CN104547388A - Traditional Chinese medicine composition for treating alzheimer disease, as well as preparation method and application thereof - Google Patents
Traditional Chinese medicine composition for treating alzheimer disease, as well as preparation method and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/89—Cyperaceae (Sedge family)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/537—Salvia (sage)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
Abstract
The invention discloses a traditional Chinese medicine composition for treating alzheimer disease, as well as a preparation method and application thereof. The composition is prepared from the following medicinal raw materials in parts by weight: 25-35 parts of waternut herb, 15-25 parts of earthworm, 10-20 parts of salviae miltiorrhizae, 10-20 parts of radix paeoniae rubra, 5-10 parts of pseudo-ginseng, 5-15 parts of fructus polygoni orientalis and 5-15 parts of licorice root. The traditional Chinese medicine composition has the effects of promoting blood circulation to remove stasis, and boosting qi and dredging collaterals, and the curative effect is satisfactory by means of clinical verification. Furthermore, an objective basis is provided to further research and development of a novel traditional Chinese medicine for treating alzheimer disease by studying the alzheimer disease resisting effect and action mechanism of the traditional Chinese medicine composition for an alzheimer disease animal model.
Description
Technical field
The present invention relates to a kind of Chinese medicine composition and its production and use, particularly a kind of Chinese medicine composition being used for the treatment of Alzheimer and its production and use, the invention belongs to technical field of Chinese medicines.
Background technology
Along with the development of society and the aging of population, the disease of the mental health aspect of old people becomes increasingly conspicuous, and especially endanger the most serious Alzheimer patient and get more and more, the morbidity of Alzheimer increases with the growth at age.At present, the sickness rate male of Alzheimer is 30.5 ‰ in the world, and women is 48.2 ‰, and the U.S.'s more than 85 years old dementia incidence reaches 47.2 ‰.In western countries, Alzheimer replaces apoplexy just gradually, occupies first of neuropathy.Patient's sum of China's Alzheimer accounts for No. 1 in the world, anticipates the year two thousand fifty, and old people and the man at an advanced age of China's over-65s and more than 80 years old will account for 35 ‰ and 22 ‰ of total population, and when the time comes, AD patient can reach 25,000,000.Present China more than 60 years old population, more than 10 ‰, strides into aging country.The sickness rate of China's over-65s elderly population Alzheimer reaches 4.5 ‰, within more than 75 years old, be more than 11 ‰, 85 years old higher than 30 ‰, estimate that Alzheimer patient about has 5,400,000 ~ 7,200,000, wherein about have 0.1 ‰ ~ 7.2 ‰ for advanced dementia, monitoring property must be obtained and look after; About have 2 ‰ ~ 15 ‰ for light moderate dementia, patient lives dependent life.This disease seriously affects the quality of life of old people, brings white elephant to country, society and family.From now on along with the increase of aging population, number of patients also can continue to increase.Therefore, be a problem that can not be ignored to the control of AD disease.World's each side research is a lot of, but so far still without effective remedy measures.Chinese medicine contains number of chemical composition due to it, can for multiple target spots of disease, in the Complex Diseases of this multifactor, many pathology target spot for the treatment of Alzheimer, have unique advantage.
A large amount of clinical and experimental data displays, Chinese medicine is delaying to study in Alzheimer Process Protection neurocyte, inhibited oxidation stress and anti-excitatory toxicity etc. to achieve certain progress.Many research shows potentiality and the superiority of Chinese medicine Alzheimer.If simple Chinese medicine effectively can control early stage Alzheimer disease symptoms, the toxic and side effects of Western medicine can be avoided, greatly strengthen the compliance of patient consumes, thus be long-term treatment Alzheimer, effective control Alzheimer process lays the foundation, and this is perhaps the developing direction of Chinese medicine Alzheimer.
Summary of the invention
For existing issue, technical problem to be solved by this invention there is provided a kind of Chinese medicine composition for the treatment of Alzheimer.Full side has the merit of blood circulation promoting and blood stasis dispelling, regulating qi and dredging collateral, through clinical verification satisfactory effect.
In order to achieve the above object, the technology used in the present invention means are:
A kind of Chinese medicine composition for the treatment of Alzheimer of the present invention, is characterized in that being made up of following crude drug: Herba Eleocharitis, Pheretima, Radix Salviae Miltiorrhizae, Radix Paeoniae Rubra, Radix Notoginseng, Fructus Polygoni Orientalis, Radix Glycyrrhizae.Wherein, Herba Eleocharitis returns spleen, kidney channel, effect of tool heat-clearing and toxic substances removing, diuresis, sending down the abnormal ascending QI; Pheretima is cold in nature, salty in the mouth, returns liver, stomach, lung, urinary bladder channel, heat clearing away suppressing the hyperactive liver, relieving spasm by subduing liver-wind, dredging collateral eliminating impediment; Radix Salviae Miltiorrhizae nature and flavor are bitter, and be slightly cold, GUIXIN, Liver Channel, have stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, the effect of the relieving restlessness that clears away heart-fire; Radix Paeoniae Rubra bitter in the mouth, is slightly cold, and returns Liver Channel, clearing away heat and cooling blood, blood circulation promoting and blood stasis dispelling; Radix Notoginseng is warm in nature, acrid in the mouth, significant blood circulation promoting and blood stasis dispelling, subduing swelling and relieving pain; Fructus Polygoni Orientalis tool falls apart blood eliminating mass, removing food stagnancy pain relieving, the effect of inducing diuresis to remove edema, and be in harmonious proportion all tastes with Radix Glycyrrhizae, compatibility plays the merit of blood circulation promoting and blood stasis dispelling, regulating qi and dredging collateral altogether.
Chinese medicine composition of the present invention, preferably, is made up of each crude drug of following weight portion: Herba Eleocharitis 25-35 part, Pheretima 15-25 part, Radix Salviae Miltiorrhizae 10-20 part, Radix Paeoniae Rubra 10-20 part, Radix Notoginseng 5-10 part, Fructus Polygoni Orientalis 5-15 part and Radix Glycyrrhizae 5-15 part.
Preferred, described Chinese medicine composition is made up of each crude drug of following weight portion: Herba Eleocharitis 30 parts, Pheretima 20 parts, Radix Salviae Miltiorrhizae 15 parts, Radix Paeoniae Rubra 15 parts, Radix Notoginseng 8 parts, Fructus Polygoni Orientalis 10 parts and 10 parts, Radix Glycyrrhizae.
Described crude drug adds the adjuvant needed for preparations shaping after pulverizing, make various preparations suitable clinically according to pharmaceutical preparation conventional method.Described preparation comprises water decoction, capsule, pill, granule, tablet or oral liquid.The preparation method of invention formulation belongs to conventional formulation method, and at this, there is no need to go into details.
Compositions of the present invention just has the merit of blood circulation promoting and blood stasis dispelling, regulating qi and dredging collateral entirely, through clinical verification satisfactory effect.Meanwhile, the present invention uses advanced experimental technique means to carry out further investigated to Chinese medicine composition anti-Alzheimer disease effects anb Mechanism.Proposition of the present invention will for develop mechanism of action clear and definite and safely and effectively anti-Alzheimer disease new Chinese medicine lay the foundation, therefore, there is important theory significance and wide application prospect.
Accompanying drawing explanation
Fig. 1 is neuromal ultrastructure observed result;
Fig. 2 is that RT-PCR method detects β-APPmRNA expression of results in Hippocampus;
Upper caption: Marker:661nt Ladder; 1,2,3,4,5,6,7 is β-actin internal reference;
Lower caption: Marker:271nt Ladder; 1,2,3,4,5,6,7 groups are respectively normal group, sham operated rats, model group, high, medium and low dosage group and piracetam group;
Fig. 3 is that RT-PCR method detects β-APPmRNA expression of results in Hippocampus;
Note: Marker:313nt Ladder; 1,2,3,4,5,6,7 groups of difference Normal group, sham operated rats, model group, high, medium and low dosage group and piracetam groups;
Fig. 4 is that RT-PCR method detects IL-6mRNA expression of results in Hippocampus;
Note: Marker:384nt Ladder; 1,2,3,4,5,6,7 groups of difference blank groups, sham operated rats, model group, high, medium and low dosage group and piracetam groups;
Fig. 5 is that RT-PCR method detects TNF-α mrna expression result in Hippocampus.
Note: Marker:398nt Ladder; 1,2,3,4,5,6, the expression of 7 groups of difference Normal group, sham operated rats, model group, high, medium and low dosage group and piracetam group TNF α mRNA.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of embodiment 1 Chinese medicine composition
Be made up of each crude drug of following weight: Herba Eleocharitis 30g, Pheretima 20g, Radix Salviae Miltiorrhizae 15g, Radix Paeoniae Rubra 15g, Radix Notoginseng 8g, Fructus Polygoni Orientalis 10g and Radix Glycyrrhizae 10g.
The preparation of embodiment 2 Chinese medicine composition
Be made up of each crude drug of following weight: Herba Eleocharitis 25g, Pheretima 25g, Radix Salviae Miltiorrhizae 10g, Radix Paeoniae Rubra 20g, Radix Notoginseng 10g, Fructus Polygoni Orientalis 5g and Radix Glycyrrhizae 15g.
The preparation of embodiment 3 Chinese medicine composition
Be made up of each crude drug of following weight: Herba Eleocharitis 35g, Pheretima 15g, Radix Salviae Miltiorrhizae 20g, Radix Paeoniae Rubra 10g, Radix Notoginseng 5, Fructus Polygoni Orientalis 15g and Radix Glycyrrhizae 10g.
The preparation of embodiment 4 Chinese medicine composition
Be made up of each crude drug of following weight: Herba Eleocharitis 30g, Pheretima 15g, Radix Salviae Miltiorrhizae 150g, Radix Paeoniae Rubra 20g, Radix Notoginseng 10g, Fructus Polygoni Orientalis 10g and Radix Glycyrrhizae 15g.
Embodiment 5 is in Chinese medicine composition of the present invention application clinically
Chinese medicine composition used is prepared in accordance with the following methods:
Each crude drug by following weight takes: Herba Eleocharitis 30g, Pheretima 20g, Radix Salviae Miltiorrhizae 15g, Radix Paeoniae Rubra 15g, Radix Notoginseng 8g, Fructus Polygoni Orientalis 10g and Radix Glycyrrhizae 10g.Crude drug distilled water immersion 1h, decocting 2 times, each 30min, twice decocting liquid mixing, Rotary Evaporators is concentrated into 50mL respectively, 4 DEG C of preservations, makes water decoction, for following research:
1. data and method
The routine patient of 1.1 inclusion criteria 85 is and is in hospital and the patients with Alzheimer disease of out-patient treatment in my institute in October ,-2015 in November, 2012.Patient diagnosis is according to diagnosis of Alzheimer disease standard in " Chinese Spirit classification of diseases scheme and diagnostic criteria (CCMD-2) ".
1.2 physical data establish matched group and treatment group.In table 1
Table 1 liang group clinical data
1.3 Therapeutic Method treatment group application Chinese medicine compositions 1 dose/day 20 days are a course for the treatment of, totally 3 courses for the treatment of; Matched group Chinese medicine composition.Two groups was all 1 course for the treatment of with 12 weeks.Close observation patient ECG change during medication, makes regular check on hepatic and renal function, hematuria is conventional, compare two groups of remission status.
2. observed result
Table 2 liang group patient MMSE, ADL scoring is compared
Analyze through Ridit, between two groups, have significant difference (P<0.05).
Test example 2 pharmacological experiment
Chinese medicine composition used is prepared in accordance with the following methods:
Each crude drug by following weight takes: Herba Eleocharitis 30g, Pheretima 20g, Radix Salviae Miltiorrhizae 15g, Radix Paeoniae Rubra 15g, Radix Notoginseng 8g, Fructus Polygoni Orientalis 10g and Radix Glycyrrhizae 10g.Crude drug distilled water immersion 1h, decocting 2 times, each 30min, twice decocting liquid mixing, Rotary Evaporators is concentrated into 50mL respectively, 4 DEG C of preservations, makes water decoction, for following research:
Adopt A β
1-40neuronal damage in rat brain is brought out in hippocampus injection, copies AD rat model.Morris water maze method is utilized to observe the change of AD rat behavior; Use the change of transmission electron microscope observing AD rat brain Vascular epidermal growth factor; Adopt euzymelinked immunosorbent assay (ELISA) to detect the change of A β 1-40 and β-APP content in serum, RT-PCR method detects the expression of β-APPmRNA, IL-1 β mRNA, IL-6mRNA, TNF-α mRNA in Hippocampus, carries out especially by following experiment:
1. laboratory animal
12 monthly age Wistar male rats, body weight 350 ± 20g, Heilongjiang University of Chinese Medicine's Experimental Animal Center provides.A β
1-40(Amyloid β-protein, Fragment1-40, Sigma company), the anti-A β of rabbit, IL-1 β, IL-6, TNF-α radioimmunological kit are purchased from Beijing Fu Rui biotech firm.Diethylpyrocarbonate (Promega company of the U.S.), Trizol RNA extracts reagent (American I nvitrogen company), RT-PCR kit (Promega company of the U.S.), upstream and downstream primer, design of primers uses Primer5.0 software design, and Shanghai Jie Beisi Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080 synthesizes.
2. key instrument equipment
Rat brain stereotaxic instrument SN-2 (Japan), Morris water maze (institute of Materia Medica,Chinese Academy of Medical Sciences), Leica-2135 microtome (German Leica), tissue processor (German Leica), biological tissue embedding machine (German Leica), type exhibition sheet machine (German Leica), sheet machine (German Leica) baked by type, Nikon E600 type microscope (Japanese Nikon), transmission electron microscope (Holland).DNA thermal cycler Eppendof 100 type (Germany), DU640 foranalysis of nucleic acids instrument (U.S.), numeral gel imaging system Chemi Imager4000 (Algpha Innotech Corporation), running gel image analysis system (Backman company), low-temperature and high-speed centrifuge CR21 (FDAC), electrophresis apparatus DYY-8B (Beijing 61 company), ice machine SIM-F124 (SANYO GS), superclean bench SW-CJ-IF (Suzhou).
3. experimental technique
3.1 animal screenings and grouping
After rat raises one week under laboratory steady-state conditions, first animal is carried out Morris water maze primary dcreening operation, to measure the acquisition capability of rat to water maze Learning and Memory.Carry out according to Morris experimental technique.Experiment is divided into two parts:
3.1.1 orientation navigation experiment
For measuring the learning capacity of rat to water maze.Last 5 days, from the 1st day, every natural gift upper and lower noon trained 1 time.During training, Stochastic choice place of entry, puts into water by rat towards pool wall, and rat finds and climbs up platform required time (incubation period) record, and each training time is 60 seconds.If rat did not find platform in 60 seconds, must platform be caused, be at this moment designated as 60 seconds incubation period, be allowed to condition at after each training on platform and stop 15 seconds.
3.1.2 space search experiment
Ability to platform space position memory after platform is found for measuring rat association, namely underwater platform was removed at the 6th day, random selecting one place of entry, at same place of entry, rat is put into water towards pool wall, record its number of times striding across original platform relevant position in 60 seconds and the time of first time by platform.
Pass through primary dcreening operation, remove congenital dementia (meansigma methods > 50s incubation period) and the bad person of swimming position, qualified rat 98 is divided into normal group, sham operated rats, model group at random, Chinese medicine composition group, QI invigorating group, group of invigorating blood circulation and piracetam group, often organizes each 15.
3.2AD rat model animal coping
By A β
1-40be diluted to 5g/L with physiological saline solution, be placed on 7d in 37 DEG C of calorstats, put into-20 DEG C of refrigerators after forming aggregatory peptides for subsequent use.Latter 48 hours of water maze test, model group rats lumbar injection 10% chloral hydrate (3ml/kg) is anaesthetized, with reference to bag new people " rat brain stereotaxic atlas ", the head of rat is fixed on stereotaxic instrument, cut off local hair, sterilization tailing edge sagittal line cuts skin, and otch is about 1.5cm, pushes periosteum open exposure skull to both sides.Determine hippocampus injection point: 3mm after bregma, center line is other to the right opens 2mm, DV 3.5mm.Punching on skull by dental burr, bore thin skull and do not bore, is slightly firmly saturating skull with microsyringe, slowly injection A β
1-40has injected in solution 2ul (10ug), 5min, let the acupuncture needle remain at a certain point 5min, dentistry mudding lives skull hole, and gentamycin is dripped in art district, and skin suture is also sterilized, and intramuscular injection penicillin 100,000 unit every day 1 time, continuous 7 days, protects from infection.Sham operated rats injects equivalent aseptic double-distilled water, all the other same model group.Single cage is raised completely clear-headed to rat.
3.3 medicine preparation and administrations
It is as follows that each group of rat 7d after modeling starts drug treatment:
Chinese medicine composition high, medium and low dosage group: the former side of Chinese medicine composition of the present invention (Herba Eleocharitis, Pheretima, Radix Salviae Miltiorrhizae, Radix Paeoniae Rubra, Radix Notoginseng, Fructus Polygoni Orientalis, Radix Glycyrrhizae), concentrated decocting liquid becomes the crude drug concentration of 2g/ml according to a conventional method, after sterilizing, cold preservation is for subsequent use prepares decocting liquid, every milliliter contained 2.2 grams (2.2g/ml), 4 DEG C save backup.High dose group gavage gives 5.0g/kgd, and middle dosage group gavage gives 2.5g/kgd, and low dose group gavage gives 1.25g/kgd, continuous 21d.
Piracetam group: piracetam tablet, 400mg/ sheet (by Northeast Pharmaceutical Factory), pulverizes and adds distilled water and be made into l0mg/ml suspension.Gavage gives 0.1g/kgd, day 1 time, continuous 21d.
Normal group, sham operated rats and model group rats give isometric(al) normal saline, day 1 time, continuous 21d.
4 observation index
4.1Morris water maze behavioristics measures
Morris water fan official tests with reference to related documents operation
[1], each group rat respectively after 2d and treatment after surgery 2d carry out Morris water maze behavioristics and detect, be total to 5d.Method is with 3.1.
4.2 Neuropathologies are observed
4.2.1 the preparation of cerebral tissue specimen
Each group of rat detects pneumoretroperitoneum in last behavioristics and injects 10% chloral hydrate (3ml/kg) anesthesia, open breast fully expose heart and tear pericardium off, through left ventricle row aorta intubate, fixing intubate also cuts off right auricle, after first rinsing the liquid clarification of flowing out to right auricle with normal saline 200ml, pour into fixing again with 4% paraformaldehyde PBS (pH value 7.2-7.4,4 DEG C) 200m1, broken end gets brain.Get 3mm before and after injection point and do coronal section through fixing, paraffin embedding, the many covers of continuous coronal tissue slice, thick 5 μm of sheet, mounts on the clean slide through poly-D-lysine process, and in 60 DEG C of baking boxs, baking put into 4 DEG C of Refrigerator stores after 2 hours.
4.2.2 Ultrastructural observation
Get the large cerebellar tissue of hippocampal injection district 2mm, just fix 24h with 4% glutaraldehyde, after 0.10/ osmic acid, fix 90 minutes, conventional embedding.Repair block lmm size, cut into slices under ultramicrotome, each specimen does 2 copper mesh, drags for sheet as far as possible, and the plumbous two dyeing of uranium, observes under transmission electron microscope and take pictures.
4.3 euzymelinked immunosorbent assay (ELISA) detect A β 1-40 and β-APP content in serum
Each group of rat, after last Behavior test, through heart extracting blood, leaves standstill 2h, 2500 revs/min of centrifugal 10min, separation of serum, puts standby inspection in-20 DEG C of refrigerators.Euzymelinked immunosorbent assay (ELISA) (ELISA) is adopted to detect the content of A β 1-40 and β-APP in each group of rat blood serum.Operating procedure is strictly undertaken by the requirement of test kit description.
4.4RT-PCR method detects β-APPmRNA, IL-1 β mRNA, IL-6mRNA, TNF-α mrna expression in Hippocampus
4.4.1 cerebral tissue process
Each group of rat at the appointed time, through aorta quick filling normal saline 200m1, flush out blood in brain, ice ware takes out brain fast, discard olfactory bulb, cerebellum and low brain stem, get each 1.5mm brain before and after needle track and do coronal section, peel off right side Hippocampus, put into the standby inspection of the mid-liquid nitrogen container of cryopreservation tube.In operating process, various liquid adds 1%DEPC water, operating theater instruments, vessel all through 1%DEPC water soaking, with inactivating endogenous organized enzyme.
4.4.2 detection method
4.4.2.1 total serum IgE is extracted
1. hippocampal homogenates: in carrying out on ice.Get 100mg hippocampal tissue, put into glass homogenizer, add Trizol reagent 1ml and grind;
2. 5 minutes are hatched in 25 DEG C of water baths;
3. moved in 1.5ml centrifuge tube by homogenate, add chloroform 0.2ml, mix and shake 15 seconds, room temperature places 5 minutes;
4. 4 DEG C centrifugal (10000r/min) 15 minutes
5. moved in 1.5ml centrifuge tube by upper strata colourless liquid, add 0.5ml isopropyl alcohol, room temperature is placed after 10 minutes and is mixed;
6. 4 DEG C centrifugal (10000r/min) 15 minutes;
7. supernatant is removed.Add 75% ethanol 1ml.4 DEG C centrifugal (7500r/min) 5 minutes.Repeat this step 2 time;
8. vacuum drying 10 minutes;
9. measuring with calculating the RNA that carries after water dissolution, making A260/A280 ratio remain on more than 1.8.-70 DEG C of cryogenic refrigerators are preserved.
4.4.2.2RNA reverse transcription
1. get above-mentioned specimen RNA2 μ g, add the following mixing material of total amount 20 μ l.
2. 10000 ~ 12000r/m in, centrifugal about 1 minute.
3. incubation 45 minutes synthesis cDNA in 42 DEG C of water baths;
4. 10 minutes deactivation reverse transcriptases in 95 DEG C of water baths.
4.4.2.3DNA polymerase chain reaction (PCR)
1. the following mixed liquor of total amount 25 μ l is added in each pcr amplification pipe.
2. shake, slightly centrifugal above-mentioned mixed liquor.
3. PCR amplification instrument amplification.
Reaction condition: degeneration: 94 DEG C 30 seconds
Annealing: 57 DEG C 60 seconds
Extend: 68 DEG C 60 seconds
After doing 36 circulations altogether, 72 DEG C extend 5 minutes.
4.4.2.4RT-PCR product analysis
Get pcr amplification product 10 μ l with 1.5% agarose gel, 100V electrophoresis 60min, GoldView dyeing, under uviol lamp observed result, take pictures.Kodak2.0 software is adopted to carry out scanning analysis and date processing to PCR primer agarose gel electrophoresis.Computing formula:
Relative quantity=product electrophoretic band density/β-actin electrophoretic band density × 100%
4.4.3 statistical analysis
Data represent with means standard deviation, and statistical analysis adopts SPSS10.0 software to do t inspection.
5 experimental results
5.1 orientation navigation experiment
Different times treated rats in Morris water maze performance test result incubation period is as shown in table 1:
Table 1 different times treated rats in Morris water maze performance tests incubation period
(S)
Note: sham operated rats compares with Normal group, * P<0.01; Compare with model group,
Δp<0.01;
5.2 space search experiments
The number of times result that different times respectively organizes rat spanning platform is as shown in table 2:
Table 2 different times respectively organizes the number of times of rat spanning platform
Note: sham operated rats compares with Normal group,
*p<0.01; Compare with model group,
Δp<0.01,
#p<0.05;
5.3 neuron transmission electron microscope observing results
Chinese medicine composition of the present invention to AD rat model neuromal ultrastructure observed result as shown in Figure 1.As can be seen from the figure:
Normal group and sham operated rats: observe hippocampus neuronal cell dense arrangement, nucleus is clear, and nuclear membrane is clear and full, and mitochondrion, endoplasmic reticulum are clear, and have abundant ribose granule in endochylema, lysosome is less; Blood capillary tube chamber is smooth, has no edema, and basement membrane double-decker is clear.
Model group: observe hippocampal neuron distribution disorders, nuclear membrane is unclear, and structure of mitochondria disorder is unintelligible, membrane portions merges caves in, lysosome showed increased; Blood capillary tube chamber surrounding basement membrane double-decker is unintelligible, pericapillary edema, astrocyte serious swelling.
Treatment group: observe hippocampal neuron distribution normal, nuclear membrane is clear, and chromatin is clear, and mitochondrion is more clear, and intracytoplasmic ribose granule increases, and lysosome is less, compares without significant change with normal cell; Blood capillary tube chamber peripheral edema alleviates, astrocyte mild swelling.
5.4 euzymelinked immunosorbent assay (ELISA) detect A β 1-40 content in serum
Chinese medicine composition of the present invention is to A β in AD rat model serum
1-40the impact of content is as shown in table 3 below:
Table 3 Chinese medicine composition of the present invention is to A β in AD rat model serum
1-40the impact of content
Note: compare with normal group, * P<0.05, * * P<0.01; Compare with model group,
Δp<0.05,
Δ Δp<0.01.
Table 3 result shows, and model group compares with normal group, A β in serum
1-40content has remarkable rising, and difference has significant (P<0.01); Drug therapy is after 28 days, and high dose group, middle dosage group, low dose group and piracetam group compare with model group, A β in serum
1-40content obviously declines, and difference has significant (P<0.05, P<0.01).
5.5 euzymelinked immunosorbent assay (ELISA) detect β-APP content in serum
The adjustment impact of Chinese medicine composition of the present invention on β-APP content in AD rat model serum is as shown in table 4:
Table 4 Chinese medicine composition of the present invention affects the adjustment of β-APP content in AD rat model serum
Note: compare with normal group, * P<0.05, * * P<0.01; Compare with model group,
Δp<0.05,
Δ Δp<0.01.
Table 4 result shows, and model group compares with normal group, and in serum, β-APP content has remarkable rising, and difference has significant (P<0.01); Drug therapy is after 28 days, and high dose group, middle dosage group, low dose group and piracetam group compare with model group, and in serum, β-APP content obviously reduces, and difference has significant (P<0.05, P<0.01).
5.6RT-PCR method detects β-APPmRNA in Hippocampus and expresses
In RT-PCR method detection Hippocampus, β-APPmRNA expression of results is as shown in Fig. 2 and table 7.
The change of table 7 β-APP/ β-actin mRNA RT-PCR electrophoresis product gray scale scanning ratio
Note: compare with normal group, * P<0.05, * * P<0.01; Compare with model group,
Δp<0.05,
Δ Δp<0.01; Compare with high dose group,
▲p<0.05
Fig. 2 result shows that RT-PCR product electrophoretic band is all visible under uviol lamp: respectively organize the brightness of β-actin electrophoretic band consistent, amplified fragments is 661nt; β-APPmRNA is 271nt fragment, but the brightness of each group electrophoretic band has notable difference.Chinese medicine composition of the present invention compares minimizing with the expression of piracetam group β-APPmRNA with model group, and difference has significant (P<0.05 or P<0.01).
5.7RT-PCR method detects IL-1 β mrna expression in Hippocampus
In RT-PCR method detection Hippocampus, β-APPmRNA expression of results is as shown in Fig. 3 and table 8.
The change of table 8 IL-1 β/β-actin mRNA RT-PCR electrophoresis product gray scale scanning ratio
Note: compare with normal group, * P<0.05, * * P<0.01; Compare with model group,
Δ Δp<0.01;
Fig. 3 result shows that RT-PCR product electrophoretic band is all visible under uviol lamp: respectively organize the brightness of β-actin electrophoretic band consistent, amplified fragments is 661nt; IL-1 β mRNA is 313nt fragment, but the brightness of each group electrophoretic band has notable difference.Compare with model group, the high, medium and low dosage group of Chinese medicine composition of the present invention and piracetam group are expressed and are reduced, and difference has significant (P<0.05 or P<0.01).
5.8RT-PCR method detects IL-6mRNA in Hippocampus and expresses
In RT-PCR method detection Hippocampus, IL-6mRNA expression of results is as shown in Fig. 4 and table 9.
The change of table 9IL-6/ β-actin mRNA RT-PCR electrophoresis product gray scale scanning ratio
Note: compare with normal group, * P<0.05, * * P<0.01; Compare with model group,
Δ Δp<0.01; Compare with high dose group,
▲p<0.05;
Fig. 4 result shows that RT-PCR product electrophoretic band is all visible under uviol lamp: respectively organize the brightness of β-actin electrophoretic band consistent, amplified fragments is 661nt; IL-6mRNA is 384nt fragment, but the brightness of each group electrophoretic band has notable difference.Compare with model group, the high, medium and low dosage group of Chinese medicine composition of the present invention and piracetam group are expressed and are reduced, and difference has significant (P<0.05 or P<0.01).
5.9RT-PCR method detects TNF-α mrna expression in Hippocampus
In RT-PCR method detection Hippocampus, TNF-α mrna expression result is as shown in Fig. 5 and table 10.
The change of table 10 TNF α/β-actin mRNA RT-PCR electrophoresis product gray scale scanning ratio
Note: compare with normal group, * P<0.05, * * P<0.01; Compare with model group,
Δ Δp<0.01; Compare with high dose group,
▲p<0.05;
Fig. 4 result shows that RT-PCR product electrophoretic band is all visible under uviol lamp: respectively organize the brightness of β-actin electrophoretic band consistent, amplified fragments is 661nt; TNF-α mRNA is 398nt fragment, but the brightness of each group electrophoretic band has notable difference.Compare with model group, the high, medium and low dosage group of Chinese medicine composition of the present invention and piracetam group are expressed and are reduced, and difference has significant (P<0.05 or P<0.01).
The present invention uses advanced experimental technique means to carry out further investigated to Chinese medicine composition anti-Alzheimer disease effects anb Mechanism.Proposition of the present invention will for develop mechanism of action clear and definite and safely and effectively anti-Alzheimer disease new Chinese medicine lay the foundation, therefore, there is important theory significance and wide application prospect.
Claims (5)
1. treat a Chinese medicine composition for Alzheimer, it is characterized in that this Chinese medicine composition is made up of each crude drug of following weight portion: Herba Eleocharitis 25-35 part, Pheretima 15-25 part, Radix Salviae Miltiorrhizae 10-20 part, Radix Paeoniae Rubra 10-20 part, Radix Notoginseng 5-10 part, Fructus Polygoni Orientalis 5-15 part and Radix Glycyrrhizae 5-15 part.
2. Chinese medicine composition as claimed in claim 1, is characterized in that this Chinese medicine composition is made up of each crude drug of following weight portion: Herba Eleocharitis 30 parts, Pheretima 20 parts, Radix Salviae Miltiorrhizae 15 parts, Radix Paeoniae Rubra 15 parts, Radix Notoginseng 8 parts, Fructus Polygoni Orientalis 10 parts and 10 parts, Radix Glycyrrhizae.
3. Chinese medicine composition as claimed in claim 1 or 2, is characterized in that the adjuvant added after described crude drug is pulverized needed for preparations shaping, makes various preparations suitable clinically according to pharmaceutical preparation conventional method.
4. Chinese medicine composition as claimed in claim 3, is characterized in that described preparation comprises water decoction, capsule, pill, granule, tablet or oral liquid.
5. the purposes of the Chinese medicine composition described in any one of claim 1-4 in preparation treatment Alzheimer disease drugs.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017014162A1 (en) * | 2015-07-21 | 2017-01-26 | Ｗｅｌｌ Ｓｔｏｎｅ 有限会社 | Amyloid β fibril-decomposing agent, therapeutic/prophylactic agent for disease associated with amyloid β fibrillogenesis, and food composition for treating/preventing disease associated with amyloid β fibrillogenesis |
| JP2017025019A (en) * | 2015-07-21 | 2017-02-02 | Ｗｅｌｌ Ｓｔｏｎｅ 有限会社 | AMYLOID-β FIBER DECOMPOSER, THERAPEUTIC AND PROPHYLACTIC AGENTS OF DISEASE ATTRIBUTED TO AMYLOID-β FIBROGENESIS, AND FOOD COMPOSITIONS FOR TREATMENT AND PREVENTION OF DISEASE ATTRIBUTED TO AMYLOID-β FIBROGENESIS |
| CN107405366A (en) * | 2015-07-21 | 2017-11-28 | 井石有限会社 | The agent of amyloid beta fiber hydrolization, curative/preventive medicine of the disease as caused by the fibrosis of amyloid beta and treatment are with/prevention food compositions |
| AU2016296962B2 (en) * | 2015-07-21 | 2021-10-28 | Well Stone Co. | Amyloid beta fibril decomposing agent, and therapeutic or preventive agent and therapeutic or preventive food composition for diseases caused by amyloid beta fibril formation |
| CN107405366B (en) * | 2015-07-21 | 2022-07-22 | 井石有限会社 | Amyloid beta-fibrinolytic agent, and therapeutic/prophylactic agent for diseases caused by amyloid beta-fibrosis |
| CN105055872A (en) * | 2015-07-30 | 2015-11-18 | 黑龙江儒泰生物制药有限责任公司 | Traditional Chinese medicine composition for treating alzheimer disease as well as preparation method and application thereof |
| CN105055872B (en) * | 2015-07-30 | 2019-01-15 | 黑龙江儒泰生物制药有限责任公司 | Chinese medicine composition and its preparation method and application for treating Alzheimer disease |
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Application publication date: 20150429 |