CN102697791A - Application of hederagenin in preparation of medicine for resisting senile dementia - Google Patents
Application of hederagenin in preparation of medicine for resisting senile dementia Download PDFInfo
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- CN102697791A CN102697791A CN2012102055616A CN201210205561A CN102697791A CN 102697791 A CN102697791 A CN 102697791A CN 2012102055616 A CN2012102055616 A CN 2012102055616A CN 201210205561 A CN201210205561 A CN 201210205561A CN 102697791 A CN102697791 A CN 102697791A
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Abstract
The invention discloses the application of hederagenin or the medical salt thereof in the preparation of medicine for resisting senile dementia, cranial nerve damage and aging. According to the invention, the hederagenin or the medical salt thereof is used for treating the diseases, the curative effect is excellent, the side effect is small, the security is high, and a novel selection is provided for treating senile cerebral diseases.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of helexin or its officinal salt and in preparation anti-senile brain dementia, anti-Cranial nerve injury as birth trauma and antidotal medicine, use as active component.
Background technology
Senile brain dementia is claimed Alzheimer again, is the not bright constitutional degeneration brain degenerative disease of the cause of disease.A lot of diseases are in the geratic period, the onset of hiding, and the course of disease is slow and irreversible, is main with the intelligence infringement clinically.Pathological change is mainly the cortex diffuse atrophy, and ditch returns broadening, and the ventricles of the brain enlarge, and neuron reduces in a large number, and visible senile plaque, pathological changes such as neurofibril knot, and choline acetylase and acetyl choline content significantly reduce.Onset was once called as presenile dementia at 65 years old with the former, or presenile dementia, had with sick family history more, and PD is very fast, and temporal lobe and parietal lobe lesion are more remarkable, and aphasia and apraxia are often arranged.Alzheimer mainly shows as the brain cell of the extensive death, particularly basal ganglia region of brain cell.Under the normal condition, the fiber that basal ganglia region is sent projects brain and memory and cognitive relevant cortex, and it discharges acetylcholine.The formation of impermanent memory must have the participation of acetylcholine, and the patient compares acetylcholine transferase with the normal person content reduces 90% than the normal person.Find that through dissecting NFT is widely arranged in patient's brain, aixs cylinder is tangled and is formed senile plaque.Contain downright bad neurocyte fragment, aluminum, unusual albumen in the senile plaque, amyloid-beta excessive buildup in the patients with Alzheimer disease brain.
The pathogenesis of having generally acknowledged mainly contains two kinds: 1, because the protein ingredient that causes unusually of amyloid precursor protein spills cell membrane, cause NFT and cell death, gene is positioned at chromosome No. 21.2, relevant with the gene of apo E (APO-E4), APO-E4 increases the function that can resist APO-E2 or APO-E3.APO-E4 reduces the stability of neuron membrane, causes NFT and cell death.APO-gene pure is higher than the ill probability of heterozygote.
Still lack comparatively ideal medicine and Therapeutic Method to diseases such as senile brain dementia, Cranial nerve injury as birth traumas at present.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point provides a kind of helexin or its officinal salt in preparation anti-senile brain dementia, anti-Cranial nerve injury as birth trauma and antidotal medicine, to use.
The present invention realizes through following technical scheme:
Helexin or its officinal salt can be used in the medicine of the anti-senile brain dementia of preparation.Helexin or its officinal salt can be used in the medicine of the anti-Cranial nerve injury as birth trauma of preparation.Helexin or its officinal salt can be used in the antidotal medicine of preparation.Helexin or its officinal salt day for human beings use dosage is 0.2～1.8g in this medicine, and administration number of times can be 2-3 time/day.
Helexin (Hederagenin), and another name (3beta, 4alpha)-3,23-Dihydroxyolean-12-en-28-oicacid; Hederagenin, molecular formula C
30H
48O
4, can derive from commercially available or prepare by number of ways, molecular structure is following:
Helexin of the present invention or its officinal salt can become various dosage forms with pharmaceutically acceptable preparing carriers as active component.The dosage form of described medicine is any dosage form of pharmaceutically approving.
Above-mentioned pharmaceutically acceptable carrier comprises the adjuvant of oral formulations adjuvant or parenteral administration.Route of administration can be oral, injection, topical etc.According to technical scheme of the present invention, said composition can be oral formulations or injection preparation, and wherein oral formulations comprises capsule, soft capsule, granule, oral liquid, tablet, drop pill etc.Used adjuvant comprises: conventional adjuvants such as starch, sucrose, lactose, Icing Sugar, glucose, mannitol, xylitol, Polyethylene Glycol, isopropyl alcohol, tween 80, glycerol, propylene glycol, microcrystalline Cellulose sodium, dextrin, cyclodextrin, sodium chloride, vitamin C, cysteine, citric acid, sodium thiosulfate, sodium sulfite, stearate and gelatin; Preparation later stage preparation technology and equipment all belong to the routine techniques of pharmaceutical field; The present invention does not limit this, so will not detail at this.
With prior art beneficial effect more of the present invention: the present invention shows that first helexin has repair to the inductive PC-12 cell injury of A β 25-35 model; And the protective effect of the cholinergic system injured mice that (270mg/Kg, 90mg/Kg and 30mg/Kg) helexin monomer of having investigated three kinds of dosage is caused the lumbar injection scopolamine; Compare with model group; Administration high dose administration group (270mg/Kg) can improve learning and memory abilities in aging mice significantly, increase SOD vigor, the interior TChE level of reduction brain in the cerebral tissue, and the mice cholinergic system damage that scopolamine caused is had the certain protection effect.Therefore, helexin or its officinal salt are used for resisting senile brain dementia, anti-Cranial nerve injury as birth trauma and defying age to have remarkable result as active component.
The specific embodiment
Embodiment 1 helexin is to A β
25-35The repair of inductive PC-12 cell injury model
This experiment is intended to explore the activity that helexin possesses external anti-AD.
1, material and method:
Washing liquid: 10g K
2Cr
2O
7, 16ml H
2O, the dense H of 200ml
2SO
4Mixed preparing;
D-Hank ' s balanced salt solution: NaCl 4.00g, KCl 0.20g, Na
2HPO
412H
2O 0.067g, KH
2PO
40.03g, NaHCO
30.175g, phenol red 0.01g, tri-distilled water 500mL regulates pH to 7.0～7.4;
PBS balanced salt solution: NaCl 8.00g, KCl 0.20g, Na
2HPO
412H
2O 3.49g, KH
2PO
40.20g tri-distilled water 1000mL regulates pH to 7.2 ~ 7.4;
RPMI 1640 culture medium: RPMI Medium 1640 dehydrated mediums (GIBCO Invitrogen) 10.4g, NaHCO
32.0g, penicillin (4,000,000 U units are commercially available) 0.06g, streptomycin (1,000,000 U units are commercially available) 0.1006g, tri-distilled water 900mL preparation, sucking filtration degerming;
NBCS: (Newborn Calf Serum, GIBCO Invitrogen Corporation, New Zealand)-20 ℃ of freezing preservations, room temperature is thawed during use;
Cell nutrient solution: RPMI 1640 culture medium solutions that contain 10% NBCS;
0.25% tryptic digestive juice:, USA) be dissolved in the PBS balanced salt solution 0.22 μ m filter filtration sterilization with trypsin Trypsin (1:250) AMRESCO;
MTT:3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt, Thiazolyl blue, Nanjing Sheng Xing Bioisystech Co., Ltd; Precision takes by weighing MTT 20.0mg and joins in the cillin bottle, in cillin bottle, adds 4mL PBS, on magnetic stirrer, stirs 1.5h, and it is dissolved fully, filtration sterilization, and 4 ℃ keep in Dark Place in the refrigerator.
Cells frozen storing liquid: get in RPMI 1640 culture medium 85mL to the 100mL serum bottle that contain 10% NBCS, add DSMO to scale;
A β
25-35: Amyloid β-protein Fragment 25-35, and Sigma-Aldrich (St.Louis, MO, USA); Precision takes by weighing A β
25-351.0mg be mixed with 75 μ mol/L with aseptic tri-distilled water, with the filtration of 0.22 μ m sterile filters, packing ,-20 ℃ frozen; Face with preceding and in 37 ℃ of cell culture incubators, hatched 24 hours;
Helexin reference substance (the HPLC normalization method is measured, and purity is greater than 98%).
The helexin medicinal liquid: precision takes by weighing helexin 4.72mg, i.e. 0.01mmoL.Consider the poorly soluble of aglycon, the DMSO that adds 100 μ L earlier is made into the high concentration mother solution of 100mmol/L.Adopting a small amount of method of repeatedly diluting repeatedly to use culture medium that it is diluted respectively when carrying out administration is 100 μ mol/L, 50 μ mol/L, 25 μ mol/L, 12.5 μ mol/L, 6.25 μ mol/L, 3.125 μ mol/L.This operation will notice that the content of DMSO in the cell inoculation plate is controlled at below 3 ‰.Rat adrenal gland pheochromocyte oncocyte (PC-12 cell), Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences provides.Helexin (purity is 94.85%); The relation of cell culture and the mensuration, MTT colorimetry absorbance and the cell concentration that change liquid, passage, cell cryopreservation, cell recovery, cell counting, cell attachment rate is measured and is all undertaken by conventional method.
The method for preparing of helexin: it is 90% soak with ethanol 2h that Radix Dipsaci medicinal material coarse powder (akebin D content is 4%) is used concentration, heating and refluxing extraction 3 times, and ethanol is doubly measured to the Radix Dipsaci medical material and is weighed 30 times; Extraction time is 6h; Each 2h merges 3 times extracting solution, filters; Filtrate recycling ethanol does not obtain crude extract to there being the alcohol flavor, and the yield of crude extract is about 6%, the rate of transform 98% of akebin D;
Adding concentration is 95% ethanol and concentrated hydrochloric acid in crude extract; Stir, obtain acid hydrolysis solution, make that the hydrochloric acid volume fraction is 7% in the acid hydrolysis solution; The ethanol volume fraction is 50% in the acid hydrolysis solution; Solid-liquid ratio is 1:30 (containing the 1g crude drug in every 30ml acid hydrolysis solution), and acidolysis temperature is 80 ℃, and the acidolysis time is 4h.The solution that obtains after the acidolysis reclaims ethanol to there not being the alcohol flavor, puts coldly at ambient temperature, and deposition is filtered, precipitate, through the low amounts of water washing to neutral, drying under reduced pressure under 65 ℃ of conditions, the helexin sample, helexin purity 94.85%.
The preparation of helexin reference substance solution: precision takes by weighing helexin reference substance 1.25mg, with dissolve with methanol and be settled to 5ml, shakes up, and promptly gets the reference substance solution that concentration is 250 μ g/ml.
The preparation of helexin sample solution: the helexin sample adds small amount of methanol as in the evaporating dish, and ultrasonic dissolution is transferred in the volumetric flask with clean dropper, and methanol constant volume shakes up, and promptly gets the sample liquid that concentration is 0.2g crude drug/ml.The accurate 2.5ml sample liquid of drawing of pipet is to the 10mL volumetric flask, and methanol constant volume shakes up to graduation mark, and 0.45 μ m filtering with microporous membrane promptly gets need testing solution, and concentration is 0.05g crude drug/mL.
Helexin HPLC detection method: chromatographic condition: chromatographic column: phenomenex-C
18Post (4.6mm * 25cm, 5 μ m) and pre-column; Mobile phase: acetonitrile-0.1% aqueous formic acid=64:36; Detect wavelength: 212nm; Sample size: 20 μ l; Flow velocity: 1.0ml/min; Column temperature: 30 ℃.With this understanding, the helexin in the sample all can be separated with other composition chromatographic peaks preferably, and separating degree is greater than 1.5 between adjacent peak, and peak shape is good, and theoretical cam curve is calculated by the helexin peak and is not less than 10000.
2, cell proliferation vitality test:
(1) the cell fishplate bar is cultivated: get well-grown PC-12 cell, clean with the PBS balance solution, process cell suspension through 0.25% trypsinization, it is 1 * 10 that cell concentration is regulated in the counting back
5Individual/mL.In above-mentioned 96 porocyte culture plates, except that each hole around the cell plates, 100 μ L are inoculated in every hole with this cell suspension inoculation.96 each hole of porocyte culture plate respectively add 100 μ L D-hank ' s balanced salt solutions.Culture plate behind the inoculating cell is placed 5%CO
2, cultivate 24h in 37 ℃ of constant incubators, subsequent use.
(2) cell divides into groups: outside the hole around 96 porocyte culture plates are removed, all the other each holes are according to column split.Begin from the secondary series of cell plates, be made as blank control group successively; Model group; Administration group 1; Administration group 2; Administration group 3 or the like, two administration concentration of each administration group, each administration concentration are provided with 3 multiple holes.
(3) modeling and administration: after the cell fishplate bar has been cultivated 24h,, according to following method each group cell is carried out modeling and administration, and put into 5%CO according to 96 porocyte culture plate grouping situation
2, cultivate 48h in 37 ℃ of constant incubators, subsequent use.
1. blank control group: every hole adds water-containing cell nutritional solution 20 μ L, adds the aseptic tri-distilled water of 30 μ L again, cultivates 48 hours.
2. model group: every hole adds water-containing cell nutritional solution 20 μ L, adds 30 μ L A β again
25-35(75 μ mol/L) solution makes A β
25-35Final concentration in the cell culture hole is 15 μ mol/L, cultivates 48 hours.
3. administration group: every hole adds the pastille cell nutrient solution 20 μ L of variable concentrations, adds 30 μ L A β again
25-35(75 μ mol/L) solution makes A β
25-35Final concentration in the cell culture hole is 15 μ mol/L, cultivates 48 hours.
3, determination step: after 48h had been cultivated in each group cell modeling and administration, every hole added respectively 20 μ L of 0.5%MTT solution respectively, continues to put into 5%CO
2, cultivate 4h in 37 ℃ of constant incubators.In superclean bench, carefully sop up the liquid in every hole in the 96 porocyte culture plates, in each hole, adding 150 μ L DMSO solution respectively, jolting 10min on the culture plate agitator immediately, the mensuration OD of the most rearmounted ELIASA 570nm place value.
4, experimental result: through investigating the effect of the beta induced PC12 cell injury of variable concentrations helexin antagonism A; We find to work as helexin under 12.5 μ mol/L, 6.25 μ mol/L, three concentration of 3.125 μ mol/L; The effect of antagonism A β damage is to a certain degree arranged, and have remarkable antagonism (P＜0.05) under the concentration of 6.25 μ mol/L, 3.125 μ mol/L.Experimental result is seen table 1.
The beta induced PC12 cell injury of the anti-A of table 1 helexin experimental result
(
#Expression relatively has significant difference with blank control group; * expression is compared p＜0.05 with model group)
The anti-AD of embodiment 2 helexin probes into acute AD at the body pharmacodynamics and causes the mice dementia at phantom type scopolamine hydrobromide
1, reagent and test kit:
Helexin (purity is 94.85%, and method for making is with embodiment 1); Scopolamine hydrobromide (purchasing) in Nanjing Zelang Pharmaceutical Technology Inc.;
Galanthamine hydrobromide (Shaanxi Kang Sheng Bioisystech Co., Ltd);
Folium Ginkgo extract (the Xuzhou triumphant Semen Ginkgo of perseverance Products Co., Ltd);
Coomassie brilliant blue protein determination kit (bio-engineering research institute is built up in Nanjing);
Acetylcholinesterase is measured test kit (bio-engineering research institute is built up in Nanjing);
Superoxide dismutase testing cassete (bio-engineering research institute is built up in Nanjing);
Mice amyloid beta 1-40 enzyme-linked immunologic detecting kit (Shanghai Jiang Lai).
2, animal
ICR kind mice, male and female half and half, body weight 18-22g (Green Dragon mountain provides)
3, the preparation of experimental solutions:
(1) helexin solution (27.0mg/mL, 9.0mg/ml and 3.0mg/ml)
Taking by weighing an amount of helexin powder uses 0.5% CMC-Na aqueous solution to be mixed with the solution of concentration as 27.0mg/ml, 9.0mg/ml and 3.0mg/ml.
(2) Folium Ginkgo extract solution (6.0mg/mL)
Taking by weighing an amount of ginkgo leaf extract powder uses 0.5% CMC-Na aqueous solution to be mixed with the solution of concentration as 6.0mg/ml.
(3) galanthamine hydrobromide solution (0.6mg/ml)
Taking by weighing an amount of galanthamine hydrobromide powder uses 0.5% CMC-Na aqueous solution to be mixed with the solution of concentration as 0.6mg/ml.
(4) scopolamine hydrobromide solution (2mg/ml)
Taking by weighing an amount of galanthamine hydrobromide powder uses 0.9% CMC-Na aqueous solution to be mixed with the solution of concentration as 2mg/ml.
4, confirming of dosage:
(1) confirming of mice dosage: adopt the body surface area method, be scaled mice dosage through human dosage.
(2) confirming of helexin dosage: the dosage of helexin is divided into three ranks, and high dose administration group is 0.27g/kg, and middle dosed administration group is 0.09g/kg, and low dosage administration group is 0.03g/kg.Dosage is according to being the dosage of MD in the body experiment.
(3) confirming of Western medicine positive drug control group dosage: the referrer of " American Pharmacopeia " the 31st edition regulation galanthamine hydrobromide uses dosage to be 8-24mg60Kg
-1Day
-1Therefore the galanthamine hydrobromide raw material consumption of 20g mice is 24 * 0.003mg=0.072mg0.02Kg
-1Day
-1So the mice dosage that adopts the body surface area method to convert is 0.072mg ÷ 0.02Kg=3.6mgKg
-1Day
-1The mice dosage that this experiment is adopted is 6.0mgKg
-1Day
-1
(4) confirming of Chinese medicine positive drug control group dosage: 2010 editions " the human dosage of a regulation Folium Ginkgo extract of Chinese pharmacopoeia is 240mg60Kg
-1Day
-1Because the Folium Ginkgo extract consumption of 20g mice is 240 * 0.003mg=0.72mg0.02Kg
-1Day
-1So the mice dosage that adopts the body surface area method to convert is 0.72mg ÷ 0.02Kg=36mgKg
-1Day
-1The mice dosage that this experiment is adopted is 60mgKg
-1Day
-1
5, experimental technique:
(1) experiment is divided into groups
After the mice adaptability raised for 1 week, be divided into 7 groups at random, 12 every group, male and female half and half.Group is respectively normal control group (blank control group); Model control group (model group); Western medicine positive controls (Western medicine positive drug group); Chinese medicine positive controls (Chinese medicine positive drug group); High dose administration group (high group); Middle dosed administration group (middle group); Low dosage administration group (low group)
(2) modeling and administration
1) normal control group
A. 10 AM irritate stomach 0.5%CMC-Na aqueous solution (the administration volume, 0.1ml/10g)
B. give CMC-Na solution fortnight continuously, measure the same day since the 15th day until biochemical indicator, continuous five days, last was irritated stomach to behind the CMC-Na solution 20 minutes, the normal saline of lumbar injection 0.9%.(volume injected, 0.1ml/10g)
2) model control group
A. 10 AM irritate stomach 0.5%CMC-Na aqueous solution (the administration volume, 0.1ml/10g)
B. give CMC-Na solution fortnight continuously, measure the same day since the 15th day until biochemical indicator, continuous five days, last was irritated stomach to behind the CMC-Na solution 20 minutes, lumbar injection scopolamine solution.Modeling in first day, first dose doubles, and 2mg/kg remains four days 1mg/kg.(volume injected, 0.1ml/10g).
3) Western medicine positive drug group
A. 10 AM irritate stomach galanthamine hydrobromide solution (the administration volume, 0.1ml/10g)
B. successive administration fortnight is measured the same day since the 15th day until biochemical indicator, and continuous five days, behind the last gastric infusion 20 minutes, lumbar injection scopolamine solution.Modeling in first day, first dose doubles, and 2mg/kg remains four days 1mg/kg.(volume injected, 0.1ml/10g).
4) Chinese medicine positive drug group
A. 10 AM irritate stomach Folium Ginkgo extract solution (the administration volume, 0.1ml/10g)
B. successive administration fortnight is measured the same day since the 15th day until biochemical indicator, and continuous five days, behind the last gastric infusion 20 minutes, lumbar injection scopolamine solution.Modeling in first day, first dose doubles, and 2mg/kg remains four days 1mg/kg.(volume injected, 0.1ml/10g).
5) high dose administration group
A. 10 AM is irritated stomach helexin solution, and concentration is 27mg/ml.(the administration volume, 0.1ml/10g)
B. successive administration fortnight is measured the same day since the 15th day until biochemical indicator, and continuous five days, behind the last gastric infusion 20 minutes, lumbar injection scopolamine solution.Modeling in first day, first dose doubles, and 2mg/kg remains four days 1mg/kg.(volume injected, 0.1ml/10g).
6) dosed administration group in
A. 10 AM is irritated stomach helexin solution, and concentration is 9mg/ml.(the administration volume, 0.1ml/10g)
B. successive administration fortnight is measured the same day since the 15th day until biochemical indicator, and continuous five days, behind the last gastric infusion 20 minutes, lumbar injection scopolamine solution.Modeling in first day, first dose doubles, and 2mg/kg remains four days 1mg/kg.(volume injected, 0.1ml/10g).
7) low dosage administration group
A. 10 AM is irritated stomach helexin solution, and concentration is 3mg/ml.(the administration volume, 0.1ml/10g)
B. successive administration fortnight is measured the same day since the 15th day until biochemical indicator, and continuous five days, behind the last gastric infusion 20 minutes, lumbar injection scopolamine solution.Modeling in first day, first dose doubles, and 2mg/kg remains four days 1mg/kg.(volume injected, 0.1ml/10g).
8) time limit of modeling and administration and frequency
The administration time limit is 19 days, and administration frequency is for once a day.The modeling time limit is 5 days, and the modeling frequency is for once a day.
6, experimental index is measured
(1) learning and memory power index
The measurement operation of Y labyrinth: the person's character that had not only kept in dark place and stimulated but also be afraid of electricity irritation according to mice, with reference to Yang Feng
[1]Method carry out the Y maze experiment.The Y labyrinth is made up of three identical arms, and the angle between arm and the arm is 120 °, becomes I district, II district and III district respectively.In each district, bright lamp district is the place of safety, and all the other promptly have electricity irritation not have photostimulation for the electric shock district, have photostimulation not have electricity irritation.Before the experiment mice is put into one of them district, Y labyrinth, no power is not turned on light, and lets it adapt to 5-10 minute, and the ability of photostimulation and place of safety is distinguished to train mice in irregular then adjustment place of safety.The disposable behavior of running away to the place of safety of 10s became correct response after mice was shocked by electricity, and was wrong reaction on the contrary.After it ran away to the place of safety, light continued 15s, let the bright lamp of its learning and memory district be the place of safety.
[2]Trained to mice earlier in first day, every the irregular adjustment of mice place of safety 10-20 time carried out learning and memory in second day to test, and write down the correct number of times of every mice.Experimental data adopts mean ± standard deviation
expression, and carries out t check between variance analysis and group with SPSS 13.0 statistics softwares.P＜0.05 thinks to have significant difference.
(2) keep away the measuring principle and the operation of dark experiment
To keep away dark experimental box according to the dark person's character of becoming of mice and be divided into bright, dark two Room, and pass to 20V voltage in the bottom, darkroom, it is the round hole about 3cm that a diameter is set between bright, dark two Room.When beginning experiment, will put into earlier and keep away camera bellows and adapt to 3min, become dark person's character according to mice; Make mouse head aim at the hole and be put into bright chamber on one side; If mice is because the dark habit of happiness gets into the darkroom, the electrical current of the bottom mice of can shocking by electricity impels it to run away to bright chamber from the darkroom.
[3]
With above-mentioned operation, all mices were trained in first day every training 5 minutes.After 24 hours, carried out learning and memory and test in promptly second day.Test period, the record mice gets into the time in darkroom for the first time from bright chamber, and this time becomes incubation period, can be escaped from the darkroom by behind the mice of shocking by electricity.Write down the number of times that in 5 minutes shocked by electricity in mice entering darkroom, this number of times becomes errors number.The achievement that two data of incubation period and errors number are tested as learning and memory jointly.
(3) mice administration and modeling arrangement between study and memory ability test period
Mice continues administration between the ability of learning and memory test period, and after the administration 20 minutes, except that the normal saline of normal group lumbar injection equivalent, all the other respectively organized the scopolamine solution of lumbar injection respective volume, and modeling is after 30 minutes, the test of beginning behavioristics.Modeling scopolamine injection consumption doubled for first dose in first day, and concentration is 2mg/ml.All the other behavioristics's test period injection concentrations are 1mg/ml.
(4) the bright albuminometry of coomassie is measured the cerebral tissue total protein
Principle: protein molecule has-NH
3 +Group, when henna Coomassie brilliant blue developer adds in protein standard liquid or the sample, the anion on the Coomassie brilliant blue dyestuff and albumen-NH
3 +In conjunction with, make solution become blueness, can calculate protein content through measuring absorbance.
(5) the TNB method is measured TchE vigor in the mouse brain tissue
Principle: Acetylcholinesterase (True choline esterase) hydrolysis acetylcholine generates choline and acetic acid; Choline can generate TNB (symmetrical trinitrobenzene with the reaction of sulfydryl developer; Sym-Trinitrobenzene) yellow compound; Carry out colorimetric assay according to shade, the quantity of hydrolyzate choline can be reacted the vigor of acetylcholine esterase.
Present universally acknowledged cholinergic nerve system is the major avenues of approach of learning and memory, and cholinergic neurotransmitter and learning and memory and senile dementia dysmnesia relation are very close.
[4]Acetylcholinesterase is the main enzyme of catalysis acetylcholine hydrolyzation, and AD patient's neurofibrillary tangles is free on the outer acetylcholine esterase active of neuron and raises, and makes acetyl choline content reduction in the cortex, thereby causes patient's ability of learning and memory to go down.So the vigor of measuring the in-house Acetylcholinesterase of mouse brain has certain directive function to the effect whether the investigation medicine has anti-AD.
(6) the WST-1 method is measured the SOD vigor in the mouse brain tissue
WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2; 4-disulfophenyl)-2H-tetrazolium; Monosodi um salt); WST-1 can produce water miscible Jia Za fuel (formazan dye) with the superoxide anion reaction that xanthine oxidase catalysis produces, and this reactions step can be suppressed by SOD.Through can calculate the enzyme activity of SOD to the colorimetric analysis of WST-1 product.
(7) double antibody sandwich method is measured amyloid beta 1-40 in the mouse brain tissue
Principle: what this test kit adopted is the level of mice amyloid beta 1-40 in double-antibody sandwich enzyme linked immunosorbent assay (ELISA) working sample.Mice amyloid beta 1-40 (A β 1-40) antibody sandwich microwell plate with purification; Process insolubilized antibody; In the microwell plate that encapsulates monoclonal antibody, add amyloid beta 1-40 (A β 1-40) successively; With amyloid beta 1-40 (the A β 1-40) antibodies of HRP labelling, form antibody-antigen-hrp-antibody complex again, through thoroughly adding substrate TMB colour developing after the washing.TMB changes into blueness under the catalysis of HRP enzyme, and under the effect of acid, finally is converted into yellow.The depth of color and the amyloid beta 1-40 in the sample (A β 1-40) are proportionate.Under 450nm, measure absorbance (OD value) with ELIASA, through mice amyloid beta 1-40 in the standard curve calculation sample (A β 1-40) concentration.
7. experimental result
(1) learning capacity and memory ability are investigated
The Y maze experiment: through the Y labyrinth learning and memory abilities in aging mice is investigated, the number of times of escaping the place of safety after being shocked by electricity with mice is as investigating index.Experimental result shows that mice is through behind the lumbar injection scopolamine, and the model group mice is when second day learning and memory, and with compared with normal, ability of learning and memory descends.And in the result of positive controls and administration group; Mice not only administration but also take under the situation of modeling; Compare the model group ability of learning and memory in mice and obtained tangible improvement, wherein the correct number of times of galantamine group and senior middle school's dosed administration group improves obviously (P＜0.01), sees table 2.
Table 2 mice Y maze learning memory ability test result
(compare #p＜0.05 with normal group;
##P＜0.01; Compare * p＜0.05 with model group; * p＜0.01)
(2) keep away camera bellows:
Through keeping away camera bellows learning and memory abilities in aging mice is investigated, with mice by the latent time before being shocked by electricity and the total errors number in 5 minute testing time as investigating index.Experimental result shows that mice is through after the modeling of lumbar injection scopolamine, and latent time of model group mice and compared with normal shorten, and 5 minutes test period errors number increase learning capacity decline.And in the result of positive controls and administration group, mice not only administration but also take under the situation of modeling, comparing the model group ability of learning and memory in mice has had tangible improvement.Wherein galantamine group and Folium Ginkgo extract group and high dose administration group latent time obviously are longer than model group; Errors number obviously is less than model group (P＜0.01); See table 3; And low dosage administration group mice latent time and errors number all are superior to model group, but there is not significant difference in difference.
Table 3 mice is kept away dark experiment test ability of learning and memory result
(compare #p＜0.05 with normal group;
##P＜0.01; Compare * p＜0.05 with model group; * p＜0.01)
To sum up, we reach a conclusion through behavioristics's test learning and memory abilities in aging mice, and mice is through after the modeling of lumbar injection scopolamine, and the model group learning and memory abilities in aging mice obviously descends than normal group.And in the mensuration result of positive drug control group and administration group, positive drug control group and senior middle school's dosed administration group can both be improved the modeling learning and memory abilities in aging mice in various degree.
(3) the TchE vigor is investigated the result in the cerebral tissue:
In this result of experiment, with the normal group mice relatively, TChE vigor significantly raise (P＜0.05) in the model group mouse brain tissue of lumbar injection scopolamine modeling.In galantamine group, administration among dosed administration group and the low dosage administration group result, mice not only administration but also take under the situation of modeling, decline (P＜0.01 of significance is arranged all with respect to the TChE vigor in the model group mouse brain tissue; P＜0.01; P＜0.01); Among Folium Ginkgo extract group and the administration high dose administration group result, mice not only administration but also take under the situation of modeling, significant decline (P＜0.05 is also arranged with respect to the TChE vigor in the model group mouse brain tissue; P＜0.05).
Above experimental result shows; Under the situation of lumbar injection scopolamine modeling; Give the rising that the high, normal, basic dose groups of mice galantamine, Folium Ginkgo extract and administration all can significantly be resisted TChE vigor in the caused mouse brain tissue of damage simultaneously respectively, see table 4.
TChE vitality test result in table 4 mice 10% brain tissue homogenate
(compare #p＜0.05 with normal group;
##P＜0.01; Compare * p＜0.05 with model group; * p＜0.01)
(4) the SOD vigor is investigated the result in the cerebral tissue:
This experiment reflects the antioxidant levels in the mouse brain through measuring the activity of the superoxide dismutase (SOD) in the cerebral tissue.The result shows that total SOD vigor and normal group reduce (P＜0.05) more significantly in model group mice 0.2% brain tissue homogenate's liquid of lumbar injection scopolamine.And take mice both administration under the situation of modeling at Folium Ginkgo extract, the total SOD vigor of high dose administration group result, raising (P＜0.05 of significance is all arranged with respect to the SOD vigor in the model group mouse brain tissue; P＜0.05).And low dose group in galantamine group and the administration, SOD vigor in the mouse brain tissue homogenate and model group relatively do not have significance difference (P＞0.05).This experimental result proves; Folium Ginkgo extract and administration high dose administration group can significantly suppress the decline of total SOD vigor in the mouse brain tissue due to the scopolamine; Improve the antioxidant levels in the mouse brain, galantamine and middle low dosage administration group then do not have the obvious suppression effect, see table 5.
SOD vitality test result in table 5 mice 0.2% brain tissue homogenate
(compare #p＜0.05 with normal group;
##P＜0.01; Compare * p＜0.05 with model group; * p＜0.01
(5) amyloid beta 1-40 content is investigated the result in the cerebral tissue:
Measured the content of respectively organizing the amyloid beta 1-40 in mice 10% brain tissue homogenate's liquid after the administration modeling in this experimentation.Experimental result shows, raises to some extent through the A β 1-40 content in the model group mouse brain tissue of lumbar injection scopolamine, does not see table 6 but relatively have the significance difference with normal group.Other positive drug groups are compared with model group with the A β 1-40 content in the administration group mouse brain tissue, through T check, there was no significant difference.Hence one can see that, and this modeling method is the content of the A β 1-40 in the elevation model group mouse brain tissue significantly.Analyze its reason, supposition is shaped on the pass with the molding machine of scopolamine, and scopolamine is the modeling approach with the cholinergic system in the reversible destruction cerebral tissue mainly as the m receptor blocker, and is not high with the proteic rising dependency of β appearance starch.
A β 1-40 result in table 6 mice 10% brain tissue homogenate
Compare #p＜0.05 with normal group;
##P＜0.01; Compare * p＜0.05 with model group; * p＜0.01
Embodiment 3 gets the helexin for preparing according to embodiment 1 method, adds capsule adjuvant commonly used, is prepared into capsule by conventional preparation technology.
Embodiment 4 gets the helexin for preparing according to embodiment 1 method, adds oral liquid adjuvant commonly used, is prepared into oral liquid by conventional preparation technology.
Embodiment 5 gets the helexin for preparing according to embodiment 1 method, adds tablet adjuvant commonly used, is prepared into tablet by conventional preparation technology.
Embodiment 6 gets the helexin for preparing according to embodiment 1 method, adds granule adjuvant commonly used, is prepared into granule by conventional preparation technology.
List of references
[1] Yang Feng, Wang Daning etc. taurine is to dying the influence of plumbous uncle's body weight and learning and memory. [J]. industrial hygiene and occupation disease .2003,29 (5): 281-283
[2] Shi Junxia, Zong Jun etc. the extraction process of Semen Platycladi and composition Study thereof. master thesis
[3] Pan Baolong, Niu Qiao etc. Chronic Aluminum Exposure is to the influence of mice cognitive function and A β expression. master thesis
[4] Chen Qin. the research on anti-senescence experimental technique. Beijing: Chinese Medicine science and technology publishing house; 1996; 135.
Claims (5)
1. helexin or its officinal salt application in the medicine of the anti-senile brain dementia of preparation.
2. helexin or its officinal salt application in the medicine of the anti-Cranial nerve injury as birth trauma of preparation.
3. helexin or its officinal salt application in the antidotal medicine of preparation.
4. according to claim 1,2 or 3 described application, it is characterized in that in the described medicine that it is 0.2～1.8g that helexin or its officinal salt day for human beings are used dosage.
5. according to claim 1,2 or 3 described application, it is characterized in that any dosage form of dosage form for pharmaceutically approving of described medicine.
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| CN105481935A (en) * | 2015-11-21 | 2016-04-13 | 吉林省中医药科学院 | Hederagenin derivatives and application of same in preparation of drugs used for preventing and treating senile dementia |
| US10493118B2 (en) * | 2016-09-29 | 2019-12-03 | Macau University Of Science And Technology | Triterpenoid obtainable from hedera helix for treatment of neurodegenerative diseases |
| CN114794014A (en) * | 2022-06-09 | 2022-07-29 | 辽宁中医药大学 | Animal model for establishing Alzheimer disease yin deficiency syndrome and/or Alzheimer disease non-yin deficiency syndrome by coprophilous fungus transplantation method, evaluation and application |
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| CN1909910A (en) * | 2003-10-10 | 2007-02-07 | Sk化学株式会社 | Triterpene compounds which are effective on improvement of brain function |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105481935A (en) * | 2015-11-21 | 2016-04-13 | 吉林省中医药科学院 | Hederagenin derivatives and application of same in preparation of drugs used for preventing and treating senile dementia |
| CN105481935B (en) * | 2015-11-21 | 2019-06-07 | 吉林省中医药科学院 | Hederagenin derivative and its application in the drug of preparation prevention and treatment senile dementia |
| US10493118B2 (en) * | 2016-09-29 | 2019-12-03 | Macau University Of Science And Technology | Triterpenoid obtainable from hedera helix for treatment of neurodegenerative diseases |
| CN114794014A (en) * | 2022-06-09 | 2022-07-29 | 辽宁中医药大学 | Animal model for establishing Alzheimer disease yin deficiency syndrome and/or Alzheimer disease non-yin deficiency syndrome by coprophilous fungus transplantation method, evaluation and application |
| CN114794014B (en) * | 2022-06-09 | 2023-07-21 | 辽宁中医药大学 | Fecal bacteria transplanting method for establishing animal model, evaluation and application of Alzheimer's disease yin deficiency syndrome and/or Alzheimer's disease non-yin deficiency syndrome |
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