CN112618538B

CN112618538B - Preparation method of 1,6-O, O-diacetyl inula flower lactone and application of 1, 6-O-diacetyl inula flower lactone in preparation of anti-Alzheimer disease drugs

Info

Publication number: CN112618538B
Application number: CN202011507157.5A
Authority: CN
Inventors: 汤江江; 黄兰芳; 郭聪; 高锦明; 项萍
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2023-04-25
Anticipated expiration: 2040-12-18
Also published as: CN112618538A

Abstract

The invention discloses a preparation method of 1,6-O, O-diacetyl inula flower lactone and application thereof in preparing an anti-Alzheimer disease medicament. According to the invention, after the OABL is used for carrying out intraperitoneal injection on the AD model mouse, the precipitation of the A beta amyloid in the hippocampus and cerebral cortex of the AD model mouse is obviously reduced; correspondingly, the OABL has more obvious improvement of learning and memory capacity in the water maze experiment of the AD model mice. The preparation method comprises the steps of extracting an ethanol extract of a dried inflorescence of the inula europaea with ethyl acetate to obtain an ethyl acetate extract; then separating the ethyl acetate extract to obtain 1,6-O, O-diacetyl inula flower lactone, and recrystallizing to obtain the high-purity 1,6-O, O-diacetyl inula flower lactone.

Description

Preparation method of 1,6-O, O-diacetyl inula flower lactone and application of 1, 6-O-diacetyl inula flower lactone in preparation of anti-Alzheimer disease drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of 1,6-O, O-diacetyl inula flower lactone and application thereof in preparation of anti-Alzheimer disease drugs.

Background

The Inula europaea is a biennial or perennial plant, phototropic, golden petals, upright growing, approximately 15-75 cm higher, camptothectic loam and clay, and is commonly used for treating tumors, digestive system diseases, bronchitis, antibiosis and virus infection (including hepatitis virus), wherein inflorescences can be used for treating respiratory tract inflammation such as intestinal tract diseases, bronchitis and the like. 1,6-O, O-diacetyl inula flower lactone (OABL) is reported to be obtained by extracting an Inula europaea inflorescence with ethanol and separating the Inula europaea inflorescence from chloroform, and the separation process requires normal-phase silica gel, reverse-phase chromatography, gel separation technology and the like, and is laborious and time-consuming.

The prior art shows that the anti-tumor activity and anti-inflammatory activity of OABL are more prominent. OABL inhibitionThe phosphorylation of Bcl-2 by breast cancer cell BRK-p53 is blocked and the cell cycle G2/M is blocked. Furthermore, OABL induces apoptosis by activating Caspasts-3, 8 and 9, release of cytochrome C in mitochondria, inhibiting proliferation of leukemia cells HL-60. Anti-inflammatory Activity exhibited by OABL inhibition of Nitric Oxide (NO) and prostaglandin 2 (PGE) in macrophage RAW264.7 by inhibition of synthesis of nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) ₂ ) Is a synthesis of (a). In addition, OABL is effective in activating endothelial cells, and can effectively inhibit macrophage adhesion to endothelial cells by reducing the expression of vascular cell adhesion molecule-1 (VCAM-1), plasma Osteopontin (OPN), MMP-9, and tenascin-c, a macromolecular glycoprotein in the extracellular matrix. However, the effects of OABL on alzheimer's disease have not been reported.

Disclosure of Invention

The invention is based on new research findings, and firstly provides application of 1,6-O, O-diacetyl inula flower lactone in preparing an anti-Alzheimer disease medicament.

The medicine based on the application comprises 1,6-O, O-diacetyl inula flower lactone and pharmaceutical auxiliary materials. The pharmaceutical auxiliary materials can be pharmaceutical auxiliary materials which do not influence the efficacy or contribute to the efficacy exertion after being reasonably selected from auxiliary materials commonly used in the pharmaceutical field. The medicine dosage form can be injection type.

The invention also provides a preparation method of the 1,6-O, O-diacetyl inula flower lactone. To this end, a method is provided comprising: extracting the ethanol extract of the dried inflorescence of the inula flower with ethyl acetate to obtain an ethyl acetate extract; and then separating the ethyl acetate extract to obtain the 1,6-O, O-diacetyl inula flower lactone.

Preferably, the separation comprises the step of adopting a normal phase silica gel column, petroleum ether and ethyl acetate mixed solution as an eluent for separation.

The clinical characteristics of Alzheimer's disease are mainly manifested by cognitive and behavioral disorders such as spatial learning and memory. Overcoming the disease has been a medical problem to be solved. A large number of national drug enterprises including the large heads of the pyroxenes, the predators, the ghanins, the smith and the like, have been combined on the drug for Alzheimer's disease, and the billions of money are burned without work. It follows that the search for a new drug or method for treating AD remains a significant proposition. The etiology and pathogenesis of AD is currently unknown, and abnormal deposition of amyloid beta (aβ) is recognized as the last common pathway leading to AD. New anti-AD drugs against the etiology of aβ have been continuously tested clinically, but have not been reported successfully. According to the invention, after the OABL is used for carrying out intraperitoneal injection on the AD model mouse, the precipitation of the A beta amyloid in the hippocampus and cerebral cortex of the AD model mouse is obviously reduced; correspondingly, the OABL has more obvious improvement of learning and memory capacity in the water maze experiment of the AD model mice. Based on the above, OABL can be used for preparing anti-Alzheimer disease drugs.

Drawings

FIG. 1 shows a compound of the invention ¹ H NMR (400 mhz, cdcl 3) spectra;

FIG. 2 shows a compound of the invention ¹³ C NMR(100MHz,CDCl ₃ ) A map;

FIG. 3 shows the results of purity analysis for the isolation of compounds of the present invention;

FIG. 4 shows the results of an OABL-improved 5XFAD mouse cognitive function experiment;

FIG. 5 is an experimental result of OABL alleviating the deposition of mouse amyloid beta (Abeta);

FIG. 6 shows the results of an experiment for OABL to reduce the expression of inflammatory proteins in AD model mice.

Detailed Description

Unless otherwise indicated, the terms herein are to be understood based on knowledge of one of ordinary skill in the relevant art.

The following examples are further illustrative of the invention and are not intended to be limiting thereof.

The experimental materials and reagents used in the following examples are all commercially available products. The OABL used in the examples below may be commercially available or may be obtained using the methods provided by the present invention.

The OABL preparation method of the invention is implemented by the following examples:

weighing 10kg of dried inflorescence of Inulae flos, adding industrial ethanol 20L, soaking for 3 hr, adding zeolite, reflux extracting with ethanol for 3 times, 20L each time for 4 hr, discarding residues, recovering ethanol phase, and concentrating extract;

adding water (2L) with three volumes, extracting with ethyl acetate (3 x 2L), and concentrating under reduced pressure to obtain ethyl acetate phase extract, namely ethyl acetate phase extract, with a volume of 0.4Kg;

directly loading the ethyl acetate phase extract on a normal phase silica gel column (100-200 meshes, 20 cm. Times.140 cm), and sequentially using petroleum ether: 5. 7: 5. 1: 1L each for elution; wherein in petroleum ether, ethyl acetate 7:5, separating out crystals, and recrystallizing with ethanol to obtain pure OABL; the structural formula of the identified 1,6-O, O-diacetyl inula flower lactone (OABL) is shown as formula (I), and the identification result is shown in figures 1 and 2;

simultaneously, purity analysis is carried out on the separated substances: analysis by Waters 1525series with Evaporative Light Scattering Detector (ELSD), 10min gradient,80%MeOH:20%H ₂ O to 100％MeOH；t _R = 4.164min,99.27%at ELSD; HPLC analysis of purity>99% and the results are shown in FIG. 3.

Example 1: OABL improves 5XFAD mouse cognitive function

Morris water maze test

The AD model mice used were 5XFAD transgenic mice purchased from Jackson laboratories (JAX) in the United states, which produced Swedish (K670N/M671L) mutations in the Amyloid Precursor Protein (APP), florida (I716V) and London (V717I) mutations in proprotein 1 (PSEN 1).

All mice were fed and drunk freely at 22+ -2deg.C and humidity 50+ -10%, were fed for 12h light/dark period, were fed to 6 months of age, and were started to experiment, all operations followed the national institutional guidelines and laboratory animal guidelines;

the experiments were divided into 4 groups, a blank control group (WT group), an OABL control group (WT-OABL group), an AD model group (5 XFAD group), an OABL intervention group (5 XFAD-OABL group), wherein the mice used in the WT group and the WT-OABL group were wild-type healthy mice, and the mice used in the 5XFAD group and the 5XFAD-OABL group were 5XFAD transgenic mice; WT-OABL group and 5XFAD-OABL group were intraperitoneally injected with the natural compound OABL of formula (I) at a dose of 20 mg/Kg/day, and the control group was given a solvent for 3 weeks after the continuous administration, and after the end of the administration period, morris water maze test was performed, specifically referring to Morris water maze test method, for measuring learning and memory ability of mice.

The Morris Water Maze (MWM) maze is a circular pool (150 cm in diameter and 50cm in height) filled with water (24+ -1) deg.C, and is divided into 4 quadrants, and a platform with a diameter of 10cm is placed 1cm under one of the quadrants. Mice were trained continuously for 4 days, 4 quadrants per day; during training, the mice were placed in the water facing the wall and allowed to stand at the station for 10s if they found the station within 60 s. If a station cannot be found, it is brought to the station and allowed to stand for 10s. On day 5, a positional navigational test was performed in which mice were placed in water from the proximal quadrant of the platform towards the wall for 60s and the latency of the mice from entering water to the platform was recorded.

2. Experimental results

As shown in fig. 4A, in the Morris water maze test, the escape latency of the mice from the water inlet to the upper platform is extremely remarkably increased in the AD model group compared with the blank group, and after the 5XFAD mice are subjected to OABL treatment, the escape latency is remarkably reduced, and as shown in fig. 5B, a trace diagram of the positioning cruising of the mice on the 5 th day reveals that the natural compound OABL of the formula (I) can improve the learning and memory capacity of the AD model mice.

Example 2: OABL reduces AD model mouse amyloid-beta (Abeta) deposition

1. Immunofluorescence assay

After the mice are sacrificed, the brain tissues of the mice are immediately fixed by 10% neutral buffer formaldehyde for more than 24 hours, the tissues are taken out from the fixing solution, the tissues of the target parts are leveled by a surgical knife in a fume hood, the tissues are gradually dehydrated in a series of gradient ethanol solutions (70, 80, 95 and 100 percent, v/v) and embedded into paraffin blocks, and 4 mu m thick tissue sections are cut on paraffin sections for immunofluorescence experiments.

The sections were placed in a repair box filled with EDTA antigen retrieval buffer (PH 8.0) (china, seville) and subjected to antigen retrieval in a microwave oven, after antigen retrieval, blocked with 3% bsa for 30min at room temperature, incubated overnight with a primary antibody to aβ1-42 protein (us, CST) at 4 ℃, followed by incubation with a fluorescent secondary antibody (china, seville) at room temperature in the absence of light for 50min. DAPI counterstain, sealing, and sections were observed under a fluorescence microscope and images were acquired.

2. ELISA assay

After the mouse brain cortex tissue is fully cracked by protein lysate (containing protease inhibitor), a brain cortex tissue protein sample is obtained, an antibody is coated by a micro-pore plate, a standard substance and a protein sample are added, the mixture is incubated for 2 hours at 4 ℃, a plate is washed, a Human Amyloid beta (aa 1-42) antibody is added, the mixture is incubated for 2 hours at 4 ℃, the plate is washed, a substrate solution is added, the mixture is incubated for 30 minutes at a dark room temperature, a stop solution is added (R & D SYSTMS) and an enzyme-labeled instrument is used for detecting absorbance value within 30 minutes. The absorbance values measured were converted to the concentration of Human Amyloid beta (aa 1-42) by a standard curve.

3. Immunoblotting experiments

After the mouse brain cortex tissue is fully lysed by using protein lysate (containing protease inhibitor), a brain cortex tissue protein sample is obtained, protein quantification is carried out by using a BCA protein quantification kit (Chinese, tiangen), protein samples are separated by SDS-PAGE electrophoresis after boiling denaturation, then membrane transfer is carried out, primary antibody (US, CST) of BACE-1 protein is used for incubation at 4 ℃ overnight, then 5% nonfat milk powder is used for blocking, secondary antibody (US, SAB) of the corresponding species is used for incubation, and then a membrane scanner is used for detecting the relative expression level of the protein.

4. Experimental results

According to the immunofluorescence experimental result, as shown in fig. 5A, compared with the blank control group, the deposition of beta amyloid in the brain tissue of the mice (pink part in the figure) is obviously increased, and after the 5XFAD mice are treated by OABL, the deposition of beta amyloid in the brain tissue of the mice is obviously reduced; (in FIG. 5A, "GD, CA1, CA2, cortex Prl" represents the mouse hippocampal tissue GD region, CA1 region, CA2 region, CA3 region, respectively; according to the experimental results of the enzyme-linked immunosorbent assay, as shown in fig. 5B, the deposition of beta-amyloid in the brain tissue of the mice is significantly increased, and the deposition of beta-amyloid in the cortex tissue of the mice is significantly reduced after the 5 xfd mice are subjected to OABL treatment, compared with the blank control group.

The above results show that the native compound OABL of formula (I) can reduce amyloid β deposition in brain cortex and hippocampal tissues of AD model mice.

The inventors further explored the possible mechanism of reducing amyloid β by the natural compound OABL of formula (I), beta amyloid precursor protein lyase 1 (BACE-1) was a key enzyme for amyloid β production, and according to the immunoblotting experimental results, as shown in fig. 5C and 5D, the expression of BACE-1 in mouse brain cortex tissue was significantly increased in AD model group compared to the blank control group, and the expression of BACE-1 in mouse brain cortex tissue was significantly decreased after treatment with the natural compound OABL of formula (I), revealing that the possible mechanism of reducing amyloid β by the natural compound OABL of formula (I) in brain tissue was to inhibit expression of beta amyloid precursor protein lyase 1 (BACE-1). (GAPDH is an internal control of Western Blot protein normalization in FIG. 5C).

Example 3: OABL reduces expression of inflammatory proteins in AD model mice

1. Immunofluorescence assay

The procedure was as in example three, immunofluorescence experiments were performed using primary antibodies to GFAP, IBA-1 protein (USA, CST) at 4deg.C overnight, then using secondary antibodies to fluorescence (China, seville), incubated at room temperature in the dark for 50min, counterstained with DAPI, blocked, sectioned under a fluorescence microscope and images were acquired.

2. Immunoblotting experiments

After the mouse cerebral cortex tissue is fully cracked by protein lysate (containing protease inhibitor), a cerebral cortex tissue protein sample is obtained, protein quantification is carried out by a BCA protein quantification kit, protein samples are separated by SDS-PAGE electrophoresis after boiling denaturation, then membrane transfer is carried out, the mice are incubated overnight at 4 ℃ by using primary antibodies of GFAP and IBA-1 proteins, then blocked by using 5% nonfat milk powder, then incubation is carried out by using secondary antibodies of corresponding species, and then the relative expression level of the proteins is detected by using a membrane scanner.

3. Experimental results

According to the immunofluorescence test results, as shown in fig. 6A and 6B, the expression of GFAP protein (red marked part in the figure) and IBA-1 protein (green part in the figure) in the brain tissue of the mice was significantly increased, and after OABL treatment of 5XFAD mice, GFAP protein was significantly decreased in the brain tissue of the mice, (in fig. 6A and 6B, "GD, CA1, CA2, cortex Prl" respectively represents GD region, CA1 region, CA2 region, CA3 region and cerebral Cortex region of the hippocampal tissue of the mice), and the inventors verified the above results by immunoblotting test, as shown in fig. 6C, D, E, F, that the expression levels of GFAP protein and IBA-1 protein were decreased in the brain tissue of the AD model mice after OABL treatment of the natural compound of formula (I). The above results show that the natural compound OABL of formula (I) can inhibit the expression of neuroinflammation in AD model mice.

Claims

Application of 1.1,6-O, O-diacetyl inula flower lactone in preparing anti-Alzheimer disease medicine; the preparation method of the 1,6-O, O-diacetyl inula flower lactone comprises the following steps:

weighing 10kg of dried inflorescence of Inulae flos, adding industrial ethanol 20L, soaking for 3 hr, adding zeolite, reflux extracting with ethanol for 3 times, 20L each time for 4 hr, discarding residues, recovering ethanol phase, and concentrating extract;

adding water with the volume of three times, extracting with ethyl acetate, and concentrating under reduced pressure to obtain ethyl acetate phase extract, namely ethyl acetate phase extract, of 0.4Kg;

directly loading the ethyl acetate phase extract on a normal phase silica gel column, and sequentially using petroleum ether to prepare ethyl acetate 9: 5. 7: 5. 1: 1L each for elution; wherein in petroleum ether, ethyl acetate 7: and 5, separating out crystals, and recrystallizing with ethanol to obtain 1,6-O, O-diacetyl inula flower lactone.