CN105770384A - Medicinal composition extract and applications thereof - Google Patents
Medicinal composition extract and applications thereof Download PDFInfo
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- CN105770384A CN105770384A CN201410771264.7A CN201410771264A CN105770384A CN 105770384 A CN105770384 A CN 105770384A CN 201410771264 A CN201410771264 A CN 201410771264A CN 105770384 A CN105770384 A CN 105770384A
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Abstract
The invention discloses a medicinal composition extract for preventing and treating Alzheimer's disease (AD), and in particular relates to an ethanol extract of a medicinal composition. The medicinal composition comprises panax ginseng, radix ophiopogonis and the fruits of Chinese magnoliavine at the weight ratio of 9 to 9 to 6. Compared with the water decoction extract of the medicinal composition, the ethanol extract disclosed by the invention can obviously delay the paralysis symptoms of an AD model, and therefore, the ethanol extract has good efficacies of preventing and treating the AD, and has no toxicity.
Description
Technical field
The present invention relates to a kind of medical composition extractive, be specifically related to this pharmaceutical composition and extract
The effect of thing preventing and treating Alzheimer's disease, belongs to the field of Chinese medicines.
Technical background
Along with the prolongation of human longevity, geriatric disease has become what the interior urgent need of world wide was captured
One difficult problem, and Alzheimer's disease (AD disease) is most commonly seen geriatric disease, it
Mainly show as patient's memory disorders, cognitive disorder and phrenoblabia, to patient and patient home
Bring white elephant.Present stage does not also have any medicine can cure AD disease, mesh completely
Before drug main AD to be delayed disease morbidity on the market or alleviate the symptom of AD disease, Er Qiexian
There is stronger side effect in stage for the medicine of AD disease, and price is the highest.
Caenorhabditis elegans, as a kind of model organism, is widely used in screening treatment alzheimer '
The medicine of silent disease (AD disease), fact proved, proves to have to delay in online worm experiment in vivo
The active medicine of AD disease morbidity, also has preferable curative effect, such as Memantine pair in the mankind
The Caenorhabditis elegans of AD disease model has the effect delaying morbidity, and Memantine is finally by as spy
Effect medicine, is used for treating severe AD disease in the mankind.Caenorhabditis elegans can be used for screening the mankind
The reason of disease medicament is: the gene relevant to AD disease has conservative, at beautiful hidden bar line
Worm all has with the mankind, and drug target is similar.
The result of the present invention proves: ginseng, the tuber of dwarf lilyturf, the medicine that proportioning is 9:9:6 of the fruit of Chinese magnoliavine
Composition decocting liquid significantly delays AD model Caenorhabditis elegans to benumb, but this drug regimen
Thing decocting liquid shows the toxicity of suppression Caenorhabditis elegans growth, shows the druggability of decocting liquid
Poor.
And present invention application alcohol extract and the method for stepwise elution, obtain active height, nontoxic
The medical composition extractive of property.
Summary of the invention
It is an object of the invention to provide a kind of medical composition extractive, be specifically related to this compound and carry
Take ginseng in thing, the tuber of dwarf lilyturf, the weight ratio of the fruit of Chinese magnoliavine are 9:9:6, another object of the present invention
Being to provide the preparation technology of a kind of medical composition extractive, another object of the present invention is to carry
For a kind of medical composition extractive in preparation preventing and treating Alzheimer's disease (AD disease) medicine
Application.
The present invention is practiced by following technical proposals.
A kind of medical composition extractive of the present invention, wherein this pharmaceutical composition extracts
Preparation method is as follows: uses alcohol immersion ginseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine, is heated to reflux, and filters,
Obtaining medicinal extract after reduced pressure concentration, be completely dissolved medicinal extract with distilled water, upper macroporous resin adsorption, with not
With the ethanol solution gradient elution of concentration, eluent concentrates, after being dried and get final product.
A kind of medical composition extractive of the present invention, alcohol immersion raw material in the preparation
The every 300mL of medicinal material is 1h.
A kind of medical composition extractive of the present invention, described in the preparation is heated to reflux
Twice, reflux 3h every time.
A kind of medical composition extractive of the present invention, macroreticular resin described in the preparation
Model be D101.
A kind of medical composition extractive of the present invention, ethanol described in the preparation is molten
The product of absorption on liquid gradient elution macroreticular resin, in solution, the concentration of ethanol is followed successively by 0%,
30%, 50%, 70%, 100%.
A kind of medical composition extractive of the present invention, wash-out macropore described in the preparation
On resin during adsorbed product, the product that the present invention uses is that 100% ethanol is from macropore tree
The product obtained is eluted on fat.
The present invention is that a kind of medical composition extractive is at preparation preventing and treating Alzheimer's disease (AD
Disease) application in medicine, described AD disease is by overexpression amyloid-beta (A β)
Cause.Cascading hypothesis according to A β, the overexpression of A β and cascade reaction cause AD disease
Major reason.
Accompanying drawing explanation
In figure, C represents blank, and P is the memantine of positive control, i.e. 0.1mM,
WD represents ginseng, the tuber of dwarf lilyturf, and fruit of Chinese magnoliavine weight ratio is the pharmaceutical composition decocting liquid of 9:9:6；
EE represents ginseng, the tuber of dwarf lilyturf, and fruit of Chinese magnoliavine weight ratio is the medical composition extractive of 9:9:6,
The crude drug concentration of WD and EE is all 24g/L.
Fig. 1: pharmaceutical composition processes the paralysis curve of Caenorhabditis elegans CL4176.In figure
Abscissa is the time that temperature is increased to after 25 DEG C, and ordinate is do not benumb beautiful hidden
Rhabditida accounts for the percentage of all Caenorhabditis elegans.In same time point, the show that EE processes
The percentage not having paralysis in beautiful hidden rhabditida CL4176 is significantly higher than the beautiful hidden of WD process
Rhabditida CL4176.
Fig. 2: pharmaceutical composition processes the half paralysis time of Caenorhabditis elegans CL4176
(PT50).In figure, abscissa is the group processed, and ordinate is PT50, words different in figure
Matrix shows significant difference between any two, the Caenorhabditis elegans CL4176's that EE processes
PT50It is considerably longer than the PT of the Caenorhabditis elegans CL4176 that WD processes50, the show of drug-treated
Beautiful hidden rhabditida CL4176 half paralysis time (PT50) the longest, show that it delays paralysis
Act on the strongest.
Detailed description of the invention
Below in conjunction with specific embodiment technical scheme is described in further detail, but this
Bright protection domain is not limited to the following stated.
The preparation of embodiment one pharmaceutical composition decocting liquid
Material: ginseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine are bought in the Lanzhou chain Limited Liability of Hui Rentang medicine company
Company.
The preparation method of pharmaceutical composition decocting liquid:
A, weigh ginseng 9g, the tuber of dwarf lilyturf 9g, fruit of Chinese magnoliavine 6g；
B, measure the distilled water immersion ginseng of 100mL, the tuber of dwarf lilyturf, fruit of Chinese magnoliavine 30min；
C, the mixture of medicinal material Yu water is heated to boiling；
After d, boiling, 30min stops heating, after treating its cooling, by gauze and funnel mistake
Filter；
E, use distilled water are the volume constant volume of liquid to 50mL；
F, centrifugal liquid are to remove precipitation, and the condition being centrifuged is 12000r/min, 4, DEG C from
Heart 10min, is centrifuged and pours out supernatant afterwards, be the most again centrifuged, then incline
Pour out supernatant；
G, using the aseptic filter filtering liquid medicine of 0.22 μm, the liquid after filtration is with sterilized
Centrifuge tube preserve, sterile liquid medicine is placed in 4 DEG C preservation.
The preparation of embodiment two medical composition extractive
Material: with embodiment one.
The preparation method of medical composition extractive:
Weigh ginseng 90g, the 90g tuber of dwarf lilyturf, fruit of Chinese magnoliavine 60g, by 300mL alcohol immersion 1h,
Being heated to reflux twice, reflux 3h every time, filters, is concentrated under reduced pressure to give pharmaceutical composition medicinal extract.
Being completely dissolved pharmaceutical composition medicinal extract with distilled water, upper macroporous resin column is adsorbed, and uses second
Alcoholic solution gradient elution, in solution, the concentration of ethanol is followed successively by 0%, and 30%, 50%, 70%,
100%, eluent concentrates, and is dried.Weigh each concentration ethanol and elute the quality of product
Summation is 19.6g.
Wherein, the model of big pore resin column is D101 type.The product that the present invention uses is
Get off the product obtained with 100% ethanol elution
Embodiment three pharmaceutical composition delays the effect that Caenorhabditis elegans CL4176 benumbs
1, biomaterial
(1) Caenorhabditis elegans CL4176 is purchased from Caenorhabditis Genetics Center；
Its genotype is smg-1 (cc546) I；dvIs27[(pAF29)myo-3p::A-Beta
(1-42)::let-？3'UTR)+(pRF4) rol-6 (su1006)] X, after temperature is raised to 25 DEG C, elegant
Beautiful hidden rhabditida CL4176 expresses amyloid-beta (A β) in muscle, thus fiber crops occurs
Numbness phenotype.
(2) Escherichia coli OP50 (uracil leaky mutant), purchased from Caenorhabditis
Genetics Center, as the food of Caenorhabditis elegans.
2, reagent
(1) (1) memantine, chemical name: MEM
Amine hydrochlorate, molecular formula: C12H21N HCl, molecular weight is about 216, purchased from lark prestige science and technology,
CAS:41100-52-1, as positive drug.The preliminary experiment of early stage uses 1 μM, 10 μMs,
The memantine of 0.1mM and 1mM processes nematode respectively, and result shows: the salt of 0.1mM
Acid Memantine has the best effect delaying Caenorhabditis elegans to benumb, and does not shows and press down
The toxicity of Caenorhabditis elegans processed growth, so the end of the memantine used in the present embodiment
Concentration is 0.1mM.
(2) solid NGM (Nematode Growth Medium) medium component and making
(as a example by 1 liter):
| Composition | Content |
| NaCl | 3.00g |
| K2HPO4 | 2.34g |
| KH2PO4 | 17.23g |
| Peptone | 2.50g |
| Agar | 17.00g |
| Supplement H2O is extremely | 1000mL |
After solid NGM culture medium prepares, high-pressure constant temp sterilizing 20min at 121 DEG C,
5g/L cholesterol 1mL, 1M MgSO is added under aseptic operating platform41mL,1M CaCl2
1mL, shakes up, and pours sterilizing culture dish while hot into.Stand and wait culture medium solidifying, standby.
(3) M formula of liquid
| Na2HPO4 | 6.00g |
| KH2PO4 | 3.00g |
| NaCl | 5.00g |
| 1M MgSO4 | 1.00mL |
| Supplement H2O is extremely | 1000mL |
After preparing M liquid, high-pressure constant temp sterilizing 20min at 121 DEG C, treat that it cools down,
Put at room temperature, standby.
(4) preparation of Caenorhabditis elegans lysate
The NaClO liquid taking 0.32mL is dissolved in the M liquid of 4.68mL, adds 5mL1M
NaOH solution, be Caenorhabditis elegans lysate.
(5) the NGM culture medium of not drug containing (blank) is prepared
Draw 1mLM liquid, be dissolved in the NGM culture medium after 9mL sterilizing, then training
Foster base is poured in the culture dish of three 30mm respectively.
(6) preparation NGM culture medium containing memantine (positive control)
Weighing 0.022g memantine to be dissolved in 10mLM liquid, the concentration of memantine is i.e.
For 10mM, it is taken out the liquid of 1mL, is dissolved in 9mLM liquid, now memantine
Concentration be 1mM.After again the 1mM memantine of 1mL being dissolved in 9mL sterilizing
In NGM, the concentration of memantine, just for 0.1mM, is averagely fallen this NGM culture medium
Enter in the culture medium of 3 30mm.
(7) preparation NGM culture medium containing medical composition extractive
After pharmaceutical composition medicinal extract is added to macroporous resin adsorption, it is followed successively by with concentration of alcohol
The aqueous solution of 0%, 30%, 50%, 70% and 100% elutes absorption respectively on macroreticular resin
Product, the product that each eluant solution is got off dispenses with round-bottomed flask respectively, and weighs sky
The quality of round-bottomed flask.The product rotary evaporation that each eluant solution is got off, treat solution all by
After removing, weighing the quality sum of round-bottomed flask and product, the difference of the quality of twice is exactly every
The product quality that individual eluant solution gets off, the product quality that all eluant solutions are got off is added,
It is the quality of the medical composition extractive that 240g proportioning is 9:9:6.
Weigh the quality sum of all products is 19.6g, i.e. the crude drug amount of 1g extract is
240g/19.6=12.24g.
The crude drug concentration of extract is set to 24g/L.24g/12.24=1.96g, i.e. 1.96g/L carry
Take thing being equivalent to crude drug concentration is 24g/L.
Weigh the product of 0.147g100% ethanol elution, be dissolved in the aseptic M liquid of 7.5mL,
Fully vortex, and ultrasonic dissolution 30min, now the concentration of medical composition extractive is
19.6g/L, is designated as liquid 1
Take 1mL liquid 1 and be dissolved in the NGM culture medium after 9mL sterilizing, then drug regimen
Thing extract ultimate density in NGM is respectively 1.96g/L, is equivalent to pharmaceutical composition
The crude drug concentration of extract is respectively 24.0g/L.NGM culture medium is averagely poured into three
In the culture dish of 30mm.
(8) preparation NGM culture medium containing pharmaceutical composition decocting liquid
The compound decocting liquid of crude drug concentration 24g/50ml drawing 1mL is dissolved in the M liquid of 1mL,
Being designated as liquid 2, the crude drug concentration of liquid 2 is 240g/L.
Take 1mL liquid 2 and be dissolved in the NGM culture medium of 9mL, then decocting liquid is in NGM
Crude drug concentration be 24g/L.NGM culture medium is poured in the culture dish of three 30mm.
3, instrument
4, step is implemented
(1) cultivation of Caenorhabditis elegans:
Caenorhabditis elegans is connected on the solid NGM plate scribbling Escherichia coli OP50, so
It is placed in the insulating box of 16 DEG C cultivation, when Caenorhabditis elegans grows to adult stage, carries out
Synchronization process.
(2) Caenorhabditis elegans synchronizes:
Select containing a large amount of adults and the NGM culture medium that has part worm's ovum to hatch,
With M liquid, Caenorhabditis elegans is swept away from culture medium, transfer in centrifuge tube, stand and make show
Beautiful hidden rhabditida free settling, at the bottom of pipe, abandons supernatant.Depending on Caenorhabditis elegans amount how much to centrifugal
Adding Caenorhabditis elegans lysate in pipe, on whirlpool misfortune agitator, vibration is treated beautiful for 5-7 minute
Stop vortex during hidden rhabditida complete rupture, and be sub-packed in 1.5mL centrifuge tube, wash with M liquid
Worm's ovum three times, then the ovum of Caenorhabditis elegans be applied directly to scribble OP50 containing medicine
NGM culture medium on.
(3) pharmaceutical composition delays the effect that Caenorhabditis elegans CL4176 benumbs:
Synchronize after Caenorhabditis elegans CL4176, its ovum is coupled with not drug containing,
It is 24g/L medical composition extractive containing 0.1mM memantine, containing crude drug concentration
NGM culture medium, it is that the NGM of 24g/L pharmaceutical composition decocting liquid cultivates containing crude drug concentration
Base, is placed in 3.5d at 16 DEG C culture medium, then placing 34h at media transfer to 25 DEG C,
Then Caenorhabditis elegans muscular paralysis phenotype is started counting up, every 2h number once, until
All Caenorhabditis elegans are all benumbed.When paralysis refers to mechanical stimulus Caenorhabditis elegans health,
Caenorhabditis elegans can not move or only head movement.
The ordinate of Fig. 1 represents the percentage that the Caenorhabditis elegans do not benumbed accounts for, same
One time point, this value is the biggest, shows the Caenorhabditis elegans ratio not having paralysis in this process group
Example is the biggest, i.e. medicine delays the effect benumbed the strongest, the effect of medical treatment Alzheimer's disease
The strongest.Fig. 2 represents the half paralysis time of the Caenorhabditis elegans CL4176 of each process group
(PT50), half paralysis time (PT50) the longest, show that nematode is benumbed the slowest, i.e. medicine
The effect that thing delays nematode to benumb is the strongest, and the effect of medical treatment Alzheimer's disease is the strongest.
Pharmaceutical composition decocting liquid (WD) significantly delays Caenorhabditis elegans CL4176 to benumb
(Fig. 1: P < 0.05), and its effect delaying CL4176 to benumb is significantly stronger than prior art
Memantine (Fig. 1: C, vs.P.p > 0.05；WD.p < 0.05. Fig. 2: P, PT50=40.46
± 0.40vs.WD, PT50=41.09 ± 0.16.p > 0.05).Pharmaceutical composition decocting liquid is described
(WD) effect of preventing and treating Alzheimer's disease is significantly better than prior art memantine.
Medical composition extractive (EE) significantly delays Caenorhabditis elegans CL4176 paralysis (figure
1:P < 0.001), and its effect delaying CL4176 to benumb is significantly stronger than prior art hydrochloric acid
Memantine (Fig. 1: C, vs.P.p > 0.05；EE.p < 0.001. Fig. 2: P, PT50=40.46
± 0.40vs.EE, PT50=43.40 ± 0.05.p < 0.001).Medical composition extractive is described
(EE) effect of preventing and treating Alzheimer's disease is significantly better than prior art memantine.
Medical composition extractive (EE) significantly delays Caenorhabditis elegans CL4176 to benumb
(Fig. 1: P < 0.001), and do not show the toxicity of suppression Caenorhabditis elegans growth,
Pharmaceutical composition decocting liquid (WD) significantly delay Caenorhabditis elegans CL4176 paralysis (Fig. 1:
P < 0.05), but show the toxicity of suppression Caenorhabditis elegans growth.And drug regimen
The effect that thing extract (EE) delays CL4176 to benumb is significantly stronger than pharmaceutical composition decocting liquid
(WD) (Fig. 2: EE, PT50=43.40 ± 0.05vs.WD, PT50=41.09 ± 0.16.
p<0.001).Illustrate that the effect of medical composition extractive (EE) preventing and treating Alzheimer's disease shows
Work is better than pharmaceutical composition decocting liquid (WD).
Claims (8)
1. a medical composition extractive, it is characterised in that: ginseng in this pharmaceutical composition,
The tuber of dwarf lilyturf, the weight ratio of the fruit of Chinese magnoliavine are 9:9:6.
2. a kind of medical composition extractive as claimed in claim 1, it is characterised in that
The preparation method of described medical composition extractive is as follows: use alcohol immersion ginseng, the tuber of dwarf lilyturf,
The fruit of Chinese magnoliavine, is heated to reflux, and filters, and obtains medicinal extract, be completely dissolved leaching with distilled water after reduced pressure concentration
Cream, upper macroporous resin adsorption, with the ethanol solution gradient elution of variable concentrations, eluent concentrates,
After being dried and get final product.
3. a kind of medical composition extractive as claimed in claim 2, it is characterised in that:
The every 300mL of alcohol immersion raw medicinal material is 1h.
4. a kind of medical composition extractive as claimed in claim 2, it is characterised in that:
Being heated to reflux twice, each time is 3h.
5. a kind of medical composition extractive as claimed in claim 2, it is characterised in that:
The model of macroreticular resin is D101 type.
6. a kind of medical composition extractive as claimed in claim 2, it is characterised in that:
During the product adsorbed on ethanol solution gradient elution macroreticular resin, in solution, the concentration of ethanol depends on
Secondary is 0%, 30%, 50%, 70%, 100%.
7. a kind of medical composition extractive as claimed in claim 6, it is characterised in that:
Described eluent is 100% ethanol.
8. a kind of medical composition extractive as described in any one of claim 1-7 is in preparation
Application in preventing and treating Alzheimer disease drug.
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ID=56374597
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Application publication date: 20160720 |