CN113842406A - Application of mulberry aqueous extract in preparation of medicine for treating Alzheimer's disease - Google Patents
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Abstract
The invention discloses a new application of a mulberry aqueous extract, in particular to an application of the mulberry aqueous extract in preparing a medicament for treating Alzheimer disease, and belongs to the field of biological medicines. The mulberry aqueous extract acts on a pathologic model of the Alzheimer's disease caenorhabditis elegans, and is found to have a remarkable treatment effect on the caenorhabditis elegans of the pathologic model of the Alzheimer's disease, such that the muscle paralysis phenotype of the Alzheimer's disease caenorhabditis elegans is remarkably delayed, and the mulberry aqueous extract has the potential of treating neurodegenerative diseases.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an application of a mulberry aqueous extract in preparation of a medicine for preventing or treating Alzheimer disease.
Background
Alzheimer's Disease (AD) is a progressive degenerative disease of the nervous system with occult onset. Clinically, it is characterized by generalized dementia such as memory impairment, aphasia, disuse, agnosia, impairment of visual-spatial skills, dysfunction in executive functioning, and personality and behavioral changes.
AD is the most common and predominant degenerative disease of the brain, with a significant annual upward trend in incidence. Most data show a prevalence of about 5% over 65 years and 20% over 85 years. The disease is slow in onset or latent, and patients and their families often say it is unclear when the disease is onset. A few patients develop rapid and distinct symptoms after physical illness, fracture or mental irritation. Women are more than men (female: male is 3: 1). Mainly manifested by decline of cognitive function, mental symptoms and behavioral disorders, and gradual decline of daily living ability. The deterioration degree of cognitive ability and physical function is divided into three periods according to which physical and psychological health of old people is seriously damaged and life quality is influenced, deep pain is caused to patients, heavy burden is brought to families and society, and the three periods become serious social problems and cause general attention of governments and medical circles of various countries.
As a major type of senile dementia, the pathological features of Alzheimer's disease include degeneration of local brain, especially hippocampal and cortical neurons, intracellular neurofibrillary tangles and extracellular senile plaque deposits[1,2]. Modern medicine has studied the pathogenesis of AD for many years, but the pathogenesis of the AD is still unclear due to its complex etiology, and in the research on the pathogenesis of AD, many evidences indicate that a β (β -amyloid, a major component of senile plaque) is a major pathological link that induces the development of AD. First, amyloid plaques are formed by the deposition of a β, which is caused by a dysbolic disorder of its own clearance and degradation, and a large number of amyloid plaque deposits are found in the brain of almost all AD patients[3]. Secondly, A beta can promote nerve cell apoptosis[4]. Thirdly, the A beta can also activate the glial cells, release cytokines and inflammatory reaction mediators and generate inflammatory reaction, and the A beta is used as a stimulator to accelerate the death of nerve cells by activating peripheral microglia to generate cytokines and immune and inflammatory responses of the nervous system, so that the memory decline and the cognitive disorder are caused. Thus, amyloid beta has become a recognized target for screening anti-AD drugs. The causes of AD are complex and changeable, the disease course is long, the pathogenesis is complex, and long-term administration is requiredAt present, the main anti-Alzheimer disease drug memantine is expensive, and has obvious side effects of nausea, vomiting, diarrhea, hypodynamia, hallucinations, consciousness chaos, dizziness, headache, tiredness and the like after being taken, and the condition of patients can only be relieved but not cured. Until now, no specific medicine exists for treating AD, most of the medicines are still in the stage of basic research and clinical trials, and scientists are still continuously screening anti-AD medicines.
The Chinese medicine has been used for 5000 years in China[5]The traditional Chinese medicine also plays a role in improving the life quality of the old and promoting the health of human beings. Mulberries, also known as mulberries, jujubes, mulberries, etc., are mature fruits of mulberry (Morus albaLinn.) belonging to the family of moraceae. Modern medical clinical evidence: the mulberry has good effects of nourishing heart, liver and kidney, nourishing blood and dispelling wind. Has obvious curative effects on reducing blood fat, relieving neurasthenia, arteriosclerosis, sexual function weakness, deafness and dim eyesight, early white beard and hair, internal heat diabetes, blood deficiency constipation and rheumatic arthralgia. The mulberry contains a substance named resveratrol, and the component of resveratrol has been proved in-vivo and in-vitro experiments on the neuroprotective effect of human body, and can improve cognitive ability[6]. However, resveratrol is difficult to dissolve in water and easy to dissolve in organic solvent, and the extraction efficiency of the water extraction method is low[7]The extraction rate of the resveratrol extracted by water at 100 ℃ reaches 0.1713 percent[8]。
Caenorhabditis elegans (Caenorhabditis elegans) is a good model organism for the study of AD. It is cheap and easy to culture; the generation period is short, the number of offspring is large, a large number of individuals with consistent backgrounds can be obtained, the experimental repeatability is guaranteed, and experiments are carried out by adopting a large sample size, so that the influence of individual differences is eliminated. Therefore, the transgenic caenorhabditis elegans strain CL4176 is used as a pathological model for screening the medicines for preventing or treating the Alzheimer disease to evaluate the effect of the mulberry extract on preventing or treating the Alzheimer disease.
The invention provides an application of a mulberry aqueous extract in preparation of a medicine for treating Alzheimer's disease. The inventor surprisingly discovers that the mulberry aqueous extract disclosed by the invention has the effect of resisting Alzheimer disease after screening a large number of traditional Chinese medicine compositions by adopting a caenorhabditis elegans AD pathological model, and can be used for preparing a medicine for treating Alzheimer disease. Experiments in the embodiment prove that the curative effect of the mulberry aqueous extract on treating the Alzheimer disease is obviously better than that of the ready-made memantine hydrochloride.
Reference to the literature
[1]Mattson MP.Pathways towards and away from Alzheimer’s disease.Nature,2004,430； 631-639.
[2]Hardy J,Selkoe D J.The amyloid hypothesis ofAlzheimer’s disease:progress andproblems on the road to therapeutics.Science,2002,297:353-356.
[3]Fu HJ,Liu B,Frost JL,et al.Amyloid-beta immunotherapy for Alzheimer’s disease[J]. CNS Neurol DisordDrud Targets,2010,9(2):197-206.
[4]Nelson TJ,Alkon DL.Oxidation of cholesterol by amyloid precursor protein and beta-amyloidpeptide[J].J Biol Chem,2005,280(8):7377-7387.
[5]YuY.,Dosanjh L.,Lao L.,et al.Cinnamomum cassia Bark in two herbal formulas increase life span in Carnohabditis elegans via insulin signaling and stress response pathways[J].PLOS ONE,2010,5(2).
[6] The research progress of resveratrol [ J ] Chinese herbal medicine 2016,47(14):2568-2578.
[7] The extraction and detection method of resveratrol is developed [ J ] food and machinery, 2013,29(02): 234-.
[8] Orthogonal experimental method is preferably the extraction process of resveratrol in giant knotweed rhizome [ J ] in Shizhen national medicine (2006 (04): 594-595).
Disclosure of Invention
The invention discloses an application of a mulberry aqueous extract in preparation of a medicine for treating Alzheimer's disease.
Preferably, the mulberry is dried mature fruit of Moraceae plant mulberry (Morus albalin.).
The preparation method of the mulberry aqueous extract comprises the following steps:
soaking Mori fructus in water, boiling, filtering, mixing filtrates, and centrifuging to obtain Mori fructus water extract.
The dosage form of the mulberry aqueous extract is one of tablets, capsules, powder, syrup, granules, medicinal granules, pills, mixtures or dropping pills.
The experiment is carried out by taking caenorhabditis elegans as a pathological model of Alzheimer's disease, and the result shows that: the mulberry aqueous extract has a remarkable treatment effect on caenorhabditis elegans suffering from Alzheimer's disease. The mulberry aqueous extract has a remarkable inhibiting effect on the paralytic phenotype caused by the intramuscular A beta overexpression of the AD caenorhabditis elegans, namely the paralytic phenotype of the Alzheimer's disease caenorhabditis elegans can be remarkably delayed, and the mulberry aqueous extract has the potential of treating the Alzheimer's disease and can be applied to the preparation of the medicine for treating the Alzheimer's disease.
The technical solution of the present invention is further described in detail with reference to the following specific examples, but the scope of the present invention includes, but is not limited to, the following.
Drawings
FIG. 1 shows the effect of aqueous extracts of Mori fructus at various concentrations on the retardation of paralysis caused by AD C.
FIG. 2A shows an HPLC chromatogram of Resveratrol (Resveratrol) at 360 nm.
FIG. 2B shows HPLC chromatograms of CA (chlorogenic acid), Isoquercitrin (Isoquercitrin), Rutin (Rutin), Astragalin (Astragalin) at 360 nm.
Figure 2C shows an HPLC chromatogram of a mulberry aqueous extract MB recorded at 360 nm.
Detailed Description
Example selection of a pathological model
The transgenic caenorhabditis elegans strain CL4176 is used for screening pathological models for preventing or treating Alzheimer disease to evaluate the mulberry extract for preventing or treating the Alzheimer disease. The nematode strain can grow normally at 15 deg.C, when transferred to 25 deg.C1-42Is expressed and accumulated in the muscle of the nematode, and leads the nematode to lose the motor ability and be paralyzed. The addition of the test drug delayed the muscle paralysis phenotype, with the results expressed as the number of muscle paralysis nematodes as a percentage of the total number of the population of test nematodes. The higher the nonparallel nematode ratio, the more significant the anti-AD effect of the drug.
EXAMPLE II preparation method of aqueous extract of Mulberry
Taking 5g of dried and mature mulberry, adding 200mL of deionized water, soaking for 30min, heating, boiling and keeping for 30min, naturally cooling to room temperature, filtering, separating filtrate and filter residue, soaking the filter residue for 20min with 200mL of deionized water, heating to boil with strong fire, heating for 25min, naturally cooling to room temperature, filtering, discarding the filter residue, taking the filtrate, centrifuging the filtrate at 12000rpm for 10min, combining the two filtrates, diluting to 100mL to obtain 50.00mg/mL of mulberry aqueous extract (based on crude drug), and diluting by 10 times and 100 times to obtain 5.00mg/mL of mulberry aqueous extract (based on crude drug) and 0.50mg/mL of mulberry aqueous extract (based on crude drug).
EXAMPLE therapeutic Effect of three different concentrations of aqueous Mulberry extract on the treatment of C.elegans CL4176
1. Biological material
(1) Caenorhabditis elegans CL4176 was purchased from Caenorhabditis Genetics Center; for transgenic lines, specifically expressing human a β in muscle at 25 ℃ under induction, a β aggregates in muscle tissue, eventually leading to nematode paralysis, the present example employed caenorhabditis elegans line CL4176 as a pathological model for screening drugs for preventing or treating alzheimer's disease.
(2) Escherichia coli OP50 (uracil leaky mutant), purchased from Caenorhabditis Genetics Center, as a feed for C.elegans.
2. Reagent
(1) Memantine hydrochloride, chemical name: 1-amino-3, 5-dimethyladamantane hydrochloride of formula: C12H 21N. HCl, available from carbofuran technologies, CAS: 41100-52-1; memantine hydrochloride is a non-competitive NMDA receptor antagonist for the treatment of moderate to severe alzheimer dementia, and the literature reports that the paralytic phenotype of the transgenic nematode strain CL4176 is significantly inhibited, which is used as a comparative positive drug in this example.
(2) Solid NGM (Newatode Growth Medium) medium composition and preparation (taking 1 liter as an example):
after the solid NGM culture medium is prepared, sterilizing at 121 deg.C under high pressure and constant temperature for 20min, adding 5g/L cholesterol 1mL, 1M MgSO41mL,1M CaCl21mL was shaken well and poured hot into a sterilized 9cm plate, approximately 20 mL/plate. Standing for cooling and solidifying the culture medium for later use.
(3) M9 buffer solution formula
| Composition (I) | Content (wt.) |
| NaCl | 4.97g |
| Na2HPO4 | 5.96g |
| KH2PO4 | 2.99g |
| MgSO4 | 0.12g |
| Supplement H2O to | 1L |
(4) Preparation of lysate: a6.4% NaClO solution and a 1M NaOH solution were mixed in a volume ratio of 1: 1.
(5) Preparing a memantine hydrochloride solution with optimal drug effect concentration: memantine (2.0 mg) was weighed out, and 186.04. mu.L of dimethyl sulfoxide (DMSO) was added to prepare a 50mM solution (10.75 mg/ml).
3. Preparing an NGM flat plate:
(1) preparing NGM culture medium (not containing agar, magnesium sulfate, calcium chloride and cholesterol) according to NGM formula, subpackaging the culture medium into 6 conical flasks as 6 groups, wherein each conical flask is filled with 20ml of NGM culture medium liquid, adding 0.343g of agar into each conical flask, sealing with sealing film, and sterilizing at 121 deg.C for 20 min.
(2) When the medium temperature was lowered to 80 ℃, 20. mu.L of 1M magnesium sulfate, 20. mu.L of 1M calcium chloride, and 20. mu.L of 5mg/mL cholesterol were added to each flask. Then, 2mL of deionized water sterilized and cooled to room temperature, 20. mu.L of dimethyl sulfoxide DMSO, 2mL of the above-mentioned 50.00mg/mL mulberry aqueous extract (based on crude drug), 2mL of the above-mentioned 5.00mg/mL mulberry aqueous extract (based on crude drug), 2mL of the above-mentioned 0.50mg/mL mulberry aqueous extract (based on crude drug), 20. mu.L of the above-mentioned 50mM memantine hydrochloride solution, and 1.98mL of deionized water sterilized and cooled to room temperature were added to each Erlenmeyer flask, respectively, and after thoroughly mixing, the mixture was divided into 3 cm-diameter culture dishes, and about 4mL of culture medium was added to each culture dish, and each set of 4 culture dishes was counted as a control group (H), respectively2O), a control group (DMSO), a drug group (MB-H), a drug group (MB-M), a drug group (MB-L), and a positive control group (MJG).
Standing for culture medium solidification. Liquid E.coli OP50 was spread evenly on the medium as feed for the nematodes.
4. Carrying out the step
(1) And (3) culturing nematodes:
the nematodes were plated on solid NGM plates coated with E.coli OP50 and then incubated in an incubator at 15 ℃ and synchronized when they reached adult growth.
(2) Nematode synchronization:
selecting NGM culture medium containing a large amount of imago and part of nematode eggs which have been hatched, flushing the nematodes from the culture medium by using M9 buffer solution, transferring the nematodes to a centrifuge tube, standing the nematode to enable the nematode to freely settle to the bottom of the tube, and discarding the supernatant. And (3) adding the lysate into the centrifuge tube according to the amount of the nematode, oscillating for 5-7min on a vortex stirrer, stopping vortex when the nematode is completely broken, subpackaging the nematode into 1.5mL centrifuge tubes, and washing nematode eggs for three times by using an M9 solution.
(3) And (3) counting nematode paralysis:
transferring the synchronized nematode eggs subpackaged in the centrifuge tubes to different groups of culture dishes, culturing 60 nematodes in each culture dish, setting 4 culture dishes in each group as parallel, and culturing at 15 ℃ for 3 days until the L3 stage of the nematodes. In order for the nematodes to express the A.beta.protein, the L3 phase nematodes were transferred to 25 ℃ for induction and the number of paralyzed nematode sticks started to be counted after 34 h. Counting every two hours until the nematodes are paralyzed to about 80% (paralysis means that the nematodes cannot move or only the head moves when the nematode body is mechanically stimulated). The results are shown in FIG. 1.
FIG. 1 shows the non-paralyzed percentage of different concentrations of aqueous mulberry extract to C.elegans CL4176
The ordinate of FIG. 1 shows the percentage of C.elegans which are not paralyzed, and at the same time point, a larger value indicates a larger proportion of C.elegans which are not paralyzed in the treated group, i.e., a stronger effect of the drug in delaying paralysis.
Wherein the final concentration of each group of drug-containing culture medium is as follows: the high concentration group (MB-H) of the mulberry aqueous extract contains 5.00mg/mL of mulberry (based on the crude drug amount); the concentration group (MB-M) of the mulberry aqueous extract contains 0.50mg/mL of mulberry (calculated by crude drug); the low concentration group (MB-L) of the mulberry aqueous extract contains 0.05mg/mL of mulberry (calculated by crude drug); memantine control group (MJG) contained Memantine hydrochloride at 50. mu.M.
The experiment result shows that the DMSO solvent does not produce additional influence on the experiment, the mulberry aqueous extract containing 5.00mg/mL, 0.50mg/mL and 0.05mg/mL has obvious inhibition effect on the paralytic phenotype caused by the overexpression of A beta in the muscle of the AD caenorhabditis elegans, compared with the group with the highest drug effect, the group with high concentration and medium concentration of the mulberry has more obvious effect on delaying the paralytic phenotype of the Alzheimer's disease caenorhabditis elegans, and has the capability of more obviously treating the Alzheimer's disease.
Example HPLC fingerprint of an aqueous extract of Mori fructus
HPLC analysis was performed using Agilent 1260 and C18 columns (250 mm. times.4.6 mm). Preparing mulberry water extract MB (10mg/mL), standard CA (chlorogenic acid), Isoquercitrin, Rutin (Rutin), Astragalin (Astragalin) (1mg/mL) and Resveratrol (Resveratrol) (1 mg/mL). The extract and standards were dissolved in water/methanol and the solution was filtered through a 0.22 μm filter head. Mobile phase a (acetonitrile) and mobile phase B (water containing 0.2% phosphoric acid) were used at a flow rate of 1.0 ml/min. The column temperature was maintained at 30 ℃ and the injection volume was 20. mu.L, and the signal was recorded by UV spectroscopy at 360 nm.
The gradient elution conditions were as follows.
The MB component is analyzed by selecting five related compounds of CA (chlorogenic acid), Isoquercitrin, Rutin, Astragalin and Resveratrol, and the retention time is 9.210min, 13.774 min, 13.087min, 15.013min and 18.317min respectively. FIG. 1A shows an HPLC chromatogram of Resveratrol (Resveratrol) at 360 nm. FIG. 1B shows HPLC chromatograms of CA (chlorogenic acid), Isoquercitrin (Isoquercitrin), Rutin (Rutin), Astragalin (Astragalin) at 360 nm. FIG. 1C shows an HPLC chromatogram of recorded MB at 360 nm. The standard substance is consistent with the comparison of the extract spectrums and can be used as an index of the mulberry extract, and the comparison chromatogram does not have resveratrol in MB, which indicates that the resveratrol does not play an anti-AD role in the mulberry water extract.
Claims (4)
1. An application of a mulberry aqueous extract in preparing a medicament for treating Alzheimer's disease.
2. The use according to claim 1, wherein the mulberry is dried ripe fruit of Moraceae plant Mulberry (Morus alba Linn.).
3. The use of claim 1, wherein the mulberry aqueous extract is prepared by a method comprising: soaking Mori fructus in water, boiling, filtering, mixing filtrates, and centrifuging to obtain Mori fructus water extract.
4. The use of claim 1, wherein the mulberry aqueous extract is in a dosage form of one of a tablet, a capsule, a powder, a syrup, a granule, a pill, a mixture or a drop pill.
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Citations (3)
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| CN106924350A (en) * | 2017-04-07 | 2017-07-07 | 龙文杰 | Mulberry extract and preparation method and application thereof |
| CN110016084A (en) * | 2018-01-08 | 2019-07-16 | 中国科学院上海药物研究所 | A kind of mulberry fruit proteoglycan, preparation method and the usage |
| CN110615821A (en) * | 2019-09-17 | 2019-12-27 | 西北大学 | Mulberry extract, extraction and separation method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106924350A (en) * | 2017-04-07 | 2017-07-07 | 龙文杰 | Mulberry extract and preparation method and application thereof |
| CN110016084A (en) * | 2018-01-08 | 2019-07-16 | 中国科学院上海药物研究所 | A kind of mulberry fruit proteoglycan, preparation method and the usage |
| CN110615821A (en) * | 2019-09-17 | 2019-12-27 | 西北大学 | Mulberry extract, extraction and separation method and application thereof |
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Application publication date: 20211228 |