CN115093427B - Medicine for treating Alzheimer disease and related diseases causing cognitive disorder - Google Patents

Medicine for treating Alzheimer disease and related diseases causing cognitive disorder Download PDF

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CN115093427B
CN115093427B CN202210859179.0A CN202210859179A CN115093427B CN 115093427 B CN115093427 B CN 115093427B CN 202210859179 A CN202210859179 A CN 202210859179A CN 115093427 B CN115093427 B CN 115093427B
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王�琦
谢旻臻
芦俊中
谷思琪
吴浩田
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Abstract

The invention provides a medicament for treating Alzheimer disease and related diseases causing cognitive impairment, which comprises alkaloid shown in a formula I and a medicinal composition of the alkaloid shown in the formula I and metformin. The alkaloid shown in the formula I has stronger acetylcholinesterase inhibition activity, can improve the cognitive dysfunction of mice, improve the learning and memory capacity of the mice, prevent the amyloid deposition in the brains of AD mice and slow down the disease development process of AD; meanwhile, the expression of the autophagy-related factor LC3I protein in brain tissue can be promoted, and the growth promoting effect on bifidobacterium SHQ1 is obvious. The above effects are more remarkable for the pharmaceutical composition of the alkaloid shown in the formula I and the metformin.
Figure DDA0003757227960000011

Description

Medicine for treating Alzheimer disease and related diseases causing cognitive disorder
Technical Field
The invention belongs to the field of medicines, relates to a medicine for treating Alzheimer disease and related diseases causing cognitive impairment, and particularly relates to a novel alkaloid, a pharmaceutical composition of the novel alkaloid and metformin, and application of the novel alkaloid in preparation of medicines for preventing and treating Alzheimer disease and related diseases causing cognitive impairment.
Background
Alzheimer's Disease (AD), also known as primary senile dementia, is a common degenerative Disease of the central nervous system, with memory dysfunction, language decline, and loss of cognitive function as the major clinical symptoms. The condition of AD progressively worsens, progressing from initial short-term memory impairment to dementia, with consequent loss of body function and ultimately death. According to statistics, the number of AD patients in the world reaches 0.5 hundred million at present, accounting for 50% -75% of all dementia cases, 1 person is increased every 3 seconds, the number of AD patients is estimated to be increased by more than 3 times in 2050, and the number of AD patients in China reaches 2800 ten thousand. The explosive growth of the number of people suffering from AD places a heavy burden and a difficult challenge on patients, families and society.
Alkaloid compounds such as berberine have the function of obviously inhibiting the activity of acetylcholinesterase, and are one of the most promising candidate drugs for treating AD [ Jiang Y, gao H, turdu G. Traditional Chinese medicinal herbs as potential AChE inhibitors for anti-Alzheimer's disease: A review. Bioorganic Chemistry 2017, 75. A series of studies in recent years have shown that alkaloid components inhibit the growth of pathogenic bacteria and regulate intestinal flora disturbances [ Dey A, bhattacharya R, mukherjee A, pandey DK. Natural products against Alzheimer's disease: pharmaco-therapeutics and biotechnology intermediates, biotechnology Advances 2017,35 (2): 178-216 ]. In addition, the combined effect of berberine and bifidobacterium of 300 diabetic patients is observed, the blood sugar reducing effect of the combined group patients is more remarkable than that of single berberine or single bifidobacterium administration group, the berberine can promote the proliferation of bifidobacterium and has obvious synergistic effect [ Ming J, xu SY, liu C, liu XY, jia AH, ji QH. Efficiency and safety of bifidobacterium and berberine in peptide with hyaluronic acid: study protocol for a random regulated control of three 2018,9 ]. Meanwhile, berberine alkaloids can promote the growth of strains such as bifidobacterium, lactobacillus and the like (P <0.01, the growth rate is respectively increased by about 40 percent and 35 percent), improve cognitive dysfunction [ Hussain G, rasul A, anwar H, aziz N, razzaq A, wei W, ali M, li J, li XM, role of plant derived alkaloids and the hair mechanism in neurogenetic disorders, international Journal of Biological Sciences 2018,14 (3): 341-357; trexiang, taojinhua, jiangzuo, weixiao Yan, xujun, qian Renwan, chaoan, yellow river Caoan. Berberine reverses the development of diseases such as rectal cancer, AD by promoting the growth of probiotics, regulating the flora balance of patients, improving the function of intestinal microecology [ Kumar A, thotakucura PL, tiwar BK, krishna R.target identification in Bacillus subtilis approach and expression analysis of host-pathogen protein-protein interactions. BMC Microbiology 2016,16 ]. Berberine can also simultaneously target amyloid deposition, hyperphosphorylation of Tau protein, autophagy clearance to alleviate cognitive decline in AD Mouse models, but its mechanism of action for treating AD is still actively being explored [ Chen Y, chen YL, liang YB, chen HD, ji XY, huang m. Beta. Major cognitive definer in an Alzheimer's Disease Model by targeting bone Tau phosphorylation and autophagic clearance 2020. Biomedicine & Pharmacotherapy 2020, 121.
Before 2019, only 5 drugs developed by the above-mentioned therapeutic mechanism were approved by FDA and marketed globally, including tacrine (side effect too large and now unused), donepezil, rivastigmine, galantamine and memantine; the natural medicine huperzine A developed in China for treating AD is clinically applied, however, the medicines can only slightly improve the cognitive dysfunction of patients, the effect is not obvious, and the progress of AD cannot be prevented or delayed.
Disclosure of Invention
The invention aims to solve the defects of the existing single berberine and analogues thereof or metformin in clinical curative effect and treatment range, and provides a novel alkaloid which has better curative effect than a single compound on Alzheimer disease and related diseases causing cognitive impairment, and a pharmaceutical composition of the novel alkaloid and metformin.
In one aspect, the invention provides a novel alkaloid.
The novel alkaloid 2-hydroxy-4-methoxy-7,8-dihydro-2H- [1,3] dioxane [4,5-g ] oxetano [2',3':5,6] isoquinolo [3,2-a ] isoquinolin-6-ium (2-hydroxy-4-methoxy-7, 8-dihydro-2H- [1,3] dioxolo [4,5-g ] oxo [2',3':5,6] isoquinon oligo [3,2-a ] isoquinolin-6-ium, DMQ) provided by the invention has a structural formula shown in formula I:
Figure BDA0003757227940000031
the novel alkaloids of formula I above can be prepared by extraction:
pulverizing Coptidis rhizoma, and mixingExtracting with 90-98% (or 95%) ethanol under reflux, collecting extractive solution, extracting the residue with 65-75% (or 70%) ethanol, collecting extractive solution, concentrating the extractive solution until no ethanol smell exists, mixing the extracts, adjusting pH to 9.8-10.2 (or 10), extracting with chloroform, concentrating the chloroform solution to obtain chloroform layer, separating with silica gel column, wherein CH is used 2 Cl 2 1% aqueous ammonia System/MeOH (CH) 2 Cl 2 Mixed with methanol containing 1% by volume of ammonia) was gradient eluted (elution procedure: CH (CH) 2 Cl 2 -MeOH 0-30min volume ratio of 15, 30min-60min volume ratio of 10, 60min-90min volume ratio of 8, 90min-120min volume ratio of 6, 120min-150min volume ratio of 4.
In the method, the weight ratio of the volume of the 90-98% ethanol solution to the weight of coptis chinensis can be 5-8mL to 1g, and specifically can be 5mL.
The reflux extraction may be performed a plurality of times, specifically, may be performed twice.
The weight ratio of the volume of the 65-75% ethanol to the weight of the coptis root can be 5-8mL.
The chloroform extraction may be performed a plurality of times, specifically three times.
The volume of chloroform used in each time is 1.0-1.2 times, specifically 1.0 time, of the volume after pH adjustment. Liquid phase investigation shows that the obtained chloroform layer alkaloid has rich component types and balanced trace components, so that the chloroform layer is separated.
The novel alkaloids of formula I can also be prepared according to the following synthetic scheme by a process comprising the following steps:
(1) 2- (1, 3-benzodioxol-5-yl) ethylamine and 2, 2-dimethoxyacetaldehyde react under the action of trifluoroacetic acid to generate an intermediate 1;
(2) The intermediate 1 and 4-bromo-2, 5-dimethoxybenzaldehyde are subjected to reduction reaction under the catalysis of sodium triacetoxyborohydride under an acidic condition to be coupled into an intermediate 2;
(3) The intermediate 2 is reacted in an organic solvent by utilizing trifluoromethanesulfonic acid to obtain an intermediate 3;
(4) Then carrying out demethoxylation reaction to generate an intermediate 4;
(5) The intermediate 4, N-dimethylformamide and butyl lithium are subjected to an aldehyde reaction to obtain an intermediate 5;
(6) Performing demethylation reaction on the intermediate 5 to obtain an intermediate 6;
(7) The intermediate 6 is subjected to oxidation reaction and cyclization to obtain the novel alkaloid shown in the formula I;
Figure BDA0003757227940000051
in the step (1) of the above method, the molar ratio of 2- (1, 3-benzodioxol-5-yl) ethylamine to 2, 2-dimethoxyacetaldehyde may be 1.1 to 3.
The reaction is carried out in an organic solvent, which may be specifically dichloromethane.
The reaction temperature can be 18-45 ℃ and the reaction time can be 0.5-3 h.
In the step (2) of the above method, the molar ratio of the intermediate 1 to the 4-bromo-2, 5-dimethoxybenzaldehyde may be 1.1 to 2.
The reaction temperature can be 102-171 ℃, and the reaction time can be 4-6 h.
The reaction is carried out in the presence of acetic acid.
The reaction is carried out in an organic solvent, which may be specifically methanol.
In the step (3), the organic solvent may be dichloromethane.
The molar ratio of the intermediate 2 to the trifluoromethanesulfonic acid can be 1.5-3.0.
The reaction temperature can be 80-110 ℃, and the reaction time can be 2-4 h.
In the step (4), the demethoxylation reaction is performed in an organic solvent, and the organic solvent may be ethanol.
The demethoxy reaction is shown in I 2 And potassium acetate.
Intermediates 3 and I 2 The molar ratio of the potassium acetate can be 1:1.1-1.5:1 to 20.
The reaction temperature can be 100-140 ℃ and the reaction time can be 10-20 h.
In the step (5), the molar ratio of the intermediate 4 to N, N-dimethylformamide and butyl lithium may be 1: 1.2-2.5: 1.0-1.5.
The reaction temperature can be 80-110, and the reaction time can be 3-4 h.
In the above process step (6), the demethylation reaction is carried out in the presence of aluminum chloride and potassium iodide.
The molar ratio of the intermediate 5 to the potassium iodide and the aluminum chloride can be 1:1 to 5.0:1.0-1.2.
The reaction temperature can be 20-35 ℃, and the reaction time can be 2-3 h.
In the above method, step (7), the oxidation cyclization is carried out in the presence of p-toluenesulfonic acid under UV.
The reaction temperature of the oxidation cyclization can be 150-200 ℃, and the time can be 7-8 h.
The application of the novel alkaloid shown in the formula I in the preparation of the medicine for preventing and treating Alzheimer disease and related diseases causing cognitive impairment also belongs to the protection scope of the invention.
In another aspect, the present invention provides a pharmaceutical composition comprising a novel alkaloid of formula I as an active ingredient.
The pharmaceutical composition may further comprise pharmaceutically acceptable carriers and adjuvants.
In addition, the invention also provides a pharmaceutical composition which takes the novel alkaloid shown in the formula I and metformin as active ingredients. Compared with a single medicament, the composition can obviously shorten the experimental latency of the mouse, obviously prolong the times of passing through a platform and the time of swimming in a quadrant, obviously improve the learning and memory capacity of the mouse, and simultaneously reduce the amyloid deposition in the brain of the AD mouse; can further promote the expression of the autophagy-related factor LC3I protein in brain tissues; has obvious effect of promoting the growth of bifidobacterium SHQ 1. The experimental results show that: the novel alkaloid shown in the formula I and the metformin can generate a synergistic interaction effect after being combined, and can be used for preparing medicines for preventing and treating Alzheimer disease and related diseases causing cognitive impairment.
The pharmaceutical composition provided by the present invention has a weight ratio of the novel alkaloid represented by formula I to metformin of 1.1 to 1, preferably 1.
The application of the pharmaceutical composition in preparing the medicines for preventing and treating the Alzheimer disease and the related diseases causing the cognitive impairment also belongs to the protection scope of the invention.
Advantageous effects
The novel alkaloid shown in the formula I has stronger acetylcholinesterase inhibition activity, and the drug effect of the novel alkaloid is superior to that of berberine and a positive drug galanthamine. Mouse experiments show that the novel alkaloid shown in the formula I, the novel alkaloid shown in the formula I and the metformin composition can improve cognitive dysfunction of mice, improve learning and memory abilities of the mice, prevent amyloid protein deposition in brains of AD mice and slow down the disease development process of AD mice; meanwhile, the novel alkaloid shown in the formula I and the metformin composition can also promote the expression of autophagy-related factor LC3I protein in brain tissue, and the effect of the composition on promoting the expression is more obvious. In addition, the novel alkaloid shown in the formula I and the metformin composition have obvious growth promotion effect on bifidobacterium SHQ1, and the pharmaceutical composition has more obvious promotion effect than a single novel alkaloid. The mechanism research shows that the novel alkaloid shown in the formula I, the novel alkaloid shown in the formula I and the metformin composition can improve the cognitive dysfunction of AD mice by acting on metabolites such as guanine, uridylic acid, 12-oxo-plant dienoic acid, pipecolic acid and the like to regulate a histidine metabolic pathway, a riboflavin metabolic pathway, nicotinate and a nicotinamide metabolic pathway.
In a word, the novel alkaloid shown in the formula I and the metformin composition can be used for treating Alzheimer disease and related diseases causing cognitive impairment, have synergistic effect, and have good application prospects.
Drawings
FIG. 1 is a scheme showing the synthesis scheme of novel alkaloids.
FIG. 2 is a schematic representation of DMQ 1 H-NMR spectrum (400 MHz, methanol-d 4).
FIG. 3 is a schematic representation of DMQ 13 C-NMR spectrum (100 MHz, methanol-d 4).
FIG. 4 shows the DEPT135 spectrum of DMQ (100 MHz, methanol-d 4).
FIG. 5 is a DEPT90 spectrum (100 MHz, methanol-d 4) of DMQ.
FIG. 6 is a schematic representation of DMQ 1 H- 1 HCOSY spectrum (400 MHz, methanol-d 4).
FIG. 7 shows the HMBC spectra (400 MHz, methanol-d 4) of DMQ.
FIG. 8 shows the HSQC spectra of DMQ (400 MHz, methanol-d 4).
FIG. 9 is HR-ESI for DMQ (+) -MS spectrum.
FIG. 10 is an IR spectrum of DMQ.
FIG. 11 is a graph showing the results of experiments on the AChE inhibitory activity of DMQ.
Figure 12 is a statistical plot of Barnes maze latency times for each group of mice (n =8, DMQ group (novel alkaloids); MET group (metformin); DMQ + MET group (novel alkaloids and metformin combinations)). P <0.01, P <0.001 compared to the Model.
Figure 13 is a statistical plot of the Barnes maze target quadrant percentage for each group of mice (n =8, DMQ group (novel alkaloid); MET group (metformin); DMQ + MET group (novel alkaloid and metformin combination)). P <0.01, P <0.001 compared to the Model group.
Figure 14 is a Barnes maze roadmap for each group of mice.
FIG. 15 shows congo red staining patterns of paraffin sections of mouse brains of various groups (a. Control group; b. Model group; c. DMQ group (novel alkaloid), d. MET group (metformin), e. DMQ + MET group (novel alkaloid and metformin combination)).
FIG. 16 shows the protein expression levels of LC3I in brain tissue of different groups of mice. Comparison with Control group<0.05，**P<0.01; ratio to Model groupIn comparison, the method has the advantages that, ## P<0.01。
FIG. 17 is the OD of Bifidobacterium SHQ1 cultured for 24h after single administration of novel alkaloid and pharmaceutical composition 600nm The value is obtained.
FIG. 18 is a schematic diagram of metabolic pathway analysis.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 preparation of Compounds of formula I
1.1 extracting DMQ from coptis:
pulverizing Coptidis rhizoma, reflux-extracting with 5 times volume of 95 (volume ratio)% ethanol solution twice, collecting extractive solution, extracting residue with 70 (volume ratio)% ethanol solution once, collecting extractive solution, concentrating the obtained extractive solution until no ethanol smell exists, mixing extracts, adjusting pH to 10, extracting with chloroform, concentrating the obtained chloroform solution to obtain chloroform layer, and separating with silica gel column, wherein CH is used for separating the chloroform layer 2 Cl 2 1% aqueous ammonia System in MeOH (CH) 2 Cl 2 Mixed with methanol containing 1% ammonia by volume) was subjected to gradient elution by the following procedure: CH (CH) 2 Cl 2 -MeOH 0-30min volume ratio of 15, 30min-60min volume ratio of 10, 60min-90min volume ratio of 8, 90min-120min volume ratio of 6, 120min-150min volume ratio of 4.
1.2 preparation of Compound (DMQ) of formula I with reference to the synthetic scheme shown in FIG. 1.
(1) Mixing 2- (1, 3-benzodioxol-5-yl) ethylamine and 2, 2-dimethoxyacetaldehyde in a molar ratio of 1.1 in dichloromethane, and reacting for 2.5h at 30 ℃ under the action of trifluoroacetic acid to generate an intermediate 1;
(2) Mixing the intermediate 1 and 4-bromo-2, 5-dimethoxybenzaldehyde in a molar ratio of 1.1 in methanol, and carrying out coupling reaction at 150 ℃ for 6h in the presence of acetic acid under the catalysis of sodium triacetoxyborohydride to generate an intermediate 2;
(3) Mixing the intermediate 2 with trifluoromethanesulfonic acid in a molar ratio of 1 in dichloromethane, and reacting at 100 ℃ for 4h to obtain an intermediate 3;
(4) In ethanol, intermediate 3 is in I 2 Carrying out demethoxylation reaction (reaction at 120 ℃ for 18 h) in the presence of potassium acetate to generate an intermediate 4; wherein intermediates 3 and I 2 And the molar ratio of the potassium acetate is 1:1.2: 10;
(5) Intermediate 4 was reacted with dimethylformamide and butyllithium in a molar ratio of 1:2.0:1.2, mixing, and carrying out an hydroformylation reaction at 100 ℃ for 4 hours to obtain an intermediate 5;
(6) Demethylating the intermediate 5 in the presence of aluminum chloride and potassium iodide (reacting for 2.5h at 30 ℃), wherein the molar ratio of the intermediate 5 to the potassium iodide to the aluminum chloride is 1:4.0:1.1, intermediate 6 is obtained;
(7) Under UV irradiation, the intermediate 6 undergoes oxidation cyclization in the presence of p-toluenesulfonic acid at the reaction temperature of 180 ℃ for 7 hours to obtain the novel alkaloid shown in the formula I.
Of the product 1 H NMR data and 13 the C NMR data are shown in tables 1 and 2:
table 1: of DMQ 1 H NMR data (400MHz, methanol-d) 4 ,δin ppm,J in Hz)
Figure BDA0003757227940000101
Table 2: of DMQ 13 C NMR data (100MHz, methanol-d) 4 ,δin ppm)
Figure BDA0003757227940000102
Figure BDA0003757227940000111
FIGS. 2 to 10 show the structural confirmation data of the obtained compounds.
The structural analysis of the compound is:
Figure BDA0003757227940000112
example 2 study of AChE hydrolysis inhibiting Activity of Compounds of formula I
AChE inhibitory Activity
AChE (ec 3.1.1.7) belongs to the superfamily of folded proteins of hydrolases, which also includes butylphthalocyanine enzyme, fusidic acid enzyme, lipase, etc. with high homology thereto. One of the major causes of Alzheimer's Disease (AD) is insufficient levels of acetylcholine, and several AChE inhibitors are currently used clinically to increase the concentration of ACh between synapses by inhibiting ACh hydrolysis, to alleviate the symptoms of AD patients, and to improve the learning, memory and cognitive levels of the patients.
Preparation of solutions
ATCI (thioacetyl choline iodide) solution: a2 mM ATCI solution was prepared by adding 28.9mg ATCI to 50mL deionized water.
Tris solution: weighing a proper amount of Tris, adding deionized water to prepare a solution with the concentration of 30mM, and adjusting the pH to be about 8.0.
DTNB (5, 5' -dithiobis (2-nitrobenzoic acid)) solution: 6mg of DTNB was dissolved in 50mL of Tris solution to prepare a 0.3mM DTNB solution.
AChE (acetylcholinesterase) solution: AChE was added to a Tris solution at 2U/mL.
Preparation of a solution of a compound of formula I: a proper amount of the compound shown in the formula I is weighed and added with DMSO to prepare a stock solution of the drug to be tested with the concentration of 2 mM.
Preparing a berberine solution: weighing appropriate amount of berberine, adding DMSO to prepare berberine stock solution with concentration of 2 mM.
Preparation of positive control solution: weighing a proper amount of the positive drug galanthamine, and adding DMSO to prepare a positive drug stock solution with the concentration of 2 mM.
Experimental methods
The DTNB solution and the AChE solution were mixed at a ratio of 176.5.
Placing in a microplate reader in dark, shaking for 10s, measuring absorbance at absorption wavelength of 405nm every 1min for 10min.
And taking the time of each compound to be tested as an abscissa and the absorbance as an ordinate, and performing linear fitting. The corresponding slope is calculated. The magnitude of the slope reflects the rate of reaction, i.e., enzyme activity. The activity of the compound is expressed by inhibition rate, and the higher the inhibition rate value is, the better the activity is. The specific formula is as follows:
inhibition (%) = [1- (average of absorbance and time obtained 10 times-slope of DMSO blank) ] × 100%
The results of the experiment are shown in FIG. 11.
As can be seen from fig. 11: the novel alkaloid shown in the formula I has stronger activity of inhibiting acetylcholinesterase, and the inhibition activity is more remarkable than that of positive medicines galanthamine and berberine.
EXAMPLE 3 pharmacodynamic Studies of treatment of AD
1. Test materials and methods.
1. Experimental drugs and reagents
APPswe/PS1 at 6 months of age and the mice, SPF grade, 40, were purchased from Nanjing Junceae bioengineering, inc., and the animals were raised at Harbin medical university college.
2. The preparation method of the composition comprises the following steps:
the weight ratio of the novel alkaloid to the metformin in the composition is 1.
3. Experimental methods
(1) Animal grouping and dosing: after one week feeding of APPswe/PS1 mice, the experiment was divided into 5 groups in total: control group; model group; dmq group (novel alkaloids); met group (metformin); dmq + MET group (novel alkaloid and metformin composition in a weight ratio of 1. The administration was performed 1 time per day (once daily, the dose of the novel alkaloid per administration was 10mg/kg of mouse body weight, metformin was 12mg/kg of mouse body weight, and the Control group and Model group were given physiological saline of the same volume) for 6 weeks.
(2) And (3) behavioral detection: barnes maze experiments were performed 6 weeks after dosing.
The Barnes maze method was used to perform spatial learning and memory function tests on experimental mice. The Barnes maze was designed based on the "happy-dark" habit of experimental mice, which were allowed to find the corresponding holes in the dark box by some external stimulus at the prescribed time. The whole test is divided into two stages, namely a training stage and a testing stage, and after a period of continuous training, the total route, the speed and the time of entering a camera bellows and the frequency of entering an error hole are recorded so as to investigate the spatial reference memory capacity of the camera bellows. The matters required for the experiment are as follows: the randomness of the hole distribution and the positions of the maze are different after each experiment and two consecutive days, but the position of the target box cannot be changed in the process. When the animal loiters at the opening and the head extends into the opening, the eyes of the experimental mouse are lower than the edge of the platform to judge the escape error.
(3) Collection and preparation of samples
a. Fecal samples of each group of mice administered for the last time, taken over 0-24 hours, were sacrificed and brain tissue was stripped off by decapitation.
b. Naturally air drying feces, and grinding 0.5g into powder; the brain tissue was homogenized and 0.1g was taken. Ultrasonically extracting feces with 10 mL and 2mL of methanol for 30min, centrifuging after extraction, collecting supernatant, blow-drying with nitrogen, redissolving with 200 μ L of methanol, and filtering with 0.22 μm microporous membrane. The fecal samples were cryopreserved at-80 ℃.
c. Frozen feces samples are taken, 5 times of physiological saline (w/v) is added to the feces samples to be placed on ice for tissue homogenization, the feces samples are centrifuged at 4 ℃ and 13000rpm for 15min, 80 mu L of supernatant is absorbed, 240 mu L of precooled methanol is added, vortex oscillation is carried out for 1min, the feces samples are placed on ice for standing for 20min and then centrifuged at 4 ℃ and 12000rpm for 15min, and 150 mu L of supernatant is absorbed to a sample injection vial. In addition, 10. Mu.L of the above-prepared fecal sample was precisely aspirated to obtain a fecal QC sample.
(4) Pathology detection
Congo red dyeing: fixing the hippocampal tissue with 4% paraformaldehyde, embedding in paraffin, dehydrating, making 1 slice with a freezing microtome, staining with Congo red, and observing the brain tissue morphological expression and blood vessel amyloid deposition of mice.
2. The experimental results are as follows:
the pharmacological effects of the novel alkaloid and the pharmaceutical composition shown in formula I can improve the learning and memory abilities of AD mice, as shown in FIG. 12, compared with the Control group, the experimental latency of the Model group mice is obviously longer than that of the Control group mice, the experimental latency of the novel alkaloid group mice is obviously shorter than that of the Model group mice, the experimental latency of the MET group mice is not obviously changed than that of the Model group mice, and the experimental latency of the composition group mice is further shortened than that of the Model group mice. Figure 13 shows that the percentage of target quadrant in Model group mice was significantly reduced, compared to the percentage of target quadrant in both novel alkaloid group mice and pharmaceutical composition group mice, the cognitive dysfunction in mice was significantly improved, and the percentage of target quadrant in MET group mice was relatively reduced. The maze roadmap of the novel alkaloid group mouse and the pharmaceutical composition group mouse in fig. 14 is simpler than that of the Model group mouse, which shows that the learning and memory ability of the mouse is improved.
FIG. 15 shows that slight red amyloid plaque deposition appears in the brains of Model group mice, and the fibrosis plaques in the brains of the mice of the novel alkaloid group mice and the pharmaceutical composition group mice are negative, which indicates that the two groups can prevent the amyloid deposition in the brains of AD mice, thereby slowing down the disease progression of AD mice.
Example 4 Effect on autophagy of nerve cells
1. Test materials and methods.
1. Experimental drugs and reagents
Newborn sterile APPswe/PS1 mice, SPF grade, 32, purchased from Nanjing Junceae bioengineering GmbH, and animals bred at Harbin medical university college of medicine.
2. The preparation method of the composition comprises the following steps:
the weight ratio of the novel alkaloid to metformin in the composition is 1.2.
3. Experimental methods
(1) Animal grouping and dosing: after one week feeding of APPswe/PS1 mice, the experiment was divided into 4 groups in total: control group; model group; dmq group (novel alkaloids); dmq + MET group (novel alkaloid and metformin composition in a weight ratio of 1. The administration was performed 1 time per day (once daily, the dose of the novel alkaloid per administration was 10mg/kg of mouse body weight, metformin was 12mg/kg of mouse body weight, and the Control group and Model group were given physiological saline of the same volume) for 6 weeks.
(2) Effect on autophagy of nerve cells
a. Primary neural cell model: anesthetizing the mouse, cutting off the head to strip the brain tissue, cutting the cortex hippocampus on two sides, completely removing the meninges and the vascular tissue, digesting with pancreatin, repeatedly blowing, standing for 3min, taking the supernatant suspension, passing through the membrane to remove the unseparated tissue block, centrifuging at 1000rpm for 5min, and discarding the supernatant. Adding stop solution, re-suspending, centrifuging at 1000rpm for 5min, discarding supernatant, and repeating twice. At 5X 10 5 The density per mL is planted in culture flasks. Replacing the culture solution every 3d, culturing till 2-3 generations, and carrying out subsequent experiments.
Western Blot: extracting total protein of cell and tissue sample, adding protein loading buffer solution and RIPA cell lysate, diluting, and denaturing at 100 deg.C for 5min. Protein samples were separated by SDS-PAGE and transferred to PVDF membrane. 5% milk was blocked for 2h at room temperature, incubated primary antibody for 2h at room temperature, the membrane was washed 3 times with TBS-T buffer, and then incubated with secondary antibody labeled with an infrared fluorescent dye for 1h. And detecting the protein expression condition by using a double-color infrared fluorescence imaging system, and calculating the relative gray value of the band of each sample and the beta-actin.
2. Results of the experiment
By comparing the expression of LC3I in brain tissue 6 weeks after the administration of the Control group, model group, DMQ + MET group mice (n = 8), it was found that: compared with the Control group mice, the novel alkaloid and metformin composition can promote the expression of autophagy-related factor LC3I protein in brain tissues, and the effect of the pharmaceutical composition on promoting the expression is more obvious (figure 16).
Example 5 Effect on the growth of Bifidobacterium SHQ1
1. The experimental method comprises the following steps:
(1) Preparation of culture medium, diluent and administration solution:
preparation of a culture medium: beef extract 0.3g, peptone l g, glucose 1g, sodium chloride 0.5g, cysteine hydrochloride 0.04g, water 100mL, pH value adjusted to 7.0, subpackaging, and sterilizing at 115 deg.C for 15min.
(2) Preparing a diluent: 4.5g of monopotassium phosphate, 6g of disodium hydrogen phosphate, 0.5g of cysteine hydrochloride, 0.5mL of tween-80, l000 mL of distilled water and 1g of agar, subpackaging, and sterilizing at 221 ℃ for 20min.
(3) Preparation of novel alkaloid and composition solutions: dissolving the medicine in double distilled water, and sterilizing at 115 deg.C for 15min. Under the in vitro anaerobic condition, the bifidobacterium SHQ1 is inoculated in a slant culture medium according to the inoculation amount of 0.1 percent, and after being cultured for 5 days, the bifidobacterium is subcultured on a new slant for 72 hours. The solutions of the administration groups (0.5 mg/mL,1.0mg/mL,2.0 mg/mL) were added at different concentrations, and diluted 10-fold with the diluent, and the control group was a blank medium. Each group is provided with three multiple holes, the OD value is measured once at the wavelength of 600nm at the interval of 2h by using a full-automatic growth curve analyzer, a growth curve is drawn, and the specific growth rate is calculated.
2. Results of the experiment
As shown in fig. 17, the bifidobacterium SHQ1 strain was cultured in vitro at the early stage, and compared after being added with the novel alkaloid and the composition to be cultured for 24h, the result found that the novel alkaloid and the composition have an obvious effect (P < 0.01) on promoting the growth of the bifidobacterium SHQ1, and the pharmaceutical composition has a more significant effect (P < 0.01) than a single novel alkaloid.
Example 6 mechanism of action for the treatment of Alzheimer's disease
1. The experimental method comprises the following steps:
a. the detection method comprises the following steps: the chromatographic column is ACQUITY
Figure BDA0003757227940000171
CSHTM C18 (2.1 mm. Times.100 mm, 1.7 μm), mobile phase A0.1% formic acidSolution (v/v), mobile phase B acetonitrile, elution gradient as follows: 0-3min,1% mobile phase B;4-7min,15% mobile phase B;8-15min,45% mobile phase B; 16-24min,60% mobile phase B;25-39min,70% mobile phase B;30-31min,100% mobile phase B;32-35min,1% mobile phase B. The sample introduction amount is 5. Mu.L, the flow rate is 0.3mL/min, and the column temperature is 40 ℃. The amount of feces samples (feces samples collected in 3 (3) of example 3) was 5. Mu.L, 10. Mu.L of each of the 3 groups of blank group, model group, and administration group (DMQ group) was mixed to prepare a quality control sample, 3 ions of different polarities were selected from them for methodological evaluation, and their peak areas and retention times were extracted for methodological verification. And continuously feeding a quality control sample for analysis to detect the repeatability of the analysis method. To ensure the stability and reproducibility of the system, 1 QC sample was added every 3 samples for a total of 4 injections in the analysis sequence.
Collecting feces sample with Orbitrap Fusion Lumos mass spectrometer, adopting heated electrospray ionization (HESI) method, positive and negative ion conversion acquisition mode, and scanning method Full Scan/dd-MS 2 (ii) a The spraying voltage anode and cathode are respectively 3.5KV and 2.5KV; the capillary temperature was 325 ℃; the sheath gas volume flow is the flow rate 50arb; the volume flow of the auxiliary gas is 10arb; resolution of 120 000; the collection range is m/z 100-800; HRMS 2 The intensity threshold is 2.0 × 10 4 ；HRMS 2 Scanning high-energy collision dissociation to 20%, 35% and 50%, and the resolution is 30000; the collision induced dissociation was 15%, 30%, 45% with a resolution of 30000.
b. And (3) data analysis and processing: importing mass spectrum original data into Compound distributor 3.0 software, selecting an 'unused molecules workflows' module to perform noise reduction, peak identification, matching and normalization processing on the original data, and setting relevant parameters as follows: mass tolerance is 5ppm, maximum shift is 0.2min, minimum intensity is 1000000, the generated two-dimensional data matrix containing mass-to-charge ratio, retention time and peak area is led into SIMCA 14.0 software to carry out multivariate variable data statistical Analysis, including Principal Component Analysis (PCA), orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), VIP >1 is screened based on OPLS-DA, t test p is less than 0.05, and variables satisfying the following two conditions: first, the metabolites are differential metabolites between the Control group and the Model group; second, the metabolite was significantly recalled after DMQ treatment in the DMQ panel compared to the Model panel. The variables screened above were identified by searching the database mzcloud, chemSpider, etc., using the "unused strategies workflow" module of the Compound discover 3.0 software. Pathway analysis is carried out on different metabolites of each sample by adopting Metabionalyst 5.0 (https:// www. Metabionalyst. Ca /), and the pathway with pathway impact >0.1 is screened as the relevant pathway for treating the Alzheimer disease by the novel alkaloid.
2. Results of the experiment
As shown in table 3, 10 VIP >1 and p <0.05 potential biomarkers were finally screened from the S-plot: guanine, adenine, uridylic acid, methyl isonicotinate, pipecolic acid, 12-oxovegetable dienoic acid, stearamide, hydrolyzed fumonisin B1, 2' -deoxyadenosine, and the like.
TABLE 3 marker screening
Figure BDA0003757227940000181
As shown in figure 18, table 4, the Metaboanalyst 5.0 screen results for the riboflavin metabolic pathway, the histidine metabolic pathway, the nicotinate and the nicotinamide metabolic pathway, revealing that the novel alkaloids may act on these 3 metabolic pathways to treat AD.
TABLE 4 Metabolic pathways
Figure BDA0003757227940000182
Figure BDA0003757227940000191
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (7)

1. The alkaloid shown in the formula I has the following structural formula:
Figure FDA0003988758470000011
2. a process for the preparation of an alkaloid of formula I according to claim 1, comprising the steps of:
crushing the coptis root, performing reflux extraction by using a 90-98% ethanol solution, collecting an extracting solution, extracting filter residues by using a 65-75% ethanol solution, collecting the extracting solution, concentrating the obtained extracting solution until no alcohol smell exists, combining extractum, adjusting the pH value of the extractum to 9.8-10.2, performing chloroform extraction, concentrating the obtained chloroform solution to obtain a chloroform layer, and separating the obtained chloroform layer by using a silica gel column, wherein CH is adopted 2 Cl 2 Gradient elution is carried out in a/MeOH-1% ammonia water system, and the obtained fraction is separated by Sephadex LH-20 column chromatography to obtain the alkaloid shown in the formula I.
3. The preparation method according to claim 2, wherein the volume of the 90% -98% ethanol solution to the weight ratio of coptis chinensis is 5-8mL; the weight ratio of the volume of the 65% -75% ethanol to the coptis root is 5-8mL.
4. A process for the preparation of an alkaloid of formula I according to claim 1, comprising the steps of:
(1) 2- (1, 3-benzodioxol-5-yl) ethylamine and 2, 2-dimethoxyacetaldehyde react under the action of trifluoroacetic acid to generate an intermediate 1;
(2) The intermediate 1 and 4-bromo-2, 5-dimethoxybenzaldehyde are subjected to reduction reaction under the catalysis of sodium triacetoxyborohydride under an acidic condition to be coupled into an intermediate 2;
(3) The intermediate 2 is reacted in an organic solvent by utilizing trifluoromethanesulfonic acid to obtain an intermediate 3;
(4) Then carrying out demethoxylation reaction to generate an intermediate 4;
(5) The intermediate 4, N-dimethylformamide and butyl lithium are subjected to an aldehyde reaction to obtain an intermediate 5;
(6) Performing demethylation reaction on the intermediate 5 to obtain an intermediate 6;
(7) The intermediate 6 is subjected to oxidation reaction and cyclization to obtain the alkaloid shown in the formula I;
Figure FDA0003988758470000021
5. the method according to claim 4, wherein the method is prepared according to the reaction equation shown below:
Figure FDA0003988758470000031
6. the production method according to claim 4,
in the method step (1), the molar ratio of 2- (1, 3-benzodioxol-5-yl) ethylamine to 2, 2-dimethoxyacetaldehyde is 1.1-3; the reaction temperature is 18-45 ℃ and the reaction time is 0.5-3 h;
in the method step (2), the molar ratio of the intermediate 1 to the 4-bromo-2, 5-dimethoxybenzaldehyde is 1.1-2; the reaction temperature is 102-171 ℃, and the reaction time is 4-6 h; the reaction is carried out in the presence of acetic acid;
in the step (3), the molar ratio of the intermediate 2 to the trifluoromethanesulfonic acid is 1.5-3.0; the reaction temperature is 80-110 ℃, and the reaction time is 2-4 h;
in method step (4), the demethoxy reaction is carried out in step I 2 And potassium acetate; intermediates 3 and I 2 And the molar ratio of the potassium acetate is 1:1.1-1.5:1 to 20; the temperature of the reactionThe temperature is 100-140 ℃, and the time is 10-20 h;
in the step (5), the molar ratio of the intermediate 4 to the N, N-dimethylformamide and butyl lithium is 1: 1.2-2.5: 1.0-1.5; the reaction temperature is 80-110 ℃, and the reaction time is 3-4 h;
in process step (6), the demethylation reaction is carried out in the presence of aluminum chloride and potassium iodide; the molar ratio of the intermediate 5 to potassium iodide and aluminum chloride is 1:1 to 5.0:1.0-1.2; the reaction temperature is 20-35 ℃ and the reaction time is 2-3 h;
in the method step (7), the oxidation cyclization is carried out under UV and in the presence of p-toluenesulfonic acid; the reaction temperature of the oxidation cyclization is 150-200 ℃, and the time is 7-8 h.
7. Use of an alkaloid of formula I according to claim 1 for the preparation of a medicament for the prevention and treatment of alzheimer's disease and related diseases leading to cognitive impairment.
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