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CN116284038B - Limonin compound Munronin V, preparation method, derivative, pharmaceutical composition and application thereof - Google Patents

Limonin compound Munronin V, preparation method, derivative, pharmaceutical composition and application thereof Download PDF

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CN116284038B
CN116284038B CN202211535032.2A CN202211535032A CN116284038B CN 116284038 B CN116284038 B CN 116284038B CN 202211535032 A CN202211535032 A CN 202211535032A CN 116284038 B CN116284038 B CN 116284038B
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methanol
munroninv
dichloromethane
solution
pharmaceutical composition
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CN116284038A (en
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晏英
罗荣灿
冉晓倩
姚永刚
郝小江
汤磊
王丹
彭明友
张�雄
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Kunming Institute of Zoology of CAS
Guizhou Medical University
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Guizhou Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
    • C07D493/20—Spiro-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract

The invention provides a limonin compound MunroninV, a preparation method, a derivative, a pharmaceutical composition and application thereof, and relates to the technical field of medicines. The limonin compound MunroninV with the structure shown in the formula I, the derivatives (pharmaceutically acceptable salts, esters and stereoisomers) of the limonin compound and the pharmaceutical composition all have the effect of removing pathological microtubule-associated protein Tau by activating autophagy, so that the limonin compound MunroninV has the effect of preventing and treating the onset of Alzheimer's disease, and has good value and application prospect in preparing anti-Alzheimer's disease medicines. The invention takes the coptis chinensis as the preparation raw material of limonin compound MunroninV, has simple operation, mild preparation condition, wide raw material source and low production cost, and is suitable for industrial production.

Description

Limonin compound Munronin V, preparation method, derivative, pharmaceutical composition and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a limonin compound MunroninV, a preparation method and application thereof, a limonin compound derivative and application thereof, a pharmaceutical composition and application thereof.
Background
Alzheimer's Disease (AD), commonly known as senile dementia, is one of the most common neurodegenerative diseases occurring in the elderly, and is a medical problem in all countries of the world. Our country has over ten million AD patients, the most abundant world of patients, and this number is also rapidly rising as the population ages. The "strengthening of preventive interventions such as senile disability and senile dementia" has been listed as a major task for "promoting healthy chinese construction comprehensively" in fourteen-five periods. AD is currently not a good therapeutic drug, and its care and treatment will place a heavy burden on the home and society. Based on the important role that autophagy-lysosome (ALP) defects play in the progression of AD, activation of ALP to remove aberrant protein aggregates has been considered a promising therapeutic strategy for AD. Searching for a lead compound for activating ALP is expected to promote AD drug development.
Rhizoma Coptidis (Munronia HENRYI HARMS) also called rhizoma anemones Altaicae or rhizoma Coptidis (rhizoma picrorhizae), belonging to family Meliaceae (MELIACEAE) genus Annula (Munronia), about 6 kinds. The whole plant of Coptis Fugu Linn can be used as medicine for treating traumatic injury, rheumarthritis, stomach ache, flatulence and abdominal pain, common cold and fever, malaria. However, no report on the treatment of Alzheimer's disease by Coptis Fuguchi is found in the prior art.
Disclosure of Invention
Accordingly, the invention aims to provide a limonin compound Munronin V, a preparation method, a derivative, a pharmaceutical composition and application thereof, and the limonin compound MunroninV provided by the invention has the activity of preventing and treating the onset of Alzheimer's disease.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a limonin compound MunroninV, which has a structure shown in a formula I:
The invention provides a preparation method of limonin compound Munronin V, which comprises the following steps:
(1) Reflux-extracting the whole coptis chinensis with ethanol water solution with the volume fraction of 90-95%, and collecting an extracting solution; concentrating the extracting solution to obtain an extract;
(2) Dissolving the extract, mixing the extract with 60-80 mesh silica gel, performing normal phase silica gel column chromatography solid phase extraction, eluting with petroleum ether, petroleum ether-ethyl acetate and dichloromethane-methanol respectively, and collecting 8 parts of eluates, which are named as Fr1, fr2, fr3, fr4, fr5, fr6, fr7 and Fr8 in sequence; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate is 20:1-3:1; the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol is 30:1-3:1;
(3) Performing small-pore resin chromatographic separation on the Fr5, and performing first gradient elution, wherein the first gradient elution comprises the following steps: sequentially eluting with 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, and collecting 6 eluate (Fr 5-1, fr5-2, fr5-3, fr5-4, fr5-5 and Fr 5-6);
(4) Subjecting the Fr5-4 to C18 reverse phase silica gel column chromatography, and performing second gradient elution, wherein the second gradient elution comprises the following steps: sequentially eluting with 50% methanol water solution, 70% methanol water solution, 90% methanol water solution and anhydrous methanol, collecting 5 parts of eluates, and sequentially named Fr5-4a, fr5-4b, fr5-4c, fr5-4d and Fr5-4e;
(5) Carrying out normal phase silica gel column chromatographic separation on the Fr5-4c, and carrying out third gradient elution, wherein the third gradient elution comprises the following steps: sequentially eluting with 30% of dichloromethane-methanol solution, 40% of dichloromethane-methanol solution, 50% of dichloromethane-methanol solution, 60% of dichloromethane-methanol solution and anhydrous methanol, and collecting 5 parts of eluates named Fr5-4c-1, fr5-4c-2, fr5-4c-3, fr5-4c-4 and Fr5-4c-5;
(6) Separating and concentrating Fr5-4c-3 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic liquid every 5mL, combining 22-29 parts of chromatographic liquid, and concentrating the obtained combined chromatographic liquid to obtain the limonin compound MunroninV; the conditions of Sephadex LH-20 methanol gel column chromatography include:
Eluent: analytically pure methanol;
Gel column size: 16mm x 180mm;
Flow rate of the eluent: 0.3mL/min;
The eluent dosage is as follows: 1.2L.
Preferably, in step (1), the number of times of reflux extraction is 3, and the time of single reflux extraction is 2h.
The invention provides a limonin compound derivative, which comprises pharmaceutically acceptable salts, esters or stereoisomers of limonin compound MunroninV; the limonoid MunroninV is limonoid MunroninV described in the above technical scheme or limonoid MunroninV prepared by the preparation method described in the above technical scheme.
The invention provides a pharmaceutical composition, which is characterized by comprising a pharmaceutical active ingredient and pharmaceutically acceptable auxiliary materials;
The pharmaceutical active ingredient comprises limonoid Munronin V in the technical scheme, limonoid Munronin V prepared by the preparation method in the technical scheme and one or more limonoid derivatives in the technical scheme.
Preferably, the mass fraction of the active ingredients in the medicine composition is 0.1-99%.
Preferably, the dosage form of the medicament comprises a liquid formulation, a solid formulation, a spray or an aerosol.
The invention also provides application of the limonoid MunroninV in the technical scheme, the limonoid MunroninV prepared by the preparation method in the technical scheme, the limonoid derivative in the technical scheme or the pharmaceutical composition in the technical scheme in preparation of anti-Alzheimer disease drugs.
The limonin compound MunroninV with the structure shown in the formula I can clear pathological microtubule-related protein Tau by activating TFEB (transcription factor EB) -mediated autophagy and inhibit Tau pathology, so that the limonin compound MunroninV has the activity of preventing and treating the onset of Alzheimer's disease, and has good value and application prospect in preparing anti-Alzheimer's disease medicines.
The invention provides a preparation method of limonin compound MunroninV. The preparation method provided by the invention uses the rehmannia glutinosa Libosch as a preparation raw material, is simple to operate, mild in preparation condition, wide in raw material source, low in production cost and suitable for industrial production.
The limonin compound derivative (pharmaceutically acceptable salt of limonin compound Munronin V, ester of limonin compound MunroninV and stereoisomer of limonin compound Munronin V) and the pharmaceutical composition provided by the invention enable the limonin compound MunroninV to be capable of clearing pathological microtubule-associated protein Tau by activating TFEB (transcription factor EB) -mediated autophagy, inhibiting accumulation of Tau pathology, having activity of preventing and treating Alzheimer disease, and having good value and application prospect in preparing anti-Alzheimer disease drugs.
Drawings
FIG. 1 is a 1 HNMR spectrum of limonoids MunroninV;
FIG. 2 is a 13 CNMR profile of limonoids MunroninV;
FIG. 3 is a HSQC spectrum of limonoids MunroninV;
FIG. 4 is a HMBC spectrum of limonoids MunroninV;
FIG. 5 is a 1H-1 H COSY spectrum of limonoids MunroninV;
FIG. 6 is a ROESY spectrum of limonoids MunroninV;
FIG. 7 is a HRESIMS pattern of limonoids MunroninV;
FIG. 8 is an IR spectrum of limonoids MunroninV;
FIG. 9 is a UV spectrum of limonoids MunroninV;
FIG. 10 shows results of the limonoid MunroninV in promoting autophagy, wherein A is the results of flow cytometry analysis of HM-mCherry-GFP-LC3 cells treated with limonoid MunroninV, calculating the percentage of 10000 cells expressing GFP or/and mCherry protein, rapamycin (Rap) as positive control; b is to quantify only mCherry point cells in A, rap is positive control, bafilomycinA1 (BAFA 1) is negative control; representative images of HM MCHERRY-GFP-LC3 cells treated with limonoid MunroninV at different concentrations at a scale of 10 microns. D is the statistical result of C;
FIG. 11 is a graph showing the results of the removal of Tau protein pathology by the promotion of autophagy by limonoid MunroninV, wherein A is the morphology of U251-MAPTP S cells treated with limonoid MunroninV (2.5. Mu.M, 10. Mu.M and 40. Mu.M) or not with limonoid MunroninV for 24 hours; B-E are Western blotting results and quantification results of protein levels of Tau P301S, LC-II/LC 3-I, SQSTM, TFEB, P-TFEB (Ser 122) and CTSB in U251-MAPT P301S cells;
FIG. 12 is a graph showing the results of limonoids MunroninV in promoting the nucleation of TFEB, wherein A is a representative image of HM-TFEB-GFP cells treated with limonoids MunroninV at different concentrations, and B is a quantification of the proportion of TFEB in the nucleus.
Detailed Description
The invention provides a limonin compound MunroninV, which has a structure shown in a formula I:
The invention provides a preparation method of limonin compound Munronin V, which comprises the following steps:
(1) Reflux-extracting the whole coptis chinensis with ethanol water solution with the volume fraction of 90-95%, and collecting an extracting solution; concentrating the extracting solution to obtain an extract;
(2) Dissolving the extract, mixing the extract with 60-80 mesh silica gel, performing normal phase silica gel column chromatography solid phase extraction, eluting with petroleum ether, petroleum ether-ethyl acetate and dichloromethane-methanol respectively, and collecting 8 parts of eluates, which are named as Fr1, fr2, fr3, fr4, fr5, fr6, fr7 and Fr8 in sequence; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate is 20:1-3:1; the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol is 30:1-3:1;
(3) Performing small-pore resin chromatographic separation on the Fr5, and performing first gradient elution, wherein the first gradient elution comprises the following steps: sequentially eluting with 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, and collecting 6 eluate (Fr 5-1, fr5-2, fr5-3, fr5-4, fr5-5 and Fr 5-6);
(4) Subjecting the Fr5-4 to C18 reverse phase silica gel column chromatography, and performing second gradient elution, wherein the second gradient elution comprises the following steps: sequentially eluting with 50% methanol water solution, 70% methanol water solution, 90% methanol water solution and anhydrous methanol, collecting 5 parts of eluates, and sequentially named Fr5-4a, fr5-4b, fr5-4c, fr5-4d and Fr5-4e;
(5) Carrying out normal phase silica gel column chromatographic separation on the Fr5-4c, and carrying out third gradient elution, wherein the third gradient elution comprises the following steps: sequentially eluting with 30% of dichloromethane-methanol solution, 40% of dichloromethane-methanol solution, 50% of dichloromethane-methanol solution, 60% of dichloromethane-methanol solution and anhydrous methanol, and collecting 5 parts of eluates named Fr5-4c-1, fr5-4c-2, fr5-4c-3, fr5-4c-4 and Fr5-4c-5;
(6) Separating and concentrating Fr5-4c-3 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic liquid every 5mL, combining 22-29 parts of chromatographic liquid, and concentrating the obtained combined chromatographic liquid to obtain the limonin compound MunroninV; the conditions of Sephadex LH-20 methanol gel column chromatography include:
Eluent: analytically pure methanol;
Gel column size: 16mm x 180mm;
Flow rate of the eluent: 0.3mL/min;
The eluent dosage is as follows: 1.2L.
In the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise.
The invention adopts ethanol water solution with the volume fraction of 90-95 percent to reflux and extract the whole coptis root of the double leaf, and collects the extracting solution; concentrating the extract to obtain extract.
In the invention, the whole plant of the Coptis multocida is preferably obtained from Xingyi City of Guizhou province. In the invention, the whole golden thread of the double leaf is preferably dried and crushed before use, obtaining the coptis root powder of the compound leaf; the invention has no special limitation on the temperature and time of the drying, and the drying conditions of the coptis chinensis in the field are adopted; the invention is not particularly limited to the pulverization, and the pulverization is carried out until the particle size of the obtained Coptis chinensis powder is 80-100 meshes.
In the present invention, the temperature of the reflux extraction is preferably 60 ℃; the times of reflux extraction are preferably 3 times, and the time of single reflux extraction is preferably 2 hours; the amount of the ethanol aqueous solution is not particularly limited, and the amount of the ethanol aqueous solution for extracting the whole golden thread of the compound leaf, which is known to a person skilled in the art, is only required, and in the specific embodiment of the invention, the volume of the ethanol aqueous solution adopted for each reflux extraction is 15L based on the mass of the golden thread powder of the compound leaf of 9 kg. In the present invention, the volume fraction of ethanol in the aqueous ethanol solution is preferably 91 to 94%, more preferably 92 to 93%.
The conditions for the concentration are not particularly limited, and the concentration conditions well known to those skilled in the art may be employed.
After the extractum is obtained, the extractum is dissolved and then mixed with 60-80 meshes of silica gel, normal phase silica gel column chromatography solid phase extraction is carried out, petroleum ether-ethyl acetate and methylene dichloride-methanol are respectively used for eluting, 8 parts of eluent are obtained, and the eluent are named as Fr1, fr2, fr3, fr4, fr5, fr6, fr7 and Fr8 in sequence; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate is 20:1-3:1; the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol is 30:1-3:1.
In the invention, the dosage of each eluent is preferably 8L in the solid phase extraction process of the normal phase silica gel column chromatography; the flow rate of the eluent is preferably 10mL/min.
After Fr5 is obtained, the Fr5 is subjected to small-pore resin chromatographic separation, and the first gradient elution is carried out according to the following procedures: sequentially eluting with 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, and collecting 6 eluate, which are named Fr5-1, fr5-2, fr5-3, fr5-4, fr5-5 and Fr5-6.
In the invention, the purpose of the small pore resin chromatographic separation is to carry out preliminary refining on the Fr5 rich in limonoid compounds, fr5-5 and Fr5-6 are rich in residual chlorophyll and other small polar compounds, and Fr5-4 is rich in limonoid, lignin and other types of compounds.
After Fr5-4 is obtained, the Fr5-4 is subjected to C18 reverse phase silica gel column chromatographic separation, and the second gradient elution is carried out according to the following procedures: sequentially eluting with 50% methanol water solution, 70% methanol water solution, 90% methanol water solution and anhydrous methanol, and collecting 5 parts of eluates named Fr5-4a, fr5-4b, fr5-4c, fr5-4d and Fr5-4e.
In the present invention, the mass ratio of the components Fr5-4 to the C18 reverse phase silica gel is preferably 1:50.
In the present invention, the amount of each eluent used in the second gradient elution is preferably 7L; the flow rate of the eluent is preferably 8mL/min.
In the invention, the purpose of the C18 reverse phase silica gel column chromatographic separation is to distinguish limonin and lignin compounds, and further realize the enrichment of target limonin compounds.
After Fr5-4c is obtained, normal phase silica gel column chromatography separation is carried out on the Fr5-4c, and the third gradient elution is carried out according to the following procedures: eluting sequentially with 30% of dichloromethane-methanol solution, 40% of dichloromethane-methanol solution, 50% of dichloromethane-methanol solution, 60% of dichloromethane-methanol solution and anhydrous methanol, and collecting 5 parts of eluates named Fr5-4c-1, fr5-4c-2, fr5-4c-3, fr5-4c-4 and Fr5-4c-5.
In the invention, the purpose of the normal phase silica gel column chromatography separation is to carry out preliminary refining on Fr5-4c fractions rich in limonoid compounds and lignin compounds, fr5-4c-1 and Fr5-4c-2 are rich in residual chlorophyll and other small polar compounds, fr5-4c-4 and Fr5-4c-5 are rich in limonin with larger polarity and other types of compounds such as lignin; fr5-4c-3 contains limonin compound at high concentration.
After Fr5-4c-3 is obtained, separating and concentrating the Fr5-4c-3 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic liquid every 5mL, combining 22-29 parts of chromatographic liquid, and concentrating the obtained combined chromatographic liquid to obtain the limonin compound MunroninV; the conditions of Sephadex LH-20 methanol gel column chromatography include:
Eluent: analytically pure methanol;
Gel column size: 16mm x 180mm;
Flow rate of the eluent: 0.3mL/min;
The eluent dosage is as follows: 1.2L.
In the invention, ephadex LH-20 methanol gel chromatography is mainly separated according to the difference of molecular weight, plant secondary metabolites with the molecular weight larger than 600 flow out before 20 parts of chromatographic liquid, plant secondary metabolites with the molecular weight smaller than 450 flow out after 30 parts of chromatographic liquid, and 22-29 parts of chromatographic liquid are mainly used for enriching the secondary metabolites with the molecular weight between 450 and 600, thus obtaining the limonin compound MunroninV.
The invention provides a limonin compound derivative, which comprises pharmaceutically acceptable salts, esters or stereoisomers of limonin compound MunroninV; the limonoid MunroninV is limonoid MunroninV described in the above technical scheme or limonoid MunroninV prepared by the preparation method described in the above technical scheme. The pharmaceutically acceptable salts, esters and stereoisomers of the limonoids Munronin V are not particularly limited, and pharmaceutically acceptable salts, esters and stereoisomers well known to those skilled in the art are employed.
The invention provides a pharmaceutical composition, which comprises a pharmaceutical active ingredient and pharmaceutically acceptable auxiliary materials; the pharmaceutical active ingredient comprises limonoid Munronin V in the technical scheme, limonoid MunroninV prepared by the preparation method in the technical scheme and one or more limonoid derivatives in the technical scheme; the mass fraction of the pharmaceutical active ingredient in the pharmaceutical composition is preferably 0.1-99%, more preferably 1-50%.
In the present invention, the pharmaceutically acceptable auxiliary materials preferably include one or more of solid auxiliary materials, semisolid auxiliary materials and liquid auxiliary materials. In the present invention, the dosage form of the pharmaceutical composition preferably includes a liquid formulation, a solid formulation, a spray or an aerosol. In the present invention, the liquid preparation preferably includes an injection, a suspension, an emulsion, a solution or a syrup; when the dosage form is a liquid formulation, the pharmaceutically acceptable excipients preferably include one or more of a dispersing medium, a suspending agent, a wetting agent, a flocculating agent and a deflocculating agent. In the present invention, the solid preparation preferably includes a tablet, a capsule, a granule or a granule; when the dosage form is a tablet, the pharmaceutically acceptable excipients preferably include one or more of fillers, adsorbents, binders, lubricants, dispersants, wetting agents, and disintegrants; when the dosage form is a capsule, the pharmaceutically acceptable excipients preferably comprise a capsule material; when the dosage form is a granule, the pharmaceutically acceptable auxiliary materials preferably comprise one or more of diluents, absorbents, wetting agents, binders and flavoring agents; when the dosage form is a granule, the pharmaceutically acceptable auxiliary materials preferably comprise one or more of an excipient and a flavoring agent.
In the present invention, the route of administration of the pharmaceutical composition preferably includes injection, oral administration, sublingual administration or mucosal dialysis; the injection preferably comprises intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection.
The invention provides application of the limonoid MunroninV in the technical scheme, the limonoid MunroninV prepared by the preparation method in the technical scheme, the limonoid derivative in the technical scheme or the pharmaceutical composition in the technical scheme in preparation of anti-Alzheimer disease drugs. In the present invention, the application is preferably: limonin compound MunroninV can remove pathological microtubule-associated protein Tau by activating TFEB (transcription factor EB) -mediated autophagy, inhibit accumulation of Tau pathology, and have activity of preventing and treating Alzheimer disease.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Drying and crushing the whole plant of Coptis chinensis Franch (collected from Guizhou Xingyi) to obtain whole plant powder of Coptis chinensis Franch with the particle size of 80-100 meshes; mixing the whole coptis root powder of the double-leaf rehmannia and an ethanol water solution with the volume fraction of 90-95%, carrying out reflux extraction for 3 times at the temperature of 60 ℃, merging the extracting solutions, and concentrating to obtain an extract; the mass of the golden thread whole plant powder is 9kg, the volume of ethanol water solution adopted by each reflux extraction is 15L, and the mass of the extract is 1.2kg.
(2) Dissolving the extract, mixing the extract with 60-80 mesh silica gel, performing normal phase silica gel column chromatography solid phase extraction, eluting with petroleum ether, petroleum ether-ethyl acetate (20:1-3:1) and dichloromethane-methanol (30:1-3:1) respectively, and collecting 8 parts of eluents, which are named as Fr1, fr2, fr3, fr4, fr5, fr6, fr7 and Fr8 in sequence; the mass ratio of the extract to the silica gel for sample mixing is 1:3, and the mass ratio of the extract to the separation silica gel (100-200 meshes) is 1:12; the amount of each eluent was 8L, and the flow rate of the eluent was 10mL/min.
(3) Performing small-pore resin chromatographic separation on the Fr5, and performing first gradient elution, wherein the first gradient elution comprises the following steps: sequentially eluting with 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, and collecting 6 eluate (Fr 5-1, fr5-2, fr5-3, fr5-4, fr5-5 and Fr 5-6); the purpose of the small pore resin column chromatography is to carry out preliminary refining on the fraction Fr5 rich in limonoid compounds, fr5-5 and Fr5-6 are rich in residual chlorophyll and other small polar compounds, and Fr5-4 is rich in limonoid, lignin and other types of compounds.
(4) Performing C18 reverse phase silica gel column chromatographic separation on the Fr5-4, and performing second gradient elution, wherein the second gradient elution comprises the following steps: sequentially eluting with 50% methanol water solution, 70% methanol water solution, 90% methanol water solution and anhydrous methanol, collecting 5 parts of eluates, and sequentially named Fr5-4a, fr5-4b, fr5-4c, fr5-4d and Fr5-4e; wherein the mass ratio of Fr5-4 to C18 reverse phase silica gel is 1:50; the amount of each eluent was 7L, and the flow rate of the eluent was 8mL/min.
(5) Carrying out normal phase silica gel column chromatographic separation on the Fr5-4c, and carrying out third gradient elution, wherein the third gradient elution comprises the following steps: eluting sequentially with 30% of dichloromethane-methanol solution, 40% of dichloromethane-methanol solution, 50% of dichloromethane-methanol solution, 60% of dichloromethane-methanol solution and pure methanol, and collecting 5 parts of eluates named Fr5-4c-1, fr5-4c-2, fr5-4c-3, fr5-4c-4 and Fr5-4c-5. The purpose of normal phase silica gel column chromatography is to carry out preliminary refining on fractions rich in limonin and lignin compounds Fr5-4c, fr5-4c-1 and Fr5-4c-2 are rich in residual chlorophyll and other small polar compounds, fr5-4c-4 and Fr5-4c-5 are rich in limonin with larger polarity and other types of compounds such as lignin; fr5-4c-3 increases the concentration of limonin compounds.
(6) Separating and concentrating Fr5-4c-3 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic liquid every 5mL, combining 22-29 parts of chromatographic liquid, and concentrating to constant weight to obtain limonin compound Munronin V (abbreviated as compound 1) shown in formula I; the conditions of Sephadex LH-20 methanol gel column chromatography include:
Eluent: analytically pure methanol;
Gel column size: 16mm x 180mm;
Flow rate of the eluent: 0.3mL/min;
The eluent dosage is as follows: 1.2L.
Structural characterization of limonoids Munronin V:
Fig. 1 is a 1 H NMR spectrum of limonoid MunroninV, fig. 2 is a 13 C NMR spectrum of limonoid MunroninV, fig. 3 is a HSQC spectrum of limonoid Munronin V, fig. 4 is a HMBC spectrum of limonoid MunroninV, fig. 5 is a 1H-1 H COSY spectrum of limonoid MunroninV, fig. 6 is a roosy spectrum of limonoid Munronin V, fig. 7 is a hresis spectrum of limonoid Munronin V, fig. 8 is an IR spectrum of limonoid MunroninV, and fig. 9 is a UV spectrum of limonoid MunroninV.
TABLE 1 1 H NMR (600M) and 13 C NMR (150M) data for limonoids MunroninV (CDCl 3, delta units: ppm, J units: hz)
As can be seen from table 1 and fig. 1 to 9, the structure data of limonoids MunroninV are as follows:
[ alpha ] 2 D 1 +7.89 (c 0.14, meOH), white amorphous powder, dark spots under irradiation of ultraviolet 254nm, no fluorescence under irradiation of 365nm, and black gray color after spraying 8% ethanol-concentrated sulfuric acid. High resolution mass spectrometry: m/z621.2677[ M+Na ] +,(cald for C33H42O10 Na, 621.2670), the molecular weight was determined to be 598, the molecular formula was C 33H42O10, and the unsaturation degree was 13. 1H-NMR、13 C-NMR and HSQC data showed 8 methyl [δH 1.17(3H,s),δC 15.4;δH 1.23(3H,s),δC 14.7;δH 1.67(3H,s),δC 34.4;δH 1.47(3H,s),δC 23.5;δH 1.49(3H,s),δC 19.9;δH 1.72(3H,d,J＝7.2Hz),δC 14.7;δH 1.81(3H,s),δC 12.2;δH 1.85(3H,s),δC 20.9],4 methylene groups, 10 methine groups [δH 5.00(1H,dd,J＝3.2,11.8Hz),δC 71.3;δH 2.56(1H,brd,J＝11.8Hz),δC 47.9;δH3.12(1H,brd,J＝15.2Hz),δC 45.8;δH 5.17(1H,brd,J＝10.8Hz),δC 76.3;δH 3.63(1H,s),δC 56.0;δH 2.84(1H,dd,J＝5.8,12.2Hz),δC 42.5;δH 7.07(1H,s),δC 140.0;δH 5.97(1H,s),δC 111.4;δH 7.30(1H,t,J＝6.2Hz),δC 142.8;δH 6.30(1H,q,J＝5.6Hz),δC136.8],11 quaternary carbons. Further analysis of the one-dimensional data suggests the presence of four ester carbonyl groups (δ C 169.2, 172.1, 169.1, 168.3), 1 substituted double bond (δ C 129.5, 136.8), 1 β -furan ring (δ H 7.07,5.97,7.30,δC 122.0, 140.0, 111.4, 142.8) in the molecule. The removal of the four carbonyl groups, three double bonds, 8 unsaturations occupied by one epoxy group, and the remaining 5 unsaturations suggest that the compound is a pentacyclic system. The compound is shown to be limonin compound MunroninV with a structure shown in a formula I. The planar structure of the compound was finally confirmed by two-dimensional nuclear magnetic techniques (HSQC, HMBC and 1H-1 H COSY). The steric structure was initially identified by ROESY and finally calculated by ECD, including confirmation of its absolute configuration.
Test example 1
Activity evaluation
(1) Construction of U251 cells stably expressing mutant MAPTP S (MAPTMUT) Gene
The CDS region of MAPT gene with tag was cloned into PLVX vector of Lenti-X Tet-On advanced inducible expression System (Clontech). By using the site-directed mutagenesis PCR method, MAPTP S mutations were introduced into PLVX-MAPTP S vectors, respectively.
U251 cells were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS). U251 cells stably expressing mutant MAPT P301S were constructed according to the instructions of the Lenti-X Tet-On advanced inducible expression System (Clontech). The responsive lentiviral system consisted of mutant PLVX-MAPT construct, packaging plasmid psPAX (Addgene, england, 12260) and envelope plasmid PMD2.G (Addgene, england, 12259), whereas the regulated lentiviral system consisted of PLVX-Tet-On-Advanced vector, psPAX2 and PMD2.G. Lentiviral supernatant from HEK293T cells for infection of U251 cells, the ratio of responsive lentiviral to regulator lentiviral was 4:1. Cells were selected in growth medium containing 1. Mu.g/mL puromycin.
(2) Construction of HM cells with stable expression of TFEB Gene
The CDS region of the TFEB gene with the tag label was cloned into PLVX vectors. The HM cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS). The response lentiviral system consisted of PLVX-TFEB construct, packaging plasmid psPAX (adedge, england, 12260) and envelope plasmid pmd2.G (adedge, england, 12259), and lentiviral supernatants of psPAX and pmd2.G were produced by HEK293T cells for infection of HM cells at a 4:1 ratio of response lentivirus to regulator lentivirus. Cells were selected in growth medium containing and 1. Mu.g/mL puromycin.
(3) Flow cytometer analysis
HM MCHERRY-GFP-LC3 cells stably expressed triple fusion proteins (red fluorescent protein (mCherry), green Fluorescent Protein (GFP) and autophagosome marker LC 3), and can directly reflect the intensity of autophagy flux for detecting the biological activity of compound 1. HM MCHERRY-GFP-LC3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 37℃incubator, 5% carbon dioxide and 95% humidity. HM MCHERRY-GFP-LC3 cells were cultured in 12-well plates and the compounds were added directly to the culture (10. Mu.M and 40. Mu.M). After 24 hours, cells were harvested, fixed with 4% pfa (paraformaldehyde) and then examined by flow cytometry to check if autophagy flux was enhanced. Data was analyzed using FlowJo software (FlowJo, LLC).
(4) Western blotting analysis (Western blotting)
Cell lysates were prepared using protein lysis buffer (Beyotime institute of biotechnology, P0013). Protein concentration was determined using BCA protein assay kit (Beyotime institute of biotechnology, P0012). About 20. Mu.g of total protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, L1620177Rev D). The membrane was blocked with 5% (w: v) skim milk for 2 hours at room temperature. The membranes were incubated with the target Tau(1:1000,cell signaling technology,46687S)、TFEB(1:1000;cell signaling technology,4240S)、p-TFEB Ser122(1:1000;cell signalingtechnology,86843S),CTSB(1：1000;Affinity,AF5189),SQSTM1(1：1000,Elabscience,E-AB-62289),LC3(1：1000,Proteintech,14600-1-AP),GAPDH(1：20000,Affinity,AF7021), overnight at 4 ℃. The membrane was then washed 3 times with TBST(Tris buffered saline[Servicebio,G0001]with0.1％Tween 20[Sangon Biotech(Shanghai)Co.,Ltd,HB09BA0007]) minutes each, and then incubated with peroxidase-conjugated anti-mouse (KPL; 474-1806; 1:10000) or anti-rabbit IgG (KPL; 474-1516;1:10000; KPL) for 1 hour at room temperature. The target protein was detected using ECLWESTERN BLOT detection kit (Millipore, WBKLS 0500). The Westernblot of GAPDH was used as an internal control to measure protein levels of target genes. Density measurements of the target proteins were assessed by imageJ software (National Institutes ofHealth, bethesda, maryland, USA).
FIG. 10 shows results of the limonoid MunroninV in promoting autophagy, wherein A is the results of flow cytometry analysis of HM-mCherry-GFP-LC3 cells treated with limonoid MunroninV, calculating the percentage of 10000 cells expressing GFP or/and mCherry protein, rapamycin (Rap) as positive control; b is to quantify only mCherry point cells in A, rap is positive control, bafilomycinA1 (BAFA 1) is negative control; representative images of HM MCHERRY-GFP-LC3 cells treated with limonoid MunroninV at different concentrations at a scale of 10 microns; d is the statistical result of C.
FIG. 11 is a graph showing the results of the removal of Tau protein pathology by the promotion of autophagy by limonoid MunroninV, wherein A is the morphology of U251-MAPTP S cells treated with limonoid MunroninV (2.5. Mu.M, 10. Mu.M and 40. Mu.M) or not with limonoid MunroninV for 24 hours; B-E are Western blot results showing protein levels of Tau P301S, LC-II/LC 3-I, SQSTM, TFEB, P-TFEB (Ser 122) and CTSB in U251-MAPT P301S cells, data shown are representative Western blot results (B and D) and quantification of protein levels (E and G) based on 3 independent experiments; c is Westernbolt statistics.
FIG. 12 is a graph showing the results of limonoids MunroninV in promoting the nucleation of TFEB, wherein A is a representative image of HM-TFEB-GFP cells treated with limonoids MunroninV at different concentrations, and B is a quantification of the proportion of TFEB in the nucleus.
As shown in figures 10-12, the limonin compound MunroninV with the structure shown in the formula I can inhibit Tau pathology by activating autophagy mediated by TFEB (transcription factor EB), and shows that the limonin compound has the activity of preventing and treating the onset of Alzheimer's disease.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (7)

1. A limonoid Munronin V or a pharmaceutically acceptable salt thereof, the limonoid Munronin V having a structure shown in formula I:
2. the method for preparing limonin compound MunroninV as claimed in claim 1, comprising the steps of:
(1) Reflux-extracting the whole coptis chinensis with ethanol water solution with the volume fraction of 90-95%, and collecting an extracting solution; concentrating the extracting solution to obtain an extract;
(2) Dissolving the extract, mixing the extract with 60-80 mesh silica gel, performing normal phase silica gel column chromatography solid phase extraction, eluting with petroleum ether, petroleum ether-ethyl acetate and dichloromethane-methanol respectively, and collecting 8 parts of eluates, which are named as Fr1, fr2, fr3, fr4, fr5, fr6, fr7 and Fr8 in sequence; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate is 20:1-3:1; the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol is 30:1-3:1;
(3) Performing small-pore resin chromatographic separation on the Fr5, and performing first gradient elution, wherein the first gradient elution comprises the following steps: sequentially eluting with 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, and collecting 6 eluate (Fr 5-1, fr5-2, fr5-3, fr5-4, fr5-5 and Fr 5-6);
(4) Subjecting the Fr5-4 to C18 reverse phase silica gel column chromatography, and performing second gradient elution, wherein the second gradient elution comprises the following steps: sequentially eluting with 50% methanol water solution, 70% methanol water solution, 90% methanol water solution and anhydrous methanol, collecting 5 parts of eluates, and sequentially named Fr5-4a, fr5-4b, fr5-4c, fr5-4d and Fr5-4e;
(5) Carrying out normal phase silica gel column chromatographic separation on the Fr5-4c, and carrying out third gradient elution, wherein the third gradient elution comprises the following steps: sequentially eluting with 30% of dichloromethane-methanol solution, 40% of dichloromethane-methanol solution, 50% of dichloromethane-methanol solution, 60% of dichloromethane-methanol solution and anhydrous methanol, and collecting 5 parts of eluates named Fr5-4c-1, fr5-4c-2, fr5-4c-3, fr5-4c-4 and Fr5-4c-5;
(6) Separating and concentrating Fr5-4c-3 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic liquid every 5mL, combining 22-29 parts of chromatographic liquid, and concentrating the obtained combined chromatographic liquid to obtain the limonin compound MunroninV; the conditions of Sephadex LH-20 methanol gel column chromatography include:
Eluent: analytically pure methanol;
Gel column size: 16mm x 180mm;
Flow rate of the eluent: 0.3mL/min;
The eluent dosage is as follows: 1.2L.
3. The method according to claim 2, wherein in the step (1), the number of times of the reflux extraction is 3, and the time of the single reflux extraction is 2 hours.
4. A pharmaceutical composition, which is characterized by comprising a pharmaceutical active ingredient and pharmaceutically acceptable auxiliary materials;
The pharmaceutically active ingredient is limonoid Munronin V and/or pharmaceutically acceptable salt thereof according to claim 1.
5. The pharmaceutical composition according to claim 4, wherein the mass fraction of the pharmaceutically active ingredient in the pharmaceutical composition is 0.1-99%.
6. The pharmaceutical composition according to claim 4 or 5, wherein the dosage form of the drug is selected from the group consisting of a liquid formulation, a solid formulation, a spray or an aerosol.
7. Use of limonin compound Munronin V of claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of any one of claims 4-6 in the preparation of an anti-alzheimer's disease medicament.
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Munronin V with 7/7/6 tricarbocyclic framework from Munronia henryi harms inhibits tau pathology by activating autophagy;Yan, Ying等;《Organic & Biomolecular Chemistry》;20221206;第21卷(第3期);第514-519页 *

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