CN112592351B

CN112592351B - Eleusine indica trilactone A, medicine for treating Alzheimer disease, preparation method and application

Info

Publication number: CN112592351B
Application number: CN202011432002.XA
Authority: CN
Inventors: 郝小江; 姚永刚; 邸迎彤; 罗荣灿; 唐小涵
Original assignee: Kunming Institute of Botany of CAS; Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Botany of CAS; Kunming Institute of Zoology of CAS
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2022-02-22
Anticipated expiration: 2040-12-07
Also published as: CN112592351A

Abstract

The invention provides a gooseberry trilactone A, a medicine for treating Alzheimer disease, a preparation method and application, and belongs to the technical field of medicines; the tinospora edulis trilactone A disclosed by the invention can influence the metabolic pathway of Amyloid Precursor Protein (APP), can reduce the expression capacity of beta-amyloid A beta 42 and A beta 40, can promote the conversion of APP to a non-A beta metabolic pathway, shows a certain capacity of resisting the generation of beta-amyloid A beta, and has the activity of preventing and treating the Alzheimer's disease.

Description

Eleusine indica trilactone A, medicine for treating Alzheimer disease, preparation method and application

Technical Field

The invention relates to the technical field of medicines, in particular to a gooseberry trilactone A, a medicine for treating Alzheimer disease, a preparation method and an application.

Background

Harrisonia perforata (Bl.) Merr. is a plant of the genus Harrisonia of the family Simaroubaceae (Simaruceae), which is a global plant of four species, all distributed in tropical Africa, tropical Asia and oceania. In China, gooseberry is mainly distributed in Fujian, Guangdong and Hainan provinces; it is common in shrubs and spars at low altitudes. Gooseberry has the effects of clearing heat and preventing malaria, is bitter in taste and cold in nature and is commonly used for malaria. However, to date, no reports of the gooseberry trilactone a compound and its activity have been found in the prior art.

Disclosure of Invention

The invention aims to provide the tinospora edulis trilactone A, the medicine for treating the Alzheimer disease and the preparation method and the application thereof, wherein the tinospora edulis trilactone A can influence the metabolic pathway of Amyloid Precursor Protein (APP), not only can reduce the expression capacity of beta-amyloid protein Abeta 42 and Abeta 40, but also can promote the conversion of the APP to non-Abeta metabolic pathways, shows a certain capacity of resisting the generation of the beta-amyloid protein Abeta, and has the activity of preventing and treating the Alzheimer disease.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a gooseberry trilactone A shown in a structure of a formula I;

the invention also provides a preparation method of the gooseberry trilactone A in the scheme, which comprises the following steps:

1) ethanol water solution with the volume fraction of 90-95% is adopted to carry out reflux extraction on the gooseberry branches, and the extracting solution is collected; concentrating the extracting solution to obtain an extract; re-suspending the extract in water to obtain a crude extract;

2) carrying out first extraction on the crude extract by adopting petroleum ether, removing the petroleum ether extraction part, and collecting residues; performing second extraction on the remainder by using ethyl acetate, and collecting an ethyl acetate part to obtain an ethyl acetate extract;

3) subjecting the ethyl acetate extract to small pore resin chromatography with a first gradient elution, wherein the first gradient elution is performed by the following procedure: sequentially eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, collecting 5 eluates, and sequentially naming as Fr1, Fr2, Fr3, Fr4 and Fr 5;

4) subjecting said Fr4 to normal phase silica gel column chromatography with a second gradient elution, said second gradient elution being performed by the procedure: sequentially eluting by using a petroleum ether-ethyl acetate solution with the volume fraction of 10%, a petroleum ether-ethyl acetate solution with the volume fraction of 20%, a petroleum ether-ethyl acetate solution with the volume fraction of 30%, a petroleum ether-ethyl acetate solution with the volume fraction of 40% and pure ethyl acetate, and collecting and obtaining 5 parts of eluents which are sequentially named as Fr4-1, Fr4-2, Fr4-3, Fr4-4 and Fr 4-5;

5) subjecting said Fr4-2 to C18 reverse phase silica gel column chromatography with a third gradient elution, said third gradient elution being performed by the procedure: sequentially eluting with 30% methanol aqueous solution, 40% methanol aqueous solution, 45% methanol aqueous solution, 50% methanol aqueous solution and anhydrous methanol, collecting 5 eluates, and sequentially naming as Fr4-2-1, Fr4-2-2, Fr4-2-2, Fr4-2-4 and Fr 4-2-5;

6) separating and concentrating the Fr4-2-4 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic solution per 5mL, and combining 30-35 parts of chromatographic solution to obtain combined chromatographic solution; the Sephadex LH-20 methanol gel column chromatography conditions include:

gel column size: 20mm by 200 mm;

the flow rate of the eluent is 0.3 mL/min;

the eluent is absolute methanol;

the dosage of the eluent is as follows: 1L;

7) performing semi-preparative high performance liquid chromatography separation on the combined chromatographic solution, eluting by using an acetonitrile aqueous solution with the volume fraction of 32%, and collecting an eluent of 20.1-21.1 min, wherein the eluent contains eleusine indica trilactone A;

the conditions for the semi-preparative high performance liquid chromatography in step 7) include:

a chromatographic column: waters XSelect CSH C-18column (5 μm, 10X 250 mm);

the mobile phase comprises: mobile phase a and mobile phase B; the mobile phase A is purified water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 68%; the volume fraction of the mobile phase B is 32%;

the flow rate of the mobile phase is 3.0 mL-min^-1；

The column temperature was 25 ℃;

the amount of sample was 30. mu.L each time.

The invention also provides a medicine for treating the Alzheimer disease, which comprises the elactonia solani or the pharmaceutically acceptable salt, ester or stereoisomer thereof.

Preferably, the mass fraction of the gooseberry trilactone A or the pharmaceutically acceptable salt, ester or stereoisomer thereof in the medicine is 0.1-99%.

The invention also provides application of the gooseberry trilactone A in the scheme in preparation of a medicine for treating the Alzheimer disease.

Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the drug is selected from one of a liquid preparation, a solid preparation, a spray and an aerosol.

The invention provides a gooseberry trilactone A shown in a structure of a formula I; the tinospora edulis trilactone A disclosed by the invention can influence the metabolic pathway of Amyloid Precursor Protein (APP), has the capacity of reducing the expression of beta-amyloid A beta 42 and A beta 40, shows a certain beta-amyloid A beta generation resistance capacity, and has the activity of preventing and treating the onset of Alzheimer's disease.

Drawings

FIG. 1 is a morphological diagram of U251-APP cells treated with tinospora cordifolia trilactone A, where A is the control group, B is the 10 μ M tinospora cordifolia trilactone A treated group, and C is the 40 μ M tinospora cordifolia trilactone A treated group;

FIG. 2 is a graph of the levels of Abeta 42/Abeta 1-42 and Abeta 40/Abeta 1-40 in the supernatant of U251-APP cells treated with tinospora vulgaris trilactone A (10. mu.M; 40. mu.M), where A is the decrease in Abeta 42 content after treatment with tinospora vulgaris trilactone A and B is the decrease in Abeta 40 content after treatment with tinospora vulgaris trilactone A;

FIG. 3 is a Western blot Western blot of U251-APP cells treated with Harrisonia perforatum trilactone A, wherein A is a representative Western blot result graph, and B is a statistical graph of results of three independent repeated experiments;

FIG. 4 is a Principal Component Analysis (PCA) of U251-APP cells treated with tinospora bovis trilactone A;

FIG. 5 is a KEGG pathway analysis of U251-APP cells treated with gooseberry trilactone A (40 μ M);

FIG. 6 is the mRNA and protein levels of alpha-secretase-associated components ADAM9/10/17 treated by Eleusine indica trilactone A, wherein A is the gene transcription level value derived from RNA-seq data, B is the result verified by an experimental quantitative PCR experiment, C is a representative Western blot result graph, and D is a statistical graph of the results of three independent repeated experiments.

Detailed Description

The invention provides a gooseberry trilactone A (Harpertrioate A) shown in a formula I structure, which is limonin with a novel chemical structure;

the invention also provides a preparation method of the gooseberry trilactone A shown in the structure of the formula I in the scheme, which comprises the following steps:

gel column size: 20mm by 200 mm;

the flow rate of the eluent is 0.3 mL/min;

the eluent is absolute methanol;

the dosage of the eluent is as follows: 1L;

a chromatographic column: waters XSelect CSH C-18column (5 μm, 10X 250 mm);

the flow rate of the mobile phase is 3.0 mL-min^-1；

The column temperature was 25 ℃;

the amount of sample was 30. mu.L each time.

Firstly, ethanol water solution with the volume fraction of 90-95% is adopted to carry out reflux extraction on the gooseberry branches, and an extracting solution is collected; concentrating the extracting solution to obtain an extract; re-suspending the extract in water to obtain a crude extract; in the invention, the gooseberry branches are preferably collected from Hainan; in the invention, the gooseberry branches are preferably dried and crushed to obtain crushed materials, and the crushed materials are mixed with ethanol water solution for reflux extraction.

In the present invention, the particle size of the pulverized material is preferably 80 to 100 mesh; the temperature of the reflux extraction is preferably 50-70 ℃, and further preferably 60 ℃; the reflux extraction frequency is preferably 3 times; the volume of the ethanol aqueous solution used for each reflux extraction is preferably 100L, based on 100kg of the mass of the pulverized product.

In the invention, in the process of reduced pressure concentration, extract is obtained and solvent (ethanol water solution with volume fraction of 90-95%) is recovered; the condensing temperature of the reduced pressure concentration is preferably 4 ℃, and the pressure of the reduced pressure concentration is preferably 0.2-0.25 MPa; the mass of the extract is preferably 4-5 kg, and more preferably 4.5kg, calculated by taking the mass of the gooseberry branches as 100 kg; the mass ratio of the extract to the water is preferably 1: (7.5 to 12).

After the crude extract is obtained, carrying out first extraction on the crude extract by adopting petroleum ether, removing the petroleum ether extraction part, and collecting residues; performing second extraction on the remainder by using ethyl acetate, and collecting an ethyl acetate part to obtain an ethyl acetate extract; the number of times of the first extraction is preferably 3, and the volume ratio of the petroleum ether to the crude extract in each first extraction is preferably 1: 1; the number of times of the second extraction is preferably 3, and the volume ratio of ethyl acetate to the residue in each second extraction is preferably 1: 1.

in the present invention, petroleum ether is used to remove low-polarity compounds, and ethyl acetate is used to enrich the component of eleusine indica trilactone a.

After obtaining the ethyl acetate extract, carrying out small-pore resin chromatographic separation on the ethyl acetate extract, and carrying out first gradient elution, wherein the first gradient elution is carried out by the following procedures: eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol in sequence, collecting 5 eluates, and sequentially naming Fr1, Fr2, Fr3, Fr4 and Fr 5. In the present invention, the ratio of the mass of the ethyl acetate extract to the volume of the small pore resin is preferably 1 kg: 4L; the volume of the eluent adopted by each gradient in the first gradient elution is preferably 20L, and the flow rate of the eluent is preferably 20 ml/min.

In the present invention, Fr1 is rich in sugars, inorganic salts and other highly polar impurities; fr2 is rich in glucoside, polyhydroxy substituted phenolic compounds, etc. and other relatively polar impurities; fr3 is rich in lignin compounds; fr4 is rich in limonin and quassin compounds; fr5 is rich in chlorophyll and other small polar compounds.

After Fr4 was obtained, the present invention performed normal phase silica gel column chromatography on Fr4 with a second gradient elution, which was performed by the following procedure: eluting by sequentially adopting 10% by volume of petroleum ether-ethyl acetate solution, 20% by volume of petroleum ether-ethyl acetate solution, 30% by volume of petroleum ether-ethyl acetate solution, 40% by volume of petroleum ether-ethyl acetate solution and pure ethyl acetate, and collecting and obtaining 5 parts of eluents which are sequentially named as Fr4-1, Fr4-2, Fr4-3, Fr4-4 and Fr 4-5. In the present invention, the mass ratio of Fr4 to silica gel is preferably 1: 20; the volume of eluent used in each gradient in the second gradient elution is preferably 4L, and the flow rate of the eluent is preferably 10 ml/min.

In the invention, normal phase silica gel column chromatography aims at carrying out primary refining on fractions rich in limonin and a quassin compound Fr4, Fr4-1 is rich in residual chlorophyll and other small polar compounds, Fr4-3 and Fr4-4 are rich in other types of compounds such as quassin, limonin and lignin with larger polarity; fr4-2 increases the concentration of limonin compounds.

After Fr4-2 is obtained, the invention carries out C18 reverse phase silica gel column chromatography separation on the Fr4-2, and carries out third gradient elution, wherein the procedure of the third gradient elution is as follows: sequentially eluting with 30% methanol aqueous solution, 40% methanol aqueous solution, 45% methanol aqueous solution, 50% methanol aqueous solution and anhydrous methanol, and collecting 5 eluates named as Fr4-2-1, Fr4-2-2, Fr4-2-2, Fr4-2-4 and Fr 4-2-5.

In the present invention, the mass ratio of the Fr4-2 to the C18 reverse phase silica gel is preferably 3: 100, respectively; the volume of the eluent used in each gradient in the third elution is preferably 5L, and the flow rate of the eluent is preferably 10 ml/min.

In the invention, the purpose of the reversed phase silica gel column chromatography is to distinguish limonin and quassin compounds with different oxygen contents, thereby further achieving the purpose of enriching the target limonin.

After Fr4-2-4 is obtained, the invention adopts Sephadex LH-20 methanol gel column chromatography to separate and concentrate Fr 4-2-4; collecting one part of chromatographic solution per 5mL, and combining 30-35 parts of chromatographic solution to obtain combined chromatographic solution; the Sephadex LH-20 methanol gel column chromatography conditions include: gel column size: 20mm by 200 mm; the flow rate of the eluent is 0.3 mL/min; the eluent is absolute methanol; the amount of eluent is as follows, wherein the weight of the Fr4-2-4 is 0.5 g: 1L of the compound.

The Sephadex LH-20 methanol gel chromatography is mainly used for separating according to the difference of molecular weights, plant secondary metabolites with the molecular weight more than 600 flow out before 30 bottles, plant secondary metabolites with the molecular weight less than 450 flow out after 35 bottles, and the 30 th to 35 th parts of chromatography liquid are mainly used for enriching the secondary metabolites with the molecular weight between 450 and 600.

After the chromatography solutions are combined, the combined chromatography solution is preferably dissolved in a chromatographic methanol solution to obtain a methanol chromatographic dissolved solution; the volume of the chromatographic methanol solution is preferably 1ml, based on the mass of the combined chromatographic solutions being 0.1 g.

After obtaining a chromatographic methanol solution, the invention carries out semi-preparative high performance liquid chromatography separation on the methanol chromatographic dissolved solution, adopts acetonitrile water solution with the volume fraction of 32 percent to carry out elution,and collecting the eluent for 20.1-21.1 min, wherein the eluent contains the gooseberry trilactone A. In the present invention, the conditions for the semi-preparative high performance liquid chromatography separation include: a chromatographic column: waters XSelect CSH C-18column (5 μm, 10X 250 mm); the mobile phase comprises: mobile phase a and mobile phase B; the mobile phase A is purified water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 68%; the volume fraction of the mobile phase B is 32%; the flow rate of the mobile phase is 3.0 mL-min^-1(ii) a The column temperature was 25 ℃; the amount of sample was 30. mu.L each time.

In the present invention, the semi-preparative HPLC separation is preferably performed by Agilent 1260 HPLC (Agilent, USA).

In the invention, the mass fraction of the gooseberry trilactone A or the pharmaceutically acceptable salt, ester or stereoisomer thereof in the medicine is preferably 0.1-99%, more preferably 0.5-90%, and even more preferably 5-20%.

The invention also provides application of the gooseberry trilactone A in the scheme in preparation of a medicine for treating the Alzheimer disease. In the present invention, the medicament preferably further comprises pharmaceutically acceptable excipients; the auxiliary materials preferably comprise diluents and/or fillers; the auxiliary materials comprise one or more of solid auxiliary materials, semi-solid auxiliary materials and liquid auxiliary materials.

In the invention, the dosage form of the medicine is selected from one of a liquid preparation, a solid preparation, a spray and an aerosol; the liquid preparation comprises injection, suspension, emulsion, solution or syrup; the solid preparation comprises tablets, capsules, granules or medicinal granules.

In the present invention, the administration route of the drug preferably includes injection, oral administration, sublingual administration or mucosal dialysis; the injection preferably comprises intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection. In some embodiments of the present application, the daily dose of the pharmaceutical composition administered to a subject in need thereof is preferably 0.1 to 100mg/kg body weight, and more preferably 0.5 to 20mg/kg body weight, in terms of eleusine indica trilactone a, its pharmaceutically acceptable salt, ester, or stereoisomer.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1) The gooseberry plant sample is collected from 100kg of gooseberry twigs. Drying and crushing small twigs of Eleusines indica into powder of 80-100 meshes, heating and refluxing the powder by using an ethanol water solution with the volume fraction of 95%, wherein the extraction temperature is 60 ℃, the volume of the ethanol water solution used in each extraction is 100L, heating and refluxing the powder for three times, and combining the extracting solutions.

2) And concentrating the obtained extracting solution under reduced pressure, recovering the solvent, and concentrating to obtain an extract, wherein the condensation temperature is 4 ℃, and the pressure is 0.2-0.25 MPa. And adding 50L of water into the concentrated crude extract, suspending and dispersing, sequentially extracting for three times by using 50L of petroleum ether, removing the petroleum ether extraction part, collecting the residues, and extracting for three times by using 50L of ethyl acetate to obtain an ethyl acetate extract.

3) After obtaining the ethyl acetate extract, the invention performs small-pore resin (dosage is 4L) chromatographic separation on the ethyl acetate extract, and performs first gradient elution, wherein the procedure of the first gradient elution is as follows: eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol sequentially at a flow rate of 20ml/min and a gradient elution volume of 20L, collecting 5 fractions of eluates, and sequentially naming the eluates as Fr1 (rich in other large polar impurities such as sugar and inorganic salt), Fr2 (rich in other large polar impurities such as glycoside and polyhydroxy-substituted phenolic compound), Fr3 (rich in lignin compound), Fr4 (rich in limonin and lignin compound), and Fr5 (rich in chlorophyll and other small polar compounds).

Small-pore resin material: MCI, MB 100-40/75(FUJI SILYSIA CHEMICAL LTD)

Pore resin column size: 150mm 900 mm;

the flow rate of the eluent is 20 mL/min;

the amount of each gradient eluent was: 20L.

4) After Fr4(20g) was obtained, the present invention performed normal phase silica gel column chromatography on Fr4 with a second gradient elution, which was performed by the following procedure: separating with 20 times of silica gel (400g), eluting with 10% petroleum ether-ethyl acetate solution, 20% petroleum ether-ethyl acetate solution, 30% petroleum ether-ethyl acetate solution, 40% petroleum ether-ethyl acetate solution and pure ethyl acetate in sequence, wherein each gradient elution volume is 4L, the flow rate is 10ml/min, collecting 5 fractions of eluates, and sequentially naming the eluates as Fr4-1, Fr4-2, Fr4-3, Fr4-4 and Fr 4-5.

Normal phase silica gel material: 200-300 meshes, Qingdao ocean factory;

normal phase silica gel column size: 60mm by 400 mm;

the flow rate of the eluent is 10 mL/min;

the amount of each gradient eluent was: 4L.

5) After Fr4-2(3g) was obtained, the present invention performed C18 reverse phase silica gel column chromatography on Fr4-2 with a third gradient elution, which was performed by the following procedure: separating with 66.6 times of C18 reverse phase silica gel (200g), sequentially eluting with 30% methanol aqueous solution, 40% methanol aqueous solution, 45% methanol aqueous solution, 50% methanol aqueous solution and anhydrous methanol, collecting 5 eluates, and sequentially naming Fr4-2-1, Fr4-2-2, Fr4-2-2, Fr4-2-4 and Fr 4-2-5.

Reversed phase silica gel material: lichroprep RP-18gel (40-63 μm; Merck, Darmstadt, Germany).

Size of reverse phase silica gel column: 30mm x 400 mm;

the flow rate of the eluent is 10 mL/min;

the amount of each gradient eluent was: 5L.

6) After Fr4-2-4(0.5g) is obtained, the invention adopts Sephadex LH-20 methanol gel column chromatography to separate and concentrate the Fr 4-2-4; collecting one part of chromatographic solution per 5mL, and combining 30-35 parts of chromatographic solution to obtain combined chromatographic solution; the Sephadex LH-20 methanol gel column chromatography conditions include:

gel material (Sephadex LH-20): 40-70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden

Gel column size: 20mm by 200 mm;

the flow rate of the eluent is 0.3 mL/min;

the eluent is absolute methanol;

the dosage of the eluent is as follows: 1L of the compound.

Combining chromatographic solutions (0.1g), concentrating and dissolving in 1ml of chromatographic methanol solution to obtain a sample solution with the concentration of 0.1g/ml, carrying out semi-preparative high performance liquid chromatography separation on the sample solution, eluting by using an acetonitrile aqueous solution with the volume fraction of 32%, and collecting an eluent of 20.1-21.1 min, wherein the eluent contains eleusine indica trilactone A (17 mg).

In the present invention, the conditions for the semi-preparative high performance liquid chromatography separation include: a chromatographic column: waters XSelect CSH C-18column (5 μm, 10X 250 mm); the mobile phase comprises: mobile phase a and mobile phase B; the mobile phase A is purified water; the mobile phase B is acetonitrile; the volume fraction of the mobile phase A is 68%; the volume fraction of the mobile phase B is 32%; the flow rate of the mobile phase is 3.0 mL-min^-1(ii) a The column temperature was 25 ℃; the amount of sample was 30. mu.L each time.

The structural data of Harpertrioate a from the semi-preparative hplc separation were as follows:

colorless transparent bulk crystals (MeOH); [ alpha ] to]₂₇ ^D-2.83(c 0.14,MeOH)；

And (3) structure confirmation process: positive ion HR-ESI-MS (positive) shows m/z 525.2097[M+Na]⁺，(cald for C₂₇H₃₄O₉Na, 525.2095) has molecular weight of 502 and molecular formula of C₂₇H₃₄O₉The unsaturation degree was 11.¹H-NMR and¹³the C-NMR spectrum data show that 27 carbon signals including 5 methyl groups, 5 methylene groups, 8 methine groups and 9 quaternary carbons exist in the molecule. Further analysis of the one-dimensional data suggests the presence of 3 ester carbonyl groups (. delta.) in the molecule_C174.2, 170.3, 168.9), 1 beta-furan ring (. delta.) -_H 6.37，7.42，7.39，δ_C142.9, 141.2, 120.3, 110.0), 1 substituted double bond (. delta.) in the presence of a catalyst_C130.8, 132.0), 1 methoxy (δ)_H 3.73，3H，s；δ_C52.8), and 4 tertiary methyl groups (. delta.))_H 0.99，1.08，1.29，1.57；δ_C17.3, 21.9, 32.8, 23.2). The remaining unsaturation, excluding the 6 unsaturations occupied by the three carbonyl, three double bonds, suggests that the compound is a pentacyclic system. The compound is indicated to be a limonin compound. The planar structure of the compound is finally processed by two-dimensional nuclear magnetic technology (¹H-¹H COSY, HMBC and HSQC). Its three-dimensional structure was preliminarily identified by ROESY and finally by X-ray single crystal diffraction techniques, including confirmation of its absolute configuration.

Crystallization conditions are as follows: acetone is adopted: water (10: 1), dissolving the sample in a 1ml liquid phase bottle, covering the bottle mouth with a plastic film, pricking 2 small holes on the film, and volatilizing at room temperature to obtain a plurality of crystals. Testing an instrument: bruker Smart Apex CCD diffractometer equpped with graphite-monochromated Mo K α radiation.

Crystal data: can be downloaded freely in the Cambridge crystal center (website: http:// www.ccdc.cam.ac.uk /), crystal number: CCDC-2028648.

Spectroscopic data for Harpertrioate a:

IR(KBr)ν_max 3436,2984,2953,2926,2896,2853,1727,1631,1461,1436,1384,1248,1213,1168,1122,1031,990cm^–1；ESI-MS(+)m/z 525[M+Na]⁺；HR-ESI-MS(+)m/z 525.2097[M+Na]⁺(calcd for C₂₇H₃₄O₉Na，525.2095)；¹H NMR(DMSO-D₆,500MHz)δ：4.03(1H,d,J＝6.7Hz,H-1),3.34(1H,o,H-2a),2.50(1H,o,H-2b),2.82(1H,d,J＝8.8Hz,H-5),2.62(1H,d,J＝17.3Hz,H-6a),2.17(1H,dd,J＝17.3,8.8Hz,H-6b),5.62(1H,br s,H-5),4.15(1H,m,H-11),1.35(1H,dd,J＝11.5,5.4Hz,H-12a),1.46(1H,dd,J＝11.5,10.3Hz,H-12a),3.67(1H,d,J＝18.6Hz,H-15a),2.65(1H,d,J＝18.6Hz,H-15b),5.41(1H,br s,H-17),0.89(3H,s,H-18),1.05(3H,s,H-19),7.66(1H,br s,H-21),6.47(1H,s,H-22),7.69(1H,br s,H-23),1.18(3H,s,H-28),1.53(3H,s,H-29),2.23(1H,d,J＝13.6Hz,H-30a),2.12(1H,d,J＝13.6Hz,H-30b),3.63(3H,s,O-CH₃),4.41(1H,d,J＝6.1Hz,OH)；¹³C NMR(DMSO-D₆,125MHz)δ：72.9(C-1),37.8(C-2),170.7(C-3),83.8(C-4),41.6(C-5),34.1(C-6),174.0(C-7),130.6(C-8),131.9(C-9),45.0(C-10),63.4(C-11),35.7(C-12),38.8(C-13),74.6(C-14),32.2(C-15),168.7(C-16),77.7(C-17),17.2(C-18),21.0(C-19),120.7(C-20),141.2(C-21),110.2(C-22),143.5(C-23),32.5(C-28),22.4(C-29),40.8(C-30),51.7(O-CH₃)。

example 2

The injection is prepared by adding injection water into the gooseberry trilactone A prepared by the method of the example 1 and the auxiliary materials used in the conventional injection, finely filtering, encapsulating and sterilizing.

Example 3

Dissolving the gooseberry trilactone A prepared in example 1 in sterile water for injection, stirring to dissolve, filtering with a sterile suction filter funnel, performing sterile fine filtration, subpackaging with 2 ampoules, freeze-drying at low temperature, and performing sterile melt-sealing to obtain the powder injection.

Example 4

And respectively mixing the separated gooseberry trilactone A and an excipient in a weight ratio of 9: 1, adding excipient, and preparing into powder.

Example 5

The gooseberry trilactone A is prepared according to the method of the example 1, and the weight ratio of the gooseberry trilactone A to the excipient is 1: 5, adding excipient, granulating and tabletting.

Example 6

The gooseberry trilactone A is prepared according to the method of the example 1, and the gooseberry trilactone A is prepared into oral liquid according to the conventional oral liquid preparation method.

Example 7

The gooseberry trilactone a is prepared according to the method of example 1, and the weight ratio of the gooseberry trilactone a to the excipient is 5: 1, adding excipient, and preparing into capsule, granule or electuary.

Example 8

The gooseberry trilactone a is prepared according to the method of example 1, and the weight ratio of the gooseberry trilactone a to the excipient is 3: 1, adding excipient, and preparing into capsule, granule or electuary.

Test example 1

The pharmacological effect test result of the tinospora edulis trilactone A prepared in example 1 on the Alzheimer disease resistance is as follows:

experimental methods

Cell culture and processing

The U251-APP cell line is a human glioma cell line capable of stably expressing a human APP mutant (APP-p.K670N/M671L). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified air incubator at 37 ℃ with 5% CO₂And 95% humidity. Cells were seeded in 6-well plates in growth medium (RPMI-1640 medium containing 10% fetal bovine serum). Eleusine indica trilactone A (10. mu.M and 40. mu.M, respectively) was added directly to the medium and cells were harvested 24h after drug treatment.

Abeta ELISA assay

The content of beta-amyloid (beta-amyloid, Abeta, which is a key pathological marker of Alzheimer's disease) in the supernatant of U251-APP cells, including Abeta 40/Abeta 1-40 and Abeta 42/Abeta 1-42, was measured by using a commercial ELISA kit (E-EL-H0542c for Abeta 40, E-EL-H0543c for Abeta 42), and the specific experimental procedures were carried out according to the specifications of the ELISA kit.

Western blot analysis

The method comprises the following specific steps:

1) extracting the protein: the proteins in the cells were extracted using western from BYttrium (Beyotime Biotechnology) and IP lysates.

2) The protein concentration was determined: we used the BCA protein quantification kit to measure the U251-APP cellular protein, and the extracted protein was used for subsequent detection.

3) Sample adding pretreatment: each sample was loaded at 20. mu.g based on protein concentration. Firstly, adding a protein denaturant of anhydrous mercaptoethanol, denaturing at 95 ℃ for 6min, and standing on ice for 5min after the denaturation is finished.

4) Loading and carrying out electrophoresis: the denatured protein samples were added to the corresponding gel wells and electrophoresis was started.

5) And (3) film transfer treatment: the amount of the membrane transfer solution was adjusted according to the ratio of the reagents required for 1L of the membrane transfer solution (10 Xthe membrane transfer solution: anhydrous methanol: water: 1: 2: 7), and the sponge and the filter paper were immersed in the membrane transfer solution, and the PVDF membrane was previously immersed in the anhydrous methanol for 30 seconds to activate the PVDF membrane. Preparing a membrane transfer plate (protein with negative electricity is transferred to a PVDF membrane from a positive electrode and is transferred to a PVDF membrane) according to the sequence of sponge-filter paper-glue-membrane-filter paper-sponge, putting the membrane transfer plate into a membrane transfer device, and adding the membrane transfer liquid to start membrane transfer. The film transfer process needs to be carried out under ice bath conditions.

6) Sealing treatment: the protein-transferred PVDF membrane was placed in TBST containing 5% skim milk and placed on a shaker for 2h with slow shaking.

7) Primary antibody incubation: after the end of the blocking, the PVDF membrane was gently rinsed with TBST in order to wash away the residual skim milk from the PVDF membrane. PVDF was tailored to the target protein molecular weight and corresponding antibodies (GAPDH, labeled glyceraldehyde-3-phosphate dehydrogenase; BAEC1, labeled beta secretase; PSEN1, labeled gamma secretase subunit presenilin 1; PSEN2, labeled gamma secretase subunit presenilin 2; NICTN, labeled gamma secretase subunit NICSTN; PEN2, labeled gamma secretase subunit PEN 2; APH-1, labeled gamma secretase subunit) were added and incubated overnight at 4 ℃ on a shaker.

8) And (3) secondary antibody incubation: the next day, primary antibody was recovered and the PVDF membrane was rinsed with TBST, 5 min/time, 3 times, to ensure that residual primary antibody on the PVDF membrane was washed away. The corresponding secondary antibody was then labelled according to the species source of the primary antibody (secondary antibody was formulated with 5% skim milk). Incubate on a shaker at room temperature for 1 h.

9) And (3) cleaning secondary antibody: the secondary antibody was discarded and the PVDF membrane was rinsed with TBST 5 min/time and 3 times to ensure complete washing of the residual secondary antibody from the PVDF membrane.

10) And (3) developing: the bands of the desired protein were visualized using a Bio-Rad fluorescence image analyzer.

RNA extraction, RNA sequencing (RNA-seq)

Total RNA was extracted using RNAeasy kit. The A260/A280 ratio of total RNA was measured on a NanoDrop Biophotometer, and only samples with ratios between 1.8 and 2.0 were used in subsequent experiments. The quality and integrity of the RNA samples was also assessed based on 28S and 18S rRNA bands on a 1% agarose gel. Approximately 1.5. mu.g of total RNA per sample was used to prepare the RNA sequence library. The sequencing library was generated from a NEB Next Ultra RNA library preparation kit suitable for the Illumina sequencing platform and an index code was added to the attribute sequence of each sample. The final treated library was sequenced on the IlluminaHiseq 4000 platform and paired-end reads of 150bp were generated. First, raw sequencing data was processed by trimmatic software to remove poor quality sequence (which is a quality control process, and the data was not analyzed further) reads and after filtering, clean reads were aligned to the mouse reference genome GRCm38 using STAR. Next, the insertion of aligned reads into the bam format generated by STAR is processed through the featureCounts function of the Suclean software package to assign and count the segments that are genetically uniquely mapped according to the annotation file of GRCm 38. The counts generated in the previous step were normalized and scaled using the rlogTransformation function of R-package DESeq2, which converts the count data to log2 scaling to minimize the difference between samples of the small count rows. Based on the normalized counts, Principal Component Analysis (PCA) and differential expression analysis were performed using DESeq 2R package. Differential expression was tested by DESeq2, P values were adjusted by the Benjamini & hochberg (bh) method, and genes differentially expressed between conditions were identified if P values < 0.05. Gene Ontology (GO) enrichment and kyoto protocol gene and genome encyclopedia (KEGG) pathway enrichment analysis were performed using R package clusterirprofiler with the differentially expressed gene calculated by DESeq2 as input.

Total RNA extraction, reverse transcription, Real-time quantitative PCR (Real-time quantitative PCR, RT-qPCR) detection of the expression of the corresponding gene.

1) And extracting the total RNA of the cell treated by the medicine according to the instruction of the tissue cell RNA miniprep kit.

2) The concentration of the extracted RNA was determined, and 1. mu.g of RNA was reverse-transcribed according to the instructions of the reverse transcriptase M-MLV reagent to obtain a cDNA template.

Adding 1mL of RZ cell lysate into the cells after the drug treatment, then collecting the lysate, and extracting RNA according to a total RNA extraction kit and an experimental method provided by Tiangen corporation. The concentration of the extracted total RNA is measured by a NanoDrop 2000 ultramicro spectrophotometer, and then the cDNA is synthesized by M-MLV reverse transcriptase by taking about 1.5 mu g of total RNA, wherein the specific system is as follows:

solution A:

and B, liquid B:

the prepared solution A is placed on a PCR instrument at 70 ℃ for 5min, and then is quickly transferred to ice for 5 min. Adding the solution B into the solution A, and placing the mixture on a PCR instrument at 37 ℃ for 1h to obtain cDNA.

3) The expression of corresponding genes (including ADAM10, ADAM9 and ADAM17) is detected by using a SYBR Green real-time fluorescent quantitative PCR method.

4) Specific primer information is shown in the following table.

Taking cDNA prepared according to 2) as a template, combining corresponding gene specificity quantitative primers (shown in the table below), and carrying out quantitative PCR reaction, wherein the specific reaction system is as follows:

the reaction procedure is as follows: 95 deg.C, 5min, 95 deg.C, 10s, 58 deg.C, 30s, 40 cycles. Wherein GAPDH is used as a reference gene. After the reaction is finished, taking the PCR product to draw a standard curve, and further calculating the expression quantity of the related gene through the standard curve.

RT-qPCR primers were as follows:

results

Cell analysis showed no significant change in cell morphology, indicating that the compound was not significantly toxic to nerve cells (fig. 1).

The expression level of the alzheimer's disease marker molecule in the supernatant of the gooseberry trilactone a treated cells was measured by ELISA, and it was found that the beta-amyloid a beta 42 and a beta 40 species were significantly reduced (fig. 2).

Amyloid pathway-associated proteins associated with β amyloid a β production were detected by western-blot analysis. The results show that BACE1, NCSTN and PEN2 protein levels were significantly reduced, PSEN1 was increased, and the protein levels of PSEN2 and APH1 were unchanged (fig. 3, a is a representative results graph and B is a statistical results graph).

Transcriptomics biometric analysis was performed on U251-APP cells treated with tinospora cordifolia trilactone a. From PCA analysis, it was found that the expression pattern of the gene was strongly influenced by the perforin trilactone A at a compound concentration of 40. mu.M (FIG. 4). The effect of individual genes was then examined and statistics were made for differential genes. A total of 2370 different differential genes were found between gooseberry trilactone a and the control group, 1659 of which were up-regulated and 711 of which were down-regulated. Kyoto protocol Gene and genome encyclopedia (KEGG) pathway enrichmentAnalysis shows that the differential gene of the gooseberry trilactone A is significantly enriched in the pathway of Alzheimer's disease (P ═ 4.53X 10)^-06) (FIG. 5).

Analysis of the expression levels of the relevant genes for alpha-secretase in the amyloid pathway, mRNA level examination and RT-qPCR and western blot validation: alpha-secretase-associated (ADAM9/10/17) mRNA levels were increased (FIG. 6). Panel A is derived from RNA-seq results, and panel B is RT-qPCR validation results. The C-D diagram is the protein level result obtained from the western blot experiment.

Experimental results show that the gooseberry trilactone A provided by the invention can influence the metabolic pathway of Amyloid Precursor Protein (APP), can reduce the expression capacity of beta-amyloid A beta 42 and A beta 40, can promote the conversion of APP to a non-A beta metabolic pathway, shows a certain capacity of resisting the generation of beta-amyloid A beta, and has the activity of preventing and treating the Alzheimer's disease.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. Eleusine indica trilactone A shown in a structure of a formula I;

2. a process for the preparation of the gooseberry trilactone a of claim 1 which comprises the steps of:

5) subjecting said Fr4-2 to C18 reverse phase silica gel column chromatography with a third gradient elution, said third gradient elution being performed by the procedure: sequentially eluting with 30% methanol aqueous solution, 40% methanol aqueous solution, 45% methanol aqueous solution, 50% methanol aqueous solution and anhydrous methanol, collecting 5 eluates, and sequentially naming as Fr4-2-1, Fr4-2-2, Fr4-2-3, Fr4-2-4 and Fr 4-2-5;

gel column size: 20mm by 200 mm;

the flow rate of the eluent is 0.3 mL/min;

the eluent is absolute methanol;

the dosage of the eluent is as follows: 1L;

a chromatographic column: waters XSelect CSH C-18column, 5 μm, 10X 250 mm;

the flow rate of the mobile phase is 3.0 mL-min^-1；

The column temperature was 25 ℃;

the amount of sample was 30. mu.L each time.

3. A medicament for treating anti-alzheimer's disease comprising the tinospora root trilactone a or a pharmaceutically acceptable salt thereof of claim 1.

4. The medicament according to claim 3, wherein the mass fraction of the gooseberry trilactone A or the pharmaceutically acceptable salt thereof in the medicament is 0.1-99%.

5. Use of the tinospora root trilactone a of claim 1 in the manufacture of a medicament for the treatment of anti-alzheimer's disease.

6. The use of claim 5, wherein the medicament further comprises a pharmaceutically acceptable excipient.

7. The use of claim 5, wherein the medicament is in a form selected from the group consisting of a liquid formulation, a solid formulation, a spray, and an aerosol.