CN109498703B - Application of radix codonopsis and fructus lycii composition in preparation of medicine for treating Alzheimer's disease - Google Patents
Application of radix codonopsis and fructus lycii composition in preparation of medicine for treating Alzheimer's disease Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, provides a radix codonopsitis and medlar composition and application thereof in preparing a medicine for treating Alzheimer disease, and particularly relates to application of the radix codonopsitis and medlar composition in preparing the medicine for treating Alzheimer disease. The invention adopts a caenorhabditis elegans Alzheimer disease pathological model for experiment, and the result shows that: the radix codonopsitis and medlar composition obviously delays the muscle paralysis phenotype of the caenorhabditis elegans of the Alzheimer disease, namely, the pathological characteristics of the caenorhabditis elegans like the Alzheimer disease are obviously relieved. The experiment results show that the radix codonopsitis and medlar composition provided by the invention has the potential of treating Alzheimer's disease, and can be applied to preparation of a medicine for treating Alzheimer's disease.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and provides application of a codonopsis pilosula-medlar composition in preparation of a medicine for treating Alzheimer's disease.
Background
Alzheimer's Disease (AD), a degenerative disease of the nervous system that predominates in progressive cognitive impairment and memory impairment, is one of the most common forms of senile dementia. Clinically, it is characterized by generalized dementia such as memory impairment, aphasia, agnosia, impairment of visual spatial skills, impairment of executive function, and personality and behavioral changes. There is data showing an increase in AD patients every 7 seconds worldwide. With the aggravation of the aging degree of China, AD can become one of the main diseases affecting the health of the old, and the effective prevention and treatment of AD becomes a major subject before people.
Amyloid deposition (a β protein) and neurofibrillary tangles are two major pathological features of AD. Modern medicine has studied the pathogenesis of AD for many years, but due to its complex etiology, the pathogenesis of the disease is still unclear, and the "Α β cascade hypothesis" is one of the currently widely accepted pathogenesis. The theory considers that abnormally deposited A beta in the brain of a patient directly or indirectly acts on neurons and glial cells through a series of cascade reactions such as free radical reaction, mitochondrial oxidative damage, inflammatory reaction and the like, finally leads to neuron dysfunction or death, triggers cognitive impairment and memory decline, and finally causes dementia. The frequent mutation of the Amyloid Precursor Protein (APP) gene and presenilin genes in patients with alzheimer's disease, leading to overexpression of a β in the brain, is the most compelling evidence of this theory. Thus, amyloid beta has become a recognized target for screening anti-AD drugs.
The AD has complex etiology, long course of disease and more pathogenic links, and needs to be taken for a long time, the current main anti-Alzheimer's disease drugs such as acetylcholinesterase inhibitor (such as galantamine) and N-methyl-D-aspartate receptor (NMDA receptor) antagonist (such as memantine) are expensive, the side effects such as hallucination, consciousness chaos, dizziness, headache and tiredness after taking the drug are obvious, and the disease condition of a patient can only be controlled but can not be reversed.
At present, the intervention of western medicines aiming at a certain specific pathological link of AD often causes drug resistance and serious adverse reaction. The traditional Chinese medicine composition has the characteristics of multiple active components and multiple targets, so that the prospect of screening drug candidates for treating AD from the traditional Chinese medicine composition treasury is wide.
Radix codonopsitis is a commonly used traditional tonifying medicine in China, is sweet and neutral in nature, strengthens spleen, replenishes lung, nourishes blood and promotes fluid production, and is mainly used for spleen-lung qi deficiency, anorexia, lassitude, cough, asthma, deficiency of qi and blood and the like. Modern researches show that radix Codonopsis contains chemical components such as polysaccharide, alkyne glycoside, alkaloid, triterpenes, phenylpropanoids, sterol, etc., and has various pharmacological effects of regulating immunity, regulating gastrointestinal function, resisting oxidation, resisting tumor, etc. (Liu Mei Xia, etc., 2018). The medlar is a common edible and medicinal food material in life, and has sweet and mild taste; entering liver and kidney meridians; nourishing liver and kidney, replenishing vital essence to improve eyesight, and moistening lung. Modern medical research finds that the medlar has obvious effects on reducing blood sugar, reducing blood fat, resisting tumors, resisting atherosclerosis and enhancing immunity (Zhao Ming Yu, 2018). The Chinese medicinal composition, SHENQI tablet, has effects in invigorating qi, invigorating spleen, and nourishing liver and kidney; can be used for treating deficiency of qi and blood, asthenia, deficiency of both liver and kidney, and soreness of waist and knees.
Caenorhabditis elegans (Caenorhabditis elegans) is a multifunctional drug screening and drug action mechanism research tool. 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 ensured, and the experiment is carried out by adopting a large sample amount, so that the influence of individual difference is eliminated; is highly conserved with higher organisms in gene and molecular signaling pathways (Kaletta and Hengartner, 2006). It is widely used in the field of pharmaceutical research as a bridge from the primary screening of cell in vitro horizontal drugs to the secondary screening of mouse in vivo horizontal drugs.
A humanized caenorhabditis elegans AD pathological model is established by transferring a humanized Abeta 1-42 gene into the downstream of a nematode myosin myo-3 promoter, wherein the Abeta 1-42 gene is simultaneously fused with a 3 'long untranslated region sequence (UTR), the sequence is regulated and controlled by smg-1 gene product with mRNA quality monitoring effect, smg-1 is controlled by temperature, the gene is normally expressed at the allowable temperature of 15 ℃, and the expression product is combined with the 3' UTR of the Abeta 1-42 to degrade the expressed Abeta 1-42 mRNA, so that the nematode normally grows. When transferred to the 25 ℃ limiting condition, smg-1 is inactivated, A beta 1-42 is expressed and accumulated in the muscle of the nematode, so that the nematode loses the motor ability and is paralyzed, and the AD-like pathological characteristics of the nematode muscle paralysis phenotype can be delayed by adding the tested medicament. The higher the ratio of the number of nematodes for alleviating muscle paralysis of the tested medicament to the number of the tested nematode population is, the more remarkable the anti-AD effect is. This model has been used for the screening of anti-AD disease mechanisms and anti-AD drug candidates and the study of their mechanisms of action (Link, 1995; 2001).
The invention provides a radix codonopsitis and medlar composition, which can play a synergistic effect to remarkably delay the paralytic phenotype of AD caenorhabditis elegans, and the radix codonopsitis and medlar composition has the potential of treating Alzheimer's disease and can be applied to the preparation of medicaments for treating the Alzheimer's disease.
Reference to the literature
Kaletta T,Hengartner MO.Finding function in novel targets:C.elegans as a model organism.Nat Rev Drug Discov 2006,5:387e398.
Link CD.Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.Proc Natl Acad Sci USA 1995,92:9368e9372.
Link CD.Transgenic invertebrate models of age-associated neurodegenerative diseases.Mech Ageing Dev 2001,122:1639e1649.
The pharmacological action and clinical application research of Zhao Ming Yu and matrimony vine [ J ] northern pharmacy, 2018,15(04):156.
Liumeixia, chiffon, Yuberyang and Codonopsis pilosula pharmacological action research progress [ J ]. strait pharmacology, 2018(11):36-39.
Disclosure of Invention
The invention aims to provide a codonopsis pilosula-medlar composition and application thereof in preparing a medicine for treating Alzheimer's disease.
The codonopsis pilosula-medlar composition contains the following raw material medicines in parts by weight: 1 to 9 portions of radix codonopsitis and 9 to 1 portion of medlar. The preferable weight ratio is 1:1 radix Codonopsis and fructus Lycii.
The codonopsis pilosula-medlar composition can be prepared into capsules, tablets, granules, powder, oral liquid, pills and any other dosage forms by adding proper auxiliary materials, which can be understood by researchers in the field.
The experiment is carried out by taking caenorhabditis elegans as a pathological model of Alzheimer's disease, and the result shows that: the codonopsis pilosula-medlar composition has a remarkable treatment effect on caenorhabditis elegans suffering from Alzheimer's disease. The composition 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 codonopsis pilosula and medlar composition has the potential of treating the Alzheimer's disease and can be applied to the preparation of medicaments and health-care products for treating the Alzheimer's disease.
When used alone, both dangshen and wolfberry fruit have obvious treating effect on AD nematode. In the invention, when the codonopsis pilosula and the medlar are used together, the anti-AD effect is more remarkable. The codonopsis pilosula and the medlar in the codonopsis pilosula and medlar composition provided by the invention play a synergistic effect in resisting AD.
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 is not limited to the following.
Drawings
FIG. 1A dose-dependent delay of muscle paralysis in AD C
FIG. 2 the synergistic effect of Codonopsis pilosula and Lycium barbarum for delaying the muscular paralysis of caenorhabditis elegans
FIG. 3 the composition of Codonopsis pilosula and Lycium barbarum in different weight ratios can significantly delay the muscular paralysis of AD C.elegans
Detailed Description
Example A composition of Codonopsis pilosula and Lycium barbarum can delay the muscle paralysis of AD C.elegans dose-dependently
1. Biological material
(1) Caenorhabditis elegans CL4176 was purchased from Caenorhabditis Genetics Center (CGC); for transgenic lines, muscle-specific expression of human Abeta induced at 25 ℃ temperature1-42,Aβ1-42Accumulation in muscle tissue, ultimately leading to nematode paralysis, the present example used the C.elegans CL4176 strain as a pathological model for screening anti-AD drugs.
(2) Escherichia coli OP50 (uracil leaky mutant), purchased from Caenorhabditis Genetics Center (CGC), was used as feed for C.elegans.
2. Reagent
(1) Solid NGM (Newatode Growth Medium) medium composition and preparation (taking 1 liter as an example):
composition (I) | Content (wt.) |
NaCl | 3.00g |
K2HPO4 | 2.34g |
KH2PO4 | 17.23g |
Peptone | 2.50g |
Agar-agar | 17.00g |
Supplement H2O to | 1000mL |
After preparing the solid NGM culture medium, sterilizing at 121 deg.C under high pressure and constant temperature for 20min, adding 5mg/mL cholesterol 1mL, 1M MgSO4 1mL,1M CaCl21mL was shaken well and poured hot into a sterilized 9cm plate, approximately 20 mL/plate. Standing for solidification of the culture medium.
(2) M9 liquid formula
(3) Preparation of lysate: a6.4% NaClO solution and a 1M NaOH solution were mixed in a volume ratio of 1: 1.
3. Preparing NGM flat plate containing radix codonopsis and medlar composition
Preparing a water decoction of the codonopsis pilosula and Chinese wolfberry composition:
adding 100mL of distilled water into 12g of codonopsis pilosula and 12g of Chinese wolfberry, soaking for 20min, boiling in an electric ceramic furnace, keeping boiling for 30min, stopping heating, cooling, and filtering by using multiple layers of gauze to obtain a first decoction. Adding 100mL distilled water into the obtained residue, and boiling for 30 min. Stopping heating, cooling, and filtering with multiple layers of gauze to obtain the second decoction. And combining the two decoction liquids, and fixing the volume to 55 mL.
Preparing an NGM flat plate containing a liquid medicine decoction:
adding radix Codonopsis water decoction, fructus Lycii water decoction or radix Codonopsis and fructus Lycii composition water decoction of appropriate dilution into NGM culture medium, and pouring.
The final concentration of the NGM tablet, codonopsis pilosula and medlar composition containing the medicine is respectively 10 times diluted, 20 times diluted, 40 times diluted and 80 times diluted. Standing for culture medium solidification. Coli OP50 was spread evenly on the medium as feed for 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 16 ℃ and synchronized when they reached adult growth.
(2) Nematode synchronization:
selecting NGM culture medium containing a large amount of imagoes and part of nematode eggs which have been hatched, flushing the nematodes from the culture medium by using M9 liquid, transferring the nematodes to a centrifuge tube, standing the nematode to enable the nematodes to freely settle to the bottom of the centrifuge tube, and discarding supernatant. And adding the lysate into the centrifuge tube according to the quantity of the nematode, oscillating the centrifuge tube on a vortex mixer for 5 to 7 minutes, stopping vortex when the nematode is completely broken, subpackaging the centrifuge tube with 1.5mL, and washing the nematode eggs with M9 solution for three times.
(3) Effect of radix codonopsitis and medlar composition on caenorhabditis elegans CL4176
Transferring synchronized nematode eggs subpackaged in centrifuge tubes to NGM culture dishes coated with OP50 and mixed with codonopsis pilosula-medlar compositions with different concentrations and NGM culture dishes coated with OP50 and added with sterile water with the same volume as the codonopsis pilosula-medlar compositions as blank control, wherein each culture dish has 60 nematodes and three culture dishes with each drug concentration are used as parallel, and culturing for 3 days at 16 ℃ to L3 period.
In order to express A beta, the L3 phase nematodes were induced at 25 ℃ and count of paralyzed nematode sticks started after 34 h. Counting every two hours until all nematodes are paralyzed (paralysis means that the nematodes cannot move or only the head moves when the nematode is mechanically stimulated to the body). The results are shown in FIG. 1.
The ordinate of FIG. 1 shows the percentage of C.elegans which had no paralysis, and at the same time point, the larger this value indicates a larger proportion of C.elegans which had no paralysis in the treated group, i.e.a stronger effect of the drug in delaying paralysis.
Wherein, C represents a sterile water blank control; LL for the ultra-low concentration Codonopsis pilosula and wolfberry composition group (80-fold dilution), L for the low concentration Codonopsis pilosula and wolfberry composition group (40-fold dilution), M for the medium concentration Codonopsis pilosula and wolfberry composition group (20-fold dilution), and H for the high concentration Codonopsis pilosula and wolfberry composition group (10-fold dilution).
Experimental results show that the codonopsis pilosula-medlar composition can delay the muscle paralysis of AD caenorhabditis elegans in a dose-dependent manner.
The embodiment proves that the codonopsis pilosula-medlar composition provided by the invention has the potential of resisting Alzheimer's disease, and can be applied to preparation of medicines for preventing and treating Alzheimer's disease.
Example II radix Codonopsis and fructus Lycii exert synergistic effect to delay muscular paralysis of AD C
The preparation of the decoction of the codonopsis pilosula and the medlar containing single medicines with different weights comprises the following steps:
adding distilled water 100mL into radix Codonopsis 12g, soaking for 20min, boiling in an electric ceramic furnace for 30min, stopping heating, cooling, and filtering with multiple layers of gauze to obtain the first decoction. Adding 100mL distilled water into the obtained residue, and boiling for 30 min. Stopping heating, cooling, and filtering with multiple layers of gauze to obtain the second decoction. And combining the two decoction liquids, and fixing the volume to 55 mL.
Adding distilled water 100mL into radix Codonopsis 24g, soaking for 20min, boiling in a high-grade electric ceramic furnace, keeping boiling on slow fire at low level for 30min, stopping heating, cooling, and filtering with multiple layers of gauze to obtain the first decoction. Adding 100mL distilled water into the obtained residue, and boiling for 30 min. Stopping heating, cooling, and filtering with multiple layers of gauze to obtain the second decoction. And combining the two decoction liquids, and fixing the volume to 55 mL.
Adding 12g of medlar into 100mL of distilled water, soaking for 20min, then boiling in a high-grade electric ceramic furnace, keeping boiling on low-grade slow fire for 30min, stopping heating, cooling, and filtering with multiple layers of gauze to obtain a first decoction. Adding 100mL distilled water into the obtained residue, and boiling for 30 min. Stopping heating, cooling, and filtering with multiple layers of gauze to obtain the second decoction. And combining the two decoction liquids, and fixing the volume to 55 mL.
Adding distilled water 100mL into 24g of medlar, soaking for 20min, boiling in a high-grade electric ceramic furnace, keeping boiling on low-grade slow fire for 30min, stopping heating, cooling, and filtering with multiple layers of gauze to obtain a first decoction. Adding 100mL distilled water into the obtained residue, and boiling for 30 min. Stopping heating, cooling, and filtering with multiple layers of gauze to obtain the second decoction. And combining the two decoction liquids, and fixing the volume to 55 mL.
The final concentration of the water decoctions of the two groups of radix codonopsitis is 10 times of that of the water decoction prepared initially; the final concentration of the two groups of medlar water decoction is 10 times of the initial preparation water decoction. The other operations are the same as those of the first embodiment.
Wherein, C represents a sterile water blank control; DL represents low-concentration radix Codonopsis group (12g radix Codonopsis water decoction group), and DH represents high-concentration radix Codonopsis group (24g radix Codonopsis water decoction group); GL represents a low concentration wolfberry group (12g wolfberry water decoction group), GH represents a high concentration wolfberry group (24g wolfberry water decoction group); h represents the composition group of Codonopsis pilosula and Lycium barbarum (same as example 1).
The ordinate of FIG. 2 shows the percentage of C.elegans which had no paralysis, and at the same time point, the larger this value indicates a larger proportion of C.elegans which had no paralysis in this treated group, i.e.a stronger effect of the drug in delaying paralysis.
The above examples demonstrate that the use of codonopsis pilosula or lycium barbarum (the same weight as the corresponding raw material in the composition of codonopsis pilosula and lycium barbarum or the same weight as the total weight of the composition of codonopsis pilosula and lycium barbarum) alone in this example has no effect of delaying the muscle paralysis of AD nematodes, unlike the composition of codonopsis pilosula and lycium barbarum. This indicates that radix Codonopsis and fructus Lycii can exert synergistic effect in resisting AD.
In the embodiment, the composition of the codonopsis pilosula, the Chinese wolfberry and the Chinese wolfberry in different weight proportions can obviously delay the muscle paralysis of the AD caenorhabditis elegans
The preparation of the water decoction of the codonopsis pilosula and medlar composition containing the raw materials according to different weight ratios:
the preparation of the water decoction of the traditional Chinese medicine composition containing 1 part of codonopsis pilosula and 9 parts of medlar: adding 100mL of distilled water into 2.4g of codonopsis pilosula and 21.6g of Chinese wolfberry, soaking for 20min, boiling in a high-grade electric ceramic furnace, keeping boiling for 30min on slow fire at a low gear, stopping heating, cooling, and filtering by multiple layers of gauze to obtain a first decoction. Adding 100mL distilled water into the obtained residue, and boiling for 30 min. Stopping heating, cooling, and filtering with multiple layers of gauze to obtain the second decoction. And combining the two decoction liquids, and fixing the volume to 55 mL.
The preparation of the water decoction of the traditional Chinese medicine composition containing 9 parts of codonopsis pilosula and 1 part of medlar: 21.6g of codonopsis pilosula, 2.4g of medlar and 100mL of distilled water are added, the mixture is soaked for 20min, then the mixture is boiled in a high-grade electric ceramic furnace, the mixture is kept boiling for 30min on slow fire at a low gear, the heating is stopped, and after the mixture is cooled, multiple layers of gauze are filtered to obtain a first decoction. Adding 100mL distilled water into the obtained residue, and boiling for 30 min. Stopping heating, cooling, and filtering with multiple layers of gauze to obtain the second decoction. And combining the two decoction liquids, and fixing the volume to 55 mL.
The final concentrations of the two groups of water decoctions with different weight proportions are respectively diluted by 10 times of the water decoction prepared initially; the concentration of the codonopsis pilosula and medlar composition is 10 times of that of the initially prepared water decoction, and C represents a sterile water blank control. The other operations are the same as those of the first embodiment.
Wherein G represents 1 part of radix codonopsitis and 9 parts of medlar composition group (diluted by 10 times), D represents 9 parts of radix codonopsitis and 1 part of medlar composition group (diluted by 10 times), and H represents the radix codonopsitis and medlar composition group (diluted by 10 times).
The ordinate of FIG. 3 shows the percentage of C.elegans which had no paralysis, and at the same time point, the larger this value indicates a larger proportion of C.elegans which had no paralysis in this treatment group, i.e.a stronger effect of the drug in delaying paralysis.
The above examples prove that, under the condition that the total weight is the same as that of the radix codonopsitis and medlar combined medicine, the radix codonopsitis and medlar composition containing 1 part of radix codonopsitis, 9 parts of medlar, 9 parts of radix codonopsitis and medlar 1 part of medlar can obviously delay the muscle paralysis of the AD caenorhabditis elegans and has obvious treatment effect on the AD caenorhabditis elegans. The codonopsis pilosula-medlar composition containing 1 to 9 parts of codonopsis pilosula and 1 to 9 parts of medlar by weight has the potential of resisting Alzheimer's disease, and can be applied to preparation of medicines for preventing and treating Alzheimer's disease. Wherein the ratio of radix codonopsitis and medlar is 1: the traditional Chinese medicine composition 1 has the most obvious effect of delaying the muscular paralysis of the AD caenorhabditis elegans, has the most obvious treatment effect on the AD caenorhabditis elegans, and is a codonopsis pilosula-medlar composition with the optimal weight ratio.
Claims (2)
1. The application of a radix codonopsitis and medlar composition in preparing a medicine for treating Alzheimer disease is disclosed, wherein the radix codonopsitis composition is composed of the following raw material medicines in parts by weight: 1 part of codonopsis pilosula and 1 part of medlar.
2. The use of the Codonopsis pilosula and Lycium barbarum composition as claimed in claim 1, wherein the Codonopsis pilosula and Lycium barbarum composition is in the form of capsule, tablet, granule, powder, oral liquid or pill.
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