CN109200037A - Pterostilbene is preparing the application in Antiatherosclerosis medicine - Google Patents
Pterostilbene is preparing the application in Antiatherosclerosis medicine Download PDFInfo
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Abstract
The application of the application field that the invention belongs to natural plant active components in clinic, especially Pterostilbene in preparation treatment atherosclerosis drug.Pterostilbene also known as butterfly Stilbene, are a kind of active skull cap components, belong to Verakanol derivative, are the important activity ingredients in grape, blueberry, dragon's blood product, Indian Kino tree and India's anti-diabetic herbal medicine " Bijasar ".Existing treatment atherosclerosis drug has fibrates, niacin class, Statins, unsaturated fatty acid and aspirin etc., all has apparent side effect.Pterostilbene of the invention effectively inhibits atherosclerosis by improving aorta SOD activity, reducing the effects of MDA horizontal and improvement dyslipidemia mechanism, the beneficial effect with highly effective and safe.
Description
Technical field
The invention belongs to atherosclerosis drug research technical fields, and in particular to Pterostilbene is preparing anti-atherogenic
Harden the application in drug.
Background technique
Atherosclerosis (AS) be it is a kind of there are a large amount of blood lipids to adhere under artery and its arterial blood tube wall inner membrance of branch,
With vascular wall intimal thickening, the artery class disease of yellow shape patch as congee is formed.In dyslipidemia, especially serum
Total cholesterol index or high-density lipoprotein cholesterol index etc. rise, be an important factor for causing AS lesion, actively to control
Blood lipid rises the incidence probability that can reduce AS to a certain extent.Blood lipid refers to rouge included in blood on ordinary meaning
Substance general name, including total cholesterol index (TC), triglycerides index (TG), low density lipoprotein cholesterol (LDL-
C) index, high-density lipoprotein cholesterol (HDL-C) index etc..Cholesterol and TG and internal specific proteins in blood carry
After lipoprotein is combined into lipoprotein, just can in blood plasma compatible promoting circulation of blood rouge of going forward side by side transhipment and metabolism.There is research to prove
LDL-C index is the most important lipid factor for inducing AS early stage.Although most of Hesperian blood lipid levels and blood lipid
Abnormal rate is even higher than domestic index, but along with the swift and violent promotion of social economy, compatriots' improvement of living standard and life
The increase of middle bad habit, average serum TC level are stepped up in recent years, and China's AS disease incidence is caused to rise year by year, it has also become tight
The principal disease of people's health is endangered again.
In human body anti-oxidative defense system, superoxide dismutase (SOD) is used as a kind of active material, and tool antioxygen is turned into
With, can alleviate body tissue free radical accumulation and mitigate peroxidating damage.In lipid peroxidation metabolic process, malonaldehyde
(MDA) be lipid peroxidation metabolism a kind of final product, it is toxic to body, experiment and detection in, often refer to its content, in turn
Understanding body lipid, there is a situation where peroxidating.MDA level increases, and prompts lipid peroxidation aggravation；And SOD activity reduces, instead
The decline of film projector body anti-oxidation function is eventually resulting in the result is that histiocytic damage is even dead.Therefore, in order to avoid group
The oxidative damage for knitting cell keeps suitable SOD activity, and the level for reducing MDA is necessary.
Pterostilbene (Pte) also known as butterfly Stilbene, are a kind of active skull cap components, belong to Verakanol derivative, are grape, indigo plant
Important activity ingredient in the certain kind of berries, dragon's blood product, Indian Kino tree and India's anti-diabetic herbal medicine " Bijasar ".Pte has reported tool
There are many bioactivity, such as: generating In-vitro Inhibitory Effect to the biomembrane of Candida albicans；The generation of neural cell model is protected
Shield effect；Have the function of resisting Several Kinds of Malignancy；To the increment of human body adenocarcinoma of lung A549 cell line and the influence of apoptosis；It plays a game
The protective effect etc. of stove cerebral ischemia.So far, there is not yet the report treated with Pte to AS.
Summary of the invention
The technical problem to be solved in the present invention is to provide Pterostilbenes to prepare the application in Antiatherosclerosis medicine.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
Pterostilbene of the present invention can adjust AS Rabbit Model blood lipid level, improve SOD activity, reduce MDA content.Institute
The structural formula for stating Pterostilbene is as shown in Figure 8.
Experimental method of the present invention:
1, it is grouped and is administered.
60 rabbit be grouped at random by TC with statistical method, are divided into six groups, Normal group, AS model
Group, the high, medium and low dosage group of Pte, positive control drug Atorvastatin (Ato) group.Normal group feeds normal diet, remaining is each
Group feeds high lipid food.After raising January, each administration group gives corresponding dosage drug by experimental design, and model group gives 0.5%
CMC-Na continues 8 weeks.
2, the dissection of rabbit and sample acquisition process.
Arterial blood extracting, centrifuging and taking serum are carried out to rabbit.Rabbit is dissected with scalpel, entire blood vessel exfoliation is gone out with tweezers
Come, is put into 4% paraformaldehyde fixed.Blood sample and tissue samples are saved in ultra-low temperature freezer.
3, TC, TG, HDL-C, LDL-C assay.
According to related kit, 4 kinds of lipid material contents are detected.As the result is shown with the raising of Pterostilbene dosage, high blood
Four kinds of lipid materials in rouge disease animal blood decline, and it is significant to illustrate that the drug has effects that in reduction lipid aspects,
And there are dose-dependent effects.
4, Histomorphological.
4% paraformaldehyde of aorta is fixed for 24 hours, routine paraffin wax embedded section, HE dyeing, after make pathology inspection under light microscopic
It looks into.Pte significantly can mitigate and improve aortic tissue pathological change caused by high fat diet as the result is shown.
5, the vitality test of SOD.
With SOD detection kit detection Arteries of Rabbits tissue SOD activity.Pte is able to ascend SOD vigor as the result is shown, says
Bright Pte can be obviously improved aortic tissue antioxidation system,.
6, MDA assay.
With the MDA content in the detection Arteries of Rabbits tissue homogenate of MDA detection kit.Pte can reduce MDA as the result is shown
Content.Illustrate that aortic tissue oxidative stress can be effectively reduced in Pte, to play anti-AS effect.
7, data processing and statistical analysis.
This experiment carries out data analysis using statistic software SPSS 13.0, is indicated with Mean. ± SD.Select One-way
ANOVA combination Post-Hoc (LSD method) method carries out group difference analysis.Statistical difference conspicuousness is indicated with P < 0.05
The utility model has the advantages that
1, Pterostilbene provided by the invention can be reduced AS Rabbit Model blood lipid level in a manner of dose-dependent, be subtracted
Aortic tissue pathological change caused by light and improvement high fat diet, induction SOD activity improve, and MDA content is inhibited to rise, thus
A kind of new types of therapeutic agents is provided for AS.
2, it since Pterostilbene is natural small molecule compounds, can be obtained by extraction or directly chemically synthesized method,
Thus lower production costs, are conducive to industrialized production.
Detailed description of the invention
Fig. 1 is the horizontal situation of change of AS rabbit anteserum TC after the administration of Pterostilbene described in embodiment 4, in figure, Normal group；
The rabbit group fed with chow diet；AS model group: the rabbit group fed with high lipid food；Ato group: positive control drug is given
The rabbit group that the high lipid food of Ato is fed；Pte high dose group: the rabbit group that the high lipid food of Pte 20mg/kg is fed is given；
Pte middle dose group: the rabbit group that the high lipid food of Pte 10mg/kg is fed is given；Pte low dose group: give Pte5mg/kg's
The rabbit group that high lipid food is fed.
Fig. 2 is the horizontal situation of change of AS rabbit anteserum TG after the administration of Pterostilbene described in embodiment 5, in figure, Normal group；
The rabbit group fed with chow diet；AS model group: the rabbit group fed with high lipid food；Ato group: positive control drug is given
The rabbit group that the high lipid food of Ato is fed；Pte high dose group: the rabbit group that the high lipid food of Pte 20mg/kg is fed is given；
Pte middle dose group: the rabbit group that the high lipid food of Pte 10mg/kg is fed is given；Pte low dose group: give Pte5mg/kg's
The rabbit group that high lipid food is fed.
Fig. 3 is the horizontal situation of change of AS rabbit anteserum LDL-C after the administration of Pterostilbene described in embodiment 6, in figure, normal control
Group；The rabbit group fed with chow diet；AS model group: the rabbit group fed with high lipid food；Ato group: positive control is given
The rabbit group that the high lipid food of medicine Ato is fed；Pte high dose group: the rabbit that the high lipid food of Pte 20mg/kg is fed is given
Group；Pte middle dose group: the rabbit group that the high lipid food of Pte 10mg/kg is fed is given；Pte low dose group: Pte 5mg/ is given
The rabbit group that the high lipid food of kg is fed.
Fig. 4 is the horizontal situation of change of AS rabbit anteserum HDL-C after the administration of Pterostilbene described in embodiment 7, in figure, normal control
Group；The rabbit group fed with chow diet；AS model group: the rabbit group fed with high lipid food；Ato group: positive control is given
The rabbit group that the high lipid food of medicine Ato is fed；Pte high dose group: the rabbit that the high lipid food of Pte 20mg/kg is fed is given
Group；Pte middle dose group: the rabbit group that the high lipid food of Pte 10mg/kg is fed is given；Pte low dose group: Pte 5mg/ is given
The rabbit group that the high lipid food of kg is fed.
Fig. 5 is AS Rabbit Aorta histopathology morphological change situation after the administration of Pterostilbene described in embodiment 8, in figure,
Normal group；The rabbit group fed with chow diet；AS model group: the rabbit group fed with high lipid food；Ato group: it gives
The rabbit group that the high lipid food of positive control drug Ato is fed；Pte high dose group: the high lipid food for giving Pte 20mg/kg is fed
Rabbit group；Pte middle dose group: the rabbit group that the high lipid food of Pte 10mg/kg is fed is given；Pte low dose group: it gives
The rabbit group that the high lipid food of Pte 5mg/kg is fed.
Fig. 6 is AS Rabbit Aorta tissue SOD activity change situation after the administration of Pterostilbene described in embodiment 9, in figure, normally
Control group；The rabbit group fed with chow diet；AS model group: the rabbit group fed with high lipid food；Ato group: the positive is given
The rabbit group that the high lipid food of comparison medicine Ato is fed；Pte high dose group: the family that the high lipid food of Pte 20mg/kg is fed is given
Rabbit group；Pte middle dose group: the rabbit group that the high lipid food of Pte 10mg/kg is fed is given；Pte low dose group: Pte is given
The rabbit group that the high lipid food of 5mg/kg is fed.
Fig. 7 is the horizontal situation of change of AS Rabbit Aorta tissue MDA after the administration of Pterostilbene described in embodiment 10, in figure, just
Normal control group；The rabbit group fed with chow diet；AS model group: the rabbit group fed with high lipid food；Ato group: sun is given
Property comparison medicine Ato the rabbit group fed of high lipid food；Pte high dose group: the high lipid food nursing of Pte 20mg/kg is given
Rabbit group；Pte middle dose group: the rabbit group that the high lipid food of Pte 10mg/kg is fed is given；Pte low dose group: Pte is given
The rabbit group that the high lipid food of 5mg/kg is fed.
Fig. 8 is the structural formula of Pterostilbene.
Specific embodiment
The invention will be further described with reference to embodiments.It is all that specific experiment condition is not specified in subordinate's embodiment
, it is according to normal condition well known to those skilled in the art and experimental procedure, or according to the normal condition proposed by manufacturer
And experimental procedure, these embodiments are only used for the purpose of illustration, it is in no way limited to protection scope of the present invention.
Embodiment 1: grouping
Selecting 60 healthy male rabbits at random, (the white ear hero rabbit of New Zealand, Medical University Of Anhui's Experimental Animal Center mention
For), it is marked respectively, weighing record takes blood, is centrifuged, takes serum.It is surveyed by serum TC assay kit (Nanjing is built up)
After determining serum TC, 60 rabbit are grouped at random, are divided into six groups, if Normal group, AS model group, Ato (Sigma) group
(5mg/kg), Pte (Hangzhou Great Forest Biomedical Ltd.) be high, in, small dose group (20,10,5mg/kg), totally 6 groups, every group 10
Only.
Embodiment 2: modeling and administration
Normal group gives normal diet, and AS model group and each administration group give high lipid food (containing 5% lard and 1%
Cholesterol).After 4 weeks, each administration group rabbit gives corresponding dosage by experimental design dosage stomach-filling daily in addition to giving high lipid food
Drug, drug using 0.5%CMC-Na be suspended.Then stomach-filling gives isometric 0.5% for Normal group and AS model group rats
CMC-Na, 1 time a day, totally 8 weeks.It in experimentation, weighs in weekly 1 time, adjusts dosage by changes of weight.
Embodiment 3: the dissection and sample acquisition process of rabbit
1, arterial blood extracting is successively carried out to rabbit, blood sample is centrifuged, take serum.Then injecting narcotic, to its fiber crops
After liquor-saturated, pneumatic needl is pushed into auricular vein, keeps its lethal.Rabbit is dissected with scalpel, entire blood vessel exfoliation is come out with tweezers,
It is put into 4% paraformaldehyde fixed.Collected blood sample and tissue samples are put into ultra-low temperature freezer to save, until detection
When take out.
2, blood sample is handled: the serum of preservation being taken out from ultra-low temperature freezer, trash ice is got out with ice crusher and is placed on
Serum sample is saved in foam box, presses TC, TG, HDL-C with liquid-transfering gun, the four indices such as LDL-C assay kit (build by Nanjing
At) specification requirement microplate reader detection four indices.
3, tissue samples are handled: vascular tissue's sample of preservation being taken out from ultra-low temperature freezer, is ready to ice crusher
Trash ice, which is placed in foam box, saves tissue samples, according to kit require homogenate aortic tissue, with microplate reader detection MDA and
Two indexs of SOD (the green skies Bioisystech Co., Ltd in Jiangsu).
The detection of protein concentration: it since SOD and MDA activity and content, tissue block vary in detection tissue, needs to use
Correction testing result is gone to homogenate protein concentration when test, detects (the green skies in Jiangsu using BCA determination of protein concentration kit
Bioisystech Co., Ltd).
Hematoxylin-eosin (HE) dyeing: aortic tissue sample is taken, 4% paraformaldehyde fixes 24 hours, routine paraffin wax packet
Slice is buried, is dyed with HE, and makees pathological examination under an optical microscope.
Embodiment 4:TC measurement
The present embodiment is used to measure rabbit anteserum TC horizontal.According to TC assay kit, (bio-engineering research is built up in Nanjing
Institute) specification operating procedure, if 1 table of table adds reagent, 37 DEG C of incubations 10 minutes, wavelength 510nm after mixing well, microplate reader is surveyed
Fixed each hole absorbance value.
TC/TG detecting step in 1 Small Volume Serum of table
Calculation formula: TC content (mmol/L)=(sample OD value-blank OD value)/(calibration OD value-blank OD value) × school
Quasi- product concentration
Experimental result (Fig. 1) display: compared with Normal group, the horizontal significantly raising of AS model group serum TC (P <
0.01)；Compared with AS model group, Pte high, middle dose group and Ato group serum TC level significantly reduce (P < 0.01).
Embodiment 5:TG measurement
The present embodiment is used to measure rabbit anteserum TG horizontal.According to TG assay kit, (bio-engineering research is built up in Nanjing
Institute) specification operating procedure, if table 1 adds reagent, 37 DEG C of incubations 10 minutes, wavelength 510nm after mixing well, microplate reader measurement
Each hole absorbance value.
Calculation formula: TG content (mmol/L)=(sample OD value-blank OD value)/(calibration OD value-blank OD value) × school
Quasi- product concentration
Experimental result (Fig. 2) display: compared with Normal group, AS model group serum TG levels significantly increase (P <
0.01)；Compared with AS model group, Pte high, middle dose group and Ato group serum TG levels significantly reduce (P < 0.05 or P < 0.01).
Embodiment 6:LDL-C measurement
The present embodiment is used to measure rabbit anteserum LDL-C horizontal.According to LDL-C assay kit, (biological work is built up in Nanjing
Journey research institute) specification operating procedure, if table 2 adds reagent, in wavelength 546nm after mixing well, microplate reader measures each hole and inhales
Shading value.
Calculation formula: LDL-C content (mmol/L)=[(sample A2- sample A1)-(blank A2- blank A1)]/[(standard
A2- standard A1)-(blank A2- blank A1)] ×
Calibration object concentration
Experimental result (Fig. 3) display: compared with Normal group, the horizontal significantly raising of AS model group serum LDL-C (P <
0.01)；Compared with AS model group, Pte high dose group and Ato group serum LDL-C level significantly reduce (P < 0.01).
Embodiment 7:HDL-C measurement
The present embodiment is used to measure rabbit anteserum HDL-C horizontal.According to HDL-C assay kit, (biological work is built up in Nanjing
Journey research institute) specification operating procedure, if table 2 adds reagent, in wavelength 546nm after mixing well, microplate reader measures each hole and inhales
Shading value.
2 HDL-C/LDL-C of table measures table
Calculation formula: HDL-C content (mmol/L)=[(sample A2- sample A1)-(blank A2- blank A1)]/[(standard
A2- standard A1)-(blank A2- blank A1)] × calibration object concentration
Experimental result (Fig. 4) display: compared with Normal group, AS model group Serum HDL-C is horizontal, and there was no significant difference
(P > 0.05)；But compared with AS model group, Pte high, middle dose group and Ato group Serum HDL-C it is horizontal it is significant increase (P <
0.01)。
Embodiment 8: Histomorphological
3% yellow Jackets (Hefei Mei Feng chemical industry instrument company) are injected with the dosage of 30mg/kg through rabbit auricular vein,
After being anaesthetized, injection pneumatic needl is lethal.Rabbit thoracic cavity is splitted with surgical scissors, separates aorta.Outer membrane is carefully scraped off with tweezers
After residual fat tissue, its is crosscutting, after being fixed for 24 hours with the paraformaldehyde solution that concentration is 4%, paraffin embedding carries out HE dye
Color, after make pathological examination under light microscopic.
Experimental result (Fig. 5) display: each layer structure of control group Aortic Wall in Rabbits is normal, and inner membrance is smooth, middle film elastic force
Fiber is in normal condition.The visible obvious interior skin portion cell detachment of AS model group, interior middle film is in obvious thickening phenomenon, and is convex to pipe
It is intracavitary.Furthermore, it is seen that form fibrous cap under a large amount of smooth muscle cell migration to inner membrance, subintimal foam cells and deposition
Lipid forms lipid core, partially visible calcification.It is visible on outer membrane to there is inflammatory cell to be gathered in this.In positive control Ato group
Film is still smooth, and middle film presentation thickens, and relative to model group, foam cells is significantly reduced, and has no that lipid core is formed.
Pte high, middle dose group aortic tunica intima are still smooth, and medial thickening, foam cells significantly reduces, and has no lipid core
It is formed.This result explanation, the atherosclerosis of aorta venereal disease rationality that Pte significantly can mitigate and improve high fat diet induction change.
The vitality test of embodiment 9:SOD
It takes appropriate rabbit arterial blood vessel in centrifuge tube, adds 1mL RIPA lysate (the green limited public affairs of skies biotechnology in Jiangsu
Department), ice bath homogenate is carried out using tissue refiner, after low-temperature centrifugation, takes supernatant.According to protein concentration and it is expected that albumen use
Amount detects the appropriate diluted sample of buffer with the SOD that SOD detection kit (the green skies Bioisystech Co., Ltd in Jiangsu) provides
Product.Ice bath is needed after preparing the reagent of sufficient amount according to the quantity of sample to be tested referring concurrently to specification, and ready-to-use.Make
With 96 orifice plates, sample well and various blank control wells are set.And sample to be tested and other solution are sequentially added by table 3.37 after mixing
DEG C be incubated for 30min, the OD value in each hole is measured at 450nm using microplate reader.
3 SOD vitality test of table operates table
Calculation formula: inhibition percentage=[(A blank control 1-A blank control 2)-(A sample-A blank control 3)]/
(A blank control 1-A blank control 2) × 100%
The inhibition percentage of sample need to can just be calculated from the formula SOD enzyme activity, and will in 30%~70% range
Enzyme activity unit is scaled U/mg protein.Otherwise need dilute sample until inhibiting percentage to be in zone of reasonableness.
SOD enzyme activity unit (U)=inhibition percentage/(1- inhibits percentage) in sample to be tested.
Experimental result (Fig. 6) display: compared with Normal group, the significant decrease of AS model group aorta SOD activity (P <
0.01)；Compared with AS model group, each dosage group of Pte and the significant raising (P < 0.05, P < 0.01) of Ato group aorta SOD activity.
Embodiment 10:MDA assay
It takes appropriate rabbit arterial blood vessel in centrifuge tube, adds 1mL RIPA lysate (the green limited public affairs of skies biotechnology in Jiangsu
Department), ice bath homogenate is carried out using tissue refiner, after low-temperature centrifugation, takes supernatant.According to protein concentration and it is expected that albumen use
Amount detects the appropriate diluted sample of buffer with the MDA that MDA detection kit (the green skies Bioisystech Co., Ltd in Jiangsu) provides
Product.Ice bath is needed after preparing the reagent of sufficient amount according to the quantity of sample to be tested referring concurrently to specification, and ready-to-use.It presses
Each reagent and sample are added in the EP pipe of 0.5mL, EP pipe are placed in metal bath, 100 DEG C, 15min by table 4.It is cooled to room temperature
Afterwards, it is centrifuged, takes 200 μ L supernatants in 96 orifice plates, survey absorbance in 532nm in microplate reader.MDA content in sample can basis
Standard curve calculates, and the protein content for passing through Unit Weight indicates, such as μm ol/mg albumen.
4 MDA vitality test of table operates table
Experimental result (Fig. 7) display: compared with Normal group, AS model group aorta MDA content significantly increase (P <
0.01)；Compared with AS model group, Pte high, middle dose group and Ato group aorta MDA content significantly reduce (P < 0.05).
Embodiment 11: data processing stages statistical analysis
This experiment carries out data analysis using statistic software SPSS 13.0, is indicated with Mean. ± SD.Select One-way
ANOVA combination Post-Hoc (LSD method) method carries out group difference analysis.Statistical difference conspicuousness is indicated with P < 0.05.
Claims (1)
1. Pterostilbene is preparing the application in Antiatherosclerosis medicine, the structural formula of the Pterostilbene is as follows:
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120000824A (en) * | 2010-06-28 | 2012-01-04 | 충북대학교 산학협력단 | Composition for inhibiting hyper-proliferation of vascular smooth muscle cell comprising pterostilbene as active ingredient |
| US20130296440A1 (en) * | 2012-05-01 | 2013-11-07 | ChromaDex Inc. | Pterostilbene and curcumin combination for treatment of oxidative stress and inflammation |
-
2017
- 2017-07-04 CN CN201710536856.4A patent/CN109200037A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120000824A (en) * | 2010-06-28 | 2012-01-04 | 충북대학교 산학협력단 | Composition for inhibiting hyper-proliferation of vascular smooth muscle cell comprising pterostilbene as active ingredient |
| US20130296440A1 (en) * | 2012-05-01 | 2013-11-07 | ChromaDex Inc. | Pterostilbene and curcumin combination for treatment of oxidative stress and inflammation |
Non-Patent Citations (3)
| Title |
|---|
| ELANGO BHAKKIYALAKSHMI等: "Anti-hyperlipidemic and anti-peroxidative role of pterostilbene via Nrf2 signaling in experimental diabetes", 《EUROPEAN JOURNAL OF PHARMACOLOGY》 * |
| LIN HC等: "Pterostilbene Inhibits Vascular Smooth Muscle Cells Migration and Matrix Metalloproteinase-2 through Modulation of MAPK Pathway", 《JOURNAL OF FOOD SCIENCE》 * |
| LU ZHANG等: "Pterostilbene protects vascular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivo", 《APOPTOSIS》 * |
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