Lignan compound and application thereof in preparation of butyrylcholine esterase inhibitor

Abstract

The invention discloses a lignan compound of which the structure is selected from one of the following structures: the lignan compound prepared by the invention has a remarkable inhibition effect on the activity of butyrylcholine esterase, and the binding free energy of the two lignan compounds and BChE is analyzed by performing molecular docking on the two lignan compounds and the butyrylcholine esterase. A molecular docking result proves a butyrylcholine esterase activity in-vitro inhibition experiment result, so that the lignan compound prepared by the invention has huge potential as a butyrylcholine esterase inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a lignan compound and its application in the preparation of butyrylcholinesterase inhibitors. Background Technology

[0002] genus Raphanus ( The plant is a tree, shrub or subshrub with woody rhizomes, with a total of about 50 to 70 species. It is distributed in northern Asia through the Himalayas, Malaysia, Oceania, Polynesia to the Hawaiian Islands. Among them, there are about 44 species and 5 varieties in China. It is distributed throughout the country, mainly in the area south of the Yangtze River, with the largest distribution in the southwest and South China (Flora of China, Beijing, 1999, Vol. 52(1), 292).

[0003] The more famous plant is the King of Songs ( (Also known in folk medicine as Nanling Raphanus sativus, Shanmianpi, Dimianpi, Dimiangen, etc., in the 1977 edition of the Pharmacopoeia and in traditional Chinese medicine, it is mainly used for clearing heat and detoxifying, reducing swelling and dissipating nodules, and promoting menstruation and relieving pain. It is commonly used to treat lung heat cough, mumps, scrofula, rheumatic pain, traumatic injuries, boils and carbuncles. Later, due to its strong toxic side effects, it was not included again, but it is still included in the processing standards in Zhejiang, Guangdong, Jiangxi, Shanghai and other places (People's Medical Publishing House, Pharmacopoeia of the People's Republic of China, Beijing, 1977, Vol. 2, 621.). River Raphanus sativus flower ( In traditional Chinese medicine, it is mainly used to treat edema, cough, hepatitis, schizophrenia, and infertility (Molecules 2012, 17 (6), 6424-33.). Raphanus sativus ( It has strong irritant properties and is used as an anti-tumor drug in folk medicine in southwestern China. 2006, 7 (2), 194-196.). Xiao Huang Gou ( It is a plant mainly distributed in the northwest and southwest regions of my country. In folk medicine, it is used together with the stem bark and root to treat cough, asthma and to expel phlegm (National Compendium of Chinese Herbal Medicine (Second Edition), People's Medical Publishing House, Beijing, 1996, Vol. 2, 746).

[0004] The genus *Daphne* has long been considered a plant resource with bioactive molecules, mainly containing terpenes, lignans, flavonoids, coumarins, and sterols. Furthermore, in vitro and in vivo bioactivity experiments have shown that the chemical components in *Daphne* plants possess various pharmacological activities, such as anticancer, anti-inflammatory, antiviral, anti-aging, antimalarial, and anti-mycobacterial activities. 2024, 25 (5), 563-598.).

[0005] Butyrylcholinesterase (BChE) and acetylcholinesterase (AChE), as key enzymes in the cholinesterase family, play a central role in regulating the metabolic homeostasis of the neurotransmitter acetylcholine (ACh). The activity state of these two enzymes is not only directly related to the dynamic regulation of cholinergic neurotransmission, but their abnormal expression is also closely associated with the pathological progression of various neurological diseases, including Alzheimer's disease, Parkinson's disease, and organophosphate pesticide poisoning.

[0006] Compared to AChE inhibitors, drugs targeting BChE can reduce some of the side effects associated with AChE inhibition. Because BChE is relatively less distributed in the brain and its main physiological functions differ from AChE, drugs targeting BChE may have better safety profiles. Currently, acetylcholinesterase inhibitors such as galantamine are widely used in the clinical treatment of AD patients, and there are no approved fully selective BChE inhibitors. Literature reviews have shown that some phenolic compounds have good activity against neurodegenerative diseases. 2025, 16, 1497861.).

[0007] Given that phenolic compounds are abundant in the chemical composition of plants in the genus Raphanus, and there is very little research on the chemical composition and biological activity of Rhizophora stylosa, it is urgent to conduct a systematic study on the chemical composition of Rhizophora stylosa in order to isolate compounds that can serve as inhibitors of butyrylcholinesterase. Summary of the Invention

[0008] The first objective of this invention is to provide a lignan compound.

[0009] A second objective of this invention is to provide a method for preparing the lignan compounds.

[0010] A third objective of this invention is to provide the use of the lignan compounds in the preparation of butyrylcholinesterase inhibitors.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a lignan compound having a structure selected from one of the following structures:

[0013]

[0014] The lignans were isolated and prepared from *Daphnia magna*, a plant belonging to the genus *Daphnia* of the family Thymelaeaceae.

[0015] A second aspect of the present invention provides a method for preparing lignan compounds, comprising the following steps:

[0016] The dried branches and leaves of *Hymenochloa chinensis* were cold-extracted at least three times (preferably five times) with 90-100% (preferably 95%) ethanol. The first extraction was soaked for at least 48 hours, and the subsequent extractions were soaked for at least 24 hours each. The extracts were combined and concentrated under reduced pressure to obtain the total extract.

[0017] The total extract was dissolved in an aqueous solution of 2-8% (preferably 5%) methanol, and the aqueous solution was extracted at least three times with an equal amount of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the ethyl acetate extract fraction. The extracts were then separated and purified to obtain lignan compounds I and II.

[0018] The separation and purification steps are as follows:

[0019] The ethyl acetate extract fraction was separated by normal-phase silica gel column chromatography (200-300 mesh), and gradient elution was determined by TLC. The similar components were combined by TLC and HPLC analysis to obtain 8 components, namely components Fr. A, Fr. B, Fr. C, Fr. D, Fr. E, Fr. F, Fr. G, and Fr. H.

[0020] Fr. H was decolorized by medium-pressure preparative liquid chromatography (LC) using MCI resin column chromatography, followed by fractional isocratic elution with methanol and water. TLC and HPLC analysis were performed, and similar components were combined to obtain components Fr. H-1, Fr. H-2, Fr. H-3, Fr. H-4, and Fr. H-5. Fr. H-2 was further processed by LC coupled with normal-phase silica gel column chromatography to obtain the Fr. H-2-3 subfraction, which was then separated by semi-preparative HPLC to obtain compound I. Fr. H-3 was further processed by normal-phase silica gel column chromatography to obtain component Fr. H-3-1, which was then separated by LC on a C8 reversed-phase column (10% → 50% methanol-water gradient elution) to obtain compound II.

[0021] The gradient elution conditions determined by TLC were as follows: a petroleum ether and ethyl acetate system was used, with a petroleum ether:ethyl acetate ratio of 100:0-200:1-100:1-50:1-30:1-10:1-2:1-1:1-0:100.

[0022] The conditions for fractional isocratic elution with methanol and water are: methanol:water = 10%, 30%, 50%, 70%, 90%.

[0023] The component Fr. H-2 was obtained as a subfraction Fr. H-2-3 by medium-pressure preparative liquid chromatography coupled with normal-phase silica gel column chromatography. The conditions for medium-pressure preparative liquid chromatography were: methanol:water, volume concentration 40%. 100% gradient elution.

[0024] The component Fr. H-2 was obtained by medium-pressure preparative liquid chromatography coupled with normal-phase silica gel column chromatography to obtain the Fr. H-2-3 subfraction. The normal-phase silica gel column chromatography conditions were: dichloromethane: ethyl acetate, volume ratios of 50:1, 20:1, 10:1, and 5:1 gradient elution.

[0025] The conditions for obtaining compound I by semi-preparative HPLC separation of the component were: acetonitrile:water, volume ratio 40:60.

[0026] The conditions for obtaining component Fr. H-3-1 by normal phase silica gel column chromatography were: dichloromethane:methanol, volume ratio 20:1 → 0:100 gradient elution.

[0027] A third aspect of the present invention provides the use of the lignan compound or its pharmaceutical salt in the preparation of butyrylcholinesterase inhibitors.

[0028] In a fourth aspect, the present invention provides the use of the lignan compound or a pharmaceutical salt thereof in the preparation of a medicament for treating Alzheimer's disease.

[0029] In a fifth aspect, the present invention provides a pharmaceutical preparation made from the lignan compound or its pharmaceutical salt and medically acceptable excipients.

[0030] The dosage form of the pharmaceutical preparation is selected from suspensions, granules, capsules, tablets, powders, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0031] The drug formulation can be administered orally, intravenously, via intraperitoneal injection, intramuscular injection, subcutaneously, via sublingual injection, via transdermal administration, or via rectal suppository.

[0032] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0033] This invention systematically studied *Daphnia magna*, a plant belonging to the genus *Daphnia* of the family Thymelaeaceae, and isolated two lignan compounds from it. And (-)-syringaresinol. In vitro inhibition experiments on butyrylcholinesterase activity revealed that the lignan compounds prepared in this invention have a significant inhibitory effect on butyrylcholinesterase activity, with an IC50 value of [missing information]. 50The values ​​were 8.42±0.58 μM and 3.34±0.25 μM, respectively. Furthermore, molecular docking analysis of the two lignan compounds with butyrylcholinesterase revealed their binding free energies to BChE to be -6.674 kcal / mol and -6.045 kcal / mol, respectively. The hydroxyl group at position 4 of compound I interacts with the GLU 197 group in BChE via a hydrogen bond; the benzene ring substituent at position 1 interacts with the TRP 82 group in BChE via π-π conjugation. The hydroxyl group at position 4 of compound II interacts with the GLU 197 group in BChE via a hydrogen bond. The molecular docking results corroborate the in vitro inhibition results of butyrylcholinesterase activity, thus demonstrating the great potential of the lignan compounds prepared in this invention as butyrylcholinesterase inhibitors.

[0034] The lignan compounds prepared in this invention are derived from the plant *Daphne odora*, and lignan compounds are widely distributed in plants of the Thymelaeaceae family. From a chemical perspective, lignan compounds contain multiple benzene ring substituents. Molecular docking results show that the functional groups binding to the active groups are all located on the benzene ring substituents, facilitating further structural modification. Therefore, the compounds are diverse in origin, widely distributed, and easily modified. Secondly, the lignan compounds prepared in this invention exhibit significant butyrylcholinesterase inhibitory activity, showing good potential for development into butyrylcholinesterase inhibitor drugs. Attached Figure Description

[0035] Figure 1 IC50 assay for the in vitro inhibitory activity of lignan compound I against butyrylcholinesterase 50 A diagram showing the value results.

[0036] Figure 2 IC50 assay for the in vitro inhibitory activity of lignan compound II against butyrylcholinesterase 50 A diagram showing the value results.

[0037] Figure 3 IC50 of galantamine's in vitro inhibitory activity against butyrylcholinesterase 50 A diagram showing the value results.

[0038] Figure 4 This is a schematic diagram of the three-dimensional model structure analysis of the molecular docking of lignan compound I with butyrylcholinesterase.

[0039] Figure 5 This is a schematic diagram of the three-dimensional model structure analysis of the molecular docking of lignan compound II with butyrylcholinesterase. Detailed Implementation

[0040] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] Lignan compounds I ( ) and compound II ( Preparation of )

[0043] 1. Sample Source

[0044] Small yellow chrysanthemum (Daphnia spp., Thymelaeaceae family) The samples were from Suiyang County, Zunyi City, Guizhou Province, and were provided by Kunming Zhifen Biotechnology Co., Ltd.

[0045] 2. Methods for extraction, separation and purification of chemical components

[0046] 30 kg of dried branches and leaves of *Pleurotus ostreatus* were sliced ​​and extracted five times with 60 L of 95% ethanol. The first extraction was soaked for 48 h, and the subsequent four extractions were each soaked for 24 h. The extracts were then combined and concentrated under reduced pressure using a rotary evaporator at 60 °C to obtain 4.1 kg of total extract. The 4.1 kg of total extract was dissolved in 5 L of 5% methanol aqueous solution and extracted three times with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain 2.0 kg of ethyl acetate extract. The ethyl acetate extract was mixed with silica gel and then separated by normal-phase silica gel column chromatography (200-300 mesh). TLC was used to determine the appropriate elution method: a gradient elution system of petroleum ether and ethyl acetate (petroleum ether:ethyl acetate = 100:0-200:1-100:1-50:1-30:1-10:1-2:1-1:1-0:100). TLC and HPLC analysis yielded eight fractions: Fr. A (110.5 g), Fr. B (40.4 g), Fr. C (34.8 g), Fr. D (432.9 g), Fr. E (241.3 g), Fr. F (136.1 g), Fr. G (90.3 g), and Fr. H (145.2 g).

[0047] Fr. H was preparatively precipitated under medium pressure and decolorized using MCI resin column chromatography. Fractional isocratic elution was performed with methanol and water (methanol:water = 10%, 30%, 50%, 70%, 90%). Analysis by TLC and HPLC revealed that similar components were combined to obtain fractions Fr. H-1 (5.2 g), Fr. H-2 (20.4 g), Fr. H-3 (8.6 g), Fr. H-4 (6.5 g), and Fr. H-5 (15.4 g). Fr. H-2 was further purified by preparatively precipitated under medium pressure (methanol:water, 40% v / v). Fr. H-2-3 subfraction was obtained by coupling normal-phase silica gel column chromatography (dichloromethane:ethyl acetate, v / v ratio 50:1, 20:1, 10:1, 5:1 gradient elution) with 100% gradient elution. This subfraction was then separated by semi-preparative HPLC (acetonitrile:water, v / v ratio 40:60) to obtain compound I. Fr. H-3 was separated by normal-phase silica gel column chromatography (dichloromethane:methanol, v / v ratio 20:1→0:100 gradient elution) to obtain Fr. H-3-1, which was then separated by C8 reversed-phase column medium-pressure preparative liquid chromatography (methanol-water 10%→50% gradient elution) to obtain compound II.

[0048] 3. Determination of the structures of lignan compound I and compound II

[0049] use 1 H-NMR, 13 C-NMR and ESI-MS were used to obtain the NMR and molecular weight information of the compound. By comparing the carbon and proton NMR data with known compounds, the structure of the compound was determined as shown in Formula I and Formula II.

[0050]

[0051]

[0052]

[0053]

[0054] Example 2

[0055] In vitro inhibition assay of butyrylcholinesterase activity

[0056] 1. Experimental Materials

[0057] Automated microplate reader (Bio Tek Instruments, Inc.); 0.0001 ppm balance; CO2 incubator (Sanyo, Japan); freezer (-20 °C) (Thermo Fisher, USA); ultra-low temperature freezer (-80 °C) (Thermo Fisher, USA); benchtop centrifuge (Thermo Fisher, USA); 96-well cell culture plate (Corning, USA); electric drying oven; SDJ-DP double-person laminar flow hood; pipettes (Eppendorff); biosafety cabinet (HealForce, HFsafe-1200LC); fluorescence microscope (Leica Microsystems, Germany); butyrylcholinesterase; 5,5-dimercapto-2,2-dinitrobenzene (DTNB); Tris-HCl buffer; butyrylcholine chloride; rivastigmine; sodium dodecyl sulfate (SDS).

[0058] The lignan compounds I and II prepared in Example 1.

[0059] 2. Experimental Methods

[0060] 1 U / mL butyrylcholinesterase (BChE), 5 mM DTNB, and sample solutions (lignan compound I and compound II solutions prepared in Example 1) were added to 96-well plates containing Tris-HCl buffer and incubated at 25 °C for 15 min. Then, 1 mM butyrylcholine chloride was added, and the plates were incubated at 25 °C for another 15 min. Finally, 0.4% SDS was added to terminate the reaction. The absorbance was measured at 405 nm using an enzyme-linked immunosorbent assay (ELISA) labeler. Each group had three replicates with drug concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM. The positive control, galanthamine, was administered at concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM. The IC50 of the screened drugs was calculated based on the above experiments. 50 value.

[0061] 3. Experimental Results

[0062] The experimental results for compounds I and II are as follows: Figure 1 and Figure 2 As shown, Figure 1 IC50 assay for the in vitro inhibitory activity of lignan compound I against butyrylcholinesterase 50 A diagram showing the value results. Figure 2IC50 assay for the in vitro inhibitory activity of lignan compound II against butyrylcholinesterase 50 Schematic diagram of the results. The figure shows the in vitro inhibitory activity (IC50) of butyrylcholinesterase for compounds I and II. 50 The concentrations were 8.42 μM and 3.34 μM, respectively, indicating significant in vitro inhibitory activity. Its inhibitory effect on butyrylcholinesterase was similar to that of the positive control drug galantamine (galantamine IC50). 50 The value is 1.31 μM, see Figure 3 As shown, Figure 3 IC50 of galantamine's in vitro inhibitory activity against butyrylcholinesterase 50 (Illustrative diagram of the value results.)

[0063] Example 3

[0064] 1. Experimental Methods

[0065] Download the species from the PDB database as follows: The PDB file of the AChE / BChE crystal structure was processed using AutoDock Tools and converted into a .pdbqt file. Active site prediction was performed using Sitemap. The protein was processed using the Protein Preparation module in Schrödinger, including residue repair, hydrogen bond optimization, solvent removal, and energy minimization. The target compound's 3D structure was generated using Chemdraw, and its 3D conformation was calculated using Chem 3D. Preprocessing was performed using the LigPrep module, employing an OPLS3e force field for ionization and energy minimization. The ligand file was opened using AutoDock Tools, the atom types and charges were checked, and it was converted into a .pdbqt file. The preprocessed protein and ligand were docked using the Ligand Docking module. The prepared Grid and Lig files were selected, and standard-precision docking (SP mode) was performed using the Glide module. The docking results were obtained after clicking "Run" and finally visualized in 3D using PyMOL.

[0066] 2. Experimental Results

[0067] The experimental results for compounds I and II are as follows: Figure 4 and Figure 5 As shown, Figure 4 This is a schematic diagram of the three-dimensional model structure analysis of the molecular docking of lignan compound I with butyrylcholinesterase. Figure 5This is a schematic diagram of the three-dimensional model of molecular docking of lignan compound II with butyrylcholinesterase. The diagram shows that the hydroxyl group at position 4 of compound I interacts with the GLU 197 group in BChE via a hydrogen bond; the benzene ring substituent at position 1 interacts with the TRP 82 group in BChE via π-π conjugation. Similarly, the hydroxyl group at position 4 of compound II interacts with the GLU 197 group in BChE via a hydrogen bond. The binding free energies of compounds I and II with BChE are -6.674 kcal / mol and -6.045 kcal / mol, respectively. These characteristics indicate a significant affinity between the functional groups of the compounds and the active pocket of BChE in the molecular docking simulation.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A lignan compound, characterized in that, The structure is selected from one of the following: 。 2. A method for preparing the lignan compound according to claim 1, characterized in that, Includes the following steps: The dried branches and leaves of *Gynostemma pentaphyllum* were cold-extracted with 90-100% ethanol at least three times. The first extraction was soaked for at least 48 hours, and the subsequent extractions were soaked for at least 24 hours each. The extracts were combined and concentrated under reduced pressure to obtain the total extract. The total extract was dissolved in an aqueous solution of 2-8% methanol, and the aqueous solution was extracted at least three times with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the ethyl acetate extract fraction. The extracts were then separated and purified to obtain lignan compounds I and II. The separation and purification steps are as follows: The ethyl acetate extract fraction was separated by normal-phase silica gel column chromatography. Gradient elution was determined by TLC. The similar components were combined by TLC and HPLC analysis to obtain 8 components, namely components Fr. A, Fr. B, Fr. C, Fr. D, Fr. E, Fr. F, Fr. G, and Fr. H. Fr. H was preparatively precipitated under medium pressure and decolorized using MCI resin column chromatography, followed by fractional isocratic elution with methanol and water. TLC and HPLC analyses were performed, and similar components were combined to obtain components Fr. H-1, Fr. H-2, Fr. H-3, Fr. H-4, and Fr. H-5. Fr. H-2 was further processed by preparatively precipitated under medium pressure coupled with normal-phase silica gel column chromatography to obtain the subfraction Fr. H-2-3, which was then separated by semi-preparative HPLC to obtain compound I. Fr. H-3 was further processed by normal-phase silica gel column chromatography to obtain component Fr. H-3-1, which was then separated by preparatively precipitated under medium pressure using a C8 reversed-phase column to obtain compound II.

3. The method for preparing lignan compounds according to claim 2, characterized in that, The gradient elution conditions determined by TLC were as follows: a petroleum ether and ethyl acetate system was used, with the ratio of petroleum ether:ethyl acetate being 100:0-200:1-100:1-50:1-30:1-10:1-2:1-1:1-0:

100. The conditions for fractional isocratic elution with methanol and water are: methanol:water = 10%, 30%, 50%, 70%, 90%.

4. The method for preparing lignan compounds according to claim 2, characterized in that, The component Fr. H-2 was subjected to medium-pressure preparative liquid chromatography coupled with normal-phase silica gel column chromatography to obtain the Fr. H-2-3 subfraction. The conditions for the medium-pressure preparative liquid chromatography were: methanol:water, volume concentration 40%. 100% gradient elution; The component Fr. H-2 was obtained by medium-pressure preparative liquid chromatography coupled with normal-phase silica gel column chromatography to obtain the Fr. H-2-3 subfraction. The normal-phase silica gel column chromatography conditions were: dichloromethane: ethyl acetate, volume ratios of 50:1, 20:1, 10:1, and 5:1 gradient elution.

5. The method for preparing lignan compounds according to claim 2, characterized in that, The conditions for obtaining compound I by semi-preparative HPLC separation of the component were: acetonitrile:water, volume ratio 40:60; The conditions for obtaining component Fr. H-3-1 by normal phase silica gel column chromatography were: dichloromethane:methanol, volume ratio 20:1 → 0:100 gradient elution.

6. The use of a lignan compound of claim 1 or a pharmaceutical salt thereof in the preparation of butyrylcholinesterase inhibitors.

7. The use of the lignan compound of claim 1 or its pharmaceutical salt in the preparation of a medicament for treating Alzheimer's disease.

8. A pharmaceutical preparation, characterized in that, It is made from the lignan compound of claim 1 or its pharmaceutical salt and medically acceptable excipients.

9. The pharmaceutical preparation according to claim 8, characterized in that, The dosage form of the pharmaceutical preparation is selected from suspensions, granules, capsules, tablets, powders, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

10. The pharmaceutical preparation according to claim 8, characterized in that, The drug formulation can be administered orally, intravenously, via intraperitoneal injection, intramuscular injection, subcutaneously, via sublingual injection, via transdermal administration, or via rectal suppository.

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