CN108815145A - Purposes of the Hippocampus extract 2H5M in the drug and its health care product that preparation prevents and treats neurodegenerative disease - Google Patents
Purposes of the Hippocampus extract 2H5M in the drug and its health care product that preparation prevents and treats neurodegenerative disease Download PDFInfo
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- CN108815145A CN108815145A CN201810553258.2A CN201810553258A CN108815145A CN 108815145 A CN108815145 A CN 108815145A CN 201810553258 A CN201810553258 A CN 201810553258A CN 108815145 A CN108815145 A CN 108815145A
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- neurodegenerative disease
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Abstract
The present invention relates to a kind of purposes of Hippocampus extract 2H5M in the drug, health care product or dietary supplement that preparation prevents and treats neurodegenerative disease, wherein the entitled 2'- hydroxyl -5'- methoxyacetophenone of chemistry of the Hippocampus extract 2H5M.The Hippocampus extract 2H5M is originated from Hippocampus Kuda (Hippocampus kuda Bleeler), it does not have cytotoxicity to mouse microglia, it is able to suppress the generation of NO and ROS in mouse microglia, and can reduce DNA oxidative damage.
Description
Technical field
The present invention relates to Hippocampus extract 2H5M preparation prevent and treat the drug of neurodegenerative disease, health care product or
Purposes in dietary supplement.
Background technique
Neuroinflamation is resistant to the crucial defense mechanism of infectant and neure damage in central nervous system, however,
Excessive neuroinflamation leads to neure damage, and eventually leading to neurodegenerative disease, (such as Parkinson, Alzheimer, flesh wither
Contracting lateral sclerosis).Although the hypothesis that inflammation is fallen ill as neurodegenerative disease is there is also dispute, local inflammation is persistently deposited
It can lead to neure damage, the progression of the disease of accelerans degenerative disease has been reached common understanding.Microglia is maincenter mind
Macrophage like cell through system residence plays the effect for adjusting neural inner equilibrium and injury repair.But under inflammatory conditions,
Microglia overactivity and secret out of various inflammatory mediators include nitric oxide (NO), reactive oxygen species (ROS) and it is proinflammatory because
Son, these inflammatory mediators are considered having substantial connection with the progress of neuroinflammatory conditions.
Lipopolysaccharides (LPS) is also referred to as bacterial endotoxin, and mouse Nerve microglia can be stimulated to generate inflammatory factor, led
Cause neuroinflammatory conditions.In anti-inflammatory activity experiment in vitro, the model generally used is:LPS or IFN-γ stimulation BV-2 or
RAW264.7 inhibits the expression of NO in its cell.NO plays multiple physiological function in vivo, such as adjusts vessel retraction, is beneficial to
Immunostimulation is coped with, but in higher concentrations, NO is easy and superoxide anion (O2-) mistake of the reaction generation with high cell toxicity
Oxygen nitrite (ONOO-), can also be with the protein of cell, lipid and DNA reaction.The work that the microglia of activation passes through enzyme
A large amount of active oxygen radicals (ROS) are generated with catalysis, this helps to remove cause of disease, while also activating NF- κ B as second messenger
With MAPK signal pathway, proinflammatory factor is caused further to be expressed.The release of lasting ROS and proinflammatory inflammation factor can lead to rouge
The peroxidating of the macromoleculars such as matter and Apoptosis, cause neure damage, cause the vicious circle of inflammatory reaction and neurotrosis.
Hippocampus is a kind of rare Chinese medicine, has and controls promoting blood circulation dismission, and swelling and pain relieving controls the medical values such as traumatic injury.
So far, have and study physiological function about hippocampus monomeric compound, as hormone-like effect, it is anti-oxidant, improve immunity, anti-
Tumour etc. also has a small amount of research to focus on the antiinflammatory property of hippocampus monomeric compound.Chen etc. will demonstrate that class Three's hippocampus powder
Ligroin extraction can inhibit LPS activation RAW264.7 cell generate NO and proinflammatory factor [Chen LP, Shen XR,
Chen GH,Sun FF,and Jin MN.2016. Antioxidant activity of aqueous extract from
Three-spot hippocampus[J]. Science and technology of food Industry.3:114-
116] .Ryu et al. demonstrate one of hippocampus peonol have the function of inhibit neuroinflamation [Ryu, B., Qian, Z.J.,
and Kim,S.K. 2010.Purification of a peptide from seahorse,that inhibits TPA-
induced MMP,iNOS and COX-2expression through MAPK and NF-kappa B activation,
and induces human osteoblastic and chondrocytic differentiation[J]. Chemico-
Biological Interactions,2010,184(3):413-422]。
Currently, inhibiting the excessive activation of microglia about Hippocampus extract 2H5M not yet and treating nerve with it
The effect of degenerative disease and the research of mechanism.
Summary of the invention
In order to solve above-mentioned the problems of the prior art, the present invention provides following technical schemes：
In one aspect, the present invention provides a kind of Hippocampus extract 2H5M and prevents and treats neurodegenerative disease in preparation
Drug, the purposes in health care product or dietary supplement, wherein the entitled 2'- hydroxyl -5'- of chemistry of the Hippocampus extract 2H5M
Methoxyacetophenone.
Further, the Hippocampus extract 2H5M is originated from Hippocampus Kuda (Hippocampus kuda Bleeler).
Further, the Hippocampus extract 2H5M is able to suppress the release of the inflammatory factor NO of mouse microglia.
Further, the Hippocampus extract 2H5M can remove the ROS in mouse microglia.
Further, the Hippocampus extract 2H5M is able to suppress mouse microglia DNA oxidative damage.
Further, the neurodegenerative disease includes Parkinson's disease, Alzheimer's disease, and amyotrophic lateral is hard
Change.
On the other hand, the present invention provides a kind of Hippocampus extract 2H5M and inhibits the small colloid of mouse thin in vivo or in vitro
The purposes that NO is generated in born of the same parents, wherein the entitled 2'- hydroxyl -5'- methoxyacetophenone of chemistry of the Hippocampus extract 2H5M.
Further, the generation of the NO is induced by lipopolysaccharides LPS.
On the other hand, the present invention provides a kind of Hippocampus extract 2H5M oxidisability DNA is inhibited to damage in vivo or in vitro
The purposes of wound, wherein the entitled 2'- hydroxyl -5'- methoxyacetophenone of chemistry of the Hippocampus extract 2H5M.
On the other hand, the present invention provides a kind of Hippocampus extract 2H5M purposes for removing ROS in vivo or in vitro,
Described in Hippocampus extract 2H5M the entitled 2'- hydroxyl -5'- methoxyacetophenone of chemistry.
In mediation nervous system inflammation reaction in effector cell, the microglia of activation plays a significant role.It is quiet
The microglia of breath state can regulate and control brain microenvironment, phagocyte fragment, to play immunosurveillance.When its by
When stimulating (such as infection, inflammation, ischemic), it is the state of activation (Ah meter that microglia can stimulate from quiescent condition (branch-like)
Bar sample).There are two kinds of phenotypes, i.e. M1 and M2 for the microglia of differentiation.The microglia of M1 type activation generates proinflammatory substance
(such as NO, ROS etc.) has neurotoxicity, and the microglia of M2 type activation can produce anti-inflammatory factors, play neural restoration with
The effect of remodeling.It can cause cellular damage after the neurotoxicity factor in cell excessively discharges, this phenomenon is considered as producing
During raw neurodegenerative disease one of the main reason for Neuronal cell death.Therefore, the excessive of microglia is inhibited to swash
The key mechanism living for becoming treatment neurodegenerative disease.
NO is that a kind of molecular weight is small and there is extensive gas molecule in vivo, and with neurotransmission, WeiDongLi Capsule, wound is cured
It closes, mitochondrial respiratory, apoptosis and inflammation-related.In inflammatory process, NO plays an important role, and such as adjusts relevant to inflammation
The functions such as intracellular signal transduction pathway, chemotactic factor (CF).Therefore, whether there is a mature mould of anti-inflammatory activity in screening sample
Type is the NO content measured in its BV-2 cell for whether being able to suppress LPS activation.In this experiment, using Griess reagent come
Measure the yield of NO in different experiments group.The experimental results showed that 2H5M inhibits the yield of NO and is in concentration dependent, when its concentration
When being 100 μM, the generation of NO can be effectively suppressed, therefore, experiment shows that 2H5M has anti-inflammatory activity.This anti-inflammatory power and 2H5M
Active function groups it is related, such as methoxyl group, hydroxyl and ketone group, these functional groups remove free radical by losing electronics, furthermore
Have experiments have shown that methoxyl group (- OCH3) group has high anti-inflammatory activity.On the whole, these structure features of 2H5M are shown
Its anti-inflammatory activity.
Central nervous system is the tissue for being easiest to oxidative damage.Oxygen is both to maintain a kind of indispensable object of life
Matter, but it is likely to become the killer of life.It in breathing, is normally utilized in internal 98% oxygen, but remaining 2% conversion
For very active oxygen radical.ROS has the function of certain in vivo, such as accelerates immune and signal transduction process.But in body
Excessive active oxygen radical will lead to human normal cell and tissue is destroyed, when it is intracellular can not degrade in time ROS when,
Accumulation intracellular is so as to cause oxidative stress.Local ROS, which is generated or increased, can cause inflammatory cascade effect, so as to cause a variety of diseases
Disease.Unsaturated fatty acid on active oxygen energy attack cells film and generate peroxide, while intracorporal deoxidation core can also be invaded
Acid, collagen and enzyme have destructive chain reaction so as to cause to human tissue cell, lead to various infirmitiess of age
Occur.Under normal condition, the high-caliber nicotinamide adenine dinucleotide oxidizing ferment (NOX) of microglia expression, however
NOX is caused to activate under various inflammatory factor stimulations, synthesizing a large amount of active oxygen radicals (ROS) rapidly leads to oxidative burst.ROS with
And ROS reacted with NO generate peroxynitrite by mitochondrial respiratory chain damage, lipid peroxidation, protein nitration and
The mechanism such as DNA damage lead to neure damage and apoptosis.
In addition, free radical can destroy cell component, such as DNA, protein and lipid.Wherein protein is the early stage of ROS
Target causes protein function to be damaged after protein is attacked by ROS；ROS causes membrane lipid destruction that membrane fluidity is caused to reduce,
Membrane permeability increases and the damage of memebrane protein；ROS is to the oxidative dna damage of DNA, including base modification, abasic site, chain
Fracture and DNA- protein cross.Therefore, this experiment utilizes Fenton's reaction, protects DNA with the 2H5M of various concentration, observation is different
The degree of fracture belt in group.As a result prove that Hippocampus extract 2H5M can protect DNA damage, it may be with its free radical scavenging ability
It is related.
Hippocampus extract 2'- hydroxyl -5'- methoxyacetophenone (2H5M) has the LPS BV-2 microglia activated aobvious
Protective effect is write, the release of the inflammatory factor NO for the BV-2 microglia that it can inhibit LPS to activate, and meanwhile it is thin to BV-2
The DNA of born of the same parents have protective effect, in conjunction with above may determine that 2'- hydroxyl -5'- methoxyacetophenone have antioxidant activity and
Anti-inflammatory activity.
In the present invention, term " basic " or the meaning of " complete " " substantially " is not precluded." substantially such as an ingredient
Without " Y, it is also possible to be entirely free of Y.In the case where limiting specific value, it is specific with this to refer to that the specific value has
The range to float up and down based on numerical value, floating range can be +/- the 5% of the specific value, and +/- 4%, +/- 3%, it is +/-
2%, +/- 1%, +/- 0.5%, +/- 0.2%, +/- 0.1%, +/- 0.05%, +/- 0.01% etc..If desired, " basic "
Or " substantially " can above floating range replace or deleted from present invention definition.
" containing " had both included the factor mentioned, and also allowed to include additional, uncertain factor.
" about ", " about ", " left and right " in the case where limiting specific value, refer to the specific value have with the specific number
The range to float up and down based on value, floating range can be +/- the 5% of the specific value, and +/- 4%, +/- 3%, it is +/-
2%, +/- 1%, +/- 0.5%, +/- 0.2%, +/- 0.1%, +/- 0.05%, +/- 0.01% etc..
"and/or" indicates respectively be used alone by multiple terms of its connection, mutually can also arbitrarily combine.
In the present invention, the numberical range used for simplicity not only includes its endpoint value, also includes its all son
Range and individual numerical value all within the scope of this.For example, numberical range 1-6 not only includes subrange, such as 1-3,1-4,1-
5,2-4,2-6,3-6 etc., also including individual numerical value within the scope of this, such as 1,2,3,4,5,6.
Detailed description of the invention
Fig. 1 shows the extraction flow chart of Hippocampus extract 2H5M；
Fig. 2 shows BV-2 cellular morphologies；
Fig. 3 shows influence of the various concentration sample to BV-2 cell survival rate；
Fig. 4 shows NO standard curve；
Fig. 5 shows NO burst size；
Fig. 6 shows DNA electrophoresis result；
Fig. 7 shows DCFH-ROS fluorogram.
Specific embodiment
It is described below and illustrates exemplary implementation scheme of the invention.It is discussed below described in order to clear and accurate
Exemplary implementation scheme may include preferred step, method and feature, and those of ordinary skill in the art will be appreciated that, this
A little preferred step, method and features are not the necessary conditions being within the scope of the present invention.
Used experimental method is conventional method unless otherwise specified in the following embodiments.In following embodiments
Used in material, reagent etc. be commercially available unless otherwise specified.
Embodiment 1：The preparation of Hippocampus extract 2H5M
This experiment compound used is located away from Hippocampus Kuda (the Hippocampus kuda of Zhoushan areas
Bleeler), identification is completed by Chinese Changchun University of Traditional Chinese Medicine zoologist Deng Mingdeng professor.The extraction and identification of compound
As shown in Figure 1.The structural formula of 2H5M is as follows：
Embodiment 2：The recovery of the small glioma cell of mouse (BV-2) is passed on and is frozen
2.1 cell recovery
1 cryopreservation tube for freezing BV-2 cell is taken out from liquid nitrogen container, is put into 37 DEG C of water-bath immediately and is constantly shaken
It shakes.1mL frozen stock solution is transferred in the Tissue Culture Flask equipped with 20mL complete medium in superclean bench, is put into cell training
It supports and is cultivated in case.After 4h, culture bottle is taken out, observe cellular morphology and changes liquid, continues to cultivate.
The passage of 2.2 cells
It is put into super-clean bench from Tissue Culture Flask is taken out in incubator, outwells the culture medium in culture bottle, 1mL is added
PBS rinse cell surface, is repeated twice；The digestion of 1mL pancreatin is added, culture bottle is jiggled, makes pancreatin infiltration to cell table
Face.Digestive conditions are observed under the microscope, and when cell edges diminution is rounded, stopping digestion immediately inhales and abandons pancreatin, is added and cultivates
Base gently blows and beats cellular layer, and single cell suspension is made.Part cell suspension is transferred in new culture bottle, addition 5mL is complete
Incubator is put into after culture medium to continue to cultivate.
2.3 cell cryopreservation
The culture medium abandoned in culture bottle is inhaled from the cell (convergence degree about 70-80%) for taking out logarithmic growth phase in incubator,
Pancreatin digestion is added, when cell edges diminution is rounded, stopping digestion immediately inhales and abandons pancreatin, 1mL frozen stock solution is added, gently blows and beats
It is transferred in cryopreservation tube after single cell suspension is made, is sealed with sealing compound, is placed in program temperature reduction box in -80 DEG C of refrigerator overnights,
It is transferred in liquid nitrogen container and saves for a long time again.
Embodiment 3：MTT colorimetric test
3.1 experimental principle
MTT colorimetric method is the method for detecting cell survival conditions and growth.Its principle is：Amber in living cells mitochondria
Amber acidohydrogenase can make exogenous MTT (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide) be reduced to water not
In soluble blue purple crystals first a ceremonial jade-ladle, used in libation and deposition of cells, but dead cell is without this function.Dimethyl sulfoxide (DMSO) can dissolve in cell
First a ceremonial jade-ladle, used in libation and generate purple solution, survey light absorption value under OD540nm wavelength with microplate reader.MTT forming amount and total viable cell at
Direct ratio.
3.2 experimental procedure
100 μ L BV-2 cell suspensions (deriving from embodiment 2) is added in every hole in 96 orifice plates, is put into incubator and cultivates.
It inhales afterwards for 24 hours and abandons old culture medium, the culture medium for being 10,20,50 and 100mM containing sample concentration is added, is put into incubator and continues
Culture.It inhales afterwards for 24 hours and abandons culture medium, every hole is added 100 μ L MTT solution, is put into incubator and is incubated for.96 holes are carefully sopped up after 4h
100 μ LDMSO are added in MTT solution in plate, every hole, finally survey OD540nm light absorption value with microplate reader.
In this experiment, cell is calculated as follows with respect to motility rate：
MTT experiment is a kind of rough economic experimental method, and the size of absorbance is directly proportional to total living cells quantity.Such as
Shown in Fig. 3, sample sets (10,20,50,100 μM of concentration) handle BV-2 cell for 24 hours after light absorption value and blank group light absorption value
Ratio 100% or so, it was demonstrated that under the concentration gradient, sample does not have cytotoxicity for BV-2 cell, therefore with this
Concentration gradient carries out subsequent experimental.
Embodiment 4：NO measures (LPS stimulation test)
4.1 experimental principle
This experiment measures NO content using Griess Reagent System kit (being purchased from Promega company).
Its principle is：P-aminobenzene sulfonic acid and NO2-Diazo-reaction occurs under organic acid effect, is then given birth to hydrochloric acid-naphthylamines coupling
At aubergine pigment, light absorption value is measured under the conditions of OD540nm.NO is calculated according to standard curve2 -Content to measuring NO's
Content.
4.2 experimental procedure
4.2.1 the measurement of standard curve
By 0.1M nitrous acid standard items according to kit specification be diluted to various concentration (100,50,25,12.5,
6.25,3.13 and 1.56 μM).3 column are specified in 96 orifice plates, are used for nitrite canonical reference curve.It is dense to draw 30 μ L differences
The sulfanilamide (SN) solution of 30 μ L is added in 96 orifice plates in the standard items of degree, and room temperature shading reacts 5-10min, adds 30 μ L hydrochloric acid-naphthalene
Amine aqueous solution is incubated for 5-10min in room temperature shading, surveys the absorbance under OD540nm with microplate reader.
With (100,50,25,12.5,6.25,3.13,1.56 and 0 μM) of concentration of standard items for abscissa, each concentration standard
The value of solution O D540 is ordinate after product are reacted with sulfanilamide (SN) and hydrochloric acid-naphthylamines, can obtain standard curve, as shown in Figure 4.
4.2.2 experimental group NO is measured
This time NO in measuring cell culture fluid2 -Content.It is outstanding that 100 μ L BV-2 cells are added in every hole in 96 orifice plates
Liquid (derives from embodiment 2), cultivates in the incubator.It after for 24 hours, inhales and abandons old culture medium, being added containing sample concentration is 10,20,50
With the culture medium of 100mM, it is put into and continues to cultivate in incubator.(1 μ L, 1 μ is stimulated in the aerial LPS that is added of control wells and experiment after 1h
G/mL), it is put into incubator and cultivates.After for 24 hours, it is careful draw different experiments group in 30 μ L, 96 orifice plate (blank group, control group and
Sample sets) in cell culture supernatant liquid in another 96 orifice plate.30 μ L are added into the hole containing 30 μ L cell supernatants
Sulfanilamide (SN) solution adds 30 μ L hydrochloric acid-naphthylamines solution in being stored at room temperature 5-10min, is incubated for 5-10min in room temperature shading, uses
Microplate reader surveys the absorbance under OD540nm.
Measure blank group, solution after control group is reacted with sample sets (10,20,50,100 μM) with sulfanilamide (SN) and hydrochloric acid-naphthylamines
The value of OD540nm can calculate NO in cell culture fluid using the standard curve of figure 4 above2 -Content, i.e., NO in different experiments group
Yield, experimental result are as shown in Figure 5.When no LPS is stimulated (blank group), BV-2 is in quiescent condition, generates micro
NO；After LPS stimulation for 24 hours (control group), BV-2 cell is activated, and cell generates a large amount of NO, about than the increase of blank group content
4 times；Sample 2H5M protection cell 1h is being added again with after LPS stimulation for 24 hours, the production quantity of NO and the concentration of sample are negatively correlated,
Different degrees of inhibiting effect is shown, highest inhibitory activity is observed under 100 μM of concentration, by the NO of LPS inductive formation
Content inhibit about 1.5 times, thus prove, the sample for the NO in the BV-2 cell of LPS induced activation have inhibit make
With, illustrate the sample have anti-inflammatory activity.
Embodiment 5：Agarose gel electrophoresis (DNA) experiment
5.1 extract DNA
By in Tissue Culture Flask BV-2 cell (derive from embodiment 2) and media transfer be centrifuged into 50mL centrifuge tube
(1000rmp, 10min), discards supernatant, and PBS (EDTA containing 5mM) the cleaning cell of 10mL is added, be centrifuged again (1000rmp,
10min), supernatant is removed.The sufficiently piping and druming of 350 μ L sodium acetates (0.2M) is added to centrifuge tube and uniformly, it is micro to be transferred to 1.5mL
In centrifuge tube, 25 μ L SDS (10%), 10 μ L Proteinase Ks (10mg/mL) and 25 μ L RNAase (0.5mg/mL), whirlpool are added
30s is revolved, heating (54 DEG C, 1h) in metal bath is subsequently placed in.After heating, takes out centrifuge tube and phenol is added：Chloroform：Isoamyl alcohol
=25:24:1 reagent, 410 μ L puts into a centrifuge centrifugation (12000rmp, 10min) after vibrating 30s.After centrifugation, take out
570 μ L or so pre-cooling is added in another microcentrifugal tube in centrifuge tube, the careful transparent DNA layer (about 380 μ L) in upper layer of drawing
The dehydrated alcohol of (- 20 DEG C, 15min) is centrifuged (12000rmp, 5min) after vibrating 30s again.After centrifugation, take out from
It is inhaled after heart pipe and abandons supernatant, be put in room temperature and be dried, the dissolution of 50 μ L TE water is added.Take 1 μ LDNA solution that 49 μ L are added super
In pure water, the light absorption value at 260nm and 280nm, measures DNA purity with the ratio of OD260/OD280 respectively.
After the DNA of BV-2 cell is extracted, suitable multiple is diluted, the value of its OD260 and OD280 are surveyed.When OD260 with
The ratio of OD280 proves that the DNA sample purity is higher when being 1.7-2.0.OD260=2.854, OD280 are measured in this test
=1.729, OD260/OD280=1.65 are closer to 1.7, can carry out lower continuous electrophoresis experiment.
5.2 agarose gel electrophoresis (DNA)
5.2.1 experimental principle
Agarose gel electrophoresis is the standard method of identification and purification DNA fragmentation for separating.Agarose is from agar
A kind of polysaccharide of middle extraction has hydrophily, but neutral, is a kind of good electrophoresis support.DNA is under alkaline condition
(pH=8) negatively charged, mobile to anode by gel media in the electric field, different DNA molecular segments are due to molecule and configuration
Difference, swimming rate in the electric field are also different.Goldview is a kind of nucleic acid dye, between embeddable DNA molecular base-pair
Different zone can be separated after ultraviolet light irradiates by forming fluorescent complex.
Oxidative damage to DNA is usually as caused by free radical, and the activity of these free radicals can pass through antioxidant
It reduces.Fenton reagent has extremely strong oxidability, and its essence is Fe2++H2O2→Fe3++OH-+ OH, OH have relatively strong
Oxidability.
5.2.2 experimental procedure
5.2.2.1 experimental group designs
5.2.2.2DNA electrophoresis
Weigh 0.6g agarose and be dissolved in after 60mL ultrapure water being put into micro-wave oven and heat, until agarose be dissolved as it is transparent molten
Stop heating when liquid, is placed in room temperature cooling.When agarose solution temperature is about 60 DEG C, it is slowly poured into plastic plate
In, and be rapidly inserted perpendicularly into sample comb.After 30min, one layer of glue surface (3-5mm) uniformly solidified is formed in plastic plate,
Sample comb is carefully extracted, and plastic plate is transferred in Horizontal electrophoresis tank, 500mL TAE electrophoresis is then added into electrophoresis tank
Buffer did not had glue surface 1-1.5cm.
After being sufficiently mixed 6 × DNA sample-loading buffer (3.5mL) and experimental group sample liquid (16.5mL) with liquid-transfering gun, it is added
Sample well.
Electrophoretic voltage 100V, electrophoresis time 30min are set after loading, start electrophoresis.After electrophoresis, gel is carefully taken
Out, it immerses in 5%Goldview nucleic acid dye, dyes 2h.Running gel after Labworks image acquisition and analysis software observation dyeing is simultaneously
It takes pictures, as shown in Figure 6.
2H5M is the influence detected to hydroxyl radical free radical induced damage to BV-2 cell (deriving from embodiment 2) DNA protection.Sample
Product group is the Hippocampus extract 2H5M of various concentration gradient (0,20,50 and 100 μM), and FeSO is added4And H2O2DNA is carried out brokenly
Bad, blank group is the DNA solution of BV-2 cell, and control group is free from the reaction mixture of sample.
Blank group DNA band as seen from Figure 6 containing only DNA solution is most obvious, the control without adding sample protection
Group DNA is destroyed seriously, and band very Fuzzy decentralized, experimental group can be seen that the 2H5M of various concentration gradient has in various degree DNA
Protection, wherein with the concentration of sample being in be incremented by state to the degree of protection of DNA, 2H5M concentration is higher, by Fenton's reaction shadow
Sound is smaller, makes the integrated degree of DNA band closer to blank group, the 2H5M of 100 μ g/mL can be free by hydroxyl in BV-2 cell DNA
The damage of base induction is preferably minimized.These results confirm that Hippocampus extract 2H5M has foot to the DNA damage of free radical mediated
Enough protective effects.
Embodiment 6：DCFH-DA：ROS fluorescence detection (active oxygen detection)
6.1 experimental principle
Active oxygen detection kit be it is a kind of utilize fluorescence probe DCFH-DA carry out active oxygen detection kit.DCFH-DA
Itself does not have fluorescence, can pass freely through cell membrane, into after intracellular, can generate DCFH by intracellular esterase hydrolyzed,
And DCFH cannot be by cell membrane, so that probe be made to be easy to be loaded into the cell.Intracellular active oxygen can aoxidize nothing
The DCFH of fluorescence generates the DCF for having fluorescence.Detect DCF fluorescence it is known that reactive oxygen species level.
6.2 experimental procedure
In 24 orifice plates, the BV-2 cell suspension (deriving from embodiment 2) of 200 μ L is added in every hole, is put into incubator and is cultivated.
It takes out to inhale afterwards for 24 hours and abandons culture medium in hole, the fresh culture 200 for containing different sample concentrations (10,20,50 and 100mM) is added
μ L continues to cultivate, and LPS (being purchased from Sigma company) is added after 1h, is put into and continues to cultivate in incubator.After 6h, inhales and abandons culture medium,
And with PBS rinse 3 times, the 200 μ diluted DCFH-DA of L anteserum-less substrate are then added, covers orifice plate with tinfoil and is placed in training
It supports and is incubated in case.It after 20min, inhales and abandons DCFH-DA, wash cell three times with PBS, each 5min is not entered thin with abundant removing
DCFH-DA intracellular, finally in fluorescence microscopy microscopic observation fluorescence.
This experiment is produced with the expression of ROS in ROS fluorescence probe detection BV-2 cell according to fluorescence in living cells
It is raw, it can be determined that in cell ROS content number and variation.Such as Fig. 7, by fluorescence inverted microscope it can be observed that BV-2 is thin
The discovery of born of the same parents' ROS fluorescence probe DCFH dyestuff：Blank group ROS positive cell ROS luciferase expression degree is most weak, and picture is very dim；
(control group) ROS luciferase expression degree highest after LPS stimulation, interface are very bright；In experimental group (addition various concentration 2H5M)
It can obtain, also gradually weaken as sample concentration increases ROS luciferase expression amount, wherein 100 μ g/mL group luciferase expression amount intensity
It is weak, the luciferase expression amount highest of 10 μ g/mL groups, but it still is below control group.
Activator of the present invention application LPS as microglia, using the small colloid of BV-2 of LPS stimulation in vitro culture
Cell as inflammatory model, have studied Hippocampus extract 2'- hydroxyl -5'- methoxyacetophenone (2H5M) LPS is induced it is small
The anti-neuroinflamation activity of spongiocyte.The BV-2 that Hippocampus extract 2'- hydroxyl -5'- methoxyacetophenone (2H5M) activates LPS
Microglia has significant protective effect, the release of the inflammatory factor NO for the BV-2 microglia that it can inhibit LPS to activate,
There is protective effect to the DNA of BV-2 cell simultaneously, it is anti-in conjunction with may determine that 2'- hydroxyl -5'- methoxyacetophenone has above
Oxidation activity and anti-inflammatory activity.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. drug, health care product or the diet supplement that a kind of Hippocampus extract 2H5M prevents and treats neurodegenerative disease in preparation
Purposes in agent, wherein the structural formula of the Hippocampus extract 2H5M is
2. the Hippocampus extract 2H5M according to claim 1 preparation prevent and treat neurodegenerative disease drug,
Purposes in health care product or dietary supplement, wherein the Hippocampus extract 2H5M is originated from Hippocampus Kuda (Hippocampus kuda
Bleeler)。
3. the Hippocampus extract 2H5M according to claim 1 preparation prevent and treat neurodegenerative disease drug,
Purposes in health care product or dietary supplement, wherein the Hippocampus extract 2H5M is able to suppress inflammatory in mouse microglia
The release of factor NO.
4. the Hippocampus extract 2H5M according to claim 1 preparation prevent and treat neurodegenerative disease drug,
Purposes in health care product or dietary supplement, wherein the Hippocampus extract 2H5M can be removed in mouse microglia
ROS。
5. the Hippocampus extract 2H5M according to claim 1 preparation prevent and treat neurodegenerative disease drug,
Purposes in health care product or dietary supplement, wherein the Hippocampus extract 2H5M is able to suppress the DNA of mouse microglia
Oxidative damage.
6. the Hippocampus extract 2H5M according to claim 1 preparation prevent and treat neurodegenerative disease drug,
Purposes in health care product or dietary supplement, wherein the neurodegenerative disease includes Parkinson's disease, Alzheimer's disease,
Amyotrophic lateral sclerosis.
7. the purposes that a kind of Hippocampus extract 2H5M inhibits NO to generate in vivo or in vitro, wherein the Hippocampus extract 2H5M
Structural formula is
8. the Hippocampus extract 2H5M according to claim 7 inhibits the purposes of inflammatory factor NO generation in vitro, wherein institute
The generation of NO is stated by lipopolysaccharide-induced.
9. a kind of Hippocampus extract 2H5M inhibits the purposes of DNA oxidative damage in vitro, wherein the Hippocampus extract 2H5M
Structural formula be
10. a kind of Hippocampus extract 2H5M removes the purposes of ROS in vitro, wherein the structural formula of the Hippocampus extract 2H5M is
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Citations (2)
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|---|---|---|---|---|
| CN102614156A (en) * | 2012-03-09 | 2012-08-01 | 沈阳药科大学 | Medical application of in-vivo metabolite of paeonol |
| CN108064160A (en) * | 2014-09-19 | 2018-05-22 | 庆熙大学校产学协力团 | For treating and preventing the extract of the mixture containing moutan root bark, root of Dahurain angelica root and radix bupleuri root of nerve degenerative diseases or its fraction pharmaceutical composition as active component |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102614156A (en) * | 2012-03-09 | 2012-08-01 | 沈阳药科大学 | Medical application of in-vivo metabolite of paeonol |
| CN108064160A (en) * | 2014-09-19 | 2018-05-22 | 庆熙大学校产学协力团 | For treating and preventing the extract of the mixture containing moutan root bark, root of Dahurain angelica root and radix bupleuri root of nerve degenerative diseases or its fraction pharmaceutical composition as active component |
Non-Patent Citations (4)
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| YUANYUAN ZHANG: "2′-Hydroxy-5′-methoxyacetophenone attenuates the inflammatory response in LPS-induced BV-2 and RAW264.7 cells via NF-κB signaling pathway" * |
| 薛小燕 等: "神经退行性疾病的药物治疗研究" * |
| 金鑫: "环糊精与三种药物包合过程的量子化学研究" * |
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