CN103948596A - Application for oleracein E in preparation for medicine for resisting dementia neurodegenerative diseases - Google Patents
Application for oleracein E in preparation for medicine for resisting dementia neurodegenerative diseases Download PDFInfo
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Abstract
The invention discloses an application for oleracein E in preparation for a medicine for resisting dementia, relating to an application for oleracein E in five aspects of prolonging the lives of patients with dementia, enhancing the metabolic capabilities of patients with dementia, alleviating the oxidative stresses of patients with dementia, protecting the nerve cells of patients with dementia, and improving the memory ability of patients with dementia. The invention lays the foundation for oleracein E in research and development for an innovative medicine for treating dementia neurodegenerative diseases.
Description
Technical field
The present invention relates to the medicinal application of tetrahydroisoquinoline alkaloid oleracein E (Oleracein E), relate in particular to OE in the application of preparing in anti-senile dementia neurodegenerative diseases medicine, belong to medical domain.
Background technology
Alzheimer (Alzheimer ' sdisease, AD) be a kind of chronic neurodegenerative diseases, be one of main Types of senile dementia.Its Symptoms is lost in various degree for patient's memory and cognition ability, behavioral activity generation obstacle.Old and feeble closely related with the generation of AD, there is 85 years old old man of 65 years old old man of 10% and nearly 40% can be subject to the puzzlement of senile dementia at US and European, China has 500～6,000,000 AD patients at present, and its sickness rate and mortality rate are only second to cardiovascular and cerebrovascular disease and cancer.How to prevent and treat alzheimer disease, become the hot issue that government, medical institutions and pharmaceutical industry are paid close attention to.
AD pathological characteristics is deposited as main feature with progressive Cognitive function damage, cholinergic neurons of basal forebrain regression, neurofibrillary tangle and beta amyloid peptide (A β), its pathogenesis is still not fully aware of, may be relevant with the many factors such as oxidative stress, neurotransmitter change, inflammation and gene change.At present the medicine of clinical treatment AD comprises: 1. the medicine that acts on cholinergic system: (1) cholinesterase inhibitor is the most frequently used medicine, as donepezil etc., be applicable to mild or moderate AD, and can improve symptom, but can not radical curing of disease; (2) cholinergic agonist, as xanomeline; 2. brain metabolism improving medicine: (1) brain cell activation agent: pyrrolidinone compounds medicine is as piracetam, oxiracetam, aniracetam, nefiracetam etc., there is the effect of activation, protection, reparation brain nervous cell, can resist the Brain function damage due to physics, chemical factor, improve the retrograde amnesia being caused by anoxia, effective to light moderate AD; (2) cerebral circulation improving agent: blood vessel dilating, increase tremulous pulse and tip Tissue Blood flow, thereby improve brain microcirculation, depressive emotion and attention deficit disorder to dementia patients are improved effect.3. calcium ion antagonist: as nimodipine; 4. neurocyte nutritional drugs: as cerebrolysin; 5. NSAID (non-steroidal anti-inflammatory drug): as ibuprofen; 6. neurotrophic factor; 7. free radical scavenger and antioxidant: conventional medicine has vitamin E, Folium Ginkgo extract and melatonin etc.At present, AD treatment there is no specific medicament, and Chinese medicine and natural drug can play clinical dementia effect by multipath, too many levels, many target spots, and is better than chemical synthetic drug improving aspect patient's symptom, has become the research and development focus of anti senile dementia drug.
Herba Portulacae (Portulaca oleracea L.) is widely distributed in temperate zone, the whole world and torrid areas; use as medicine food dual purpose plant in China and other many countries, modern pharmacological research shows that Herba Portulacae has many-sided effects such as antibacterial, lax skeletal muscle, excited uterus, blood fat reducing, blood sugar lowering, anti-inflammatory and antalgic, promotion wound healing, antioxidation, defying age and neuroprotective.
Except coumarin, flavone, organic acid, the compounds such as monoterpene, amide alkaloid is the important chemical composition of a class in Herba Portulacae, pyrrolo-tetrahydroisoquinoline alkaloid oleracein E (Oleracein E) is amide alkaloid [the Xiang L that applicant separates the novel structure obtaining for 2005 from Herba Portulacae, Xing DM, Wang W, et al.Alkaloids from Portulaca oleracea L..Phytochemistry, 2005, 66 (21): 2595-2601] (see following structural formula), its structure and piracetam, oxiracetam, aniracetam, the brain metabolism improving medicine structural similarity of the clinical treatment senile dementias such as pramiracetam, all there is ketopyrrolidine parent nucleus.
In earlier stage; the applicant has external free radical scavenging activity and extracorporeal neuron cytoprotection according to oleracein E and other Herba Portulacae amide alkaloid; apply for and obtained national inventing patent [to orchid; Yang Zijuan. Herba Portulacae amide alkaloid is in the application of preparing in antioxidation and Neuroprotective Agents. People's Republic of China's patent of invention, the patent No. 200810014427.1].Up to the present, in the body of oleracein E (Oleracein E), pharmacological action it be unclear that, and it has no report in the application of preparing in anti-senile dementia neurodegenerative diseases medicine.
At present, the animal model of pharmacodynamic evaluation for the treatment of medicine for senile dementia is mainly divided into following a few class: 1. cholinergic damage causes Model of Dementia: comprising that fornix-fimbria of hippocampus cuts off causes that dementia rats model, basal forebrain injection excitotoxin goose Artemisia gill fungus propylhomoserin etc. cause dementia rats model, scopolamine causes cholinergic damage and intends dementia mice model; 2. the aging Model of Dementia that causes: comprise that naturally-aged animal, Senescence-accelerated Mouse's, D-galactose subcutaneous injection cause brain aging mouse aging; 3.A β causes Model of Dementia: as APP transgenic mice, A β injection cause Model of Dementia; 4. chronic cerebral ischemia causes Model of Dementia: can be used as supplementing of AD model, be more suitable for mixed dementia; Other: comprise aluminum induction Model of Dementia, mitochondrial defects causes Model of Dementia, natural immunity Model of Dementia, Disorder Model etc. is consolidated in the memory that anoxia agent causes as sodium nitrite.Be one of main constituent of delay aging drug Herba Portulacae according to OE, and there is significant removing free radical and antioxidation; Neural cell injury to external excitatory amino acid and scarce sugared hypoxia inducible has protective effect; We select D-galactose (D-galactose, D-gal) and sodium nitrite (NaNO
2) brain aging hold concurrently memory consolidate obstacle mouse model, the anti-senile dementia effect of oleracein E is carried out to pharmacodynamic evaluation.
Summary of the invention
For above-mentioned prior art, for the deficiency of pharmacological research in oleracein E (Oleracein E) body, what the present invention illustrated is that oleracein E is in the application of preparing in anti-senile dementia neurodegenerative diseases medicine.
The present invention is achieved by the following technical solutions:
Oleracein E, in the application of preparing in anti-senile dementia neurodegenerative diseases medicine, is embodied in the following aspects:
The application of oleracein E in the preparation medicine in prolongation old dementia patients life-span.
Experiment confirms: long-term, high-dose lumbar injection D-gal/NaNO
2, cause mice hypoxic-ischemic, oxidative stress aggravation, and cause mouse life to shorten, oleracein E can significantly improve D-gal/NaNO
2the survival rate of the senile dementia mice of induction, extends its life-span (Fig. 1).
Oleracein E strengthens the application in the metabolic medicine of old dementia patients in preparation.
Experiment confirms: long-term, high-dose lumbar injection D-gal/NaNO
2, cause mice hypoxic-ischemic, cause mice sluggish metabolism, its urine amount and feces volume reduce.Herba Portulacae has diuresis and promotes the effect of gastrointestinal peristalsis, and oleracein E can significantly be alleviated D-gal/NaNO as the active component of Herba Portulacae
2the sluggish metabolism (table 1) of the senile dementia mice of induction.
The effect of oleracein E in the medicine of preparation alleviation old dementia patients oxidative stress.
Experiment confirms: long-term, high-dose lumbar injection D-gal/NaNO
2, causing the aggravation of mice oxidative stress, oleracein E can be alleviated D-gal/NaNO
2in the senile dementia mouse brain of induction, T-AOC, SOD and GSH compensatory raise, and make it recover normal level (table 2).
The application of oleracein E in the medicine of preparation protection old dementia patients neuronal damage.
Experiment confirms: long-term, high-dose lumbar injection D-gal/NaNO
2, can cause hippocampus neurons in mice cell injury.Oleracein E can improve D-gal/NaNO
2in the senile dementia Hippocampus of Mice of induction, press down the expression of apoptotic proteins Bcl-2, reduce the expression of pro apoptotic protein Bax and caspase-3, stop neuronal apoptosis, Hippocampal Neuron Cells is had to protective effect (Fig. 2, Fig. 3, Fig. 4).
The application of oleracein E in the medicine of preparation raising old dementia patients memory ability.
Experiment confirms: long-term, high-dose lumbar injection D-gal/NaNO
2, can cause mice spatial memory capacity major injury, oleracein E can improve D-gal/NaNO
2the spatial memory capacity (Fig. 4) of the senile dementia mice of induction.
In above-mentioned various application: described oleracein E refers to the raceme of oleracein E monomer or its enantiomer or enantiomer, or derivatives thereof monomer.Described neurodegenerative diseases refers to senile dementia, or other neurodegenerative diseases relevant to senile dementia, comprises Parkinson disease, Huntington chorea, amyotrophic lateral sclerosis and spinal cord muscular atrophy.
The present invention has set forth oleracein E in the application of preparing in anti-senile dementia neurodegenerative diseases medicine, for oleracein E is laid a good foundation in the original new drug exploitation of preparing in the neurodegenerative diseases such as senile dementia.
Brief description of the drawings
Fig. 1: the impact of oleracein E on D-galactose/sodium nitrite induction senile dementia mice survival rate, wherein, vehicle control: experiment contrast group; D-gal/NaNO
2: model group; PA: positive drug piracetam; OE: oleracein E; With the comparison of experiment contrast group,
* *p<0.001; With D-gal/NaNO
2model group comparison,
##p<0.01.
Fig. 2: hippocampal tissue HE coloration result, wherein, vehicle control: experiment contrast group; D-gal/NaNO
2: model group; PA: positive drug piracetam; OE: oleracein E.
Fig. 3: hippocampal tissue CA4 district neural apoptosis associated protein immunohistochemical staining, wherein, vehicle control: experiment contrast group; D-gal/NaNO
2: model group; PA: positive drug piracetam; OE: oleracein E.
Fig. 4: hippocampal tissue CA3 district neural apoptosis associated protein immunohistochemical staining, wherein, vehicle control: experiment contrast group; D-gal/NaNO
2: model group; PA: positive drug piracetam; OE: oleracein E.
Fig. 5: the impact of oleracein E on D-galactose/sodium nitrite induction senile dementia mice spatial memory capacity, wherein, vehicle control: experiment contrast group; D-gal/NaNO
2: model group; PA: positive drug piracetam; OE: oleracein E.(A) in water maze test, I～IV quadrant is treated the time (s); (B) be platform location traversing times; (C) be mouse movement trajectory diagram; Zero: original platform position (II quadrant); ●: mouse movement initial position; ●: (in figure, red point represents mouse movement initial position to mouse movement end position, blue dot represents mouse movement end position, but due to the relevant regulations of basis " guidelines for examination ", it is colored that accompanying drawing not should be, so can only represent with gray-scale map in the present invention; Again because this accompanying drawing is that correlation function software is made, be inconvenient to revise, therefore cannot distinguish red point and blue dot from gray-scale map, but this does not affect one of ordinary skill in the art's understanding, one of ordinary skill in the art in conjunction with text description with and possessed Professional knowledge be to be appreciated that the implication of this accompanying drawing); Compared with experiment contrast group,
*p<0.05; Compared with model group,
#p<0.05,
##p<0.01).
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1: the application of oleracein E aspect extending the senile dementia life-span
One, experiment material, instrument and reagent
1. laboratory animal
Eight week age 100 of healthy Male Kunming strain mice, body weight is (18.9 ± 1.5) g, is provided by Shandong Traditional Chinese Medicine University's Experimental Animal Center, the animal quality certification number is SCXK (Shandong) 20110003.Sub-cage rearing, indoor temperature is (23 ± 2) DEG C, and humidity is (50 ± 10%) RH, and 12h illumination 12h is alternately dark, ad lib drinking-water.Administration after animal conforms three days.
2. instrument
YP5001 ordinary electronic balance (Shanghai light positive Medical Instruments company limited), AL104 electronic analytical balance (prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit Instrument Ltd./Shanghai), KQ5200E type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), Arium611 pure water instrument (German Sai Duolisi group, Sartorius), pH211 type pH meter (Italian HANNA instrument plant), spacious reaction chamber, Morris water maze, automatic data collection and processing system (Shanghai Xin Ruan Information technology company limited), single track, multichannel pipettor (hundred obtain Laboratory Instruments company limited/Suzhou), IKA R104 homogenizer (the industrial commerce and trade of Beijing De Quan company limited), 5810R large high-speed refrigerated centrifuger (German Eppendorf company), 5417R miniature high-speed refrigerated centrifuger (German Eppendorf company), LG10-2.4A type generic centrifuge (Beijing Medical Centrifugal Machine Factory), thermostat water bath (instrument and meter company limited is established in Shanghai), TS-2000A decolorization swinging table (its woods Bel instrument manufacturing company limited of Haimen City), the quick vortex mixer of SZ-1 type (Jin Cheng Guo Sheng experimental apparatus factory of Community of Jin Tan County city), TU-1800 ultraviolet spectrophotometer (Beijing Puxi General Instrument Co., Ltd), M680 type microplate reader (Bio-Rad company of the U.S.), YZ1515x type constant flow pump (Baoding LanGe constant flow pump Co., Ltd), EG1150H paraffin wax embedding (German LEICA company), RM2235 microtome (German LEICA company), the roasting sheet machine (Hubei China Xiaogan Yaguang Medical-electronic Technology Ltd) in YT-7FB type biological tissue stand, BX51 microscope (Japanese OLYMPUS company).
3. reagent
D-galactose (Chemical Reagent Co., Ltd., Sinopharm Group, product batch number: 20130506), sodium nitrite (Chemical Reagent Co., Ltd., Sinopharm Group, product batch number: T20100118), piracetam (Jinan Limin Pharmaceutical Co., Ltd., product batch number: 1208177, specification: 0.4g/ sheet), oleracein E powder is (for the applicant's laboratory utilizes chemical synthesis process preparation [Liu Cejia, synthesizing of antioxidant oleracein E and derivant thereof. Shandong University, 2010], purity is 99.89%), injection normal saline (Cisen Pharmaceutical Co., Ltd.), chimera board food coloring (Shanghai Dye Inst. Co., Ltd., product batch number: 20910010), heparin sodium injection (Qianhong Biochemical Pharmaceutical Co., Ltd., Changzhou, product batch number: 120418), Coomassie brilliant blue G-250 (magnificent biological engineering company limited, product batch number: BS0481), bovine serum albumin (Shandong Ai Bo Trade Co., Ltd.), 10% chloral hydrate solution (Shandong Qilu Hospital), paraformaldehyde (Chemical Reagent Co., Ltd., Sinopharm Group, product batch number: F20090305), 30% hydrogenperoxide steam generator (economic and technological development zone, Laiyang Fine Chemical Works, product batch number: 20120310).Total antioxidant capacity (T-AOC) is measured test kit (product batch number: 20130523), superoxide dismutase (SOD) WST-1 method is measured test kit (product batch number: 20130520), catalase (CAT) is measured test kit (product batch number: 20130527), glutathion (GSH) is measured test kit (product batch number: 20130520), malonaldehyde (MDA) is measured test kit (product batch number: 20130524) all build up Bioengineering Research Institute purchased from Nanjing.Bcl-2 (product batch number: BS1511), Bax (product batch number: BS2538) and the anti-Mus polyclonal antibody of Caspase-3 (product batch number: BS1518) rabbit are provided by Ba Ao moral bio tech ltd; Instant SABC Elivision plus test kit (Mus/rabbit) is purchased from Fuzhou Maixin biotechnology Development Co., Ltd's (reagent A: reinforcing agent, product batch number: 130525404C; Reagent B: enzyme mark sheep anti mouse/rabbit igg polymer, product batch number: 130525405C); Normal goats serum working solution (product batch number: 1212K) and DAB developer (A liquid product batch number: 1313E for sealing; B liquid product batch number: 0713E) purchased from Beijing CoWin Bioscience Co., Ltd..Other common agents and chemicals are analytical pure.
4. the preparation of main agents
(1) 12.5% (g/mL) D-galactose and 0.9% (g/mL) sodium nitrite solution:
Get D-gal6.25g and NaNO
20.45g, accurately weighed, inject and make abundant dissolving with normal saline, be settled to 50mL, shake up and get final product.With 0.22 μ m membrane filtration in tool plug scale test tube, 100 DEG C of water-bath 15min, cooling after in 4 DEG C of storages, 3 days for subsequent use.
(2) 4% (g/mL) piracetam aqueous solution:
Piracetam is pulverized, get powder 0.2g, accurately weighed, adding distil water makes abundant dissolving, is settled to 5mL, shakes up.By this suspension centrifugal 10min under 4000r/min rotating speed, get supernatant and use (now with the current).
(3) oleracein E (OE) aqueous solution:
Get OE10mg, accurately weighed, add 30mL distilled water and make abundant dissolving, to obtain final product.Subpackage is stored in-20 DEG C, 3 days for subsequent use.
(4) heparin sodium anti-freezing liquid:
Use injection normal saline dilution to 10mL heparin sodium injection (2mL, 12500U), before blood sampling, in every 2mL centrifuge tube, add the heparin sodium-normal saline solution of 40 μ L dilutions, flick before use.
(5) 1mg/mL bovine serum albumin (BSA) standard solution:
Get 0.01g BSA, accurately weighed, add normal saline and make abundant dissolving, be settled to 10mL, shake up and get final product.
(6) 0.1mg/mL Coomassie brilliant blue G-250 solution:
Get 0.1g Coomassie brilliant blue G-250, accurately weighed, add 50mL95% ethanol fully to dissolve, then add 100mL85% phosphoric acid, adding distil water is settled to 1000mL, shakes up and get final product.Under room temperature, keep in Dark Place, filter before use, get subsequent filtrate and use.
(7) 0.1M phosphate buffer (pH7.4):
Get 0.78g NaCl, 29g Na
2hPO
412H
2o, 2.964g NaH
2pO
42H
2o, accurately weighed, add distilled water to make abundant dissolving, be settled to 1000mL, shake up and get final product.
(8) 4% paraformaldehyde solutions:
Take 20g paraformaldehyde, add 500mL phosphate buffer (0.1M, pH7.4), on magnetic stirring apparatus, heated and stirred makes to dissolve completely (temperature can not be too high, be controlled at 50 DEG C following), keeps in Dark Place after cooling.This reagent effect duration is about one week.
(9) the Harris haematoxylin of improvement:
Take 1g hematoxylin, add 10mL dehydrated alcohol to make abundant dissolving; Take 15g aluminium potassium sulfate (KAl (SO
4)
212H
2o), add 200mL temperature distilled water (40～50 DEG C) to make abundant dissolving; Above-mentioned two parts of solution are mixed and boiled, from fire, slowly add 0.4g mercury oxide, and constantly stir with Glass rod, reagent becomes darkviolet, and has a large amount of Bubble formations, now should be moved in cold water immediately and be cooled (if room temperature is higher, need repeatedly change water), filtered after cooling, be placed in brown little ground reagent bottle sealed storage.Before use, add 5% glacial acetic acid 4mL (or glacial acetic acid 3), have better effect.Note, this reagent effect duration can reach 1 year, and optimal use time is 1～2 month, before use, preferably again filters.
(10) 0.5% Yihong alcoholic solutions:
Take 0.5g Yihong, add 95% ethanol to make abundant dissolving, be settled to 100mL, shake up and get final product.
(11) 5% glacial acetic acid aqueous solution:
Measure 5mL glacial acetic acid, adding distil water is settled to 100mL, shakes up and get final product.
(12) 0.01M phosphate buffer (pH7.4):
Take 8g NaCl, 2.901g Na
2hPO
412H
2o, 0.296g NaH
2pO
42H
2o, adds distilled water to make abundant dissolving, is settled to 1000mL, shakes up and get final product.
(13) 0.01M citrate buffer (pH6.0):
Take 2.101g citric acid (C
6h
8o
7h
2o), adding distil water makes abundant dissolving, is settled to 100mL, shakes up to obtain A liquid; Take 7.353g sodium citrate (C
6h
8na
3o
72H
2o), adding distil water makes abundant dissolving, is settled to 250mL, shakes up to obtain B liquid.Measure respectively A liquid 18mL, B liquid 82mL, adding distil water is settled to 1000mL, shakes up and get final product.
(14) 1% hydrochloride alcohol solutions:
Measure 2mL concentrated hydrochloric acid, add 75% ethanol and be settled to 200mL, shake up and get final product.
Two, experimental procedure
1. animal grouping and processing
Animal conformed after three days, according to randomized blocks, mice was divided into 5 groups at random, was respectively experiment contrast group, D-gal/NaNO
2model group, than La Xitan positive drug (PA) matched group, OE low dose group (L-OE), OE high dose group (H-OE), 20 every group.Model group, positive drug control group and OE group mice lumbar injection every day D-gal (1.25g/kg)/NaNO
2(90mg/kg), continuous modeling 8 weeks; Experiment contrast group mouse peritoneal is injected isopyknic normal saline.Meanwhile, the OE aqueous solution of OE group mice difference gavage low dosage (3mg/kg) and high dose (15mg/kg), positive drug control group gavage PA aqueous solution (400mg/kg), experiment contrast group and model group mice be the isopyknic distilled water of gavage, successive administration 8 weeks.
2. body weight and survival rate are measured
After administration, measured Mouse Weight every three days, and the survival condition of itemized record experiment process small mouse.
3. date processing
Experimental result with
represent, and carry out statistical analysis by SPSS20 statistical software: mice survival rate data acquisition Kaplan-Meier method is analyzed, log-rank method inspection survival rate difference, has statistical significance taking P<0.05 as difference.
Three, experimental result
Animal conformed after three days, and body weight is increased to (20.7 ± 1.9) g.Mice is divided into 5 groups at random, Mouse Weight there was no significant difference between each administration group; After administration 8 weeks, Mouse Weight totally presents the trend of increase.Analyze each experimental mice body weight there was no significant difference through SPSS statistical software.
As seen from Figure 1, experimental session, except experiment contrast group, all there is animal dead phenomenon in model group and each drug treating group mice, lumbar injection D-gal/NaNO is described
2mice has been caused to serious damage, so that there are the phenomena of mortality in animal.Testing first three week, there is dead mouse phenomenon in model group successively, and other is respectively organized all without animal dead, illustrates that PA and OE can protect mice to avoid D-gal/NaNO
2the damage of induction.Along with the carrying out of experiment, model group mice still has animal dead, and dead mouse phenomenon also appears in L-OE group, PA group and H-OE group in succession.Totally it seems, experimental session, model group has 9 dead mouses, survival rate is 55%, significantly lower than experiment contrast group (
* *p=0.000); Compared with model group, the survival rate of L-OE and H-OE group mice significantly improve (
##p=0.003,
##p=0.003), its survival rate is 90%, and apparently higher than positive drug control group mice (survival rate is 75%, P=0.088).This experimental result shows, OE can resist heavy dose of lumbar injection D-gal/NaNO
2the damage of the AD mice of induction, significantly improves mouse survival rate, extends the life-span of AD mice; In addition, organize late 3 weeks because H-OE group mice starts the dead time than L-OE, show that OE high dose is being better than OE low dosage aspect the raising senile dementia life-span.
Embodiment 2: the application of oleracein E aspect the metabolism of enhancing senile dementia
One, experiment material, instrument and reagent
With embodiment 1.
Two, experimental procedure:
1. animal grouping and processing: with embodiment 1.
2. metabolic index is measured: from testing the 16th day, carry out weekly the investigation (amounting to 6 times) of a metabolic index, comprise amount of drinking water, food-intake, voided volume and the feces volume of mice in 24 hours.Specific experiment operation is as follows: after mice administration, put it in metabolic cage and (put into same metabolic cage with cage mice, every group of 4 cages), give a certain amount of food and water, ad lib drinking-water.After 24h, take out laboratory animal, weigh remaining food and water, collect simultaneously and weigh the urine amount of feces amount and excretion.When statistical analysis, the arithmetic mean of instantaneous value of the metabolic index measurement result of four cage mices is on the same group carried out to statistical analysis as the metabolic cost of this this group mice, replication 6 times.
3. date processing
Experimental result with
represent, and carry out statistical analysis by SPSS20 statistical software: between data multisample mean, relatively adopt One-way ANOVA to analyze, the Student-Newman-Keuls that relatively adopts between two between group checks, and has statistical significance taking P<0.05 as difference.
Three, experimental result
Because investigated metabolic index changes with the variation of body weight, still represent each index with the ratio of measured value and Mouse Weight.From table 1, inject for a long time D-gal/NaNO
2after, the amount of drinking water of model group mice and voided volume significantly lower than experiment contrast group (
*p=0.003,
*p=0.027), show to inject for a long time D-gal/NaNO
2significantly reduce mice metabolism rate.Compared with model group, the amount of drinking water of L-OE and H-OE group mice significantly increase (
#p=0.024,
#p=0.013), meanwhile, voided volume also significantly increase (
##p=0.009,
###p=0.000).PA group mice amount of drinking water increases to some extent, urine amount significantly increase compared with model group (
#p=0.011).This experimental result shows, OE can regulate D-gal/NaNO
2the metabolic function disorder causing, makes the metaboilic level of mice be tending towards normal.Significantly increase compared with model group except H-OE organizes feces volume in addition, (
##p=0.008), the food-intake of mice and feces volume there are no significant difference between other each group.This may have and promote that the function of enterokinesia is relevant to Herba Portulacae, and its mechanism of action waits further research.
Table 1. oleracein E is to D-gal/NaNO
2the impact (n=11～20) of induction senile dementia mouse metabolism.
(with the comparison of experiment contrast group,
*p<0.05,
*p<0.01; With model group comparison,
#p<0.05,
##p<0.01,
###p<0.001)
Embodiment 3: the effect of oleracein E aspect alleviation senile dementia oxidative stress.
One, experiment material, instrument and reagent
With embodiment 1.
Two, experimental procedure:
1. animal grouping and processing: with embodiment 1.
2. Biochemical Indexes:
(1) collection of sample and preparation
After behavioristics's experiment, animal fasting be can't help water and is spent the night.Next day, extract eyeball of mouse blood sampling, blood is splashed in the pre-anticoagulant tube of heparin, notice that unavailable tube wall scrapes blood, prevent erythroclasis, cause haemolysis, the mensuration of influence index.After blood sampling, by blood low-temperature centrifugation 10min under 3500r/min rotating speed, get supernatant (being blood plasma) in time, its subpackage is stored in-80 DEG C of refrigerators, for subsequent use.Adopt after blood, on Yu Bingtai, peeled off rapidly cerebral tissue.Wash down cerebral tissue surface bloodstain with the ice normal saline of pre-cooling, filter paper is wiped dry, shreds, and weighs.Add the ice normal saline of 9 times of volumes, under ice-water bath condition, with 14500r/min rotating speed homogenate 4 times, 10s/ time, midfeather 30s, so makes 10% brain homogenate by cerebral tissue; Again under 3500r/min rotating speed by the centrifugal 15min of brain homogenate low temperature (4 DEG C), get supernatant, subpackage is stored in-80 DEG C of refrigerators, for subsequent use.Take after cerebral tissue, win rapidly thymus, spleen, liver and kidney, reject the fatty tissue on organ, and clean surperficial bloodstain with the ice normal saline of pre-cooling, filter paper is wiped dry, weighs, and calculates organ index, and carries out statistical analysis.
(2) mensuration of cerebral tissue protein content
Adopt Coomassie brilliant blue method to carry out the mensuration of cerebral tissue protein content.Coomassie brilliant blue G-250 takes on a red color under free state, and maximum absorption wavelength is at 465nm place; When becoming cyan after it and protein bound, the maximum absorption wavelength of Coomassie brilliant blue-albumen composition is at 595nm, its absorbance is directly proportional to the content of protein, can carry out quantitatively protein by the absorbance of measuring 595nm place Coomassie brilliant blue albumen composition.
A. the drafting of standard curve:
Pipette respectively lmg/mL bovine serum albumin (BSA) titer 0 μ L, 20 μ L, 40 μ L, 60 μ L, 80 μ L, 100 μ L with micropipettor, then add respectively normal saline 100 μ L, 80 μ L, 60 μ L, 40 μ L, 20 μ L, 0 μ L, every duplicate samples adds 5mL Coomassie brilliant blue G-250 dyeing liquor again, shake up lucifuge reaction 30min under room temperature condition.Not add bovine serum albumin pipe as the zeroing of blank pipe, with lcm optical path cuvette, measure the absorbance (A) of each pipe in 595nm wavelength place.Taking bovine serum albumin content as abscissa, (unit: mg), absorbance (A) do standard curve as vertical coordinate.
B. the mensuration of cerebral tissue protein content:
Pipette respectively each mouse brain tissue samples 30 μ L with micropipettor, add normal saline 70 μ L, then add Coomassie brilliant blue G-250 dyeing liquor 5mL, lucifuge reaction 30min under room temperature condition; In 100 μ L normal saline, add 5mL Coomassie brilliant blue G-250 dyeing liquor to return to zero as blank simultaneously; With lcm optical path cuvette, measure the absorbance of each pipe in 595nm wavelength place.Calculate cerebral tissue protein content according to BSA concentration and absorbance standard curve.
(3) Biochemical Indexes
The operating procedure of the assay of the activity of AChE, T-AOC, SOD, CAT and GSH-PX and GSH, MDA is all undertaken by test kit explanation.
3. date processing
Experimental result with
represent, and carry out statistical analysis by SPSS20 statistical software: between data multisample mean, relatively adopt One-way ANOVA to analyze, the Student-Newman-Keuls that relatively adopts between two between group checks, and has statistical significance taking P<0.05 as difference.
Three, experimental result
T-AOC is the index of multiple antioxidant for clearing oxygen-derived free radicals total capacity in reflection body defense system, comprises enzymatic and non-enzymatic two individual system, is the important component part of body prevention oxidative stress damage.Table 2 shows, D-gal/NaNO
2model group mice T-AOC level be significantly higher than experiment contrast group (
*p=0.004), show modeling 8 weeks, D-gal/NaNO
2oxidative stress can cause that in mouse brain, the active compensatory of T-AOC increases; Compared with model group, the T-AOC level of PA group, L-OE and H-OE group mice obviously reduce (P=0.059,
#p=0.013,
###p=0.000) OE that, shows low dosage and high dose all can be alleviated D-gal/NaNO
2the oxidative stress causing, makes T-AOC keep normal level.
In body defense system, SOD can remove ultra-oxygen anion free radical; CAT catalyzing hydrogen peroxide decomposes generation H
2o and O
2, avoid the generation of hydroxy radical; GSH is the substrate of glutathion peroxidase (GSH-PX) and glutathione-S-transferase (GST), essential by these two kinds of enzyme decomposition of hydrogen peroxide, also can avoid the generation of hydroxy radical.SOD, CAT and GSH are the important component parts that body self is eliminated oxidative damage.Experimental result shows, D-gal/NaNO
2active and the GSH content of model group SOD in Mice compared with experiment contrast group significantly increase (
*p=0.031,
*p=0.002), show modeling 8 weeks, D-gal/NaNO
2can cause in mouse brain that the active and GSH content compensatory of SOD increases; Compared with model group, the SOD vigor of positive drug control group, L-OE and H-OE group mice and GSH content be starkly lower than model group (
#p=0.039, P=0.051,
#p=0.015; P=0.084,
#p=0.010,
##p=0.008) OE that, shows low dosage and high dose all can be alleviated D-gal/NaNO
2the oxidative stress causing, raises SOD vigor and GSH content compensatory and returns to normal level.
In each experimental mice blood plasma, SOD activity is without significant difference.In addition, the CAT vigor in model group mouse brain and blood plasma compared with experiment contrast group all significantly reduce (
*p=0.041,
*p=0.030); After administration 8 weeks, L-OE group mouse brain tissue be significantly increased with CAT vigor in blood plasma (compared with model group,
#p=0.016,
##p=0.006), in PA group mice plasma CAT vigor significantly improve (
#p=0.024).Show, low dosage OE improves the activity of CAT, reduces oxidative stress, reduces oxidative damage.Compared with model group, the CAT vigor there was no significant difference in H-OE group mouse brain and blood plasma.
Different from bibliographical information, the rising of lipid peroxidation product MDA in model group mouse brain is not observed in this experiment, and guess may be D-gal/NaNO
2the oxidative stress causing impels the non-enzymatic of the part of body and enzymatic oxidation resistance compensatory to raise, thereby has offset the degree of lipid peroxidation injury.This also may be relevant to mice sex, age and modeling degree of injury.
Table 2. oleracein E is to D-gal/NaNO
2t-AOC in the senile dementia mouse brain of induction and blood, SOD, CAT activity and GSH, the impact (n=8～17) of MDA level.
(with the comparison of experiment contrast group,
*p<0.05,
*p<0.01; With model group comparison,
#p<0.05,
##p<0.01,
###p<0.001)
Embodiment 4: the application of oleracein E aspect protection senile dementia neuronal damage.
One, experiment material, instrument and reagent
With embodiment 1.
Two, experimental procedure:
1. animal grouping and processing: with embodiment 1.
2. the morphologic detection of hippocampal tissue
(1) cardiac perfusion
After behavioristics finishes, getting 3 mouse peritoneals by each group injects 10% chloral hydrate (3mL/kg) and anaesthetizes, after anaesthetizing, be fixed on operating-table with dorsal position, open thoracic cavity, expose heart, (constant flow pump flow velocity is 18rpm to wash down fast systemic blood with normal saline, be 16.2mL/min), more slowly instil and fix (constant flow pump flow velocity is 2rpm, i.e. 1.8mL/min) with 4% paraformaldehyde solution.After extremity are stiff, carefully take out cerebral tissue, be fixed in 4% paraformaldehyde and spend the night.After 24h, cerebral tissue is taken out, after bregma, 1.5mm place draws materials backward, gets 4mm tissue and carries out routine paraffin wax embedded section.
(2) specimens paraffin embedding slices
After cerebral tissue piece is fully rinsed well with PBS, with gradient alcohol dehydration (50% ethanol 1h → 75% ethanol 1h → 85% ethanol 1h → 95% ethanol I 1h → 95% ethanol II 1h → dehydrated alcohol I 0.5h → dehydrated alcohol II 0.5h), use again dimethylbenzene transparent (anhydrous alcohol: dimethylbenzene (1:1) 30min → dimethylbenzene I 30min → dimethylbenzene II 30min), the dimethylbenzene renewing, slowly add thin wax disk(-sc) (soft wax) until saturated, move in 37 DEG C of calorstats and spend the night, carry out embedding next day.Every part of cerebral tissue paraffin sample continuous coronal section, sheet is thick is 3 μ m, gets a slice carry out conventional H E dyeing every 10 sections.
(3) HE dyeing
The section of paraffin organization sample is fully soaked to 3min after dewaxing 30min in dimethylbenzene in dimethylbenzene, again through dimethylbenzene: gradient aquation (dehydrated alcohol → 95% ethanol → 85% ethanol → 75% ethanol → 50% ethanol → distilled water after immersion 3min in dehydrated alcohol (1:1), each gradient is soaked 3min), available Harris haematoxylin redyeing after aquation, micro-Microscopic observation dye levels suitable (2min), wash away unnecessary dye liquor with tap water immediately, with 5% glacial acetic acid aqueous solution differentiation 5s, proceed to immediately and in tap water, return blue 10min, again through gradient ethanol alcoholization (50% ethanol → 75% ethanol → 85% ethanol → 95% ethanol, each gradient is soaked 5s), after alcoholization, available 0.5% Yihong ethanol liquid is redyed 20s, transit to again (95% ethanol 5s → dehydrated alcohol 5s → dehydrated alcohol: dimethylbenzene (1:1) 5s → dimethylbenzene I 2min → dimethylbenzene II 10min) in dimethylbenzene, neutral gum mounting.The morphologic variation of micro-Microscopic observation hippocampal tissue, image is by DEIAS2000P Tangier medical image analysis system acquisition.
(4) immunohistochemical staining of hippocampal tissue apoptosis-related protein Bcl-2, Bax and Caspase-3 detects
Every part of cerebral tissue paraffin sample continuous coronal section, sheet is thick is 3 μ m, gets a slice carry out immunohistochemical staining every 10 sections.Scheme is by selected brain tissue slice dewaxing aquation (dewaxing dimethylbenzene 10min → dimethylbenzene I 10min → dimethylbenzene II 10min → dimethylbenzene: dehydrated alcohol (1:1) 5min → dehydrated alcohol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min → distilled water 5min) routinely, in pressure cooker, add appropriate citrate buffer (0.01M, pH6.0) Pressure method 90s, naturally cools to room temperature.3% hydrogenperoxide steam generator soaks 20min, to remove endogenous peroxydase.Drip sealing normal goats serum working solution, incubated at room 30min; Drip Bcl-2 (1:50), Bax (1:200) or the anti-Mus polyclone of Caspase-3 (1:1600) rabbit primary antibodie, 37 DEG C add polymer intensifier after hatching 1h, incubated at room 20min, then add enzyme mark sheep anti mouse/rabbit igg polymer, incubated at room 30min.Drip appropriate DAB colour developing, micro-Microscopic observation, rinses color development stopping (Bcl-2, Bax and Caspase-3 dyeing time are respectively 10s, 4min and 2.5min) with tap water while reaching best color developing effect immediately.Section after colour developing is through haematoxylin redyeing 2min, 1% hydrochloride alcohol differentiation 5s, indigo plant is returned in washing from the beginning, transparent through dewatering (75% ethanol 5s → 85% ethanol 5s → 95% ethanol 5s → dehydrated alcohol 5s → dehydrated alcohol: dimethylbenzene (1:1) 5s → dimethylbenzene I 2min → dimethylbenzene II 10min → mounting dimethylbenzene) again, with neutral gum sealing preservation.The positive cell of micro-Microscopic observation Hippocampus Bcl-2, Bax and Caspase-3.Image is by DEIAS2000P Tangier medical image analysis system acquisition.
Three, experimental result
As shown in Figure 2, result shows HE coloration result, experiment contrast group hippocampus of mice CA
4, CA
3and CA
1district neurocyte is arranged and is compacted, dyeing homogeneous, and structural integrity, cell membrane, nuclear membrane boundary are clear; D-gal/NaNO
2model group hippocampus of mice CA
4and CA
3district is dispersed in even continuous neurocyte degeneration necrosis as seen, shows as concentrated red the dying of endochylema, karyon pyknosis, and cell membrane, nuclear membrane boundary are unclear; CA
1district's neurocyte is arranged loose, and " tomography " phenomenon appears in severe patient.With model group comparison, PA and OE administration group hippocampus of mice structure are significantly improved, CA
1district's neurocyte is arranged closely nothing " tomography " phenomenon, and CA
4and CA
3the red phenomenon of dying of the rare endochylema in district.In addition, H-OE neuroprotective is better than L-OE, because L-OE group hippocampus of mice CA
4district's endochylema is red to be dyed and CA
3district's karyon pyknocyte is obviously more than H-OE, and this may be to perform poor in the experiment of L-OE group Mice water maze, occurs one of reason of Spatial learning and memory obstacle.
As shown in Figure 3 and Figure 4, result shows immunohistochemical staining result, compared with experiment contrast group, and D-gal/NaNO
2model group hippocampus of mice CA
4and CA
3district's Bcl-2 positive cell obviously reduces, and PA and OE group hippocampus of mice Bcl-2 positive cell showed increased; In addition model group hippocampus of mice CA,
4and CA
3the visible a large amount of Bax positive cell in district, and PA and OE group only have minority positive cell.Compared with experiment contrast group, model group hippocampus of mice CA
4and CA
3district Caspase-3 high expressed, nucleus engrain; L-OE group is at CA
4and CA
3district's Caspase-3 positive cell is more, and PA and H-OE have all obviously reduced the expression of Caspase-3, and H-OE effect is better than PA.Above description of test OE high dose may be by suppressing the startup factor (as removed free radical) of neurocyte degenerative change, and blocking-up apoptosis etc. causes the signal process of neurocyte degenerative change, to D-gal/NaNO
2the senile dementia hippocampus neurons in mice cell injury of induction has been brought into play protective effect.
Embodiment 5: the application of oleracein E aspect raising senile dementia memory ability.
One, experiment material, instrument and reagent
With embodiment 1.
Two, experimental procedure:
1. animal grouping and processing: with embodiment 1.
2. behavioristics's test
Water maze laboratory is a kind of method of test experience animal Spatial learning and memory ability.This equipment is by black round pool (diameter 120cm, high 50cm), platform and recording system three part compositions.Pond is divided into 4 quadrants, and platform is placed in to the second quadrant (II quadrant) central authorities.Platform diameter is 9cm, and high 30cm above has ring grain, so that mice can be stood firm on platform.Experiment goes to water filling in pond, depth of water 30cm, platform to be positioned at the following 1cm of water surface place, and adds black food coloring, makes water be opaque state, and water temperature remains on 20～22 DEG C.Pond surrounding is hung with the blue door curtain made of cloth, is hung with in bright gay color, variform four geometric figures above as space object of reference, and position remains unchanged, for mice locating platform.Photographic head is placed in 2m place directly over pond, automatically gather animal swimming image, collected signal is directly inputted computer, by image acquisition and analytical system automatic analysis and processing, what comprise time that escape latency (animal arrives the time of platform first), swimming distance, four quadrants stop respectively and original platform region enters the parameters such as number of times.All laboratory animals are carried out the hidden platform training of continuous 5 days, carry out afterwards space exploration test, to evaluate the Spatial learning and memory ability of mice.
When hidden platform training, keep position of platform to immobilize, the training of every day divides two of upper and lower noons to carry out, put into mice the morning from I, III quadrant, put into from II, IV quadrant afternoon, accumulative total 4 times, each 120s, gives time of having a rest of 10min between twice training; When training, animal is faced to pool wall and put into gently water, record mice and find the platform time used (being escape latency).If do not find yet platform after mice 120s, guided to platform, and on platform rest 30s, be designated as 120s incubation period.When statistical analysis, the achievement using the arithmetic mean of instantaneous value of four escape latency as this mice on the same day.
Hidden platform training finishes after 24h, carries out space exploration test.Remove platform, respectively mice is put into water from four quadrants, record the time of staying of the inherent all quadrants of mice 120s and the traversing times in original platform region.When statistical analysis, using the arithmetic mean of instantaneous value of four experimental results as the same day this mice achievement carry out statistical analysis.All data record and analyze by image acquisition and analytical system, and this system is provided by Shanghai Xin Ruan Information Technology Co., Ltd.
Three, experimental result
As seen from Figure 5, through the training of five days, within the 6th day, to remove after platform, the time that each experimental mice stops at target platform quadrant (II quadrant) is all apparently higher than other quadrant, show training and study through 5 days, its spatial memory capacity all increases.Model group mice the target platform quadrant time of staying and platform location traversing times all significantly lower than experiment contrast group (
*p=0.038,
*p=0.025), show to inject for a long time D-gal/NaNO
2can damage the spatial memory capacity of senile dementia mice.Compared with model group, PA group and H-OE organize mice significantly increase in the target platform quadrant time of staying and platform location traversing times (
#p=0.025,
#p=0.049;
#p=0.023,
#p=0.048) OE that, shows high dose can improve D-gal/NaNO
2the spatial memory capacity of the senile dementia mice of induction.
In sum, OE, by alleviating oxidative stress and anti-Apoptosis of Hippocampal Neurons, has obviously improved D-gal/NaNO
2the memory ability of the senile dementia mice of induction, its effect is suitable with positive drug piracetam; But OE is better than piracetam at the successful aspect prolongation senile dementia mouse life, and the maximum dose level of OE also only has 1/27 of piracetam dosage.From dosage and life-saving aspect, OE will obviously be better than clinical senile dementia medicine piracetam.
Claims (9)
1. oleracein E is in the application of preparing in anti-senile dementia neurodegenerative diseases medicine.
2. application according to claim 1, is characterized in that: described neurodegenerative diseases refers to senile dementia, or other neurodegenerative diseases relevant to senile dementia.
3. application according to claim 2, is characterized in that: described other neurodegenerative diseases relevant to senile dementia comprises Parkinson disease, Huntington chorea, amyotrophic lateral sclerosis, spinal cord muscular atrophy.
4. the application of oleracein E in the preparation medicine in prolongation old dementia patients life-span.
5. oleracein E strengthens the application in the metabolic medicine of old dementia patients in preparation.
6. the effect of oleracein E in the medicine of preparation alleviation old dementia patients oxidative stress.
7. the application of oleracein E in the medicine of preparation protection old dementia patients neuronal damage.
8. the application of oleracein E in the medicine of preparation raising old dementia patients memory ability.
9. according to the application described in any one in claim 1～8, it is characterized in that: described oleracein E refers to the raceme of oleracein E monomer or its enantiomer or enantiomer or derivatives thereof monomer.
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| CN106955351A (en) * | 2016-01-08 | 2017-07-18 | 周艳玲 | A kind of pharmaceutical composition of anti-senile dementia |
| CN107106621A (en) * | 2014-08-27 | 2017-08-29 | 成均馆大学校产学协力团 | It is used to prevent as active component comprising Portulaca grandilora extract or its cut or treats neuroinflamation or the pharmaceutical composition of nerve degenerative diseases |
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| CN103070893A (en) * | 2013-01-10 | 2013-05-01 | 济南康众医药科技开发有限公司 | Medicine prepared by purslane and used for treating senile dementia |
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| CN106955351A (en) * | 2016-01-08 | 2017-07-18 | 周艳玲 | A kind of pharmaceutical composition of anti-senile dementia |
| CN106538996A (en) * | 2016-10-31 | 2017-03-29 | 烟台新时代健康产业有限公司 | A kind of Folium Bambosae flavone compositionss and its preparation for improving learning and memory ability |
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