CN105963245A - Preparation method and application of jasminin solution - Google Patents
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
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Abstract
The invention discloses a preparation method and an application of a jasminin solution, wherein as for the application, the jasminin is used for preparing medicines or health care products for preventing or treating Alzheimer's disease (AD). Firstly, it determines that the jasminin has an inhibitory effect on A[beta] in-vitro aggregation, and then it further detects that the jasminin, at a cellular level, has influence on the expression level of AD classical pathway-related protein; and meanwhile, the jasminin also has influence on the distribution and expression change of newborn rat primary neurons TAU-ps396; therefore, it can determine that the jasminin has an effect on preventing or treating the Alzheimer's disease; and the jasminin can be developed as the medicines or health care products for preventing and treating the AD.
Description
Technical field
The present invention relates to field of medicaments, particularly relate to the preparation method and application of a kind of jasminin solution.
Background technology
Alzheimer's disease (Alzheimer ' s disease, it is called for short AD) it is the common nervus retrogression disease of old people
Disease, with Memory and Cognition dysfunction as cardinal symptom, its typical pathological characters is formation senile plaque (senile in brain
Plaques, SP) and neurofibrillary tangles (neurofibrillary tangles, NFT).
The pathogenesis of AD is the mutual participation of multiple pathogenic factors, multiple path and molecular mechanism, in AD pathogenesis
Numerous hypothesis in, A β cascade hypothesis extensively admitted.This hypothesis thinks the poison being formed senile plaque by A β abnormal aggregation and produce
Property is probably the main cause [5,6] causing AD to occur.A β is through beta-secretase in vivo by amyloid precursor protein (APP)
Shearing with gamma-secretase and form, under normal conditions, A β metabolism keeps balance, will not produce cytotoxicity, and pathologic condition
Lower A β Developmental and Metabolic Disorder forms poisonous aggregation thus causes neurotoxicity, causes AD Pathological.
Another pathology hypothesis is in patients with Alzheimer disease body, and the Tau albumen of abnormal hyperphosphorylation is with pairing
Taenidium structure forms neurofibrillary tangles and also builds up in neuron, Tau abnormal protein in AD patients' neural's degeneration and
Learning memory disorder plays an important role in developing, and intracellular Protein tau Hyperphosphorylationof is the pathology that AD occurs the earliest
One of change.Recently some anti-AD Clinical Trials failures of external report, its reason is probably treatment time too late and medicine
Thing target spot is single.There is no the medicine that can at all treat AD both at home and abroad, the most commonly used cholinesterase inhibitor is only capable of alleviating disease
Shape, and the process of disease can not be changed, and western medicine result is not good enough, and it is easily formed drug resistance, and untoward reaction is obvious.
Jasminin has been studied as a kind of bacteriostatic activity thing, but the most not yet have any about jasminin for
The report for the treatment of AD.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide the preparation method of a kind of jasminin solution
And application, mainly it is used for jasminin preparing prevention or the medicine for the treatment of Alzheimer or health product, it is desirable to provide
A kind of new opplication of jasminin.
Technical scheme is as follows:
A kind of compound method of jasminin solution, wherein, including jasminin being dissolved in DMSO or ethanol solution, vibration
Mixing, is prepared as jasminin mother solution, and described jasminin mother solution is diluted to the element of variable concentrations by adding PBS
Fragrant bitter glycosides solution.
The compound method of described jasminin solution, wherein, the concentration of described jasminin mother solution is 5mmol/L.
The application of a kind of jasminin, wherein, is used for described jasminin preparing prevention or treatment Alzheimer
Sick medicine or health product.
Beneficial effect: first the present invention determines that jasminin is inhibited to A β aggregation in vitro, examines the most further
Measure jasminin and can affect the expression to AD classics pathway associated protein, and jasminin pair on a cellular level
Distribution and the expression change of newborn rat primary neuron TAU-ps396 also have an impact, by present invention may determine that jasminin
Having prevention or the effect for the treatment of Alzheimer, jasminin can develop into prevention and the medicine for the treatment of AD or health product.
Accompanying drawing explanation
Fig. 1 be in the present invention ThT Fluorometric assay different time points jasminin to A β aggregation in vitro fluorescence intensity change
Figure.
Fig. 2 is that in the present invention, circular dichroism detector detection jasminin becomes fibre structure variation diagram to A β.
Fig. 3 be jasminin of the present invention hatch 0h and 12h altogether with A β become fiber variation diagram.
Fig. 4 a to 4f is the gel development figure of the AD related pathways albumen that the present invention detects.
Fig. 5 a to 5i is the table of AD related pathways albumen after western blotting of the present invention detection jasminin effect
Reach variation diagram.
Fig. 6 is AD newborn mice hippocampus primary neuron figure of the present invention.
Detailed description of the invention
The present invention provides a kind of method determining that active ingredient of Chinese herbs jasminin has treatment Alzheimer, for making
The purpose of the present invention, technical scheme and effect are clearer, clear and definite, and the present invention is described in more detail below.Should be appreciated that
Specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
The present invention provides the compound method of a kind of jasminin solution, wherein, including jasminin is dissolved in DMSO or
In ethanol solution, vibration mixing, it is prepared as jasminin mother solution, by adding PBS by dilute for described jasminin mother solution
It is interpreted into the jasminin solution of variable concentrations.
In the present invention active ingredient of Chinese herbs for treating Alzheimer be Jasminin (Jasminin), Flos Jasmini Nudiflori
Element is called again jasminin, is the secoiridoid grape glycoside extracting from Oleaceae Flos Jasmini Sambac platymiscium Flos Jasmini Nudiflori
(secoiridoidglucosides), its chemical structural formula is, this monomer has certain bacteriostatic activity,
Minimal inhibitory concentration (MIC) to staphylococcus aureus is 1mg/ml, to dysentery bacterium, colibacillary minimal inhibitory concentration
For 0.5mg/ml, and there are some researches show, jasminin can induce RAW264.7 cell notable TNF secretion-α, the most also can swash
Mitogen activated protein kinase (MAPK) signal path alive.
Further, the concentration of described jasminin mother solution prepared by the present invention is usually 5mmol/L, needs logical according to experiment
Usually through adding PBS, mother solution is diluted, generally final concentration of 5 μm ol/L of jasminin of preparation, 10 μm ol/L,
20 μm ol/L, 50 μm ol/L and 100 μm ol/L and 2 mmol/L etc..
Further, the present invention also provides for the application of a kind of jasminin, wherein, is used for preparing prevention by described jasminin
Or the medicine for the treatment of Alzheimer or health product.Described jasminin can be liquid or powder.Due to tradition
Chinese medicine has Mutiple Targets, too many levels, multimode mechanism of action, and nowadays Chinese medicine has played more and more important in terms for the treatment of AD
Effect.But nowadays the research by using jasminin to treat in terms of AD not yet has been reported that, and jasminin due to
The property of medicine of its uniqueness can be as active drug or the health product preventing or treating AD..
Combine accompanying drawing below by embodiment the present invention is described in further detail.But those skilled in the art will manage
Solving, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
The present invention goes to observe element by first using sulphur production method, circular dichroism detector and atomic force microscope observation method
The action effect of fragrant bitter glycosides interaction in vitro A beta peptide aggregation, primarily determines that jasminin is inhibited, then to A β aggregation in vitro
The jasminin impact on AD classics pathway associated protein expression on detection cellular level, finally uses immunofluorescence further
The distribution of method detection newborn rat primary neuron TAU-ps396 and expression change.The present invention is by applying multiple ways and means
From molecular level to cellular level, detecting the jasminin therapeutic effect to AD, result shows active ingredient of Chinese herbs jasminin
Having the effect for the treatment of Alzheimer, jasminin can become prevention or the medicine for the treatment of AD or health product.
Embodiment 1
The ThT Fluorometric assay jasminin inhibition to A β aggregation in vitro
The present invention uses thioflavin T method to detect the jasminin inhibition to A β aggregation in vitro, thioflavin T (Thioflavin
T, ThT) it is a kind of cationic fluorescent reagent, research in 1993 finds that ThT molecule as identifying A beta structure and can be tied therewith
The fluorescent probe closed, thus ThT method becomes the Perfected process of monitoring and detection A beta peptide aggregation.Specifically, first A β sample is entered
Row pretreatment, takes A β sample 1mg, adds the hexafluoroisopropanol (HFIP) of 2ml, and vibration is dissolved overnight, takes out sample water bath sonicator
15-20 minute, nitrogen dried up, and then added appropriate DMSO(Sigma) abundant sample dissolution, water bath sonicator 15-20 minute,
-80 DEG C of sample is saved backup；Preparing various mother solution afterwards, ThT is dissolved in Tris buffer (50 mmol/L Tris-
Base, 100 mmol/L NaCl, pH 7.4) in, concentration is 1 mmol/L；The A β 1-42 processed is dissolved in 110 μ L's
In DMSO, concentration is 2 mmol/L；Jasminin is dissolved in DMSO, and concentration is 5m μm ol/L；EGCG is dissolved in ddH2O,
Concentration is 5 mmol/L；Finally using EGCG as positive controls, system cumulative volume is 100 μ l, arranges group such as table 1.
Final concentration of 50 μm ol/L of ThT, final concentration of 50 μm ol/L of A β, Jasminin final concentration sets 4 gradients and is respectively
10 μm ol/L, 20 μm ol/L, 50 μm ol/L and 100 μm ol/L.Final concentration of 50 μm ol/L of EGCG.Each for system composition is added
In 96 hole black ELISA Plate, hatching for 37 DEG C in microplate reader, detection fluorescence times point is respectively 0, and 5,15,25,45,65,85,
Fluorescent value when 115,145,205,265,325, maps, such as Fig. 1 institute according to after the fluorescent value of each group different time points obtained
Showing, Fig. 1 is the ThT Fluorometric assay different time points Jasminin variation diagram to A β aggregation in vitro fluorescence intensity, can from figure
To find out that Jasminin has obvious inhibitory action to A β aggregation in vitro, and the Jasminin of variable concentrations is to A β aggregation in vitro
Inhibition the most different, wherein the inhibition of the Jasminin of 20 μm ol/l is optimal, and 50 μm ol/L are taken second place, 100 μm ol/L
Third, the Jasminin of 10 μm ol/L poor effect after 180 minutes.
Embodiment 2
Circular dichroism spectra detection jasminin becomes the impact of fibre structure to A β
The present invention uses the impact that circular dichroism spectra detection jasminin becomes fibre structure to A β, specifically, passes through circular dichroism spectra
(circular dichroism spectra, CD) analyzes the Jasminin impact on A β secondary structure, by protein dissolution to 5
In the buffer of mmol/L PBS, pH10.0, carrying out protein quantification with BCA method, concentration is 400 μm ol/L, and Jasminin is molten
Solution is in ethanol, and concentration is 4mmol/L；EGCG is dissolved in ddH2O, and concentration is 5mmol/L.Prepare three groups of samples: (1) A β,
Final concentration of 2 mmol/L；(2) Jasminin and A β final concentration is 2 mmol/L；(3) EGCG and A β final concentration is 2
mmol/L。
Each group sample mix the most also incubated at room 12 h, uses J-815 circular dichroism spectrometer (JASCO, JAPAN) in 25 DEG C
Measuring the CD spectrum of A β, parameter arranges as follows, wave-length coverage: 255～190 nm；Scanning speed: 50 nm/min；Sensitivity: 50
mdeg；Response time: 0.25 s；Bandwidth: 1 nm；Scanning times: 3, acquired results Origin8.0 software is mapped.Such as Fig. 2
Shown in, Fig. 2 is that circular dichroism spectra detection Jasminin becomes fibre structure variation diagram to A β, from figure 2 it can be seen that β-pleated sheet exists
A characteristic peak is had at 218mm, but inconspicuous, and reason is that A beta monomers forms precipitation after hatching 12h in the solution, adds
After Jasminin or EGCG is hatched, β-pleated sheet characteristic peak disappears, and occurs a big and wide negative peak at 230mm, thus may be used
Know that Jasminin and EGCG changes A beta structure really, change the formation of β-pleated sheet.
Embodiment 3
Atomic force microscope observation jasminin becomes fibre morphology and the action effect of number change to A β
Further, the present invention also uses atomic force microscope, to observe jasminin, A β is become fibre morphology and the work of number change
By effect, specifically, A β Yu Jasminin is hatched under the conditions of 37 DEG C altogether 12h, makes final concentration of 5 μm ol/L of A β,
Final concentration of 20 μm ol/L of Jasminin, carry out AFM sample preparation in 0h, 12h sampling respectively, carry out AFM the most after drying
Scanning.Result as it is shown on figure 3, Fig. 3 is Jasminin with A β hatches 0h with 12h altogether and become the change of fibre structure and quantity,
Substantially shorten from figure 3, it can be seen that add A β formation fiber after Jasminin hatches 12h, negligible amounts, Jasminin is described
There is the effect of suppression A beta peptide aggregation.
Embodiment 4
On a cellular level with AD related pathways protein expression level after the detection jasminin effect of western blotting method
Change
The present invention is the most on a cellular level by AD related pathways after the detection Jasminin effect of western blotting method
The change of protein expression level, it includes step:
1), configuration SDS-polyacrylamide gel protein sample is carried out electrophoresis, first configure separation gel, need according to detecting
Destination protein molecular size range, calculate plastic emitting concentration；After gelling collection to be separated, sucking ethanol, configuration concentrates glue, inserts pre-
First ready comb；After gelling collection is good, upper protein sample, every porin applied sample amount 10-15 μ about g, albumen pre-dyed
Marker loading 3 μ l, loading to avoid produce bubble, during electrophoresis upper strata concentrate glue 80V voltage, when sample is to lower floor's separation gel
Time, use 120V voltage, general electrophoresis time is at about 2h, and during electrophoresis, inside groove uses newly configured 1 × SDS electrophoresis liquid, and water jacket can
By the electrophoresis liquid reclaimed.
2), using after electrophoresis pvdf membrane to carry out transferring film, specifically, gel slab is placed in electrophoretic buffer after completing by electrophoresis
Soak a few minutes.Opening offset plate, be positioned on flat board by glue, excision concentrates glue, goes out desired point according to Marker size separation
From glue and steep in electricity turns liquid.Size clip filter paper (three layers) according to taken separation gel, and according to filter paper size clip PVDF
Film, first soaks methanol by film before transferring film, avoid polluting filter paper and film as far as possible, will cut out filter paper and film is completely soaked in electricity
Swimming transfering buffering liquid stays the bubble in filter paper to drive away.Opening transfer box, the foam-rubber cushion after being impregnated with is put in transfer box wall,
Put filter paper that an electrophoretic transfer buffer soaks again on sponge.By " black offset plate sponge 3 metafiltration paper (greatly) coagulates
Glued membrane 3 metafiltration paper (little) sponge " sequence unit good (note often put a layer be required for driving bubble away), then fall some electricity
Turn liquid to film, keep sponge, filter paper, film moistening.Buffering liquid groove is loaded ice chest, the electrophoretic transfer of 4 DEG C of pre-coolings is buffered
Liquid fills electrophoresis tank.Connect transfer electrode constant current 100 mA and shift 1.5 h.Gel Coomassie brilliant blue after transferring film is dyeed
30min-1h, after destaining solution decolouring is extremely without background colour, observes whether also have remaining protein band on glue, is used for detecting transferring film
Effect, or with Ponceaux, pvdf membrane is dyeed, observes transferring film band and also before closing, wash Ponceaux off.
3) after, transferring film terminates, film is put into and 5% skim milk is closed on 37 DEG C of horizontal shaker 2h.
4), one anti-hatch, required detection antibody be diluted to suitable multiple and add in confining liquid and reach working concentration, it is ensured that
All parts of film contact with solution, general use 37 DEG C of shaking tables to hatch 2h or 4 DEG C overnight, can be according to antigen on amount of antibody and film
Amount proper extension or the time of shortening.
5), one anti-hatched after, take the film out and be positioned in clean plate, add 1 × TBST, shaking table washs
15min, changes liquid, 3 ~ 5 times repeatedly.
6), two anti-hatch, need according to experiment and the suitable ELIAS secondary antibody of design alternative and diluted concentration (5%BSA is dilute
Release), add after having washed under two anti-about 3ml room temperatures and hatch 2h in shaking table, it is ensured that all parts of film contact with solution.
7), two anti-hatched after, film bar is taken out and is positioned in clean culture dish, add 1 × TBST, on shaking table
Washing 15min, changes liquid, is repeated 3 times.
8), using Enhanced chemiluminescence to develop the color, specifically, first by two kinds of chromogenic substrate A liquid 1ml, B liquid 2 μ l enters
Row mixing, uniformly drips covering on film surface 1-2 minute, it is seen that luminous band by mixture, takes out pvdf membrane and with guarantor
Fresh film wraps pvdf membrane, wipes unnecessary nitrite ion, puts in clamping plate, is cut by X-ray to suitable size in darkroom,
Covering on pvdf membrane, clip clip, carry out tabletting exposure, the fluorescent brightness that time of exposure manifests according to albumen determines,
Also can different time repeatedly tabletting, exposure completes, and opens clip, puts in developer solution by film rapidly, after band to appear,
Can terminate, take out and put in fixative solution, till film bleach, then rinse with tap water, dry, scanning is taken pictures, and obtains
Photo is as shown in Fig. 4 a-4f.
9), data photo finishing and analysis, utilize quantity one photodensitometry software that picture is carried out gray value
Analyze, and t inspection between organizing；Utilizing Graphpad software to carry out data compilation mapping analysis, p < 0.05 has significance
Statistical significance, income analysis result is as shown in Fig. 5 a-5i.
The AD related pathways albumen that the present invention uses western blotting method to predominantly detect has APP, BACE-1,
SAPP α, sAPP β, PP2A, p-PP2A, PSD95, Tau5, Tau-ps396, Tau-ps404, Tau-ps422, by analyzing
The result obtained is as shown in Fig. 4 a-4f, Fig. 5 a-5i, from Fig. 5 a-5i it can be seen that after Jasminin effect, and AD related pathways
Expressing quantity there occurs change, specifically, after Jasminin effect, AD related pathways reduce APP, Tau-ps396,
The expression of Tau-ps404, Tau-ps422, increases the expression of PSD95, and its change has statistical significance.
Embodiment 5
After immunofluorescence observation jasminin effect, distribution and the expression of newborn rat primary neuron TAU-ps396 change
Further, newborn rat Mus primary neuron pTAU396 after the present invention uses immunofluorescence observation jasminin effect
3 Tg AD newborn rat hippocampus Primary cultured neuron specifically, are first being climbed by distribution and expression change containing cell
In 24 orifice plates of sheet, then by cultured primary neural cell (the 8th day), fix 20-30 min with 4% PFA of 1ml, with
The PBS of 4 DEG C washes 2 times, each 5 min, then adds 0.2%TritonX-100 in orifice plate, and every hole adds 500 μ l and stands 10min
After, wash 2 times with PBS, each 5min；After having washed, use PBST to prepare 10%goat antibody, close 1h for 4 DEG C, toward orifice plate
Middle addition one anti-(MAP-2: 1:10000 dilution, chicken polyclonal, Abcam:synaptophysin:1:200
Dilution, rabbit monoclonal, Abcam, 10% goat prepares), every hole adds 300 μ l, 4 DEG C of overnight incubation, adopts after hatching
Washing 3 times with PBS, each wash time is followed successively by 5min, 10min, 15min；After having washed, in orifice plate, add two resist
(goat-anti-chichen-Alexa594 or goat-anti-rabbit-Alexa488, is 1:200 and dilutes, Abcam,
10% goat preparation), every hole adds 300 μ l, incubated at room 1h, uses PBS to wash after hatching 3 times, and each wash time is successively
For 5min, 10min, 15min；Dyeing toward adding 5 μ g/ml DAPI in orifice plate after having washed, every hole adds 500 μ l again,
Dyeing time is 4min, uses PBS to wash after dyeing 3 times, and each wash time is followed successively by 5min, 10min, 15min；Then adopt
Carry out mounting by glycerol or PBS solution, fix cell climbing sheet with resinene and keep it in 4 DEG C and lucifuge, finally
Laser Scanning Confocal Microscope is used to process and analytical data.
As shown in Figure 6, Fig. 6 is that AD is newborn after Jasminin effect to the experimental result that the present invention is obtained by immunofluorescence
Mus hippocampus primary neuron figure, from fig. 6 it can be seen that adding the primary neuron dendron after Jasminin cultivates 24h
More and long, Dendritic arborization end and two matched groups are compared, and Tau-ps396 forms point-like and assembles less, and Jasminin is described
Reduce Tau-ps396 content, micro-tubular structure is played Stabilization.
In sum, first the present invention determines that jasminin is inhibited to A β aggregation in vitro, examines the most further
Measure jasminin and can affect the expression to AD classics pathway associated protein on a cellular level, and jasminin is new
Distribution and the expression change of raw Mus Mus primary neuron TAU-ps396 also have an impact, by present invention determine that jasminin has
Prevention or the effect for the treatment of Alzheimer, jasminin can develop into prevention and the medicine for the treatment of AD or health product.
It should be appreciated that the application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can
To be improved according to the above description or to convert, all these modifications and variations all should belong to the guarantor of claims of the present invention
Protect scope.
Claims (3)
1. the compound method of a jasminin solution, it is characterised in that include that jasminin is dissolved in DMSO or ethanol is molten
In liquid, vibration mixing, it is prepared as jasminin mother solution, by adding PBS, described jasminin mother solution is diluted to not
Jasminin solution with concentration.
The compound method of jasminin solution the most according to claim 1, it is characterised in that described jasminin mother solution
Concentration is 5mmol/L.
3. the application of a jasminin, it is characterised in that be used for described jasminin preparing prevention or treatment A Erci
The silent sick medicine in sea or health product.
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| US11707427B2 (en) | 2020-04-29 | 2023-07-25 | Mary Kay Inc. | Cosmetic compositions and methods |
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| CN109608510A (en) * | 2018-12-18 | 2019-04-12 | 五邑大学 | The preparation method of oleic acid glycosides -7- ethyl -11- methyl ester |
| CN109608510B (en) * | 2018-12-18 | 2021-09-03 | 五邑大学 | Preparation method of oleic acid glycoside-7-ethyl-11-methyl ester |
| US11707427B2 (en) | 2020-04-29 | 2023-07-25 | Mary Kay Inc. | Cosmetic compositions and methods |
| CN114748467A (en) * | 2022-05-01 | 2022-07-15 | 王峰 | Application of compound in preparation of medicine for treating Alzheimer's disease |
| CN115501269A (en) * | 2022-09-21 | 2022-12-23 | 南方医科大学 | Application of jasminum grandiflorum or extract thereof in preparation of medicines for treating neurodegenerative diseases |
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