (v / v), and fraction F9 eluted at a chloroform / methanol volume ratio of 80:20 is collected. Fraction F9 is then subjected to ODS-A medium-pressure column chromatography, eluted with a methanol / water-alcohol gradient from 20:80 to 80:20 (v / v), and fraction F9-4 eluted at a methanol / water volume ratio of 30:70 (v / v) is collected. Fraction F9-4 is purified by high-performance liquid chromatography to obtain 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

[0014] This invention extracts and isolates an acetylcholinesterase inhibitor from star fruit. The preparation process is controllable, convenient, environmentally friendly, and has potential economic benefits. The resulting novel alkaloid compound, 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, is stable and easily stored. It exhibits good inhibitory activity against acetylcholinesterase and holds promise as a lead compound for developing novel anticholinesterase drugs. Attached Figure Description

[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 diagram;

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

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

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

[0021] The following embodiments are further illustrations of the present invention and are not intended to limit the present invention. Simple improvements made to the present invention based on the essence of the present invention are all within the scope of protection claimed by the present invention.

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

[0023] 1.1 Plant source and identification

[0024] The fruit samples of star fruit (Averrhoa carambola Linn) used for extraction were purchased from Guangzhou Tianpingjia Fruit Wholesale Market.

[0025] 1.2 Extraction and Separation

[0026] Star fruit was homogenized with a 95% ethanol aqueous solution and then extracted for 48 hours. The extract was collected, concentrated under reduced pressure to remove organic solvents, and then the extract paste was obtained. The extract was subjected to normal-phase silica gel column chromatography, eluted with chloroform / methanol at a gradient ratio from 100:0 to 60:40 (v / v), and fraction F9 eluted at a chloroform / methanol volume ratio of 80:20 was collected. Fraction F9 was then subjected to ODS-A medium-pressure column chromatography, eluted with methanol / water / alcohol at a gradient ratio from 20:80 to 80:20 (v / v), and fraction F9-4 eluted at a methanol / water volume ratio of 30:70 was collected. Fraction F9-4 was further purified by preparative HPLC using a SunFire C18 OBD Prep column (5 μm, 19 × 250 mm) at a flow rate of 6 mL / min 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 This is the HSQC spectrum of compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Figure 5 This is the HMBC spectrum of compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Figure 6 This is the HR-EI-MS spectrum of 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 presented in Table 1 below.

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

[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 derived as shown in formula (Ⅰ), and it is named 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid:

[0033]

[0034] Example 2: Assay of 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 a substrate and 5,5-dithiobis(2-nitrobenzoic acid) as a colorimetric reagent, the inhibitory activity of the compound on the enzyme was calculated based on the absorbance of each well after the reaction.

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

[0038] b) The compound and the positive control were diluted with DMSO in equal proportions to prepare 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 not exceeding 5% of the total system):

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

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

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

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

[0044] After mixing the samples, add 20 μL of the reaction substrate thioiodoacetylcholine (1.05 mM) to each well. Incubate at 37°C for 30 min. Then, add 30 μL of sodium dodecyl sulfate solution (4%) to each sample well to terminate the reaction. Next, add 100 μL of 5,5-dithiobis(2-nitrobenzoic acid) solution to each well for color development. Measure the absorbance at 405 nm using a microplate reader. Each sample should be repeated at least 3 times. Calculate the inhibition rate using the following formula: Inhibition rate (%) = [1 - (A... 样品 –A 样品空白 ) / (A 阴性 –A 阴性空白 )]×100

[0045] The half-maximal inhibitory concentration (IC50) was calculated using SPSS 19.0 statistical analysis software. 50 value.

[0046] 2. Experimental data are shown in 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 conclusions:

[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. It has broad application potential and promising prospects.

[0051] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, 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 considered within the scope of protection of the present invention.

Claims

1. An alkaloid compound, 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, with the structure shown in formula (I), or a medicinal salt thereof: Equation (Ⅰ).

2. A method for preparing the alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid as described in claim 1, characterized in that, The 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was isolated from star fruit, and the specific steps are as follows: Extract preparation: Star fruit was homogenized with 95% ethanol aqueous solution and then extracted for 48 hours. The extract was collected, concentrated under reduced pressure to remove organic solvents, and then the extract paste was obtained. Separation and purification: The extract was subjected to normal-phase silica gel column chromatography, eluted with chloroform / methanol at a gradient ratio from 100:0 to 60:40 (v / v), and fraction F9 eluted at a chloroform / methanol volume ratio of 80:20 was collected. Fraction F9 was subjected to ODS-A medium-pressure column chromatography, eluted with methanol / water at a gradient ratio from 20:80 to 80:20 (v / v), and fraction F9-4 eluted at a methanol / water volume ratio of 30:70 was collected. Fraction F9-4 was purified by high-performance liquid chromatography to obtain 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

3. The preparation method according to claim 2, characterized in that, The star fruit mentioned includes both dried and fresh star fruit.

4. The preparation method according to claim 3, characterized in that, The star fruit mentioned above includes the peel and / or pulp.

5. The use of the alkaloid compound 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or its pharmaceutical salt as described in claim 1 in the preparation of antiacetylcholinesterase drugs.

6. An anti-acetylcholinesterase drug, characterized in that, Contains an effective amount of 6,8-dihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or a pharmaceutical salt thereof as an active ingredient, as described in claim 1.

7. The anti-acetylcholinesterase drug according to claim 6, characterized in that, It also includes excipients or carriers permitted in the formulation or drug.

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