CN105920580B - Ocean microorganism compound P-3 is preparing the application in antiaging agent - Google Patents
Ocean microorganism compound P-3 is preparing the application in antiaging agent Download PDFInfo
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- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention discloses ocean microorganism compound P-3 to prepare the application in antiaging agent.The compound can improve Cell Telomerase Activity, can significantly reduce the degree of aging of cell by the expression of increase anti-aging gene Sirt 1, have significant protective effect to the cell ageing of hypertensinⅡinduction.The new medical application of P-3 has been excavated in application of the P-3 of the present invention in the damage of preparation anti-aging and diseases related drug, has opened up a new application field, and P-3 is safe and non-toxic, pharmacological action is strong, there is good prospect in medicine.
Description
Technical field
The present invention relates to the new opplications of ocean microorganism compound P-3.
Background technique
Ocean microorganism compound P-3 is the metabolite of South Sea mangrove fungi Xylaria sp.2508
The synthesis of derivatives of xyloketal A, chemical structure are as follows:
It has been found that the activity that the compound has angiogenesis promoting by adjusting gene M MPs, and angiogenesis is in ischemic
Disease, such as coronary heart disease are of great significance in the treatment of cerebral arterial thrombosis, therefore the compound is used as ischemic disease controls
Treat the active constituent of drug.
Aging Problem is considered as one of three big world property social concern of 21 century, and aging and disease of old people are
Interpromoting relation in five elements is associated, and the two pathophysiological process having the same such as neuroendocrine disturbance, carbohydrate and disorders of lipid metabolism, is exempted from
Epidemic disease system injury etc..With the increase at age, the incidence and illness rate of some geriatric diseases are obviously increased, such as coronary disease
Disease, diabetes, atherosclerosis etc..Therefore, find one kind can effective delaying cell aging, and prevent and treat aging
Diseases related drug is of great significance to the medical treatment development in China.
Summary of the invention
The purpose of the present invention is to provide ocean microorganism compound P-3 to prepare the application in antiaging agent.
To achieve the goals above, the present invention adopts the following technical scheme:
Inventor has found that P-3 can increase the expression of cell anti-aging gene Sirt 1 by long-term research, improves cell end
Telomerase activity significantly reduces the degree of aging of cell, there is apparent protection to make the cell senescence of hypertensinⅡinduction
With can be used for preparing the drug of anti-aging and diseases associated with senescence.Especially with renin angiotensin aldosterone system
(RAAS) activation and (or) the impaired senescense and damnification being characterized of Telomerase/SIRT1 and diseases related.
Wherein it is possible to which P-3 is prepared into the various dosage forms of drug field acceptable, such as tablet, capsule.
Compared with the application of existing P-3, the invention has the following beneficial effects:
(1) the compound of the present invention P-3 has good anti-cell aging effect.
(2) the compound of the present invention P-3 is safe and non-toxic, and pharmacological action is strong, implies good prospect in medicine.
Specific embodiment
One embodiment of the present of invention is described below, but the content of present invention is not limited thereto.
The research of embodiment 1P-3 anti-cell aging
(1) materials and methods
1. drug: P-3, white powder.Be dissolved in DMSO, stock concentration 100mmol/L, be stored in 4 DEG C it is spare.
2. cell culture and model preparation:
Cell using hEPC (endothelial progenitor cells in human peripheral source) as in vitro study, 90%EGM-2 (LONZA)
+ 10% fetal calf serum (GIBCO) is in 37 DEG C of 5%CO2Incubator is incubated for, and growing to 7 days to cell can be used for testing.
Use Angiotensin II inducing cell Ageing Model: the good hEPC kind of growth conditions in 6 orifice plates, to its life
Its normal incubation medium is changed into serum free medium at long to 7 days, is added after 100nM Angiotensin II acts on 24 hours and is carried out
The detection of experimental index.
3, cytoactive detection --- CCK-8 method
100 μ L hEPC cell suspensions are inoculated in 96 orifice plates, every group uses 6 multiple holes, is put into 37 DEG C of incubator cultures, 24 is small
When after be added various concentration P-3 pre-stimulation 1 hour, add 100nM Angiotensin II handle 24 hours after, be added in every hole
10 μ L CCK-8 solution after being protected from light incubation in 37 DEG C of incubators 4 hours, are inhaled in microplate reader measurement in 450nm wavelength detecting each group
Luminosity, by each group absorbance compared with blank control group, to calculate the survival ratio of group of cells.
4, the aging degree of SA- beta galactosidase dyeing detection cell
It is cultivated hEPC7 days in 6 orifice plates, 100nM Angiotensin II is added, removes culture medium after 24 hours, is washed with PBS
2 times, every hole is added 1mL fixer room temperature and fixes, and PBS is washed 2 times after 15min, and every hole is added according to SA- β-gal staining kit
The 1mL of preparation dyes mixed liquor, at 37 DEG C, without CO2Under the conditions of be incubated overnight, taken pictures with fluorescence microscope and count cell dyeing
Rate, the cell being colored represent senile cell.
5, telomerase activation detects
In 6 orifice plates culture hEPC7 days, after P-3 incubates 1 hour in advance, the processing of 100nM Angiotensin II is added for 24 hours, centrifugation is received
Collect cell in centrifuge tube, every pipe is added 200 μ L lysates and is resuspended, and stands centrifuging and taking supernatant after 30min on ice, while taking feminine gender
Compare 85 DEG C of heating 10min.TRAP reaction is carried out, 25 μ L reaction mixtures, 1 μ L sample to be tested and right are added in each PCR pipe
Product in the same old way carry out PCR amplification.20 μ L denaturing reagents are added in corresponding each sample, 5 μ L amplified productions are incubated at room temperature 10min, in pre-
225 μ L hybridization buffers are added in every hole in coated 96 orifice plate, 100 μ L mixed liquor, 37 DEG C of shaking tables are incubated for 2h, and removal buffer is simultaneously
It is washed three times with Washing buffer, the dyeing of 100 μ L methyl biphenyl amine aqueous solutions is added in every hole, and it is whole that 100 μ L are added in every hole after 20min
Only liquid.The light absorption value at 450nm is read with Microplate (ELISA) reader.
6. the detection of expression of anti-aging gene SIRT1 albumen
The expression of SIRT1 albumen is detected using the method for western blot, and in 6 orifice plates culture hEPC7 days, P-3 incubated 1 in advance
Hour, after the processing for 24 hours of 100nM Angiotensin II is added, cell is washed three times with ice PBS, and addition contains protease inhibitors
(Cocktail) 60~80ul of lysate RIPA；5~10min of lytic cell on ice.With clean cell scraper scrape cell and
Cell pyrolysis liquid, 4 DEG C of centrifugation 12min of 12000r/min abandon precipitating, careful to draw supernatant protein liquid, -80 DEG C of preservations.Use BCA
Method is quantified, and calculates sample concentration by standard curve, with identical loading total protein concentration by sample and sample-loading buffer with
The ratio of 4:1 mixes, and 95 DEG C of warm bath 5min are denaturalized albumen under the action of sample-loading buffer.Configure polyacrylamide (SDS-
PAGE glue 5% is concentrated in) gel, separation gel 8%.Sample is added in each runway of concentration glue using Microloader, constant pressure
80V electrophoresis 90min；Separation gel is turned to be placed in together in electric turn trough after clip pack wraps with filter paper, pvdf membrane electricity consumption, turns buffering in electricity
Constant current 200mA electricity turns 90min in liquid.Electricity takes out pvdf membrane after turning, and closes in TBST of the room temperature containing 5% skimmed milk power
1h；Corresponding Sirt1 antibody is then added, using actin as internal reference, 4 DEG C of refrigerator overnights.TBST washes film 5min × 3 time, is added
Corresponding secondary antibody (HRP label, 1:1000 are diluted with antibody diluent) is incubated for 1-2h on room temperature shaker afterwards；TBST washes film 10min
× 3 times, pvdf membrane and ECL luminescent solution (A liquid: B liquid, 1:1) are mixed in darkroom, in being exposed on X-ray film, processing machine
Film then carries out gray analysis using Image J 1.39U.Sirt1 is represented with the gray level ratio of Sirt1 and internal reference actin
Expression, and the ratio of control group is set as 1, the other groups of expression height for analyzing Sirt1 in comparison.
7, it statisticallys analyze
This experiment is compared two-by-two between multiple groups quantitative data, and meets the requirement of normality and homogeneity of variance, therefore is selected
Method of the variance analysis (One-way ANOVA) as statistical analysis.Statistical analysis operation is executed using SPSS software.
(2) experimental result
1. various dose P-3 is to the protective effect such as table 1 of the AngII hEPC damage induced.
The active influence of hEPC that 1 various dose P-3 of table processing induces AngII
* the cell activity for obtaining AngII group and blank control group through one-way analysis of variance has statistical difference (P <
0.05)；
#The cell activity for obtaining 0.2 μM of processing group of P-3 and AngII group through one-way analysis of variance has statistical difference
(P < 0.05)
##The cell activity for obtaining 1 μM/5 μM/25 μM processing groups of P-3 and AngII group through one-way analysis of variance has system
Meter learns difference (P < 0.01)
Above-mentioned experimental result illustrates that P-3 has the function of protecting the damage of hEPC angiotensins, increases cells survival rate, and
Dose-dependant (0.04-25 μM) effect is presented.
The SA- beta galactosidase Study on dyeing such as table 2 of 2.P-3.
Protective capability of the 2 various dose P-3 of table to AngII induction hEPC aging
The cell ageing degree that * obtains AngII group and blank control group through one-way analysis of variance has statistical difference
(P < 0.01)；
##The cell ageing degree for obtaining 1 μM/10 μM processing groups of P-3 and AngII group through one-way analysis of variance has system
Meter learns difference (P < 0.01)；
Above-mentioned experimental result illustrates that hEPC cells in vitro AngII induction aging model constructs successfully, and aging ratio is about sky
The 360% of white control group；Meanwhile P-3 has the function of anti-hEPC aging, and dose-dependant (0.1-10 μM) effect is presented.
Effect such as table 3 of 1 μM of the 3.P-3 processing to the telomerase activation of the AngII hEPC induced.
Influence of 1 μM of the table 3P-3 processing to the telomerase activation of the AngII hEPC induced
*, which obtains AngII group and the Cell Telomerase Activity of blank control group through one-way analysis of variance, has statistics poor
Different (P < 0.01)；
#The Cell Telomerase Activity for obtaining 1 μM of processing group of P-3 and AngII group through one-way analysis of variance has statistics
Difference (P < 0.05)
Above-mentioned experimental result illustrates that 1 μM of P-3 has the function of significantly improving hEPC telomerase activation.
1 μM of 4.P-3 handles the effect such as table 4 expressed SIRT1.
The influence that SIRT1 in hEPC is expressed in 1 μM of table 4P-3 processing
* the Sirt1 expression for obtaining AngII group and blank control group through one-way analysis of variance has statistical difference (P <
0.05)；
##The Sirt1 expression for obtaining 1 μM of processing group of P-3 and AngII group through one-way analysis of variance has statistical difference
(P < 0.01)
Above-mentioned experimental result illustrates that 1 μM of P-3 has the function of significantly improving anti-aging gene Sirt1 expression in hEPC.
(3) it discusses
Research now thinks that aging is derived from the reduction of cell activity, and then leads to the aging of cell, tissue and organ,
Therefore aging is the aging of cell.As the research of Aging mechanism enters the molecule epoch, various the reason of leading to aging, include
The research fields such as free-radical theory, heat limitation, Telomerase theory and epigenetics have new development.In angiocarpy
Disease, such as hypertension, Cardiovascular Remodeling, in the pathogenic process of heart failure etc., renin-angiotensin-aldosterone system (RAAS)
Activation play an important role, wherein AngII is bioactive molecule crucial in RAAS system, AngII can by activation AT1 by
Body raises the protein expression of nadph oxidase, and inducing excessive free radical to generate leads to cell ageing；It can also be anti-by lowering
Aging gene SIRT reduces the various ways Induction of Cellular senescence such as quantity of mitochondria, characterized by the activation of RAAS system
Senescense and damnification and it is diseases related in play an important role.Therefore, in the present embodiment 1, we are thin with AngII induction
Born of the same parents' aging is that model has certain representativeness.
Telomerase and betagalactosidase activity detection are cell ageing detection means most generally acknowledged at present.Telomere is eukaryon
Special construction of the end of chromosome as cap can stablize chromosome, prevent end of chromosome fusion, protection chromosome structure
Gene and adjusting normal cell growth.Telomerase is then to aid in the molecule of telomere synthesis, keeps the length of telomere and structure steady
It is fixed, to protect chromosome.Cell aging with shortening for telomere, and when the activity of Telomerase is enough to safeguard the length of telomere
When, cell will delay senility.Beta galactosidase has an expression on human body most cells, and with the process of aging,
Expression gradually increases, therefore beta galactosidase dyeing is the index of common detection cell ageing.Our result of study hair
Existing P-3 can significantly improve cells survival rate, increase telomerase activation, reduce the cell dyeing rate of beta galactosidase, have
The effect of good anti-cell aging.
SIRT1 is also known as silent message regulatory factor relevant enzyme 1, is a kind of to go second dependent on icotinamide-adenine dinucleotide
Acylase is distributed mainly in nucleus.Research now thinks that SIRT1 is a kind of longevity gene, is current aging field
Molecules research hotspot.When cell damages, SIRT1 can be by albumen such as deacetylation p53, FOXO and Ku to mitigate
The trend of Apoptosis；In addition to this, SIRT1 can pass through control metabolism, heat consumption, fat storage, maintenance oxidation
The normal function of mitochondria stress be descended and inhibit many aspects such as inflammation to delay senescence.Research shows that inhibiting SIRT1 expression
It can promote cell ageing, cell mortality increased significantly after knocking out SIRT1；And activate the expression of its autoploid that can extend yeast
Survival period.In this research invention, it has been found that P-3 can significantly improve the expression of Sirt1 in cell, and confrontation AngII is lured
The cell ageing led.
In short, present invention research passes through detection cell survival rate, beta galactosidase dyeing, telomerase activation and anti-aging
The compound P-3 that the expression of gene Sirt1 demonstrates ocean microorganism has good anti-cell aging effect.
Claims (2)
1. ocean microorganism compound P-3 is preparing the application in antiaging agent, the following institute of the structural formula of compound P-3
Show:
2. application as described in claim 1, it is characterised in that be with renin angiotensin aldosterone system activation or telomere
The impaired senescense and damnification being characterized of enzyme/SIRT1 and diseases related.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610281054.9A CN105920580B (en) | 2016-04-28 | 2016-04-28 | Ocean microorganism compound P-3 is preparing the application in antiaging agent |
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