CN101292972B - Use of 3,4-dihydroxy coffee acyl group tartaric acid in preparing medicaments for treating dementia diseases - Google Patents
Use of 3,4-dihydroxy coffee acyl group tartaric acid in preparing medicaments for treating dementia diseases Download PDFInfo
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Abstract
The invention relates to a new medicinal use of 3, 4-dihydroxy coffee-acyl-tartaric acid, in particular to the application in the preparation of anti-Alzheimer disease drugs. The preparation is pure Chinese medicine preparation, which has rapid disappearance of clinical symptoms and significant effect.
Description
Technical field
The present invention relates to 3, the medical usage that the 4-dihydroxy coffee acyl group tartaric acid is new, especially 3, the purposes of 4-dihydroxy coffee acyl group tartaric acid in preparation treatment dementia disease medicine belongs to the Chinese medicine and pharmacy technical field.
Background technology
The present invention relates to 3, the 4-dihydroxy coffee acyl group tartaric acid be feverfew embrace the stem hardship sell dish (ripe title: the organic acid chemical compound that extracts Herba Ixeritis Sonchifoliae), structural formula is as follows:
Molecular formula: C
13H
12O
9, molecular weight: 312.
Do not see that by retrieval this material is used for the treatment of the application in the dementia disease medicine.
Summary of the invention
The invention discloses 3, the purposes of 4-dihydroxy coffee acyl group tartaric acid in preparation treatment dementia disease medicine.
The present invention is with 3, and the 4-dihydroxy coffee acyl group tartaric acid can add one or more natural or synthetic active component that have collaborative or assosting effect with it and make as main active preparation treatment dementia disease medicine.
The present invention 3, and the dementia disease medicament of 4-dihydroxy coffee acyl group tartaric acid preparation comprises any dosage form on the materia medica.
The present invention is with 3, and the 4-dihydroxy coffee acyl group tartaric acid prepares antidementia agent as main active, has tangible curative effect, and below experiment shows the therapeutic effect to dementia disease:
1 pair of extra large people's alginic acid damage nbM causes the therapeutical effect of alzheimer disease
1-1 medicine 3, the 4-dihydroxy coffee acyl group tartaric acid provides by Jilin Academy of Chinese Medicine Sciences, lot number: 20071025, purity and character: equal more than 98%, white crystals; People from sea alginic acid is the Sigma product, lot number 20070914.
80 of 1-2 animal male Wistar rats, body weight 230～250g is provided by preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SYXK-(Ji) 2003-0001.
The 1-3 test method is made rat alzheimer disease model.Ketamine 100mg/kg anesthetized rat, anesthetized rat is fixed on the stereotaxic instrument, regulating fixed pan makes front tooth than the low 2mm of internal ear line mid point, the cleaning skin of vertex is done the perpendicular otch in center, the strip off subcutaneous fascia exposes parietal bone, behind the coronal suture of both sides, get out aperture, take out broken bone bits, keep dural complete.The nbM elements of a fix: 1.4mm behind the bregma, center line outside 2.4mm, 7.4mm under the cerebral dura mater.Every side is injected 1 μ g people from sea alginic acid (normal group is given normal saline) respectively with microsyringe, inserting needle speed 0.2 μ l/min, and every side cumulative volume 1 μ l, let the acupuncture needle remain at a certain point in injection back 5min is in order to avoid medicine overflows when pulling out pin.Postoperative dental base acrylic resin powder sealing skull hole, skin suture, intramuscular injection every day benzylpenicillin 100,000 unit infection in three days.Grouping in the 3rd day after surgery, the beginning administration.The 1. group be normal group, the 2. group be model group, all irritate stomach and give distilled water 0.5ml/100g, the 3. group irritate stomach 3,4-dihydroxy coffee acyl group tartaric acid 100mg/kg.Successive administration carries out water maze test and step down test after 15 days, duration of test continues administration.Continuous 7 days of water maze test, preceding 6 days at four different place of entry of 1,2,3,4 quadrants, measure the time and the distance of swimming that rat arrives platform, remove platform on the 7th day, measure the number of times of the spanning platform of rat in 2min, in the residence time of platform area, residence time in the platform quadrant.After water maze test finishes, carry out step down test, write down the number of times that is shocked by electricity in every Mus 5min or be errors number, with this as school grade.Again do test after 24 hours, this is memory and keeps test, writes down the incubation period and the interior wrong sum of 5min that jump off platform for the 1st time.Step down test is got brain after finishing fast, and formaldehyde fixed is carried out pathologic finding.
1-4 result of the test and normal group are relatively, model group rat the 2nd day the incubation period and the distance of swimming to the 6th day arrival platform obviously prolong (P＜0.05 or P＜0.01), the number of times of the 7th day rat spanning platform in 2min, obviously reduce (P＜0.05 or P＜0.01) residence time in platform area, the errors number of the 1st day diving tower and the 2nd day errors number obviously increase (P＜0.01); With model group relatively, 3, obviously shorten 4-dihydroxy coffee acyl group tartaric acid group rat the 4th day, the 5th day and the incubation period that reached platform on the 6th day (P＜0.05); The distance of swimming of the 5th day and the 6th day arrival platform obviously shortens (P＜0.05), obviously increase (P＜0.05) residence time on the number of times of the spanning platform of the 7th day rat in 2min, the platform, rat the 1st day and the 2nd day diving tower errors number obviously reduce (P＜0.05), see Table 1,2,3,4.
Table 1.3, the 4-dihydroxy coffee acyl group tartaric acid causes the influence that the alzheimer disease rat arrives platform incubation period (s) (n=10, x ± s) to extra large people's alginic acid
Compare with model group:
*P＜0.05,
*P＜0.01
Table 2.3, the 4-dihydroxy coffee acyl group tartaric acid causes the influence that the alzheimer disease rat arrives the platform distance of swimming (cm) (n=10, x ± s) to extra large people's alginic acid
Compare with model group:
*P＜0.05,
*P＜0.01
Table 3.3,4-dihydroxy coffee acyl group tartaric acid cause influence (n=10, the x ± s) of the 7th day water maze of alzheimer disease rat to extra large people's alginic acid
Compare with model group:
*P＜0.05,
*P＜0.01
Table 4.3,4-dihydroxy coffee acyl group tartaric acid cause preventive and therapeutic effect (diving tower method, n=10, the x ± s) of rat alzheimer disease to extra large people's alginic acid
Compare with model group:
*P＜0.05,
*P＜0.01
Pathology shows: the slight dilatation and congestion of the visible meningovascular of normal control group, and the cerebral cortex neurosome is big, and nuclear is big, and it is light, even to dye, and nuclear membrane, kernel are clear.Hippocampal neurons quantity is many, is methodically arranged, and marshalling, neurosome is big, and nuclear is big, even dyeing.Basal nuclei neurocyte quantity is many, arranges more even; The visible meningovascular expansion of model group, the subregion has hemorrhage, and the lymphocyte cover is arranged around the cerebral blood vessel.Cerebral cortex has the degeneration of kitchen range shape neurocyte, necrosis, and more glial cells hyperplasia is arranged.It is big that the degeneration neurosome becomes, Nissl body limit collection, and downright bad neurocyte karyopycnosis, kernel is unclear, or the nuclear disappearance, and endochylema dyeing is dark, and cell is fusiformis.Hippocampal neurons quantity does not have significant change, is methodically arranged, and marshalling, neurosome is big, and nuclear is big, even dyeing.Basal nuclei nerve nucleus cell quantity obviously reduces, visible more degeneration, non-viable non-apoptotic cell, and neuronophagia is more; 3, all visible meningovascular dilatation and congestion of 4-dihydroxy coffee acyl group tartaric acid group, neurocyte disappears substantially in the corticocerebral necrosis region, and the necrotic area is mesh-like or has more glial nodule to form.Hippocampal neurons is methodically arranged, marshalling, no significant change.Basal nuclei nerve nucleus cell number is not seen obvious minimizing, and degeneration, non-viable non-apoptotic cell number are less, and neuronophagia is less.
Pathological examination shows: 3, and the 4-dihydroxy coffee acyl group tartaric acid all has certain improvement effect to the brain tissue pathology change that Dementia in Rats due to extra large people's alginic acid causes.
2 pairs of amyloid-betas cause the therapeutical effect of alzheimer disease
2-1 medicine 3, the 4-dihydroxy coffee acyl group tartaric acid provides by Jilin Academy of Chinese Medicine Sciences, lot number: 20071025, content and character: equal more than 98%, white crystals; A β 25-35 is the Sigma product, lot number 20071209.
80 of 2-2 animal male Wistar rats, body weight 280～310g is provided by preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SYXK-(Ji) 2003-0001.
The 2-3 test method is made rat alzheimer disease model.Ketamine 100mg/kg anesthetized rat, anesthetized rat is fixed on the stereotaxic instrument, regulating fixed pan makes front tooth than the low 2mm of internal ear line mid point, the cleaning skin of vertex is done the perpendicular otch in center, the strip off subcutaneous fascia exposes parietal bone, behind the coronal suture of both sides, get out aperture, take out broken bone bits, keep dural complete.The hippocampus elements of a fix: 3.5mm behind the bregma, center line outside 2.0mm, 2.7mm under the cerebral dura mater.Every side is injected the A β 25-35 of 5 μ l (10 μ g) aggregatory peptides respectively with microsyringe, has annotated in the 5min, and the 5min that injects that afterwards let the acupuncture needle remain at a certain point is in order to avoid medicine overflows when pulling out pin.The normal group operation method is identical, and injection is with the saline of volume.Postoperative dental base acrylic resin powder sealing skull hole, skin suture, intramuscular injection every day benzylpenicillin 100,000 unit infection in three days, grouping in the 3rd day after surgery, beginning administration.The 1. group be normal group, the 2. group be model group, all irritate stomach and give distilled water 0.5ml/100g, the 3. group irritate stomach 3,4-dihydroxy coffee acyl group tartaric acid 100mg/kg.Successive administration carries out water maze test and step down test after 15 days, duration of test continues administration.Water maze long run test 7 days, preceding 6 days, at four different place of entry of 1,2,3,4 quadrants, measure the time and the distance of swimming that rat arrives platform, removed platform on the 7th day, measure the number of times of rat spanning platform in 2min, in the residence time of platform area.After water maze test finishes, carry out step down test, and write down the number of times that is shocked by electricity in every Mus 5min or be errors number, with this as school grade.Again do test after 24 hours, this is memory and keeps test, writes down the incubation period and the interior wrong sum of 5min that jump off platform for the 1st time.Get brain fast after step down test finishes and carry out pathologic finding, observe the pathological change of Hippocampus, cortex.2-4 result of the test and normal control group ratio, the model group rat obviously prolongs (P＜0.05 or P＜0.01) the 2nd day incubation period to the 6th day arrival platform, the distance of swimming to the 6th day arrival platform obviously prolonged (P＜0.05) in the 3rd day, the number of times of the spanning platform of the 7th day rat in 2min, obviously reduce (P＜0.05 or P＜0.01) residence time in platform area, the errors number of the 1st day, the 2nd day diving tower obviously increases (P＜0.05 or P＜0.01), and the 2nd day error latence obviously shortens (P＜0.05); Compare with model group; 3; obviously shorten the incubation period that 4-dihydroxy coffee acyl group tartaric acid group water maze reached platform in the 3rd day to the 6th day (P＜0.05); the distance of swimming to the 6th day arrival platform obviously shortened (P＜0.05) in the 4th day; obviously increase (P＜0.05) residence time on the number of times of the 7th day rat spanning platform in 2min, the platform; the errors number of the 1st day, the 2nd day diving tower of rat obviously reduces (P＜0.05);, there is obvious prolongation (P＜0.05) the 2nd day error latence, sees Table 5,6,7,8.
Table 5.3, the 4-dihydroxy coffee acyl group tartaric acid causes the influence that the alzheimer disease rat arrives platform incubation period (s) (n=10, x ± s) to amyloid-beta
Compare with model group:
*P＜0.05,
*P＜0.01
Table 6.3, the 4-dihydroxy coffee acyl group tartaric acid causes the influence that the alzheimer disease rat arrives the platform distance of swimming (cm) (n=10, x ± s) to amyloid-beta
Compare with model group:
*P＜0.05
Table 7.3,4-dihydroxy coffee acyl group tartaric acid cause influence (n=10, the x ± s) of alzheimer disease rat the 7th big water maze to amyloid-beta
Compare with model group:
*P＜0.05,
*P＜0.01
Table 8.3,4-dihydroxy coffee acyl group tartaric acid cause preventive and therapeutic effect (diving tower method, n=10, the x ± s) of rat alzheimer disease to amyloid-beta
Compare with model group:
*P＜0.05,
*P＜0.01
Pathology shows: the visible cortex neurocyte of normal control group number is many, arranges evenly, and neurosome is big, nuclear is big, and nuclear membrane, kernel are clear, accidentally has a liking for the neurocyte phenomenon, does not see glial nodule and softening kitchen range.The hippocampal neurons number is many, is methodically arranged, and marshalling, neurosome is big, nuclear is big, and nuclear membrane, kernel are clear.The visible cortex neurocyte of model group number slightly reduces, and arranges inhomogeneously, and neuronal cell is degenerated, karyopycnosis, and the downright bad neurocyte of engrain is more, has a liking for the neurocyte phenomenon and sees more.The hippocampal neurons number obviously reduces, and is segmental and loses, and neurocyte is misaligned, lack unity and coherence visible a large amount of karyopycnosis, the non-viable non-apoptotic cell of engrain.3, the neural number of the visible cortex cell of 4-dihydroxy coffee acyl group tartaric acid group is not seen obvious minimizing, karyopycnosis, and the downright bad neurocyte of engrain is less, has a liking for the neurocyte phenomenon and reduces than model group.Hippocampal neurons is methodically arranged, marshalling, and cell number is not seen tangible minimizing, rare non-viable non-apoptotic cell.
Pathological examination shows: 3, and the 4-dihydroxy coffee acyl group tartaric acid can alleviate the pathological lesion of rat brain cortex and Hippocampus due to the amyloid-beta, and amyloid-beta is caused Dementia in Rats certain therapeutical effect.
3 pairs of middle cerebral artery infractions (MCAO) are intended the therapeutical effect of vascular dementia
3-1 medicine 3, the 4-dihydroxy coffee acyl group tartaric acid is provided by Jilin Academy of Chinese Medicine Sciences, lot number: 20071215, content and character: equal more than 98%, white crystals.
80 of 3-2 animal male Wistar rats, body weight 280～320g is provided by preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SYXK-(Ji) 2003-0001.
The 3-3 test method is pressed document
[8]Method is made rat MCAO and is intended vascular dementia model, and the method is to improve on the basis of cerebral ischemia reperfusion injury due to the online bolt method.With the fasting of 10% chloral hydrate (300mg/kg) intraperitoneal injection of anesthesia, it is fixing to face upward the position, cervical region is equipped with hair, after the Iodophors sterilization, operating scissors is cut off skin, clamp careful passivity with Wen Shi and separate mucosa and flesh layer, find and separate right carotid, internal carotid artery and external carotid artery, threading (No. 0 silk thread) is standby.Ligation common carotid artery and external carotid artery, cut a little otch at common carotid artery crotch 2mm place, one end is heated into spheric nylon yarn to be inserted, in the porch with silk thread with internal carotid artery and nylon yarn gently ligation fix, continue to insert nylon yarn then, can feel when locating that to line place (20mm) sense that falls through is arranged, be withdrawn into nylon yarn slightly scale (18mm) again and locate this moment, promptly finishes middle cerebral artery thromboembolism (MCAO).The silk thread of timely then ligation porch internal carotid artery is with the fixing nylon wire that inserts.In surgical field of view, spill penicillin a little, skin suture is stayed external about 1cm with nylon wire.After 2 hours, lift gently and stay external nylon yarn to resistance is arranged, realize that middle cerebral artery pours into again this moment.Only the ligation neck is total for the sham-operation rat, neck interior and external carotid artery.After the modeling success, the sign that the tired song of offside forelimb or walking are turn-taked or walked and fall to offside appears in visible animal.. postoperative gives penicillin infection, 800,000 units/kg every day.Rat normal diet drinking-water.Postoperative beginning on the 10th administration.The 1. group be normal group, the 2. group be model group, all irritate stomach and give distilled water 0.5ml/100g, the 3. group irritate stomach 3,4-dihydroxy coffee acyl group tartaric acid 100mg/kg.Postoperative the 30th day carries out water maze exercise and test, during administration always.
Learning and memory behavioristics and function of nervous system observation concrete grammar: each treated animal adopted Morris water maze laboratory observation animal learning memory behavior to learn function at after the MCAO modeling the 30th day.The Morris water maze is stainless round pool, diameter 200cm, high 50cm, depth of water 30cm, 25 ± 1 ℃ of water temperatures, pool wall are indicated 4 place of entry, thus the pond are divided into 4 quadrants, optional wherein 1 quadrant, it is 11cm that 1 diameter is placed in the center, the platform of high 29cm.The top, labyrinth is equipped with video camera and videocorder and display and is connected, and typing rats'swimming track is analyzed automatically.Continuous 7 days of water maze test positioned sea trial in preceding 6 days: rat is put into water towards pool wall, and respectively once write down its time of seeking platform in 2min and swimming path morning and afternoon.If it does not find platform in 2min, then be 120s incubation period, and draw it by the experimenter to platform with hands, allows rat stop 10s, puts back in the cage again.Carried out the space exploration test on the 7th day: the place of entry that rat was trained in the past in 6 days is put into water, write down its swimming track and time in 2min, if it does not find platform in 2min, then be 120 incubation period.And analyzing rat is at time (tP) and path (dP) and always swimming time (tT) and the always ratio in swimming path (dT) of the swimming of original platform quadrant.The space learning of analyzing rat, the variation of memory ability.
Behind the water maze, broken end is got brain, and is fixing in 4% formalin, send pathology detection.
3-4 result of the test and sham operated rats are relatively, the model group rat obviously prolongs (P＜0.05) the 3rd day incubation period to the 6th day arrival platform, the distance of swimming to the 6th day arrival platform obviously prolonged (P＜0.05) in the 4th day, and the tP/tT of rat space exploration in the 7th day and dP/dT all obviously reduce (P＜0.05 or P＜0.01); Compare with model group; 3; obviously shorten the incubation period that 4-dihydroxy coffee acyl group tartaric acid group reached platform in the 4th day to the 6th day (P＜0.05); the distance of swimming of the 5th day and the 6th day arrival platform obviously shortens (P＜0.05); the tP/tT of rat space exploration in the 7th day and dP/dT all obviously increase (P＜0.05), see Table 9,10,11.
Table 9.3, the 4-dihydroxy coffee acyl group tartaric acid is intended the influence that vascular dementia rats arrives platform incubation period (s) (n=10, x ± s) to MCAO
Compare with model group:
*P＜0.05
Table 10.3, the 4-dihydroxy coffee acyl group tartaric acid is intended the influence that vascular dementia rats arrives the platform distance of swimming (cm) (n=10, x ± s) to MCAO
Compare with model group:
*P＜0.05
Table 11.3,4-dihydroxy coffee acyl group tartaric acid are intended influence (n=10, the x ± s) of vascular dementia rats tP/tT and dP/dT to MCAO
Compare with model group:
*P＜0.05,
*P＜0.01
Pathology shows: the visible meningovascular of sham operated rats is normal, does not have expansion, congested phenomenon.Cortex neurocyte number is many, arranges evenly, and neurosome is big, nuclear is big, even dyeing, and nuclear membrane, kernel are clear, accidentally have a liking for neurological phenomena.The hippocampal neurons number is many, and cell space is big, nuclear is big, and kernel is clear; The visible cortex neurocyte of model group number reduces, and most neurocytes are the triangle of pyknosis, engrain, and nuclear membrane, kernel are unclear, see the neurocyte vacuolar degeneration more, and it is more to have a liking for neurological phenomena, and as seen softening kitchen range and glial nodule form.Hippocampal neurons obviously reduces, and lacks unity and coherence, and arranges and puts in order, and neuronophagia is more, sees the triangle cell of engrain, pyknosis more; 3, the visible cortex neurocyte of 4-dihydroxy coffee acyl group tartaric acid group number does not have obvious minimizing, and the triangle neurocyte of karyopycnosis, engrain and have a liking for the neurocyte phenomenon and reduce than model group is not seen softening kitchen range and glial nodule.The hippocampal neurons number does not have obvious minimizing, and degeneration, non-viable non-apoptotic cell lack than model group, as seen has a liking for the neurocyte phenomenon.
Pathological examination shows: 3, and the 4-dihydroxy coffee acyl group tartaric acid can alleviate the pathologic variation that middle cerebral artery infraction (MCAO) is intended vascular dementia rats to some extent, and vascular dementia is had certain therapeutical effect.
4 pairs of internal carotid arterys are injected the therapeutical effect of small embolus hyperamization pipe dementia
4-1 medicine 3, the 4-dihydroxy coffee acyl group tartaric acid is provided by Jilin Academy of Chinese Medicine Sciences, lot number: 20071025, purity and character: equal more than 98%, white crystals;
80 of 4-2 animal male Wistar rats, body weight 350～450g is provided by preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SYXK-(Ji) 2003-0001.
The 4-3 test method is according to list of references
[9～11]Make thromboembolism liquid, the blood clot of the aseptic natural drying of Wistar rat grinds classification and sieves, and selects diameter 100～200 purpose microgranules to be dissolved in the normal saline, and the suspension of making 2mg/ml is a thromboembolism liquid.Press list of references
[10]Make rat alzheimer disease model.Draw sugared intraperitoneal injection of anesthesia rat with 20% crow, cut skin in the neck, exposure left carotid and neck are interior, external carotid artery, close common carotid artery with the temporary transient folder of bulldog clamp, inject 0.3ml thromboembolism liquid (the normal control group is given isometric normal saline) from external carotid artery retrograde puncture intubate, the ligation external carotid artery is decontroled the common carotid artery folder, make embolus enter intracranial to each tremulous pulse of brain, cause many kitchen ranges property cerebral infarction by internal carotid artery.Postoperative grouping in 3 days, administration.The 1. group be normal group, the 2. group be model group, all irritate stomach and give distilled water 0.5ml/100g, the 3. group irritate stomach 3,4-dihydroxy coffee acyl group tartaric acid 100mg/kg.Successive administration carries out water maze test and step down test after 15 days, duration of test continues administration.Continuous 7 days of water maze test, preceding 6 days, at four different place of entry of 1,2,3,4 quadrants, measure the time and the distance of swimming that rat arrives platform, removed platform on the 7th day, measure the number of times of rat spanning platform in 2min, in the residence time of platform area and the residence time in the platform quadrant.After water maze test finishes, carry out step down test, write down the number of times that is shocked by electricity in every Mus 5min or be errors number, with this as school grade.Again do test after 24 hours, this is memory and keeps test, and record jumps off the incubation period of platform and the errors number in the 5min for the first time.Get brain fast after step down test finishes and fix, carry out pathologic finding.
4-4 result of the test and normal control group ratio, the model group rat obviously prolongs (P＜0.05) the 2nd day incubation period to the 6th day arrival platform, the distance of swimming of the 1st day to the 3rd day, the 5th day and the 6th day arrival platform obviously prolongs (P＜0.05), the number of times of the spanning platform of the 7th day rat in 2min, the percentage ratio that accounts for total distance of swimming in the residence time and the distance of swimming in the platform area quadrant of platform quadrant all obviously reduces (P＜0.05), the errors number of the 1st day, the 2nd day diving tower obviously increases (P＜0.01), and the 2nd day error latence obviously reduces (P＜0.05); With the model group ratio; 3; the incubation period and the distance of swimming that 4-dihydroxy coffee acyl group tartaric acid group reached platform in the 2nd day to the 6th day all shorten (P＜0.05) to some extent; the number of times of the spanning platform of the 7th day rat in 2min reaches in all obviously increases (P＜0.05) residence time of platform area; the errors number of the 1st day, the 2nd day diving tower obviously reduces (P＜0.05); the 2nd day error latence, obviously prolong (P＜0.05), sees Table 12,13,14,15.
Table 12.3, the 4-dihydroxy coffee acyl group tartaric acid is injected small embolus to internal carotid artery and is caused the influence that the alzheimer disease rat arrives platform incubation period (s) (n=10, x ± s)
Compare with model group:
*P＜0.05,
*P＜0.01
Table 13.3, the 4-dihydroxy coffee acyl group tartaric acid is injected small embolus to internal carotid artery and is caused the influence that the alzheimer disease rat arrives the platform distance of swimming (cm) (n=10, x ± s)
Compare with model group:
*P＜0.05
Table 14.3, the 4-dihydroxy coffee acyl group tartaric acid is injected influence (n=10, the x ± s) that small embolus causes the 7th day water maze of alzheimer disease rat to internal carotid artery
Compare with model group:
*P＜0.05,
*P＜0.01
Table 15.3, the 4-dihydroxy coffee acyl group tartaric acid is injected the preventive and therapeutic effect that small embolus causes the rat alzheimer disease (diving tower method, n=10, x ± s) to internal carotid artery
Compare with model group:
*P＜0.05,
*P＜0.01
Pathology shows: the visible meninges of normal group is a normal configuration, does not have obviously expansion, hyperemia.Cerebral cortex neurocyte number is many, and the cell cell space is big, and nuclear is big, and it is light to dye, and nuclear membrane, kernel are clear.The hippocampal neurons number is many, and marshalling is methodically arranged, and cell space is big, nuclear is big, and kernel is clear.Cortex, Hippocampus do not have softening kitchen range and glial nodule; Model group visible part meningovascular expansion, hyperemia, are dispersed in the necrosis of kitchen range shape at cerebral cortex and Basal ganglia pilosity, downright bad neurocyte karyopycnosis, and dyeing is dark, and kernel is unclear, and regional area has more glial cells hyperplasia, and glial nodule forms.The hippocampal neurons number obviously reduces, and neuronophagia obviously increases, the necrosis of regional area neurocyte segmental, non-viable non-apoptotic cell engrain, hypochromatosis; 3, all visible meningovascular expansion of 4-dihydroxy coffee acyl group tartaric acid, hyperemia, cerebral cortex and Basal ganglia visible multiple, be dispersed in necrosis region, with the model group no significant difference, have more glial nodule to form.The hippocampal neurons decreased number, regional area neurocyte segmental disappears, and neuronophagia is more.
Pathological examination shows: 3, and the 4-dihydroxy coffee acyl group tartaric acid is injected due to the small embolus pathological change of Dementia in Rats to internal carotid artery not to be had and obviously alleviates effect.
3 pairs of protecting neuron from acute
3-1 medicine 3, the 4-dihydroxy coffee acyl group tartaric acid provides by Jilin Academy of Chinese Medicine Sciences, lot number: 20071025, purity and character: equal more than 98%, white crystals.
3-2 animal Wistar rat, body weight 200～250g is provided by preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SYXK-(Ji) 2003-0001.The rat male and female are mated raising.
3-3 material: shears, tweezers two covers, 75% ethanol, iodine cotton balls, plate, centrifuge tube (glass or plastics), suction pipe (medicated cap), 200 mesh sieves, beaker, 96 well culture plates, horse serum, DMEM culture medium, F12, benzylpenicillin, streptomycin, 0.01% poly-D-lysine, cytosine arabinoside (final concentration is 3～5 μ mol/L or 4 μ g/ml), trypsin 0.125%～0.25%, D-Hanks liquid.
3-4 test method: in 96 well culture plates, add 0.01% poly-D-lysine in advance, inhale after the overnight incubation and abandon, clean several with D-Hanks liquid all over standby.Take out to give birth to the brood Wistar rat brain in 24 hours, carry out neurocyte former be commissioned to train foster.Step is as follows:
(1) neonate rat 75% alcohol disinfecting cuts off the scalp skull and exposes full brain, and full brain is taken out, and puts into the culture dish that ice-cold D-Hank ' s liquid is housed, and cleans 3 times.
(2) cut cerebral cortex, reject meninges and blood vessel, cerebral cortex is shredded, be cut into the fritter of 0.5mm * 0.5mm * 0.5mm with the ophthalmology tweezer.
(3) add 0.125% long-pending trypsin of pentaploid approximately, 37 ℃ of water-baths digested 15～20 minutes, and vibration once made fully contact pancreatin of tissue gently every 4 minutes.
(4) the careful suction abandoned trypsin, adds the DMEM/F12 that contains 10% horse serum and ends digestion.(it is centrifugal that the DMEM/F12 that perhaps adds 10% horse serum ends to digest the back, removes supernatant.Add the DMEM/F12 suspendible cell that contains 10% horse serum again)
(5) with the piping and druming repeatedly of piping and druming pipe, till piece of tissue dissipates substantially, become cell suspension.
(6) cross 200 eye mesh screens, collect filtrate to conical centrifuge tube.Centrifugal 5 minutes of 1000r/min abandons supernatant.
(7) cell mass is added the DMEM/F12 culture medium that contains volume fraction 10% horse serum, blow and beat into cell suspension repeatedly gently with a thin mouthful suction pipe, living cell counting number under the trypan blue dyeing mirror is adjusted cell concentration to 2 * 10
6/ ml.
(8) inoculation is gone in advance with 0.01% poly-D-lysine bag by in culture bottle of crossing or the culture plate (ware), puts in 37 ℃, 5%CO2, saturated humidity condition incubator and cultivates, and treats to change liquid behind the cell attachment in the 2nd day, and the removal dead cell changed liquid 1 time in later per 2～3 days.Behind cell culture to 3～4 day, add cytosine arabinoside (final concentration is 3～5 μ mol./L) and cultivate 24h, to suppress non-neurocyte hyper-proliferative.Begin experiment after continuing to be cultured to 7～8 days.
3-4-1 hydrogen peroxide (H
2O
2) damage model: get the neurocyte of cultivating 7～8d, be divided into 1. normal control group, do not add H
2O
2, all the other same model group; 2. H
2O
2Model group is inhaled and is removed original fluid, and adding final concentration is 100 μ molL
-1H
2O
2The DMEM culture medium of 100 μ l is at 37 ℃, 5%CO
237 ℃ of conditions of incubator experimentize behind the effect 4h; 3. drug treating group is at H
2O
22h adds the medicine of each Concentraton gradient respectively before handling.4. culture fluid matched group.Mtt assay detects cell viability: add MTT liquid 10 μ l (5mg/mL) in every hole, continue to cultivate 4h, after supernatant was removed in suction, every hole added DMSO 100 μ l, room temperature vibration 10min.After treating that the hole endoparticle dissolves fully, measure absorbance in the 57Onm place, each calculates survival rate by formula after organizing absorbance-culture fluid matched group absorbance.Cell survival rate (%)=is respectively organized OD value/matched group OD value * 100%.
3-4-2 potassium chloride (KCl) damage model: get the neurocyte of cultivating 7～8d, be divided into 1. normal control group, do not add KCl, all the other same model group; 2. the KCl model group is inhaled and is removed original fluid, and the adding final concentration is 30mmolL
-1The DMEM culture medium of KCl100 μ l is at 37 ℃, 5%CO
237 ℃ of conditions of incubator experimentize behind the effect 4h; 3. drug treating group, 2h adds the medicine of each Concentraton gradient respectively before KCl handles.4. culture fluid matched group.Mtt assay detects cell viability: add MTT liquid 10 μ l (5mg/mL) in every hole, continue to cultivate 4h, after supernatant was removed in suction, every hole added DMSO 100 μ l, room temperature vibration 10min.After treating that the hole endoparticle dissolves fully, measure absorbance in the 57Onm place, each calculates survival rate by formula after organizing absorbance-culture fluid matched group absorbance.Cell survival rate (%)=is respectively organized OD value/matched group OD value * 100%.
The 3-5 result of the test
The experimental result of rat brain neuronal cell cultures of former generation shows, 3, and 4-dihydroxy coffee acyl group tartaric acid group is to hydrogen peroxide (H
2O
2) and potassium chloride (KCl) due to the rat brain nerve cell damage all have protective effect to some extent, see Table 16.
Table 16.3, the 4-dihydroxy coffee acyl group tartaric acid is to H
2O
2And the protective effect of rat brain neural cell injury due to the KCl
Conclusion:
Cause rat alzheimer disease model at extra large people's alginic acid damage nbM, compare with normal group, model group rat the 2nd day the incubation period and the distance of swimming to the 6th day arrival platform obviously prolong (P＜0.05 or P＜0.01), the number of times of the 7th day rat spanning platform in 2min, obviously reduce (P＜0.05 or P＜0.01) residence time in platform area, the errors number of the 1st day diving tower and the 2nd day errors number obviously increase (P＜0.01); With model group relatively, 3,4-dihydroxy coffee acyl group tartaric acid 3 obviously shortens 4-dihydroxy coffee acyl group tartaric acid group rat the 4th day, the 5th day and the incubation period that reached platform on the 6th day (P＜0.05); The distance of swimming of the 5th day and the 6th day arrival platform obviously shortens (P＜0.05), obviously increase (P＜0.05) residence time on the number of times of the spanning platform of the 7th day rat in 2min, the platform, and rat the 1st day and the 2nd day diving tower errors number obviously reduce (P＜0.05 or P＜0.01); Pathology shows: 3, and 4-dihydroxy coffee acyl group tartaric acid 3, the 4-dihydroxy coffee acyl group tartaric acid has some improvement to the brain tissue pathology change that Dementia in Rats due to extra large people's alginic acid causes.
Cause rat alzheimer disease model at amyloid-beta, with normal control group ratio, the model group rat obviously prolongs (P＜0.05 or P＜0.01) the 2nd day incubation period to the 6th day arrival platform, the distance of swimming to the 6th day arrival platform obviously prolonged (P＜0.05) in the 3rd day, the number of times of the spanning platform of the 7th day rat in 2min, obviously reduce (P＜0.05 or P＜0.01) residence time in platform area, the errors number of the 1st day, the 2nd day diving tower obviously increases (P＜0.05 or P＜0.01), and the 2nd day error latence obviously shortens (P＜0.05); Compare with model group, 3, obviously shorten the incubation period that 4-dihydroxy coffee acyl group tartaric acid group water maze reached platform in the 3rd day to the 6th day (P＜0.05), the distance of swimming to the 6th day arrival platform obviously shortened (P＜0.05) in the 4th day, obviously increase (P＜0.05) residence time on the number of times of the 7th day rat spanning platform in 2min, the platform, the errors number of the 1st day, the 2nd day diving tower of rat obviously reduces (P＜0.05), and there is obvious prolongation (P＜0.05) the 2nd day error latence; Pathology shows: 3, and the 4-dihydroxy coffee acyl group tartaric acid all can alleviate the pathological lesion of rat brain cortex and Hippocampus due to the amyloid-beta to some extent, and amyloid-beta is caused the rat alzheimer disease certain therapeutical effect.
Intend vascular dementia model in middle cerebral artery infraction (MCAO), compare with sham operated rats, the model group rat obviously prolongs (P＜0.05) the 3rd day incubation period to the 6th day arrival platform, the distance of swimming to the 6th day arrival platform obviously prolonged (P＜0.05) in the 4th day, and the tP/tT of rat space exploration in the 7th day and dP/dT all obviously reduce (P＜0.05 or P＜0.01); Compare with model group, 3, obviously shorten the incubation period that 4-dihydroxy coffee acyl group tartaric acid group reached platform in the 4th day to the 6th day (P＜0.05), the distance of swimming of the 5th day and the 6th day arrival platform obviously shortens (P＜0.05), and the tP/tT of rat space exploration in the 7th day and dP/dT all obviously increase (P＜0.05); Pathology shows: 3, and the 4-dihydroxy coffee acyl group tartaric acid all can alleviate the pathologic variation that middle cerebral artery infraction (MCAO) is intended vascular dementia rats, and vascular dementia is had certain therapeutical effect.
Inject small embolus at internal carotid artery and cause rat aorta dementia model, with normal control group ratio, the model group rat obviously prolongs (P＜0.05) the 2nd day incubation period to the 6th day arrival platform, the 1st day to the 3rd day, the distance of swimming of the 5th day and the 6th day arrival platform obviously prolongs (P＜0.05), the number of times of the spanning platform of the 7th day rat in 2min, the percentage ratio that accounts for total distance of swimming in the residence time and the distance of swimming in the platform area quadrant of platform quadrant all obviously reduces (P＜0.05 or P＜0.01), the 1st day, the errors number of the 2nd day diving tower obviously increases (P＜0.01), and the 2nd day error latence obviously reduces (P＜0.05); With the model group ratio; 3; the incubation period and the distance of swimming that 4-dihydroxy coffee acyl group tartaric acid group reached platform in the 2nd day to the 6th day all shorten (P＜0.05) to some extent; the number of times of the spanning platform of the 7th day rat in 2min reaches in all obviously increases (P＜0.05) residence time of platform area; the errors number of the 1st day, the 2nd day diving tower obviously reduces (P＜0.05), and the 2nd day error latence obviously prolongs (P＜0.05).Pathology shows: 3, and the 4-dihydroxy coffee acyl group tartaric acid is injected due to the small embolus pathological change of Dementia in Rats to internal carotid artery not to be had and significantly alleviates effect.
In the experiment of rat brain neuronal cell cultures of former generation, 3,4-dihydroxy coffee acyl group tartaric acid group is to hydrogen peroxide (H
2O
2) and potassium chloride (KCl) due to the rat brain nerve cell damage all have obvious protective effect.
Good effect of the present invention is: be pure Chinese medicinal preparation, clinical symptom disappearance is rapid, and treatment dementia disease effect is remarkable.
The specific embodiment
The present invention is with 3; the 4-dihydroxy coffee acyl group tartaric acid mixes by proper proportion as pharmaceutically acceptable carrier of main active and one or more or excipient; make the pharmaceutical composition that is applicable to the different dosage form that uses clinically; for example it is mixed with and supplies intravenous; intramuscular; intraperitoneal; subcutaneous; the injection of drug administration by injection such as marrowbrain intracavity; perhaps make the tablet that is suitable for oral administration; granule; drop pill; electuary; liquid preparation; powder agent; pill; capsule and suspending agent, and the spray that is applicable to topical; aerosol; cream; ointment and suppository.
Disclosed by the invention to be used to prepare treatment dementia disease medicine be by 3 of 0.1-100%, and 4-dihydroxy coffee acyl group tartaric acid and 99.9-0% pharmaceutic adjuvant are formed pharmaceutical preparation.
To be suitable for the solution that the outer approach of gastrointestinal tract is wanted in order preparing, for example can to use distilled water, water for injection, isotonic sodium chlorrde solution or glucose solution, perhaps low solution (for example 1-100mM) phosphate buffered saline (PBS) (PBS) is as carrier or excipient.
In order to prepare tablet, the agent of powder art, suspending agent or the capsule that is suitable for oral administration, can use sucrose, galactose, corn starch, gelatin, lipid, microcrystalline Cellulose, Pulvis Talci etc. as carrier or excipient.Be suitable in the preparation of oral administration at these, also can contain other proper additive, for example solubilizing agent, disintegrating agent, lubricant, absorption enhancer, antiseptic, correctives, diluent, adsorbent, excipient, dispersant, surfactant, coloring agent etc.
Preferably pharmaceutical composition of the present invention being made route of administration is the injection that intravenous or intramuscular injection are suitable for these route of administration, for example injection, lyophilized injectable powder etc.
Claims (1)
1.
3,The purposes of 4-dihydroxy coffee acyl group tartaric acid in preparation treatment dementia disease medicine.
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