CN113956320B - Triterpenoid compound with remarkable acetylcholinesterase and butyrylcholinesterase inhibition activities and preparation method and application thereof - Google Patents

Abstract

The invention provides a triterpenoid, which is obtained by separating sporocarp of trichoderma lucidum to obtain two triterpenoids shown in formulas I and II, wherein the two triterpenoids have obvious acetylcholinesterase and butyrylcholinesterase inhibition activities and can be used as potential medicines for treating Alzheimer disease or development and application of lead compounds. The invention also provides a preparation method and application of the triterpenoid.

Description

A triterpene with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity Compounds and their preparation methods and applications

technical field

The invention belongs to the technical field of natural medicines, and in particular relates to a triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity, a preparation method and application thereof.

Background technique

Alzheimer's disease (AD) is a multifaceted neurodegenerative disease characterized by memory loss, progressive cognitive decline, and severe behavioral abnormalities. It is reported that approximately 46.8 million people worldwide suffer from AD. This number is expected to triple by 2050 as the global population ages. The pathogenesis of AD involves multiple pathways, including alkalirgic neurotransmitter deficiency, β-amyloid (Aβ) and Tau protein phosphorylation pathways. The recently announced Aβ aggregation inhibitor or tau protein aggregation inhibitor therapy has failed, and enhancing brain cholinergic neurotransmission by increasing the level of acetylcholine (ACh) is currently the most effective method for the treatment of AD. Acetylcholine in the brain is mainly hydrolyzed by cholinesterase (ChE), so inhibition of ChE has been proven to be an effective approach. There are two types of ChE in the nervous system: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Currently, the FDA has approved four acetylcholinesterase inhibitors, including tacrine, donepezil, galantamine, and rivastigmine. These acetylcholinesterase inhibitors can improve the memory and cognitive function of AD patients, but tacrine withdrew from clinical trials in the United States in 2013 due to liver toxicity, and the other three inhibitors failed to cure AD or prevent the progression of the disease . Studies have shown that BChE has a compensatory function on AChE, and AChE/BchE dual-target inhibitors are a very promising strategy for the treatment of AD.

Contents of the invention

In view of this, the object of the present invention is to provide a kind of triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity and its preparation method and application, the triterpenoid compound provided by the invention has good acetylcholine Esterase and butyrylcholinesterase inhibitory activity.

The present invention provides a triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity, comprising: a compound of formula I and/or a compound of formula II:

The present invention provides a method for preparing triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in the above technical scheme, comprising:

extracting the fruiting body of Inonotus obliquus to obtain extract;

extracting the extract to obtain an extract;

The extract is subjected to gradient elution to obtain fractions;

The fractions were developed to obtain triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity.

Preferably, the leaching solution in the leaching process is ethanol aqueous solution.

Preferably, the extraction includes:

Extraction was carried out sequentially with petroleum ether and ethyl acetate.

Preferably, the gradient elution comprises:

The extract was eluted with a first gradient of petroleum ether-ethyl acetate through a normal phase decompression silica gel column to obtain 28 fractions;

The 19th fraction was eluted with a second gradient of methanol-water through a C18 reverse-phase silica gel column to obtain 32 fractions;

The 23rd fraction was eluted with a third gradient of petroleum ether-chloroform-ethyl acetate through a normal phase silica gel column to obtain 4 fractions.

6. The method according to claim 5, wherein said unfolding comprises:

The third fraction obtained after the third gradient elution was developed by preparative TLC with petroleum ether-chloroform-isopropanol to obtain the compound of formula I.

Preferably, the gradient elution comprises:

The 11th fraction obtained after the first gradient elution was passed through a C18 reverse-phase silica gel column with methanol-water for the fourth gradient elution to obtain 33 fractions;

The 27th fraction was eluted with a fifth gradient of petroleum ether-ethyl acetate through a silica gel column to obtain 24 fractions;

The 14th fraction was separated by semi-preparative HPLC to obtain the compound of formula II.

Preferably, a C18 column is used in the semi-preparative HPLC process, acetonitrile and water are used as the mobile phase, the flow rate is 3-5 mL/min, and the detection wavelength is 200-220 nm.

The present invention provides an application of the triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in the above technical scheme in the preparation of acetylcholinesterase and butyrylcholinesterase inhibitors.

The present invention provides an application of the triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in the above technical scheme in the preparation of drugs for treating and/or preventing Alzheimer's disease.

The present invention has discovered a new class of triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity; the compound is derived from the medicinal fungus Inonotus obliquus, and is used in the development of treatment and prevention of Alzheimer's It has good application prospects in disease medicine.

Description of drawings

Fig. 1 is the ¹ H NMR spectrum of the compound of formula I structure prepared in Example 1;

Fig. 2 is the ¹³ C NMR spectrum of the compound of formula I structure prepared in Example 1;

Fig. 3 is the HSQC collection of illustrative plates of the compound of formula I structure prepared in embodiment 1;

Fig. 4 is the HMBC collection of illustrative plates of the compound of formula I structure that embodiment 1 prepares;

Fig. 5 is the ¹ H- ¹ H COZY spectrum of the compound of formula I prepared in Example 1;

Fig. 6 is the ROESY collection of illustrative plates of the compound of formula I structure prepared in embodiment 1;

Fig. 7 is the HRESIMS collection of illustrative plates of the compound of formula I structure prepared in embodiment 1;

Fig. 8 is the ¹ H NMR spectrum of the compound of formula I structure prepared in Example 1;

Fig. 9 is the ¹³ C NMR spectrum of the compound of formula II structure prepared in Example 1;

Fig. 10 is the HSQC collection of illustrative plates of the compound of formula II structure prepared in embodiment 1;

Fig. 11 is the HMBC collection of illustrative plates of the compound of formula II structure prepared in embodiment 1;

Figure 12 is the ¹ H- ¹ H COZY spectrum of the compound of formula II prepared in Example 1;

Fig. 13 is the ROESY collection of illustrative plates of the compound of formula II structure prepared in embodiment 1;

Figure 14 is the HRESIMS spectrum of the compound of formula II prepared in Example 1.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other examples improved or modified by those skilled in the art belong to the protection scope of the present invention. It should be understood that the embodiments of the present invention are only used to illustrate the technical effect of the present invention, and are not used to limit the protection scope of the present invention. In the examples, the methods used are conventional methods unless otherwise specified.

The present invention provides a triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity, comprising: a compound of formula I and/or a compound of formula II:

In the present invention, the terminal groups not given in the compound of formula I and the compound of formula II are both methyl groups.

The present invention provides a method for preparing triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in the above technical scheme, comprising:

extracting the fruiting body of Inonotus obliquus to obtain extract;

extracting the extract to obtain an extract;

The extract is subjected to gradient elution to obtain fractions;

The fractions were developed to obtain triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity.

In the present invention, the Inonotus obliquus (Inonotus obliquus), also known as chaga and chaga, is a kind of folk medicinal fungus in Russia, which is mainly produced in the alpine regions of 40°-50° north latitude. It is mainly distributed in Jilin and Heilongjiang provinces in my country. It is parasitic on white birch trees and grows for 15 to 20 years. It can withstand the extreme cold of minus 40°C. The medicinal part of Inonotus obliquus is mainly sclerotium. A large number of pharmacological experiments have shown that it has the functions of treating diabetes, anti-cancer (breast cancer, liver cancer, gastric cancer, lung cancer, cervical cancer), anti-virus, liver protection and enhancing immunity.

In the present invention, it is preferable to grind the fruit body of Inonotus obliquus before leaching.

In the present invention, the leaching solution in the leaching process is preferably an aqueous ethanol solution, and the volume fraction of the aqueous ethanol solution is preferably not less than 95%.

In the present invention, after the leaching is completed, preferably also include:

The extracted extracts are filtered, combined and concentrated to obtain extracts.

In the present invention, the extraction process preferably includes:

The extract is mixed with water to make a suspension for extraction.

In the present invention, the extraction preferably includes:

Extraction was carried out sequentially with petroleum ether and ethyl acetate.

In the present invention, the extraction preferably also includes:

In the present invention, the extraction is preferably extraction of each extraction layer until the extraction liquid is colorless.

In the present invention, after the extraction is completed, preferably also include:

The extract layer obtained after extraction with ethyl acetate was concentrated to prepare ethyl acetate extract.

In the present invention, the concentration is preferably evaporative concentration.

In the present invention, the gradient elution preferably includes:

The extract was passed through a decompressed normal-phase silica gel column, and the first gradient elution was performed with petroleum ether-ethyl acetate to obtain 28 fractions, which were marked as Fr.1-Fr.28.

In the present invention, the volume ratio of petroleum ether-ethyl acetate is preferably changed from 100:1 to 1:1 in the first gradient elution process.

In the present invention, after the first gradient elution is completed, it is preferred to further include:

The 19th fraction obtained after the first gradient elution (Fr.19, petroleum ether-ethyl acetate = 4:1 elution) was subjected to C18 reverse phase silica gel column chromatography with methanol-water for the second gradient elution , 32 fractions were obtained, which were designated as Fr.19-1 to Fr.19-32.

In the present invention, the volume ratio of methanol and water in the second gradient elution process is preferably changed from 3:7 to 1:0.

In the present invention, after the second gradient elution is completed, it is preferred to further include:

The 23rd fraction (Fr.19-23) obtained by the second gradient elution was eluted through the normal phase silica gel column with petroleum ether-chloroform-ethyl acetate for the third gradient elution, and 4 fractions were obtained, denoted as Fr .19-23-1～Fr.19-23-4.

In the present invention, the volume ratio of petroleum ether, chloroform and ethyl acetate in the third gradient elution process is preferably changed from 10:5:1 to 1:2:1.

In the present invention, after the completion of the third gradient elution, it is preferred to further include:

The third fraction (Fr.19-23-3) obtained by the third gradient elution was developed by preparative TLC with petroleum ether-chloroform-isopropanol to obtain the compound of formula I.

In the present invention, the volume ratio of the petroleum ether-chloroform-isopropanol is preferably (3～7):(3～7):(0.4～0.8), more preferably (4～6):(4～ 6): (0.5-0.7), most preferably 5:5:0.6.

In the present invention, after the first gradient elution is completed, it is preferred to further include:

The 11th fraction (Fr.11) obtained by the first gradient elution was subjected to C18 reverse-phase silica gel column chromatography with methanol-water for the fourth gradient elution to obtain 33 fractions, which were designated as Fr.11-1～ Fr. 11-33.

In the present invention, the volume ratio of methanol and water in the fourth gradient elution process is preferably changed from 1:1 to 1:0.

In the present invention, after the fourth gradient elution is completed, it is preferred to further include:

The 27th fraction (Fr.11-27) obtained after the fourth gradient elution was subjected to the fifth gradient elution with petroleum ether-ethyl acetate on a silica gel column to obtain 24 fractions, which were denoted as Fr.11- 27-1～Fr.11-27-24.

In the present invention, the volume ratio of petroleum ether and ethyl acetate is preferably changed from 40:1 to 2:1 in the fifth gradient elution process.

In the present invention, after the completion of the fifth gradient elution, it is preferred to further include:

The 14th fraction (Fr.11-27-14) obtained by the fifth gradient elution was separated by semi-preparative HPLC to obtain the compound of formula II.

In the present invention, the C18 column is preferably adopted in the semi-preparative HPLC process; acetonitrile and water are used as the mobile phase; the volume fraction of acetonitrile in the mobile phase is preferably 75-85%, more preferably 78-82%, most preferably 80%; the flow rate of the mobile phase is preferably 3-5mL/min, more preferably 3.5-4.5mL/min, most preferably 4mL/min; the detection wavelength is preferably 200-220nm, more preferably 205-215nm, most preferably 210nm.

The present invention provides an application of the triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in the above technical scheme in the preparation of acetylcholinesterase and butyrylcholinesterase inhibitors.

The present invention has no special limitation on the dosage forms of the acetylcholinesterase and butyric acetylcholinesterase inhibitors, and can be in the dosage forms such as tablets, capsules, powder injections or suspensions well known to those skilled in the art.

The present invention provides an application of the triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in the above technical scheme in the preparation of medicines for treating diseases mediated by acetylcholinesterase and butyrylcholinesterase .

In the present invention, the acetylcholinesterase and acetylcholinesterase-mediated diseases are preferably neurodegenerative diseases.

The present invention provides an application of the triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in the above technical scheme in the preparation of drugs for treating and/or preventing Alzheimer's disease.

The present invention provides a new class of triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity; the compound provided by the present invention is derived from the medicinal fungus Inonotus obliquus, and is used in the development of treatment and prevention of Alzheimer's disease. Alzheimer's disease drugs have good application prospects.

The present invention provides a class of compounds with inhibitory activity on acetylcholinesterase and butyrylcholinesterase. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.

The test materials adopted in the following examples of the present invention are all common commercial products and can be purchased in the market.

Example 1

1.1 Instruments and reagents

Bruker AV-500 superconducting nuclear magnetic resonance spectrometer (Bruker, Switzerland); Autospec300 mass spectrometer (VG, UK); analytical high-performance liquid chromatography (Agilent, USA); semi-preparative high-performance liquid chromatography (Dionex, USA) ); N-1000 (2L) vertical rotary evaporator and CA-1111 cooling water circulation device (Shanghai Ailang Instrument Co., Ltd.); SHZ-D (Ⅲ) circulating vacuum pump (Shanghai Longtuo Instrument Equipment Co., Ltd.); AS 220. R2 one ten-thousandth electronic balance (RADWAG Wagi Elektroniczne); Sephadex LH-20 gel (Merck Co.Ltd); C18 reversed-phase silica gel (20-45 μm, Japan Fuji Silysia Chemical Ltd company); column chromatography with silica gel and thin Layer chromatography silica gel plate (Qingdao Ocean Chemical Factory); deuterated reagent and chromatographic methanol (Merck, Germany); 95% ethanol, redistilled methanol, ethyl acetate, chloroform, petroleum ether, acetone and other common organic reagents (Tianjin Kemi Europe, Tianjin Fuchen, Guangzhou Guanghua and other companies).

1.2 Compound preparation and structure identification

The sample of Inonotus obliquus was purchased from Russia in June 2017, and the specimen is deposited in the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

The fruiting body of Inonotus obliquus is crushed into powder, extracted three times with ethanol aqueous solution with a volume concentration above 95%, and the obtained extract is filtered, combined and concentrated to form an extract.

Then add water and stir to make a suspension, then extract with petroleum ether and ethyl acetate successively, and extract each extraction layer until the extract is colorless; each extraction layer is evaporated and concentrated to prepare an extract for use.

Take the extract from the ethyl acetate layer, pass it through a decompression column (silica gel H), use petroleum ether: ethyl acetate (100:0 → 1:1) for system gradient elution, and concentrate and combine the obtained fractions respectively, Obtained 28 fractions (Fr.1～Fr.28)

The 19th fraction (Fr.19 (7.4g)) C18 was subjected to reverse-phase silica gel column chromatography, and methanol-water gradient elution was carried out with a concentration change of 30% to 100%, and 32 fractions (Fr.19- 1～Fr.19-32).

The 23rd fraction (Fr.19-23 (155.9mg)) was eluted through a silica gel column petroleum ether-chloroform-ethyl acetate (10:5:1→1:2:1) gradient to obtain 4 fractions ( Fr.19-23-1～Fr.19-23-4).

The third fraction (Fr..19-23-3 (11.9 mg)) was developed by preparative TLC with petroleum ether-chloroform-isopropanol (5:5:0.6) to obtain the compound of formula I.

The 11th fraction (Fr.11 (4.3g)) was subjected to methanol-water gradient elution with a concentration change of 50% to 100% by C18 reverse phase silica gel column chromatography to obtain 33 fractions (Fr.11-1 ~Fr.11-33).

The 27th fraction (Fr.11-27 (840.6mg)) was eluted with a gradient of petroleum ether-ethyl acetate (40:1→2:1) through a silica gel column to obtain 24 fractions (Fr.11- 27-1～Fr.11-27-24).

The 14th fraction (Fr.11-27-14 (105.0mg)) was separated by semi-preparative HPLC (C18 column, 80% acetonitrile/water as mobile phase, flow rate: 4mL/min, detection wavelength 210nm) to obtain Compound of formula II (3.0 mg, t _R =30 min).

Structural identification of the compound of formula I and compound of formula II prepared in Example 1 was carried out. The test results are shown in Figures 1 to 14. The high-resolution mass spectrum of the compound of formula I was m/z 481.3662[M+Na] ⁺ , and the molecular formula was C ₃₀ H ₅₀ O ₃ ; the high-resolution mass spectrum m/z of the compound of the formula II: 481.3652[M+Na] ⁺ , the molecular formula is C ₃₀ H ₅₀ O ₃ ; the ¹ H NMR (500MHz) of the compound of the formula I and the compound of the formula II ) and ¹³ C NMR (125MHz) data are shown in Table 1:

The NMR data (solvent is deuterated chloroform) of formula I structure compound and formula II structure compound of table 1

Embodiment 2 Formula I structural compound and formula II structural compound are to acetylcholinesterase and butyrylcholinesterase inhibitory activity assay

The inhibitory activity of acetylcholinesterase and butyrylcholinesterase of monomeric compounds was determined by Ellman method: 150 μL PBS buffer (pH 8.0), 10 μL test compound solution (DMSO dissolved) and 20 μL 0.1 U/mL acetylcholinesterase or butyrylcholinesterase solution, after incubating at 30°C for 15min, add 10μL 5,5'-dithio-bis-2-nitrobenzoic acid solution (DTNB, the concentration is 2mM) and 10 μL of 10 mM iodothioacetylcholine (AICI) or iodothiobutyrylcholine (BICI) solution was mixed thoroughly, and after 30 min, the absorbance value of each well was measured with a microplate reader at a wavelength of 412 nm. Tacrine was used as a positive control, DMSO was used as a negative control, and the experiment was repeated three times to obtain the average value.

The formula for calculating the inhibitory activity of acetylcholinesterase is as follows:

Inhibition rate (%)=(A2-A1)/(A2-A0)×100%;

In the formula: A0, A1, and A2 are the absorbance values of the blank group, the experimental group, and the negative group measured at 412nm, respectively. The inhibition rate at each concentration was calculated, and the IC ₅₀ value was calculated at the same time, and the test results are shown in Table 2.

The acetylcholinesterase and butyrylcholinesterase inhibitory activity of table 2 formula I structure compound and formula II structure compound

compound Acetylcholinesterase (IC₅₀±SDμM) Butyrylcholinesterase (IC₅₀±SDμM) Compound of formula I 5.92±0.20 4.12±0.16 Formula II structure compound 9.28±0.47 14.75±0.38 Tacrine 0.50±0.02 0.16±0.02

As can be seen from the above examples, the present invention has discovered a new class of triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity; It has a good application prospect in the prevention of Alzheimer's disease drugs.

What has been described above is only a preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements and modifications can also be made without departing from the principles of the present invention. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (6)

1. A triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity, comprising: formula I structural compound and/or formula II structural compound:

2. a method for preparing the triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity according to claim 1, comprising: extracting the fruiting body of Inonotus obliquus to obtain extract; extracting the extract to obtain an extract; The extract is subjected to gradient elution to obtain fractions; developing the fractions to obtain triterpenoids with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity; The extract solution in the extraction process is an aqueous ethanol solution; The extraction includes: sequentially using petroleum ether and ethyl acetate to extract; The gradient elution includes: The extract was eluted with a first gradient of petroleum ether-ethyl acetate through a normal phase decompression silica gel column to obtain 28 fractions; The 19th fraction obtained after the first gradient elution was passed through a C18 reverse-phase silica gel column with methanol-water for the second gradient elution to obtain 32 fractions; The 23rd fraction obtained after the second gradient elution was passed through a normal-phase silica gel column for third gradient elution with petroleum ether-chloroform-ethyl acetate to obtain 4 fractions. 3. The method according to claim 2, wherein said unfolding comprises: The third fraction obtained after the third gradient elution was developed by preparative TLC with petroleum ether-chloroform-isopropanol to obtain the compound of formula I. 4. The method according to claim 2, wherein the gradient elution comprises: The 11th fraction obtained after the first gradient elution was passed through a C18 reverse-phase silica gel column with methanol-water for the fourth gradient elution to obtain 33 fractions; The 27th fraction obtained after the fourth gradient elution was subjected to the fifth gradient elution with petroleum ether-ethyl acetate on a silica gel column to obtain 24 fractions; The 14th fraction obtained after removing the fifth gradient was separated by semi-preparative HPLC to obtain the compound of formula II; In the semi-preparative HPLC process, a C18 column is used, acetonitrile and water are used as the mobile phase, the flow rate is 3-5 mL/min, and the detection wavelength is 200-220 nm. 5. the application of a triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity described in claim 1 in the preparation of acetylcholinesterase and butyrylcholinesterase inhibitors. 6. A use of the triterpenoid compound with significant acetylcholinesterase and butyrylcholinesterase inhibitory activity according to claim 1 in the preparation of medicines for treating and/or preventing Alzheimer's disease.

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