CN102283858B - Application of lycium barbarum polysaccharides for preparing drugs for preventing and treating chronic stress and posttraumatic stress disorder - Google Patents

Abstract

The invention discloses application of lycium barbarum polysaccharides for preparing drugs for preventing and treating chronic stress and posttraumatic stress disorder. The application is characterized by comprising the following steps of: filling the extracted lycium barbarum polysaccharides with the purity of more than or equal to 50% in capsules or preparing the lycium barbarum polysaccharides into tablets, wherein each capsule contains 500 mg of the lycium barbarum polysaccharides, or each tablet contains 500 mg of the lycium barbarum polysaccharides; uniformly mixing the extracted lycium barbarum polysaccharides with the purity of more than or equal to 50% and corn startch; and filling the mixture into capsules or preparing the mixture into tablets, wherein each capsule contains 500 mg of the lycium barbarum polysaccharides and 100 mg of the corn starch, or each tablet contains 500 mg of the lycium barbarum polysaccharides and 100 mg of the corn starch. The application disclosed by the invention has the benefits that: the drugs are prescribed preparations composed of the lycium barbarum polysaccharides; the drugs have anti-stress effect and are capable of preventing and treating chronic stress and/or posttraumatic stress disorder and improving cognitive function; the pharmaceutical effect is clear; the safety is high; and the novel drug application selection is provided in clinical.

Description

The application of LBP in the medicine of preparation posttraumatic stress disorder

Technical field

The present invention relates to the new purposes of wolfberry fruit extract, specifically relate to the application of LBP in the medicine of preparation posttraumatic stress disorder.

Background technology

The chronic stress obstacle is meant that the individual emotion that continues that can not adjust in the face of long-term special environment changes and experienced during stable homeostasis changes and mental pressure.Its mechanism of causing a disease is: stressor stimulates body unstability attitude stress stimulation to continue or exists repeatedly; Then stress effect accumulation form the stable state strain burden; Stress related neural endocrine medium continue rising in circulation and the tissue, and then develop into stable state strain over loading with cytokine.Because the long term of a large amount of neuroendocrine media and cytokine causes each system's organ, tissue and cell function unusual.Wherein (posttraumatic stress disorder PTSD) is meant because life is on the hazard, meets with spirit, the lasting clinical syndrome of somatization that tragedy causes posttraumatic stress disorder.APA,American Psychiatric Association " had established the diagnostic criteria of PTSD among the mental disorder statistical diagnosis handbook DSM-III (third edition) in 1980.PTSD formally included " International Classification of Diseases " ICD-10 (the tenth edition) in 1993.1994 subsequently Americanism obstacle diagnosis statistical classification handbook-Di four editions (DSM-IV) return PTSD in anxiety disorder, and it is divided into three types: acute (symptom duration was less than 3 months), chronic (symptom at least continue 3 months), companion postpone onset (symptom stress after just appearance at least 6 months).It is that the pathologic of characteristic is reappeared, the avoidance of wound related thread, the height of persistence are waken up that the PTSD symptom mainly shows as the property swarmed into memory to traumatic event, and to the selective amnesia and the emotional anesthesia of wound experience, therefore the patient agonizes and can't bear.The PTSD symptom generally occurs after a few days even several months after suffering wound, and the course of disease is the several years.

The dissimilar posttraumatic stress disorder sickness rate that cause are different, have investigation to find that the general population who lives through traumatic event in the U.S. has 25%～30% people to suffer from PTSD; Some special wounds like sexual violence etc., can cause higher sickness rate.At professional health field, the PTSD because of burst accident causes also has higher sickness rate.According to APA,American Psychiatric Association's statistics, crowd's total prevalence rate of U.S. PTSD is 1%～14%, average out to 8%, and individual lifelong risk property reaches 3%～5%, and the women is about 2 times of male.The German Research result shows that ill colony is 1.3%, and Algeria shows up to 37.4%.And China's 5 will just, anticipatory remark etc. is shown as 33.89% and 23% to orphan PTSD morbidity survey situation due to the serious flood of Dongting Lake disaster area adult and the Tangshan Earthquake respectively." 5.12 " Wenchuan earthquake utmost point severely afflicated area survivor in 2008 is suffered from PTSD classical symptoms puzzlements such as insomnia, nightmare, flash back after three months people has 18.4 % approximately.PTSD is high with its sickness rate, and the course of disease is long, and weak curative effect etc. have a strong impact on trauma care, are the medical research difficult points.The generation of PTSD causes physiology disabled on the one hand patient itself, is mentally ill or the dangerous of physical disease (as: suicide due to cardiovascular disease, sleep disorder, the depression etc.) significantly increases, and patient's life quality is significantly descended; Because of patient's work functions goes down, cause heavy financial burden on the other hand to society.

Clinical drug therapy about PTSD repeatedly adopts Benzodiazepines (benzodiazepines at present; BZ) antianxiety drugs, antidepressants, selective serotonin reuptake inhibitor (SSRIs), monoamine oxidase inhibitor, atypical antipsychotic agents, mood stabilizers and anticonvulsant, sympatholytic etc.; But the prolonged application antianxiety drugs is prone to cause rely on; Withdrawal symptom appears in drug withdrawal, also damages cognitive function; Always because mechanism of drug action is different, untoward reaction is many for some other medicine.The effect of Chinese medicine in the posttraumatic stress disorder treatment paid close attention to by the medical worker always.The domestic research of having carried out at present comprises: XIAOYAO POWDER plus-minus, Fructus Alpiniae Oxyphyllae etc. all have good curative effect.

Fructus Lycii is the rare Chinese medicine of the traditional dietotherapeutic of China, and its sweet in the mouth, property are put down, and the function that has invigorating the liver and kidney, benefiting essence-blood and make eye bright in recent years, receives clinical and the great attention nutrient research field always.Pharmacology of Fructus Lycii and health-care effect and the bioactive substance LBP that wherein contains have much relations.Modern scientific research shows that LBP has the immunity of organism of adjusting, removes oxygen-derived free radicals, suppresses tumor, slow down aging, blood fat reducing, blood sugar lowering and fatigue-resisting function, has the wide development application prospect.Though good research effect is arranged in prevention and treatment chronic stress and posttraumatic stress disorder; But main still occur with compound preparation, the composition of not seeing relevant medicine, food dual-purpose use separately Fructus Lycii and wolfberry fruit extract-LBP composition prevention thereof and treatment stress report.

Summary of the invention

The purpose of this invention is to provide the application of LBP in the medicine of preparation posttraumatic stress disorder, this medicine has prevention and treatment stress, improve cognitive function.

The present invention is an active component with extract-LBP (Lycium barbarum polysaccharides) of Fructus Lycii, adds the medicament that acceptable accessories or complementary composition are prepared from.

The application of LBP of the present invention in the medicine of preparation posttraumatic stress disorder is characterized in that: the LBP of purity >=50% that extracts is incapsulated or process tablet.

Further, the LBP with extracting incapsulates, and every contains LBP 500 ㎎, or processes tablet, and every contains LBP 500 ㎎.

The application of described LBP in the medicine of preparation posttraumatic stress disorder is characterized in that: LBP and corn starch mix homogeneously with purity >=50% that extracts incapsulate or process tablet.

Further, every capsules contains LBP 500 ㎎, contains corn starch 100mg; Perhaps every contains LBP 500 ㎎, corn starch 100mg.

Beneficial effect of the present invention: the single preparations of ephedrine that medicine is made up of LBP, have anti-stress effect, can in the prevention and the treatment chronic stress with or posttraumatic stress disorder; Improve cognitive function; Drug effect is clear and definite, and is safe, provides a kind of new medication to select for clinical.

Description of drawings

Fig. 1 be contrast, stress, preventative LBP irritates harmonization of the stomach therapeutic LBP and irritates stomach group rat and refuse prisoner's reaction and matched group comparison diagram, (* P 0.05, * * P < 0.01; Compare with stress group: # P 0.05, # # P 0.01).

Fig. 2 be contrast, stress, preventative LBP irritates harmonization of the stomach therapeutic LBP and irritates 1w after the stomach group stress in rats, 2w, the stiff upright reaction of 4w relatively and the matched group comparison diagram, (* P 0.05, * * P < 0.01; Compare with stress group: # P 0.05, # # P 0.01).

Fig. 3-1. be stress back 1w water maze space exploration trajectory diagram.

Fig. 3-2. be stress back 2w water maze space exploration trajectory diagram.

Fig. 3-3. be stress back 4w water maze space exploration trajectory diagram.

Fig. 3-4. be that preventative LBP3d filling stomach stress back 1w water maze space exploration trajectory diagram.

Fig. 3-5. be that preventative LBP3d filling stomach stress back 2w water maze space exploration trajectory diagram.

Fig. 3-6. be that preventative LBP3d filling stomach stress back 4w water maze space exploration trajectory diagram.

Fig. 3-7. be to irritate stomach LBP1w water maze space exploration trajectory diagram in the back.

Fig. 3-8. be stress back LBP2w water maze space exploration trajectory diagram.

Fig. 3-9. be stress back LBP4w water maze space exploration trajectory diagram.

Fig. 4-1. be to put under house arrest the electricity irritation hippocampus of rats respectively to distinguish morphology characteristics figure (4w, HE, Nissl and Silver dyeing).

Fig. 4-2. be LBP prevention is irritated stomach and is respectively distinguished morphology and influence figure (4w, HE, Nissl and Silver dyeing) putting under house arrest the electricity irritation hippocampus of rats.

The LBP of Fig. 4-3. treatment is irritated stomach and is respectively distinguished morphology and influence figure (4w, HE, Nissl and Silver dyeing) putting under house arrest the electricity irritation hippocampus of rats.

Fig. 4-4. be matched group, stress group, stress before LBP, stress back LBP group rat hippocampus CA1 neuron density figure (mean ± standard deviation, neuron/50 mm ²).

Fig. 4-5. be matched group, stress group, stress before LBP, stress back LBP group rat hippocampus CA3 neuron density figure (mean ± standard deviation, neuron/50 mm ²).

The matched group of Fig. 4-6., stress group, stress before LBP, neuron density figure (mean ± standard deviation, neuron/50 mm that stress back LBP group rat hippocampus DG ²).

Fig. 5-1. be contrast, stress, stress before LBP, stress back LBP group rat hippocampus DG district BrdU positive cell number figure (mean ± standard deviation).

The laser confocal microscope of Fig. 5-is 2. observed the preventative low dosage of 3d and is irritated 1w Hippocampus DG district BrdU positive expression figure (top, the right is the BrdU nuclear staining, and the left side is the DAPI nuclear staining) after the stomach stress in rats.

Fig. 5-3. are 4w Hippocampus DG district BrdU positive expression figure (top, the right is the BrdU nuclear staining, and the left side is the DAPI nuclear staining) after the preventative low dosage filling of the laser confocal microscope observation 3d stomach stress in rats.

Fig. 5-4. are 1w Hippocampus DG district BrdU positive expression figure (top, the right is the BrdU nuclear staining, and the left side is the DAPI nuclear staining) after the laser confocal microscope observation high dose filling stomach stress in rats.

Fig. 5-5. are 2w Hippocampus DG district BrdU positive expression figure (top, the right is the BrdU nuclear staining, and the left side is the DAPI nuclear staining) after the laser confocal microscope observation high dose filling stomach stress in rats

Figure .5-6. is 4w Hippocampus DG district BrdU positive expression figure (top, the right is the BrdU nuclear staining, and the left side is the DAPI nuclear staining) after the laser confocal microscope observation high dose filling stomach stress in rats.

The specific embodiment

Embodiment one: the preparation of LBP

Take by weighing FRUCTUS LYCII jerky powder 100g, petroleum ether (60-90 ℃) backflow defat secondary, each 1 h.Reuse 80% alcohol reflux 2 times, each 2h is to remove monosaccharide and oligosaccharide.Medicinal residues are added 90 ℃ of water-baths of 500ml water extract secondary, merge secondary raffinate.Be evaporated to 150 ml on the rotating thin film evaporimeter, add the small amount of activated decolouring, filter.Filtrating adds 5 times of amount 95% ethanol, and placement is spent the night, and the deposition behind the sucking filtration repeats precipitate with ethanol once with 100 ml water dissolutioies.Deposition is repeatedly washed with 95% ethanol, dehydrated alcohol, acetone, ether, and 60 ℃ of dryings promptly get LBP, purity >=50%.Sieve, encapsulated, promptly get the LBP capsule; Every capsules contains LBP 500 ㎎; Perhaps process tablet, every contains LBP 500 ㎎, with addition of corn starch 100mg.

Except adopting the said method extraction; Also can adopt traditional water extraction method, quick multiple extraction methods such as enzyme extraction method, ultrasound wave, microwave-assisted water and supercritical CO 2 extraction, the distinct methods wolfberry fruit extract all have prevention and treatment chronic stress obstacle with or posttraumatic stress disorder get drug effect.

LBP also can disclosed with reference to CN 101029088A " a kind of method for preparing of LBP " prepare; Also can directly buy the commercially available prod; As: the LBP of purity >=50%, lot number: 20100409, Shaanxi Rui Kang biological engineering company limited provides; Facing the time spent adds normal saline and processes 2 mg/ml, and the medicinal liquid of 20 mg/ml concentration supplies pharmacological evaluation to use.

Below prove beneficial effect of the present invention through concrete pharmacodynamic experiment.

Experiment material:

Animal: the SD rat, male, body weight 210 ± 26g, Chongqing City Third Military Medical University the 3rd Affiliated Hospital's Experimental Animal Center provides, regular grade animal, licence: SCXK (yu) 2007017.

Reagent reagent:

LBP, purity >=50%, lot number 20100409, Shaanxi Rui Kang biological engineering company limited is produced;

Sodium chloride injection, lot number K090915, Kelun Pharm Ind Co., Ltd., Sichuan produces;

Pentobarbital sodium, Denmark's import packing;

The paraformaldehyde analytical pure, lot number 20100501, the Long Huagongshijichang of Chengdu section produces;

Dehydrated alcohol, lot number 20100509, Chongqing luxuriant industry chemical reagent company limited is produced;

Sodium dihydrogen phosphate, lot number 20091105, Chongqing Chuan Dong chemical industry Group Co.,Ltd produces;

Sodium hydrogen phosphate, lot number 90519, Chongqing game chemical reagent company limited is produced;

The sucrose analytical pure, lot number 20081113, the Long Huagongshijichang of Chengdu section produces;

Xylene, lot number 20090911, Chongqing chemical reagent factory of Chuan Dong chemical industry Group Co.,Ltd produces;

Rat BrdU monoclonal antibody, lot number B2531, U.S. Sigma provides;

BrdU, lot number 118K4835, U.S. Sigma provides;

Goat-anti mice FITC, lot number ZF-0312, company of shirt Golden Bridge provides in the import packing China.

Experimental apparatus:

Spacious test macro (U.S. Smart company), freezing microtome (CM1900 U.S. Leica), (TCSSP2 of laser confocal scanning system; Germany Leica), wax stone microtome (SM2000R, German Leica); Desk-top refrigerated centrifuge (U.S. Sigma2-16k), YLS-17B shuttle box (Shandong benefit research and development exhibition company limited), overhead cross labyrinth (U.S. Smart company); Put under house arrest electric shock case (self-control), water maze system (Chinese medical courses in general institute institute of materia medica)

Experimental example 1:LBP causes PTSD rat general state, body weight and refuses to pay the reaction observation putting under house arrest electric shock.

72 of SD rats, male, body weight 210 ± 26g, experiment is grouped into: matched group, 18; Put under house arrest the electric shock stress group, 18; LBP low dosage prevention group, 18; LBP high-dose therapy group, 18;

Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress;

LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end;

LBP high-dose therapy group is by each 20 mg/kg every day, and 18 every group, observing time point is for stress back 1w, 2w, 4w.

Refuse prisoner's reaction: the glove so that animal is never contacted are grabbed rat, observe its reaction.The standard of keeping the score: 0: be easy to grasp animal; 1: scream or avoid; 2: scream and avoid; 3: escape; 4: escape and scream; 5: attempt to bait glove; 6: the attack of initiatively jumping up.Total points is used to estimate the change of zoophobia and the competencies that conflicts.Respectively stress before, stress back 1w, 2w, 4w weighing rat body weight calculates the body weight change rate.The body weight change percentage rate=(stress back 1w, 2w, the 4w body weight-stress before body weight)/stress before body weight * 100%.Carry out two-way analysis of variance.

Experimental result is referring to Fig. 1: overview: compare with control rats, through put under house arrest+electricity irritation after rat dystropy appears, mainly show as and be slow in action, stimulate seldom to react and be prone to external world and enrage; Easy manic type (13).To irritate stomach group rat relatively more docile and quiet and irritate stomach low dose group rat and high dose in advance, cooperates any operation of research worker.

Body weight changes: compare with control rats, the body weight of putting under house arrest the electricity irritation rat obviously reduces, along with the time reduces, stress the back before the 2w body weight reduce significantly (P < 0.01).Low, high dose is irritated the stomach group and is shown as body weight and continue to increase, and the high dose weight increase amplitude of irritating the stomach group stress 4 weeks of back than low dose group obvious (P < 0.05)

Refusing prisoner's reaction changes: compare with control rats; Put under house arrest the strong reaction of refusing to pay of electric shock group; Maintenance to strange glove do one's utmost shake off (scoring is 3 minutes) or for screaming, escape (mark 4 fens), and refuse the prisoner in the time at 2w at first and react maintenance and stablized for the 4th week; Scoring on average slightly descends between group, still keeps high response.Irritating the stomach group in advance then stress back 4 all stable reaction, is 1.5 ± 0.55, high dose irritate the stomach group then stress back 1w reaction for screaming or avoid, then value is 0.33 ± 0.52 between the reaction group for extracting is easily perhaps slightly shaken off during to 4w.

LBP can remarkable tranquil stress rats emotion, reduces losing weight of posttraumatic stress disorder/chronic stress.

Experimental example 2:LBP is to the influence of the posttraumatic stress disorder property swarmed into memory.

Adopt the stiff upright behavior of rat to detect.The SD rat, male, body weight 210 ± 26g; Experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.

Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.The property swarmed into memory mainly embodies with the upright behavior of deadlock, stiff upright behavior determination: will put into reflective box through stress rats, and survey stiff upright behavior in 3 minutes.The definition of stiff upright behavior is a kind of protective behavior that is common in rodent, shows as the crouch position of mechanical formula, and waving to a certain degree can be arranged.Observing visible rat remaining muscular movement except that respiratory movement and all disappear, is the frightened way of act of expressing of rat.Continuous detecting 3min, the percentage ratio that the stiff upright time accounts for total time be deadlock immediately between percentage ratio.Carry out two-way analysis of variance.

Experimental result is referring to Fig. 2: placing oneself in the midst of when putting under house arrest the electric shock case, matched group and PTSD group rat show notable difference in deadlock immediately.Two-way analysis of variance shows the percentage of time that the stiff upright behavior of PTSD group rat continues maximum (P < 0.01); In advance low dosage irritate stomach group and PTSD organize relatively have deadlock immediately between percentage ratio obviously descend; Stress back 1w deadlock immediately between percentage ratio irritate the stomach group greater than high dose; Stress not have difference by back 2w, stress irritate the stomach group greater than high dose by back 4w.

Experimental example 3:LBP is to the free active influence of trauma stress obstacle/chronic stress rat.

Adopt spacious behavior of rat to detect.The SD rat, male, body weight 210 ± 26g, experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.With one 100 cm * 100 cm * 48 cm wooden (-packing) cases, base plate has the grid of 25 20cm * 20 cm, and illuminance is 60 lux, and barrier is set, the operating room internal sound insulation.The observer with the behavior performance of camera system record animal 5 min in spacious wooden case, comprises horizontal anomalous movement, upright number of times, modifies number of times, dull and feces volume in the behavior laboratory.Wherein walking in a horizontal state, probe into, dull and grooming behavior obtains data through the shooting analytical system, the feces volume of every animal is to represent with the granule number that open field test finishes the back defecation.

Experimental result: compare with control rats, spacious the behavior of putting under house arrest the electric shock rat is along with the time changes obviously, laterally wears lattice, upright number of times and obviously reduces (P < 0.01), modifies number of times stress back 1w, and 2w obviously reduces, the 4w zero difference.Compare with stress group, low dosage prevention group stress back 1w, and laterally wearing lattice does not have difference, but upright behavior reduces, and the behavior of washing and dressing increases (P < 0.05).Stress back 2w transverse movement obviously increase with upright behavior (P 0 .01); Wash and dress the behavior zero difference; Stress back 4 when week each item behavior all increase obviously (P < 0.01) than stress group, spacious behavior is with stress time lengthening behavior performance descending but the reduction amplitude is lower than stress group.High dose is irritated spacious behavior of stomach group to be increased obviously, and in the 4th week with matched group still variant (P < 0.05).The behavior of high dose group each item all is higher than low dosage and irritates stomach prevention group.The preventative dose of LBP can significantly reduce stress be to the influence of spacious the behavior of rat, and it is obvious than preventive effect that therapeutic is taken effect.(seeing table 1)

Table 1. LBP is to the influence of spacious experiment of PTSD rat

Compare with matched group: * P 0.05, * * P < 0.01; Compare with stress group: #P 0.05, # # P < 0.01

Experimental example 4:LBP is to the influence of trauma stress obstacle/chronic stress rat emotion.

Adopt the labyrinth behavior of the overhead cross of rat to detect.The SD rat, male, body weight 210 ± 26g, experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.Laboratory is kept quite, light is suitable, about 25 ℃ of room temperatures, and cloth is with the dull background of the high black of 2 m on every side.Test in every day 10:00～12:00 carry out.When the test beginning, rat is put to the halfpace in labyrinth, in the face of the same open arm in labyrinth.Made animal adequacy test environment before on-test, on-test, place labyrinth central authorities head towards the closure arm district animal, through its active situation of the vertical monitor log of camera monitor, and every animal testing 5 min.Middle with the wet cloth wiping labyrinth of ethanol, removing feces carries out the next round test again after cleaning with dried cloth, to reduce the phase mutual interference between the animal.Experimental index is main with percent and the percent in the open arms time of staying that gets into open arms; Probe into number of times downwards and then be reflected in the exploratory behaviour in the non-protection area in open arms and central platform district, represents animal the curiosity of foreign environment to be probed into or seek to escape lest fearing, and gets into open arms and closure arm total degree reflection animal sports ability.The impulsion that elevated plus-maze test produces animal to probe into simultaneously causes the conflict behavior of " probing into-avoid " with frightened, can reflect the anxiety of animal preferably.

Experimental result: low dosage prevention group obviously reduces (P < 0.05) than the percentage of time that control rats gets into open arms, but relatively to get into out the arm percentage high with stress group, (P 0.01).High dose is irritated the percentage of time that stomach group rat gets into open arms and is shown as first reduction, and subsequently at 2w, 4w increases (P < 0.01) gradually.Getting into aspect the percentage of open arms, high dose is irritated the stomach group and is got into number of times apparently higher than stress group (P < 0.01), and is higher than matched group (P < 0.05).But downwards the number of times of probe obviously reduces, and upright behavior reduces (P < 0.05) (seeing table 2) than stress group.

Table 2. LBP is to the influence of PTSD rat elevated plus-maze test experiment

Compare with matched group: * P 0.05, * * P < 0.01; Compare with stress group: # P 0.05, # # P < 0.01

Experimental example 5:LBP is to the influence of trauma stress obstacle/chronic stress rat cognitive function.

Adopt the behavior of rat shuttle box to detect.The SD rat, male, body weight 210 ± 26g, experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.Shop, shuttle box bottom is with the rustless steel electricity grid of 300 * 300 * 200 mm, and buzzer is equipped with in the box top.During training, rat is placed on either side in the case, buzz occurs behind 20 s, continue 15 s, give electricity irritation (current intensity 1 mA) by the bottom electrical grid behind buzzing 5 s, rat runs away to the other end after receiving electricity irritation, and the electric shock buzzing stops automatically.The record animal is initiatively being passed through the time and the time that gets shocked and the number of times of electric shock that wicket is escaped during the buzzing separately.

Experimental result: as shown in table 3; Compare with stress group, low dosage is irritated the stomach group, from initiatively avoiding achievement from stress dropping to 6.67% ± 12.11% by back 4w by back 1w 52.83% ± 41.47%; Fall is lower than stress group (P < 0.05), but the passive avoidance response indifference.High dose perfusion group is initiatively remembered achievement and is descended slowly than stress group, stress back 1w decline achievement be lower than low dose group (P < 0.05 sees table 3).The prevention of LBP and therapeutic dose can improve in various degree stress be to the infringement of rat cognitive function.

Table 3. LBP is to the influence of PTSD rat shuttle box experiment

Compare with matched group: * P 0.05, * * P < 0.01; Compare with stress group: # P 0.05, # # P < 0.01

Experimental example 6:LBP is to the influence of trauma stress obstacle/chronic stress rat spatial memory ability.

Adopt the behavior of rat Morris water maze to detect.The SD rat, male, body weight (210 ± 26), experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.

The Morris water maze of using the institute of Materia Medica,Chinese Academy of Medical Sciences development is made up of circular cistern of the iron sheet of diameter 130 cm, high 55 cm and lucite platform, and water temperature remains on 25 ℃.The pond is equally divided into 4 quadrants, but platform is positioned at some quadrant central authorities and any direction rotates.The circle pond is positioned at the light better laboratory, comprises visual cues outside the relatively-stationary labyrinth.The video camera that has display system, synchronous recording rat motor track are settled in the top, labyrinth.

Orientation navigation test (place navigation test): be used to test the acquisition capability of rat to water fan official Learning and Memory.1 d before carrying out water maze laboratory, every rat is not having free swimming 60 s in the water maze pond of escaping platform, and getting rid of has swimming capability defect rat.Experiment lasts 5 d, trains every day 4 times, each 15 min at interval.During training the experimenter to select 1 quadrant at random be that place of entry is put into water with rat, when rat climbs on the hidden platform or arrives 60 s, stop experiment.Rat is climbed up hidden platform relief, and it stops 10 s; If rat is not found platform in 60 s, then guide it to climb on the hidden platform, and let it stop 10 s.Searching platform incubation period (Escape latency, EL) behavior, record EL through rat in the image tracking system record experimentation.

Space search experiment (spatial probe test): be used to measure rat association and seek behind the platform hold facility to the platform space position memory; Promptly in the end 1 training back was removed underwater platform on the 2nd day; Optional then 1 place of entry is put into water with rat towards pool wall; The swimming distance of measuring the inherent platform quadrant of its 120 s account for the percentage ratio of total distance (Distance Percentage, DP).

Experimental result referring to Fig. 3-1 to Fig. 3-9: with stress group relatively, low dosage concern group rat seeks the platform prolongation of latency, but is shorter than stress group; The incubation period of high dose perfusion group is than stress group obviously short (P < 0.01 table 4).Stress be longer than low dose group incubation period by back 1 all platforms, but stress back 2w, 4w platform incubation period is all than low dose group and stress group shortening.The prevention of LBP and therapeutic dose can improve in various degree stress be to the infringement of rat spatial memory and stationkeeping ability.

Table 4. LBP to wound after the influence of stress rats water maze laboratory

Compare with matched group: * P 0.05, * * P < 0.01; Compare with stress group: # P 0.05, # # P < 0.01

Experimental example 7:LBP is to the influence of trauma stress obstacle/chronic stress rat hippocampus pathomorphology.

Adopt pathological staining: HE, Nissl and Holems silver staining method.The SD rat, male, body weight (210 ± 26), experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.Referring to Fig. 4-1 to Fig. 4-6.

The dyeing of haematoxylin-Yihong (HE) and Nissl (Nissl): above-mentioned each treated animal in experiment after 1% pentobarbital sodium (40 mg/kg) intraperitoneal injection of anesthesia exposes heart, through left ventricular cannulation to ascending aorta; Pour into 0.01 M PBS, 200 ml fast, (0.1mol/L pH:7.4) carries out internal fixation to pour into 4% paraformaldehyde-PB liquid 200ml subsequently; Take out the brain BIAO and BEN then, put into 4% liquid-solid fixed 24 h of paraformaldehyde, fully spend the night after the washing; Row dehydration, transparent, FFPE, crown section; Every thick 5 um of brain sheet～10 um, row HE and Nissl dyeing, transparent, sealing.Mirror is observed the Hippocampus morphological change down.All animals are all chosen section on roughly the same hippocampal formation section, whenever get 1 at a distance from 15 sections, totally 5 successively; Adopt the eyepiece grid cell counting; Every section is got 5 visuals field in DG, CA3 and CA1 respectively continuously, and meter neurocyte number is with cell number/50 mm ²The expression cell density.The change situation of Nissl body under the sight glass.Holmes silver dyes: the same zone is fixed, embedding, section, dry, soak silver, reduction, AgCl toning, fixation, dehydration, step such as transparent, and every slice thick is 10 um.Assessment cell injury semi-quantitative analysis, standards of grading are: 0: normal, not damaged; 1: damage irreversible and be less than 10%; 2: irreversible damage is 20%～50%; 3: irreversible damage is carried out qualitative processing to nerve fiber simultaneously greater than 50%.

Nissl body is observed: compare with matched group, the stress group Hippocampus is respectively distinguished the cell marshalling, the rounded or rhombus of cell space, and cell space expands, and after birth is imperfect, and Nissl body has understain in various degree, has the small amounts of cells Nissl body thoroughly to disappear.At 2w and the visible significantly Nissl body fragment (seeing Fig. 4-3) of 4w.Low dosage is irritated stomach group group Nissl body visible light dying of CA3 district neuron when 4w.See that Nissl body changes seldom but irritate the stomach group at high dose, the cell space rule, after birth is complete.

Silver dyes the observation finding: with respect to matched group, hippocampal neurons injury is more obvious, and damaging cells accounts for 11% of general cell, and scoring is 1 minute, and there is the nerve fiber axonotmesis in the CA3 district, shortens, misaligned phenomenon.Low dosage is irritated stomach prevention group damaging cells about 15%, and scoring is 1, and high dose is irritated stomach group damaging cells and accounted for 8%, and scoring is 1, and fiber is neat, has aixs cylinder to prolong and gos deep into the CA3 district.

Experimental result: the prevention of LBP and therapeutic dose can improve the pathological change that chronic stress obtains important nuclear group-hippocampal neuron of posttraumatic stress disorder rat in various degree.

Experimental example 8: LBP is to the influence of trauma stress obstacle/chronic stress rat hippocampus dentate gyrus district's cell regeneration.

(5-bromo-2'-deoxyuridine, BrdU) labelling is observed hippocampal dentate district regenerative cell detection to adopt 5-bromodeoxyuridine nucleoside.The SD rat, male, body weight (210 ± 26), experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.

After putting under house arrest the electric shock end, intraperitoneal injection 10 mg/ml BrdU solution, dosage is 100 mg/kg body weight, 2 times/day, injects continuously 3 days.Last injection is after BrdU24 hour, and with 1% pentobarbital sodium (30 mg/kg) anesthesia, dorsal position is fixed, and cuts off skin of abdomen with shears, mentions xiphoid-process with rat, cuts off rib and exposes heart with the mosquito forceps clamp along the breastbone both sides.Cut off in apex of the heart place with eye scissors, intrusion pipe is inserted left ventricle until aorta, fixing, cut off the right auricle.In aorta, inject 0.01M PBS 200 ml～250 ml fast through charge pump; It is limpid to observe the effusive liquid in right auricle; Use 4% paraformaldehyde perfusion instead, continue perfusion 40 min, observe that the rat eyes bleach, tail hardening, liver bleach, hardening gets final product.To pour into animal after the perfusion and place 4 degree refrigerators 30 min, and get brain and repair and get Hippocampus brain section, 4 degree refrigerators place 4% paraformaldehyde solution fixing after 8 hours, move into to spend the night in 20% sucrose solution to cerebral tissue.Back 2.3-5.2 mm will comprise whole DG and carry out the continuous coronal frozen section interior Hippocampus behind bregma, and slice thick 15 μ m get one for per 6, and every animal is got 8～10 and is used for BrdU dyeing.

The BrdU immunocytochemical stain:

1) frozen section, the thick 15 μ m of sheet, 0.01MPBS rinsing 3*5min;

2) 3% hydrogen peroxide is hatched 37 degree 10min down, 0.01MPBS rinsing 3*5min;

3) among the 2 mol/L HCl 37 ℃ hatch 0. 5 h;

4) the thorough rinsing 3*10 of 0.01M PBS min;

5) 37 ℃ of 20 min of 1% trypsinization;

6) 0.01M PBS rinsing 3*5 min;

7)?3?Ml/LTriton?X－100?10?min；

8) drip normal goats serum confining liquid 37 degree and hatch 15 min, get rid of unnecessary liquid, do not clean;

9) drip in the antibody (1:500) of BrdU 37 ℃ and hatch 1h, then 4 ℃ of refrigerator 48h;

10) 0.01M PBS rinsing 3 * 5min;

11) goat-anti mice FITC (1:50) lucifuge of dropping two anti-middle shirt labellings;

12) hatch 1-2 h for 37 ℃;

13) 0.01M PBS rinsing 3 * 5min;

14) DAPI nuclear staining (DAPI is 4', 6-diamidino-2-phenylindone);

15) 0.01M PBS rinsing 3 min;

16) DPX mounting, laser confocal microscope is scanning down;

17) graphical analysis: adopt Image PLUS software; Under low power 10 * object lens, focus on section earlier; Iris out count range; Switch to high power again 200 times, the counting frame counting BrdU positive cell (green fluorescence and DAPI nuclear dye overlapping) with software generates at random calculates whole DG district BrdU positive cell sum.Other gets a cover section and does blank by last method.

Experiment conclusion referring to Fig. 5-1 to Fig. 5-6: low dosage is irritated stomach prevention group stress back 1w; The BrdU positive cell in 2w and 4wDG district obviously descends than matched group; The positive cell number difference does not have the statistics implication, shows the cell proliferation damaging fixed, does not increase with the time to change.High dose is irritated the stomach group stress back 1w, and cell proliferation is suppressed, but along with the time lengthening positive cell number begins to increase progressively, and (P < 0.05), showing that LBP can obviously improve stress reduce the cell regeneration of rat hippocampus dentate gyrus district.

Experimental example 9:LBP is to the influence of trauma stress obstacle/chronic stress rat hippocampus volume.

Use serial section and calculate the method for Hippocampus volume.The D rat, male, body weight 210 ± 26g, experiment is grouped into: matched group, put under house arrest electric shock stress, LBP low dosage prevention group, LBP high-dose therapy group.Control rats irritates stomach for rat equivalent normal saline every day, and not giving stress.LBP low dosage prevention group, in electric shock preceding 3 days, every day 1 time, the set time be the morning 8:00 irritate stomach, each every 10 mg/kg, for three days on end; LBP high-dose therapy group is by each 20 mg/kg every day.Every group 24, observing time point is for stress back 1w, 2w, 4w.

In every group, select 3 brains to do Hippocampus (behind the bregma 1.40～6.70) serial section Nissl's staining, slice thickness is 40 μ m, according to the planar solid shape of rat brain collection of illustrative plates observation analysis Hippocampus.Application of SPOT software; Delineate bilateral Hippocampus profile according to Cavalier ' s principle; Drift slide 2 is 309 pixels, adds up.Three brain serial section of every group analysis BIAO and BEN, 180 brain sheets of each BIAO and BEN average analysis obtain whole Hippocampus volume.

Experimental result: stress group is compared, and measuring the Hippocampus volume behind the low dosage filling stomach prevention group 4w is 63.64 ± 1.14 um ³, with the stress group no significant difference, high dose is irritated the stomach group then obviously greater than stress group (P<0.05), less than matched group (P<0.05).

In sum:Fructus Lycii and wolfberry fruit extract LBP thereof to the ordinary circumstance of putting under house arrest posttraumatic stress disorder rat due to the electric shock, refuse to pay that reaction, body weight change, spacious free crawler behavior, anxiety, the cognitive function in the shuttle box experiment, the spatial memory in the water maze laboratory and the stationkeeping ability in overhead cross labyrinth, HE; The change experiment of pathological change, the cell regeneration of hippocampal dentate district and the Hippocampus volume of brain neuron number, Nissl body form and nerve fiber that Nissl and Holmes dyeing is shown, draw Fructus Lycii and extract LBP thereof can be used for preventing and treat chronic stress with or the treatment of posttraumatic stress disorder.The present invention is that the application of medical material Fructus Lycii of traditional drugs, food dual-purpose provides new purposes, also for prevention and treatment stress due to the drug development of damage new, available resources widely is provided.

Claims (4)

1. the application of LBP in the medicine of preparation posttraumatic stress disorder is characterized in that: the LBP of purity >=50% that extracts is incapsulated or process tablet.

2. the application of LBP according to claim 1 in the medicine of preparation posttraumatic stress disorder is characterized in that: the LBP with extracting, incapsulate, and every contains LBP 500 ㎎, or processes tablet, and every contains LBP 500 ㎎.

3. the application of LBP in the medicine of preparation posttraumatic stress disorder is characterized in that: LBP and corn starch mix homogeneously with purity >=50% that extracts incapsulate or process tablet.

4. the application of LBP according to claim 3 in the medicine of preparation posttraumatic stress disorder, it is characterized in that: every capsules contains LBP 500 ㎎, contains corn starch 100mg; Perhaps every contains LBP 500 ㎎, corn starch 100mg.

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