CN107648246A - The application of Rhoifolin, Hyperoside composition in treatment and/or prevention of neurodegenerative diseases medicine is prepared - Google Patents
The application of Rhoifolin, Hyperoside composition in treatment and/or prevention of neurodegenerative diseases medicine is prepared Download PDFInfo
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Abstract
The present invention relates to a kind of application of Rhoifolin, Hyperoside composition in treatment and/or prevention of neurodegenerative diseases medicine is prepared.
Description
Technical field
The present invention relates to a kind of Rhoifolin, Hyperoside composition to prepare treatment and/or prevention nervus retrogression disease
Application in medicine.
Background technology
Nerve degenerative diseases are caused by the central functions caused by nerve cell progressive degeneration necrosis decline
Disease, it is in exacerbation trend along with the aging of population in the world, including Alzheimer disease (Alzheimer ' s disease,
AD), Parkinson's (Parkinson ' s disease, PD), ALS (Atrophy lateral
Sclerosis, ALS), Huntington's disease (Huntington ' s disease, HD) and headstroke (Stroke) and heart and brain blood
Pipe dementia (Vascular dementia, VD).It mainly influences the cognitive function of patient and motor function, disables, fatal rate
It is high, bring greatly psychology and financial burden to patient home and society.
Current most of nerve degenerative diseases there is no the hand of the healing disease of effectively block nerves cell degenerative processes
Section, clinically mainly it can only improve symptom based on medicine symptomatic treatment and have apparent side effect.Such as levodopa conduct
Clinical treatment PD medicine, but long-term use of levodopa can bring serious adverse reaction --- in unconscious locomitivity
Fluctuation：That is dyskinesia caused by levodopa (L-dopa-induced dyskinesia, LID) and non-athletic function
Side effect.Be embodied as irritability get excited, be addicted to gambling, obsession, dopamine modulating exception syndrome etc..Therefore, one is found
The nerve degenerative diseases such as the drug therapy PD that kind securely and reliably has no side effect have far reaching significance.
Traditional traditional Chinese medicine thinks that acute turpinia leaf has clearing heat and detoxicating, reducing pharyngeal swelling, promoting blood circulation and stopping pain.For tonsillitis and larynx numbness,
Abscess of throat, sore swollen toxin, the pain of injury caused by falling and tumbling.Rhoifolin, Hyperoside are one of main components of acute turpinia leaf, Rhoifolin
Molecular formula：C27H30O14, molecular weight：578.52 structural formula is as follows：
The molecular formula of Hyperoside：C21H20O12, molecular weight：464.38 structural formula is as follows：
Do not find that acute turpinia leaf main component has the work for the treatment of and/or prevention of neurodegenerative diseases medicine at present
With.
The content of the invention
Inventors have surprisingly discovered that a kind of Rhoifolin, Hyperoside composition are for nerve degenerative diseases
With good treatment and/or prevention effect.
The present invention provides a kind of Rhoifolin, Hyperoside composition is preparing treatment and/or prevention nervus retrogression disease
Application in medicine, described Rhoifolin, Hyperoside composition are made of following weight proportion：Rhoifolin
50%~90%, Hyperoside 10%~50%.
Preferably, wherein described Rhoifolin, Hyperoside composition are made of following weight proportion：Rhus succedanea
Glycosides 60%~80%, Hyperoside 20%~40%.
Preferably, wherein described Rhoifolin, Hyperoside composition are made of following weight proportion：Rhus succedanea
Glycosides 70%, Hyperoside 30%.
Preferably, wherein, the nerve degenerative diseases are that Parkinson's, Alzheimer disease, amyotrophic lateral are hard
It is dull-witted to change disease, headstroke or cardiac and cerebral vascular.
Preferably, wherein, the nerve degenerative diseases are alzheimer disease.
Preferably, wherein, the nerve degenerative diseases are APP/PS1 alzheimer diseases.
Preferably, wherein, the nerve degenerative diseases are Parkinson's.
Preferably, wherein, the nerve degenerative diseases are Parkinson's caused by MPTP.
Preferably, wherein, the Rhoifolin, Hyperoside composition can combine preparation prevention individually or with other drugs
And/or treatment nerve degenerative diseases medicine.More importantly a kind of Rhoifolin of present invention offer, Hyperoside composition can
To overcome the side reaction drawback of existing medicine.
Brief description of the drawings
Fig. 1：Rhoifolin, the water maze laboratory datagram of Hyperoside composition for improved AD mouse.
Fig. 2, Rhoifolin, Hyperoside composition alleviate the Y maze experiment datagrams of AD mouse.
Fig. 3：Rhoifolin, the experimental data figure of Hyperoside composition for improved PD mouse suspension experiment.
Fig. 4：The experimental data figure of Rhoifolin, Hyperoside composition for improved PD mouse pole-climbing abilities.
Fig. 5：Rhoifolin, Hyperoside composition improve the experimental data figure of PD mouse swimming times.
Embodiment
Embodiment 1：The preparation of Rhoifolin, Hyperoside composition
Bee saage garden leaf 1000g is taken, adds 12 times of 70% alcohol refluxs of amount to extract 1.5 hours, filtration, filter residue adds 10 times of amounts again
70% alcohol reflux extracts 1.5 hours, filtration, merges extract solution twice, reclaims ethanol, and the aqueous solution passes through processed good D101
Macroporous resin column, eluted respectively with water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution, collect 45% ethanol elution portion
Point, be concentrated under reduced pressure, dry, obtain more than 30% Ligustrum lucidum Ait, Rhoifolin, the total glycosides of the mixing of Hyperoside, through silica gel column chromatography,
Sephadex LH-20 column chromatography for separation, merge Rhoifolin, Hyperoside fraction respectively, crystallization, obtain Rhoifolin
(1.5g, purity：98.7%), Hyperoside (1g, purity：98.2%) sterling.
Embodiment 2：
Rhoifolin (7g) is mixed with Hyperoside (3g), stirring, grinding, obtains Rhoifolin, spun gold after being well mixed
Peach glycoside composition (10g).
Embodiment 3：
Rhoifolin (9g) is mixed with Hyperoside (1g), stirring, grinding, obtains Rhoifolin, spun gold after being well mixed
Peach glycoside composition (10g).
Embodiment 4：
Rhoifolin (8g) is mixed with Hyperoside (2g), stirring, grinding, obtains Rhoifolin, spun gold after being well mixed
Peach glycoside composition (10g).
Embodiment 5：
Rhoifolin (5g) is mixed with Hyperoside (5g), stirring, grinding, obtains Rhoifolin, spun gold after being well mixed
Peach glycoside composition (10g).
Embodiment 6：The therapeutic action of Rhoifolin, Hyperoside composition to APP/PS1 alzheimer's disease mouse
1. materials and methods
1.1 experimental animals and medicine
APP/PS1 male mices and brood wild-type mice are selected in this experiment, in room temperature (22 DEG C -24 DEG C), humidity
(50%-60%), daily illumination and with raised under interlunation each 12h quiet environment, free feeding drinking-water., will before experiment
Animal adapts to 3 days as experimental situation.It is prepared by Rhoifolin, Hyperoside composition such as embodiment 2.
1.2 animal packets and administration
Mouse is randomly divided into wild type after weighing, wild type administration group, APP/PS1 model groups, APP/PS1 Rhoifolins,
Hyperoside composition 0.5g/L and 1.0g/L treatment groups, gavage capacity are 0.4ml/, every group 5.The monthly age of mouse 3 starts
Gastric infusion, it is administered continuously 6 months.
1.3 behaviouristics detect
1.3.1 water maze laboratory
Circular constant temperature pool diameter 120cm, high 50cm；Circular platform diameter 9cm, height adjustable, range 20-
35cm, this experiment setting height are 20cm, and platform can be placed in the center of any one quadrant.By all directions four direction by water
Pond is averagely divided into four quadrants (NE, SE, SW, NW), in the coloured silk of different shapes that the pool wall midpoint of all quadrants will shear
Paper is affixed on the inside of the pool wall higher than the water surface, and the label of platform is found as mouse.Pool inner wall is black, pool wall label
Position kept in whole experiment process fix.Water maze video acquisition system requires that the color of pool wall and water will be with experiment
Animal hair color forms colour contrast, and mouse movement track and every experiment parameter are recorded so as to video tracking.Depth of water 21cm,
Platform is located at underwater 1cm, and position of platform can not be seen by visually observing the water surface.And dissolve in water with white dye titanium dioxide 200g
In stir evenly, the color of water is become milky.In order to adapt to the skototaxis of rodent, laboratory hang the tight lucifuge of curtain and
Whole peripheral pool hangs the shading door curtain made of cloth, ensures inside it without light direct projection.There is temperature controller in pond, water temperature is maintained at 21-22
℃.The incandescent lamp outside pond is opened during experiment, power≤13 watt, light direct projection metope, makes metope is reflective scattered to be distributed in
In pond.
6d is lasted, 1 day not placement platform before experiment allows mouse free swimming 1min.Platform is fixed on during formal experiment
NW quadrants, positioned at underwater 1cm.The position of article and personnel in laboratory immobilizes during experiment, as animal
Space object of reference.Daily every mouse is tested 4 times, enters water from the quadrant (SE) of target quadrant offside with the 4th time for the first time, the
Secondary and third time is determined into water quadrant using randomized (not including SE and NW quadrants).The monitoring time is 60s, and mouse is in platform
Upper stop is judged to seek platform success more than 2s, and mouse enters water and is denoted as escape latency to the successful time-histories of platform is sought；If in mouse 60s not
Finding platform, then incubation period is designated as 60s.Mouse climbs up platform or experimental period and termination is then tested more than 60s；Mouse is allowed to rest
After 1min, start to test next time.Before the first time test of experiment first day, first mouse is placed on platform and stops 15s (referred to as
Adapt to), no longer give and adapt to before each test afterwards.Seek whether platform succeeds no matter being completed, mouse is placed in platform
Adapt to 10s.After having trained every time, mouse is dried with dry towel, stress caused by prevent hypothermia.
1.3.2Y maze experiment
Y maze devices：Y labyrinths make of PVC board, inside and outside wall patch black gummed paper.Totally 3 arms, each 120 degree of arm angle,
Each arm size 30cm × 8cm × 15cm (length × width × height), respectively there is a moveable dividing plate in centre, it is each in labyrinth
Different geometric figures are sticked in arm, as visual indicia.3 arms in each Y labyrinths are set at random：Strange arm (novel
Arm arm (start arm) and other arms (other arm)), are originated.Strange arm：When the 1st stage of experiment is training period
Blocked with dividing plate, opened when the 2nd stage is the test phase；Originate arm：Arm where when mouse enters labyrinth.Entirely tested
It is all always on that arm and other arms are originated in journey, and animal can freely come in and go out.Place mat wood chip in labyrinth, every time training or test
After end, the sawdust in each arm is mixed, to prevent animal residual smell interference.Pick-up lens is disposed above labyrinth at 1.5m, entirely
Process is recorded a video.
1.3.3ThT the beta-amyloyd patch of mouse brain deposition is detected
After section dewaxing aquation, 1% thioflavin (ThT) rinsing 15min is added, distilled water rinsing 5min, adds 1%
Acetate buffer solution 5min removes unnecessary color, 50% glycerine mounting, fluorescence microscopy Microscopic observation.
1.4 statistical method
Experimental data is represented with (Mean ± SEM), and statistical analysis is carried out using the softwares of Graphpad Prism 5.0.Metering
Data uses one-way analysis of variance, and P ＜ 0.05 are that difference is statistically significant.
2. experimental result
2.1 Rhoifolins, Hyperoside composition for improved AD mouse Spatial memory abilities
Mice water maze test result indicates that, the escape latency of mouse and swim distance with the training time before reaching platform
Increase significantly reduce, Rhoifolin, Hyperoside composition administration group show the preferable trend of learning ability, have statistics
Learn meaning.As shown in figure 1, Rhoifolin, Hyperoside composition group can increase mouse after platform is removed in the mesh of platform
Mark the activity time (P ＜ 0.05) of quadrant.Fig. 2 mouse Y maze experiment results illustrate that Rhoifolin, Hyperoside composition can be with
Improve residence time (P ＜ 0.05) of the mouse in novel arm.To sum up illustrate that Rhoifolin, Hyperoside composition can improve
The Spatial memory ability of AD mouse.
2.2 Rhoifolins, Hyperoside composition are deposited without influenceing on AD mouse A β's
The improvement result of ability of learning and memory is unrelated with AD amyloid plaque deposits.Experiment in vitro by Rhoifolin,
Hyperoside composition is incubated 72h with Amyloid beta1-42 albumen, is detected, found by fluorescence microplate reader after ThT dyeing
Rhoifolin, Hyperoside composition are unrelated with A β aggregation.
3. experimental data conclusion
AD clinical symptoms are that the memory disorders that gradually occur or forgetting are developed into linguistic function obstacle, visual space function
Cognitive disorder and phrenoblabia based on impaired, agnosia and appraxia and computing power obstacle.APP/PS1 alzheimer's disease mouse exist
There are within 3 months after birth AD pathological changes, A β start to deposit within 6 months.Therefore, start gavage at the monthly age of mouse 3 and give Rhus succedanea
Glycosides, Hyperoside composition, it is administered continuously 6 months.It is administered after terminating by water maze, Y Y-maze test learning and memory of little mouse energy
Power, ThT dyeing detection mouse A beta-amyloyd plaque depositions.
It is basic to determine that Rhoifolin, Hyperoside composition have the function that to improve to mouse alzheimer's disease, and this
Kind improvement result is unrelated with A β generation.
Embodiment 7：Rhoifolin, Hyperoside composition cause the therapeutic action of mouse Parkinson's to MPTP
1. materials and methods
1.1 experimental animals and medicine
From the male C57Bl/6 mouse of 10-12 week old 25-28g health, mouse is wet at room temperature (22 DEG C -24 DEG C) for this experiment
Spend (50%-60%), daily illumination and with raised under interlunation each 12h quiet environment, free feeding drinking-water.Before experiment,
Animal is adapted to 3 days as experimental situation.
MPTP is purchased from Sigma, and levodopa is purchased from Roche Group, and Rhoifolin, Hyperoside composition such as embodiment 2 are made
It is standby.MPTP is preserved on ice before administration in 30min with normal saline.
1.2 animal packets and administration
Mouse is randomly divided into normal group, MPTP (25mg/kg) model group, Rhoifolin, Hyperoside composition after weighing
5mg/kg and 10mg/kg treatment groups, levodopa treatment group, every group of 8-10 is only.MPTP (25mg/kg) presses mouse weight 0.1ml/
20g is injected intraperitoneally 5 days, and Rhoifolin, Hyperoside composition are after 1h is administered in MPTP by mouse weight 0.1ml/20g another
Ventrolateral compartment drug administration by injection 11 days, administration carry out Behaviors survey detection after terminating.Madopar (75mg/kg) presses mouse weight
0.1ml/20g gavages.
1.3 behaviouristics detect
1.3.1 suspension experiment (raction test)
Normal mouse has climbing habit, and this needs appropriate grip and limb motion harmony, therefore can use suspension
Test to test the dyskinesia of mouse after MPTP is administered.Mouse is positioned over away from ground 30cm height, Cheng Shui in experiment
The fineness degree of level state is on 0.5cm small wooden pole, records the time before landing, 3min is recorded as if more than 3min, so
After take statistics credit analysis.
1.3.2 Grasping clubglass test (Pole test)
Grasping clubglass test is to detect and understand mouse limb motor coordination situation.In a root long degree be 60cm and thickness is
One piece of sponge plate is placed on 1cm rod top, gauze is wrapped with outside rod and avoids mouse from being skidded during climbing
Problem.Mouse head is put in bar top upwards, record animal is from setting in motion to switching to the downward time (T- of head completely
Turn), and the credit that takes statistics is analysed.
1.3.3 forced swimming (Swim test)
Purpose is test mouse limb motor coordination situation.Test mice is put into a diameter of 35cm bucket,
Water temperature is 22 DEG C -25 DEG C, depth of water 20cm, record mouse mouse motionless time and time of swimming in 5 minutes swimming times,
And the credit analysis that takes statistics.
Experimental data is represented with (Mean ± SEM), and statistical analysis is carried out using the softwares of Graphpad Prism 5.0.Metering
Data uses one-way analysis of variance, and P ＜ 0.05 are that difference is statistically significant.
2. experimental result
2.1 Rhoifolins, Hyperoside composition for improved PD mouse hanging capacities
Detection mouse grippage is tested with suspension.As shown in figure 3, MPTP-PD mouse grippage weakens, on rod
Hang that the time is short, and compared with normal mouse, short there is significant difference (P ＜ 0.01) the suspension time.Giving Rhoifolin, spun gold
After the treatment of peach glycoside composition, 10mg/kg treatment groups can significantly improve the hanging capacity (P ＜ 0.01) of PD mouse, give left-handed
The suspension time (P ＜ 0.01) of PD mouse can be increased after DOPA.This shows that 10mg/kg Rhoifolin, Hyperoside combine
Thing treatment group can significantly improve the muscular tremor of PD mouse, improve mouse grippage.
In Fig. 1, (Mean ± SEM, n=8, * * P ＜ 0.01vs.control, ##P ＜ 0.01vs.MPTP-treated
group)。
2.2 Rhoifolins, Hyperoside composition for improved PD mouse pole-climbing abilities
Mouse pole-climbing ability can detect the sports coordination ability of mouse.The MPTP-PD mouse movement coordination abilities are poor, in bar
The time length to be turned around on son, there is significant difference compared with normal mouse.10mg/kg treatment groups can significantly improve PD mouse and fall
The head time, the mouse limb coordination ability (P ＜ 0.01) is improved, levodopa is also capable of the coordination that the raising mouse of conspicuousness turns around
Ability (P ＜ 0.01).
In Fig. 2, each group mouse is turned round time (Mean ± SEM, n=8, * the P ＜ of head in Grasping clubglass test
0.01vs.control, ##P ＜ 0.01vs.MPTP-treated group).
2.3 Rhoifolins, Hyperoside composition for improved PD mouse forced swimming test abilities
Mouse forced swimming test can detect the sports coordination ability and depressive symptom of mouse.As shown in Figure 3：MPTP-PD is small
Mouse shows that swimming time is short (P ＜ 0.05), and most time is in floating state, is giving Rhoifolin, Hyperoside composition
Afterwards, the Burden-Swimming Ability of KM and depressive state (P ＜ 0.05) of PD mouse can be significantly improved, levodopa treatment group can significantly improve
The sports coordination ability of PD mouse and the state (P ＜ 0.05) that is in a very depressed state.
In Fig. 3, (Mean ± SEM, n=8, * P ＜ 0.05vs.control, #P ＜ 0.05vs.MPTP-treated
group)。
4. experiment conclusion
PD clinical symptoms are dyskinesias, thus we by a series of Behaviors surveys study Rhoifolin,
Therapeutic action of the Hyperoside composition to PD.Administration is detected after terminating by suspension experiment, Grasping clubglass test, forced swim test
Mice behavior, improvement of the observation medicine to mouse Parkinson's sample behavior.
In suspension experiment, normal mouse has the habit and Climbing activities of good pawl grabbing object, and this needs appropriate
Grip controls and sports coordination, therefore can coordinate situation by hanging experiment to test the limb motion of mouse, by small
Mouse hang time and suspension active process can reflect very well mouse tremble, the feelings such as muscular rigidity, so as to for assessing mouse
Muscle strength and limbs coordination function, be a kind of good Parkinson disease mice motor function detection method.Rhus succedanea
Glycosides, Hyperoside composition and levodopa can improve the sports coordination ability of PD mouse.
In Grasping clubglass test, normal mouse can promptly invert and climb to bar bottom rapidly, and limb action is coordinated,
And there are different degrees of extension the mouse of MPTP administrations, turn-round time, pole-climbing time compared with Normal group.Rhus succedanea
Glycosides, Hyperoside composition high dose group and levodopa group can improve the reduction time of PD mouse.
Forced swim test is that one kind is used for assessing potential antidepression Curative effect.In MPTP-PD mouse, due to brain
Substantia nigra dopaminergic neuron is damaged, and shows depressive symptom in emotion, therefore by calculating mouse within a certain period of time flat
Equal swimming time assesses the depressive state of mouse, observes its limb motion for swimming process and judges mouse movement coordination energy
Power.Rhoifolin, Hyperoside composition and levodopa can improve the depressed sample symptom of PD mouse.
It is observed that although levodopa can improve MPTP-PD mouse movement abilities in Behaviors survey, increase
Mouse Muscle tensility, but restless state is presented in mouse, and Rhoifolin, Hyperoside composition are improving mouse movement function barrier
Mouse restless state will not be caused while hindering, be free from side effects compared with levodopa.In summary, Rhus succedanea is determined substantially
Glycosides, Hyperoside composition have the function that preferably to improve to mouse Parkinson's.
Applicant is experimentally confirmed, and Rhoifolin, Hyperoside composition are also in amyotrophic lateral sclerosis, brain
The nerve degenerative diseases such as wind or cardiac and cerebral vascular dementia have good treatment or prevention effect.
Claims (9)
1. a kind of Rhoifolin, Hyperoside composition answering in treatment and/or prevention of neurodegenerative diseases medicine is prepared
With described Rhoifolin, Hyperoside composition are made of following weight proportion：Rhoifolin 50%~90%, gold
Silk peach glycosides 10%~50%.
2. application according to claim 1, wherein described Rhoifolin, Hyperoside composition are matched somebody with somebody by following weight
Than manufactured：Rhoifolin 60%~80%, Hyperoside 20%~40%.
3. application according to claim 1, wherein described Rhoifolin, Hyperoside composition are matched somebody with somebody by following weight
Than manufactured：Rhoifolin 70%, Hyperoside 30%.
4. application according to claim 1, wherein, the nerve degenerative diseases are selected from Alzheimer disease, Parkinson
Disease, ALS, Huntington's disease, headstroke and cardiac and cerebral vascular are dull-witted.
5. application according to claim 1, wherein, the nerve degenerative diseases are alzheimer disease.
6. application according to claim 5, wherein, the nerve degenerative diseases are APP/PS1 alzheimer diseases.
7. application according to claim 1, wherein, the nerve degenerative diseases are Parkinson's.
8. application according to claim 7, wherein, the nerve degenerative diseases are Parkinson's caused by MPTP.
9. application according to claim 1, wherein, the Rhoifolin, Hyperoside composition can individually or and other drugs
Combination prepares prevention and/or treatment nerve degenerative diseases medicine.
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