CN108159069A - Applications of the grifolan F2 in preparing treatment hyperlipidemia, preventing or delay atherosclerosis drug - Google Patents
Applications of the grifolan F2 in preparing treatment hyperlipidemia, preventing or delay atherosclerosis drug Download PDFInfo
- Publication number
- CN108159069A CN108159069A CN201810079130.7A CN201810079130A CN108159069A CN 108159069 A CN108159069 A CN 108159069A CN 201810079130 A CN201810079130 A CN 201810079130A CN 108159069 A CN108159069 A CN 108159069A
- Authority
- CN
- China
- Prior art keywords
- grifolan
- protein
- drug
- hyperlipidemia
- atherosclerosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of applications of grifolan F2 in preparing treatment hyperlipidemia, preventing or delay atherosclerosis drug.The present invention by grifolan F2 gavages to hyperlipidemia rats, find grifolan F2 can pole significantly decrease hyperlipidemia rats serum TC, TG and LDL C contents；Based on proteomics research, it has also been found that, grifolan F2 also by the expressing quantity that raises antioxidase SOD1, Prdx 1 and HPX and lowers the expressing quantity of HSP60 and CPPED1 the occurrence and development that prevent and delay atherosclerosis.Therefore, grifolan F2 has the function of hyperlipidemia and atherosclerosis preferably prevention and auxiliary treatment.New drug for exploitation treatment hyperlipidemia and atherosclerosis lays the first stone, and actively promotes reducing blood lipid and prevention and delays the research and development of atherosclerosis biological active constituents from natural medicines.
Description
Technical field：
The invention belongs to biomedicine fields, and in particular to a kind of grifolan F2 is preparing treatment hyperlipidemia medicine
Object prevents or delays the application in atherosclerosis drug.
Background technology：
Hyperlipidemia is a kind of common and multiple metabolic disease, and one in serum is made due to lipid material metabolic disorder
Kind or several lipid contents often show as the abnormal raised hypercholesterolemia of T-CHOL (TC), glycerine three higher than normal value
The abnormal raised hypertriglyceridemia of ester (TG) or all abnormal raised combined hyperlipidemia familials of TC and TG.Hyperlipidemia is
Mankind's cerebral apoplexy, myocardial infarction etc. is caused to disable, one of the important risk factor that lethal atherosclerosis disease occurs, according to
Estimation, the current mid-aged population dyslipidemia patient groups in China are up to 100,000,000 6 thousand ten thousand.Studies have shown that cholesterol often declines 1%,
Cardiovascular disease incidence rate declines 2 to 3%, therefore, reduces blood fat especially cholesterol levels and is considered as prevention always or prolongs
One important channel of slow cardiovascular and cerebrovascular disease progress.
Application in recent years in the researchs such as angiocardiopathy, liver diseases is more and more wider, the primary process of many diseases
Protein level is happened at most drug targets, it is also gradually popular in the research of blood lipid-lowering medicine.However almost seldom
The protein science lipid-lowering effect for having research evaluation grifola frondosus Thick many candies reports that the grifola frondosus for lacking biosystem level at present is more
Sugared pharmacological basis research, limits the exploitation of wholefood new drug.Protein group is all corresponding of the expressed generation of genome
Protein.Proteomics is to study the science of all protein in body tissue cell, is with 2-DE and mass-spectrometric technique pair
The extensive parallel separation analysis of protein progress in tissue, the change of protein expression profile during comparative sample is organized before and after the processing,
It selects the marker of sample effects expression and studies its mechanism of action.Therefore, 2-DE technologies are current proteomics researches
One of mainstream technology.Choi etc. is using 2-DE technologies detection capsaicine to the differential expression situation of obese rat hepatic protein.
Using dye or silver staining visualization protein isolate point is examined, with the protein site on computer software comparative analysis gel, select and think
Significant protein site, is identified by MALDI-TOF-MS.In order to confirm the difference of grifola frondosus regulation and control rat liver protein
Different expression, we employ the two-way polyacrylamide gels (two-dimensional in proteomics
Polyacrylamide gel electrophoresis, 2-DE) binding matrix assisted laser desorption ionisation flight time mass spectrum
(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass
Spectrometry, MALDI-TOF-MS) versatility has detected difference of the grifola frondosus to hyperlipidemia rats hepatic protein
Expression.Therefore, hypolipemic function effect of the application based on proteomics assessment of levels grifolan F2.
Invention content：
The defects of the purpose of the invention is to overcome in the prior art, provides a kind of grifolan F2 and is preparing treatment
Hyperlipidemia prevents or delays the application in atherosclerosis drug.
First purpose of the present invention is to provide grifolan F2 and is preparing the application in treating hyperlipidemia.
The treatment hyperlipidemia preferably treats hypercholesterolemicagents agents object or treatment hypertriglyceridemia
Drug.
The treatment hypercholesterolemicagents agents object is preferably with the mRNA expression water for inhibiting HMGCR, ACAT2, apoB
The treatment hypercholesterolemicagents agents object of the gentle protein expression amount for lowering HMGCR2.
The treatment hypertriglyceridemia drug is preferably controlling with the mRNA expressions for inhibiting ACC1 and FAS
Treat hypertriglyceridemia drug.
Second object of the present invention be to provide it is a kind of treat hyperlipidemia, contain a effective amount of grifolan
F2 is as active constituent.
Third object of the present invention is to provide grifolan F2 and is preparing prevention or delaying atherosclerosis drug
In application.
The prevention or delay atherosclerosis drug preferably have up-regulation antioxidase SOD1, Prdx-1 and
The prevention of the expressing quantity of HPX and the expressing quantity of downward HSP60 and CPPED1 delays atherosclerosis drug.
Fourth object of the present invention is to provide a kind of prevention or delays atherosclerosis drug, containing a effective amount of
Grifolan F2 is as active constituent.
Grifolan F2 is according to China Patent No.：ZL201310733480.8, denomination of invention：Grifolan F2
Preparation method and its function of blood sugar reduction disclosed in method be prepared, grifolan F2 average molecular weight for 4.52 ×
105D, polyoses content 95.6%, protein content 3.6%, polysaccharide is mainly by glucose, mannose, xylose, galactolipin, Ah
Draw uncle sugar composition, amino acid composition be mainly proline (Pro), serine (Ser), aspartic acid (Asp), lysine (Lys),
Alanine (Ala), glutamic acid (Glu), threonine (Thr), glycine (Gly), arginine (Arg), leucine (Leu) and figured silk fabrics ammonia
Sour (Val) is β-pyranose ring heteroglycan, and contains uronic acid.
Grifolan F2 gavages to hyperlipidemia rats, are measured each group Serum TC, TG, LDL-C by the present invention
Level extracts each group rat liver RNA, and grifolan F2 is detected to rat liver differential gene using RNA-Seq technologies
The influence of expression finds that grifolan F2 can effectively reduce hyperlipidemia rats serum TC and LDL-C contents, can also reduce
Serum TG content, mechanism are the mRNA expressions and downward by inhibiting hyperlipidemia rats HMGCR, ACAT2, apoB
The protein expression amount of HMGCS2, so as to reduce serum TC level, while by inhibiting hyperlipidemia rats ACC1 and FAS
MRNA expressions reduce the synthesis of triglycerides, so as to reduce serum TG levels.Based on proteomics research it has also been found that, ash
Tree flower polysaccharide F2 also by the expressing quantity that raises antioxidase SOD1, Prdx-1 and HPX and lowers HSP60's and CPPED1
Expressing quantity and the occurrence and development for preventing and delaying atherosclerosis.Therefore, grifolan F2 to hyperlipidemia and
Atherosclerosis has the function of preferably to prevent and auxiliary treatment.For exploitation treatment hyperlipidemia and atherosclerosis
New drug lays the first stone, and actively promotes reducing blood lipid and prevention and the research of atherosclerosis biological active constituents from natural medicines is delayed to open
Hair.
Description of the drawings：
Fig. 1 is influences of the grifolan F2 to experimental rat weight；Wherein normal group of normal representation, model representative model
Group, grifolan F2 represent grifolan F2 groups；##Represent grifolan F2 groups and the poor heteropolar significantly (P of model group
<0.01)；
Fig. 2 is influences of the grifolan F2 to lipids contents in hyperlipidemia rats serum；Wherein normal representation is normal
Group, model representative model group, grifolan F2 represent grifolan F2 groups；*Representative model group and normal group comparing difference
Significantly (P<0.05),**Representative model group is with normally organizing poor heteropolar significantly (P<0.01),##Represent grifolan F2 groups with
The poor heteropolar significantly (P of model group<0.01)；
Fig. 3 is the 2-DE gel photographs of protein in rat liver tissue；A：Normal group, B：Model group, C：Grifola frondosus is more
Sugared F2 groups；
Fig. 4 is the influence that grifolan F2 expresses hyperlipidemia rats liver GAP-associated protein GAP mRNA；Wherein control
Normal group is represented, hyperlipidemia representative model groups, Grifola frondosa represent grifolan F2 groups；*It represents
Model group and normal group comparing difference significantly (P<0.05),**Representative model group is with normally organizing poor heteropolar significantly (P<
0.01),##Represent grifolan F2 groups and the poor heteropolar significantly (P of model group<0.01)；
Fig. 5 is grifolan F2 reducing blood lipid mechanism figures；Wherein,Compared with model group, grifolan F2 groups mRNA
Expression and enzyme concentration up-regulation；Compared with model group, grifolan F2 group mRNA expressions and enzyme concentration are lowered；Compared with model group, the protein expression amount up-regulation of grifolan F2 groups；Compared with model group, grifolan F2
The protein expression amount of group is lowered.
Specific embodiment：
Following embodiment is the further explanation to the present invention rather than limitation of the present invention.
Embodiment 1：
Grifolan F2 is according to China Patent No.：ZL201310733480.8, denomination of invention：Grifolan F2's
What the method disclosed in preparation method and its function of blood sugar reduction was prepared, grifolan F2 average molecular weight for 4.52 ×
105D, polyoses content 95.6%, protein content 3.6%, polysaccharide is mainly by glucose, mannose, xylose, galactolipin, Ah
Draw uncle sugar composition, amino acid composition be mainly proline (Pro), serine (Ser), aspartic acid (Asp), lysine (Lys),
Alanine (Ala), glutamic acid (Glu), threonine (Thr), glycine (Gly), arginine (Arg), leucine (Leu) and figured silk fabrics ammonia
Sour (Val) is β-pyranose ring heteroglycan, and contains uronic acid.
First, pharmacological evaluation
1.1 lipid-lowering test
1.1.1 the induction and administration of hyperlipemia model of rats
Using SPF grades of male and healthy SD rats, 6 week old, weight 120g or so, label, all rats all press SPF grades of animals
Sub-cage rearing, free water and feed keep laboratory environment health, well-ventilated, and temperature is 18-22 DEG C, and relative humidity is
50%-60%, illumination in 12 hours and the cycle at night.Adaptability is fed after a week, all rats is randomly divided into 3 groups, normally
Group, model group, grifolan F2 groups, in addition to normal rats continue feeding normal diet, other groups of rats are all changed to feeding
High cholesterol diet high in fat.Avoidance mode is taken to be administered：High cholesterol diet modeling i.e. high in fat is carried out at the same time with administration.Often it is preordained
When it is primary to the corresponding grifolan F2 of grifolan F2 group rat oral gavages, every rat oral gavage dosage is 100mg/
KgBW, normal group and the isometric distilled water of model group rats gavage.All rats give conventional free water and feed,
Experiment carries out 4 weeks.During the experiment weighs weekly a weight, observes rat daily and ingests, autonomic activities situation.
1.1.2 the materials and lipid determination of experimental animal
After last day gavage, after all Rat Fasts can't help water 12 hours, disconnected neck takes blood, at room temperature blood natural coagulation
20 minutes, 3000r/min was centrifuged 20 minutes, carefully collected supernatant, that is, serum, and serum is deposited in 4 DEG C of refrigerators every other day
With taking dissection rat of hastening after blood, take out liver rapidly, the blood stains cleaned on liver with physiological saline are simultaneously blotted above with filter paper
Physiological saline, be then divided into two parts and be stored in cryopreservation tube, cryopreservation tube be placed in it is quick-frozen in liquid nitrogen after deposit in -80 DEG C of ice
It is spare in case.
The experiment flow of 1.2 dielectrophoresis (2-DE)
1.2.1 the extraction preparation of rat liver protein and quantifying for total protein
1.2.1.1 the extraction of protein
The rat liver that step 1.1.2 is preserved is taken out from -80 DEG C of refrigerators and is placed it in the mortar of Liquid nitrogen precooler, it is fast
Speed is ground at once after pouring into liquid nitrogen, and liver is easily ground at this time, until liver becomes powdered, is without apparent particle uniformly
It can.Then the lysate of 800 μ L is added in mortar, abundant dissolved powders are simultaneously transferred to 1.5mL eppendorf (EP) centrifugations
Guan Zhong, then add mortar of lysate brush of 400 μ L, it is transferred in identical EP pipes, 4 DEG C, 12000r/m, centrifuges 10min, receive
Collect supernatant, supernatant is the protein liquid extracted.
1.2.1.2 the purifying of protein
After 5 times of vol acetones of precooling are added in the protein liquid of extraction, be vortexed concussion mixing, is placed in -20 DEG C overnight.
10000g, 4 DEG C of centrifugation 10min, abandons supernatant, 3 times of vol acetones that precooling is added in precipitation are washed repeatedly twice, at -20 DEG C
Under, 2-3 hours, 10000g centrifugation 15min abandoned supernatant, the protein precipitation being collected into drying at room temperature about 5min until
After acetone is evaporated completely completely, it is heavy to re-dissolve to add in 400 μ L aquations sample-loading buffers (not containing bromophenol blue and Bio-Lyte)
Form sediment, -80 DEG C freeze it is spare.
1.2.1.3 protein quantifies
The non-interference type quantification of protein kit produced using Shanghai life work quantifies protein after purification, and
According to the operating instruction on kit, quantitation curves are made, protein concentration is finally calculated according to standard curve.
1.2.1.4 (2-DE) experimental procedure：
First balances → the second to polyacrylamide gels → fixation and dyeing → gel to isoelectric focusing → adhesive tape
Image scanning analysis is handled with in-gel digestion
1.2.1.5 proteomic image and protein database search
Peptide fragment is by after enzymolysis, digestion, using German Bruker Dalton Autoflex speedTMMass spectrograph carries out it
Mass Spectrometric Identification.Operating parameter：Quality of scanning ranging from 700-3200Da, accelerating potential 20000V, repetition rate 200Hz,
Optimum quality resolution ratio is 1500Da, optical maser wavelength 355nm, then after being compared with standard peptide mixer, carries out signal collection.It utilizes
Identification signal peak and flexAnalysis filtering peak bases.The I and II mass spectrometric data of acquisition is integrated into BioTools
In Mascot, Uniprot databases are then searched for, identify the related protein of all matching rattus norregicus species
And inquire possible biological function.Undefined protein can be predicted its structure function by NCBI websites.
The regulating and controlling effect that 1.3 quantitative fluorescent PCRs evaluation grifolan F2 expresses lipid metabolism key enzyme mRNA
1.3.1 the extraction of total serum IgE
The cryopreservation tube equipped with rat liver that step 1.1.2 is preserved is taken out from -80 DEG C of refrigerators, adds in 1mL RNAiso
Plus is fully homogenized with homogenizer；Homogenate is transferred in centrifuge tube, is stored at room temperature 10 minutes, 12,000g 4 DEG C of centrifugations 5
Minute；Careful Aspirate supernatant is simultaneously moved into new centrifuge tube, and 200 μ L chloroforms (RNAiso Plus are added in into supernatant
1/5 volume), cover tightly centrifuge tube lid, mix to emulsifying soln and be creamy white；Be stored at room temperature 15 minutes, 12,000g 4 DEG C from
The heart 15 minutes；Careful light and handy taking-up centrifuge tube, upper strata from centrifuge：Colourless supernatant (containing RNA), middle level：White egg
White (for DNA), lower floor：Coloured organic phase, the colourless supernatant of gentle aspiration are simultaneously transferred in another new centrifuge tube,
1mL isopropanols (1 times of volume RNAiso Plus) are added in into supernatant, after the abundant mixing of the centrifuge tube that turns upside down, are stood at room temperature
10 minutes；12,000g 4 DEG C centrifuge 10 minutes, abandon supernatant, and 75% ethyl alcohol of 1mL is added in into precipitation (with RNAiso
Plus equivalent), 7,500g 4 DEG C of centrifugations carefully discard supernatant after five minutes；Centrifuge tube lid is opened, drying at room temperature precipitates a few minutes,
Add in the DEPC water dissolutions precipitation of 500 μ L.Take 2 μ L extract total serum IgE stoste, with life science it is ultraviolet/visible spectrophotometer
RNA concentration is surveyed, and RNA concentration is adjusted to debita spissitudo.
1.3.2cDNA synthesis
It is consistent to adjust each sample total RNA content, uses PrimeScriptTMRT Master Mix reverse transcription reagent box synthesizes
CDNA, reverse transcription system such as the following table 1.Reverse transcription program is：37 DEG C, 15 minutes (reverse transcription reaction)；85 DEG C, (reverse transcription in 5 seconds
The inactivation reaction of enzyme), 4 DEG C of storages.Obtained cDNA reaction solutions are added to the real-time fluorescence quantitative PCR reaction system of next step
In, addition does not exceed 1/10 (V/V) amounts of PCR reaction volumes.
1 reverse transcription system of table
1.3.3 real-time fluorescence quantitative PCR (Real-Time PCR, RT-PCR)
UsingPremix Ex TaqTM(Tli RNaseH Plus) kit carries out Real-Time PCR (RT-
PCR it) reacts.Generation double-stranded DNA is reacted by RT-PCR, this double-stranded DNA is combined with SYBR Green I sends out fluorescence, passes through inspection
It surveys in PCR reaction solutionThe fluorescence signal intensity of Green I achievees the purpose that monitor PCR product amplification amount, you can with
Relative quantification is carried out to target gene.RT-PCR reaction systems such as the following table 2, program are as follows：95 DEG C of pre-degenerations 30 seconds, 95 DEG C of denaturation
5 seconds, 58 DEG C were annealed 30 seconds, and 72 DEG C extend 30 seconds, totally 40 cycles, followed by solubility curve, 95 DEG C, and 15 seconds, 60 DEG C, 15
Second, 95 DEG C, 15 seconds.HMGCR, ACAT2, apoB, CYP7A1, FAS, ACC gene and house-keeping gene glyceraldehyde 3-phosphate dehydro-genase
The primer sequence of (Glyceraldehyde-3-phosphate dehydrogenase, GAPDH) gene is shown in Table 3.Every part of sample
Twice PCR reaction is done to repeat.
Table 2SYBR real-time PCR reaction systems
Table 3SYBR real-time PCR react primer
2nd, experimental result
1. the effect for reducing blood fat of grifolan F2
Influences of the 1.1 grifolan F2 to experimental rat weight
, from figure 1 it appears that feeding the weight of i.e. first week after a week in adaptability, each group rat body weight does not all have for we
There is apparent difference；When gavage grifolan F2 after two weeks be third week weight, compared with model group, grifolan F2
Group rat body weight pole is remarkably decreased (P<0.01), estimation has just started the last fortnight of gavage, and rat is not very to grifolan F2
It adapts to, affects food-intake, lead to weight loss, but be the weight of the 5th week at the end of to experiment, compared with model group,
Each group rat body weight is all not significantly different, and illustrates the progress with experiment, and rat slowly adapts to grifolan F2
, therefore weight is restored again.
Influences of the 1.2 grifolan F2 to lipids contents in hyperlipidemia rats serum
Grifolan F2 is continuously to experimental rat gavage after 4 weeks, as shown in Figure 2, compared with model group, grifolan
F2 can pole significantly decrease hyperlipidemia rats serum TC, LDL-C contents and restore it normal level even lower than just
Ordinary water puts down (P<0.01), and it also can pole significantly decrease hyperlipidemia rats serum TG content and restore it with just
Often organize level about the same.
2. the experiment of dielectrophoresis (2-DE)
2.1 2-DE image analysis results
Fig. 3 illustrate normal group, model group, in 3 group rat liver tissues of grifolan F2 groups protein 2-DE
Gel image scanning photo finds that every gel figure averagely contains after being analyzed by 7.0 softwares of ImageMaster 2D Platinuim
The protein spots of 1400 or so analyze the protein difference expression point (differential expression between arbitrary two groups with software calculating
Both greater than 1.5 times or more of amount), the results show that compared with model group, there is the expression quantity up-regulation of 98 protein spots in normal group
, compared with model group, the expression quantity for also having 80 protein spots in grifolan F2 groups raises, in this two groups of expression quantity
In the protein spots all raised, 23 protein spots are to repeat；Meanwhile compared with model group, there are 59 eggs in normal group
The expression quantity of white matter point is lowered, and compared with model group, is also had under the expression quantity of 74 protein spots in grifolan F2 groups
It adjusts, in the protein spots all lowered in this two groups of expression quantity, 17 protein spots are to repeat.Therefore, two histone matter
It is exactly our interested protein spots to repeat point (23+17=40), using in-gel digestion processing method that this 40 senses are emerging
The protein spots of interest are taken off from gel, and after enzymolysis, digestion, Mass Spectrometric Identification is carried out to them using MALDI-TOF-MS technologies,
Except in the case of deducting some qualification results and being same or like protein, finally, the qualification result of 20 protein has been obtained.
The Mass Spectrometric Identification result of 2.2 differential protein particles
Successful identification goes out 20 protein, this 20 protein are protein site NO.1 respectively：3- hydroxy-3-methyl glutaryls
CoA synthase (Hydroxy methylglutaryl-CoA synthase, HMGCS2), protein site NO.2：Long chain fatty acids
CoA ligase 1 (Long-chain-fatty-acid--CoA ligase 1, ACSL1), protein site NO.3：Alpha-Methyl acyl group
CoA racemase (Alpha-methylacyl-CoA racemase, AMACR), protein site NO.4：Peroxidase
(Peroxiredoxin-1, Prdx-1), protein site NO.5：Superoxide dismutase (Superoxide dismutase,
SOD1), protein site NO.6：Hemopexin (Hemopexin, HPX), protein site NO.7：S-adenosylmethionine synthase-
1 (S-adenosylmethionine synthase isoform type-1, Mat1a), protein site NO.8：Glycine betaine-homotype
Methyltransferase 1 (Betaine--homocysteine S-methyltransferase 1, Bhmt), protein site
NO.9：HSP 60 (60kDa heat shock protein, HSP60), protein site NO.10：Serine/Soviet Union's ammonia
Acid albumin phosphatase (Serine/threonine-protein phosphatase, CPPED1), protein site NO.11：Amino first
Acyl phosphate synthase (Carbamoyl-phosphate synthase, Cps1), protein site NO.12：Sarcosine dehydrogenase
(Sarcosinedehydrogenase, Sardh), protein site NO.13：Actin (ctin, Actg1), protein site NO.14：
Keratin (Keratin, type I cytoskeletal 18, Krt18), protein site NO.15：Cathepsin B
(Cathepsin B, Ctsb), protein site NO.16：ATP synthetase alphas subunit (ATP synthase subunit alpha,
Atp5a1), protein site NO.17：9 type of proteasome beta subunit (Proteasome subunit beta type-9, Psmb9), egg
White point NO.18：3 chains of tropomyosin α (Tropomyosin alpha-3chain, Tpm3), protein site NO.19：Glutamic acid half
Cystine connection enzyme adjustment subunit (Glutamate--cysteine ligase regulatory subunit, Gclm), albumen
Point NO.20：Cytochrome b5 (Cytochromeb5, Cyb5a).
The protein of identification passes through proteomics server UniProtKBC (http://www.uniprot.org/),
Classification analysis is carried out to above-mentioned protein, discovery there are 3 protein spots to be related to lipid metabolism, and 5 protein spots are related to lipid oxygen
Change, 5 protein spots are related with amino acid metabolism, and 2 protein spots participate in energetic supersession, and 3 protein spots are with cytoskeleton
Related, 2 protein spots are with protein degradation correlation.Wherein, compared with model group, normal group and grifolan F2 group all on
The protein spots that mileometer adjustment reaches have 13 (AMACR, Prdx-1, SOD1, HPX, Mat1a, Bhmt, Cps1, Sardh, Actg1,
Krt18, Ctsb, Atp5a1, Psmb9), express the protein spots of downward have 7 (HMGCS2, ACSL1, HSP60, CPPED1,
Tpm3、Gclm、Cyb5a).Specific detailed Information in Mass Spectra in relation to this 20 protein spots is referring to the following table 4.
Table 4MALDI-TOF-MS identifies rat liver albumen
Note：↑：Compared with model group, the expressing quantity of normal group and grifolan F2 groups raises；↓：With model group phase
Than the expressing quantity of normal group and grifolan F2 groups is lowered
3. realtime fluorescent quantitative PCR experiment result (RT-PCR)
As shown in Figure 4, HMGCR is important rate-limiting enzyme during Biosynthesis of cholesterol, it decides synthetic reaction
Rate also determines the main function position of the cholesterol biosynthesis amount and the extraneous internal cholesterol de novo formation of regulation and control in liver
Point, the action target spot of even more current most important hyperlipidemia clinical medicine, grifolan F2 group rat liver HMGCR's contains
Amount and mRNA expressions but significantly reduce.Therefore, we detect TC, LDL- in grifolan F2 group rat blood serums
The reduction of C levels, it is possible the reason of grifolan F2 inhibit the mRNA expressions of HMGCR and reduce containing for HMGCR
Amount；Figure 4, it can be seen that compared with normal group, the mRNA expressions of CYP7A1 extremely significantly drop in model group rats liver
Low (P<0.01)；Thus Fig. 4 is it is found that compared with normal group, and model group rats liver ACC1mRNA expressions are without significance difference
It is different, but FAS mRNA expressions significantly increase (P<0.05).Compared with model group, grifolan F2 gavages are to hyperlipemia
After disease rat, the mRNA expressions of liver ACC1 and FAS significantly reduce or even be all reduced to than normal rat ACC1 and
FAS mRNA expressions are also low；Compared with normal group, ACAT2 the and apoB contents of model group rats extremely significantly increase (P<
0.01), compared with model group, grifolan F2 gavages significantly reduce (P to rat, liver ACAT2 and apoB content pole<
0.01).The content and mRNA expressions of grifolan F2 group rat livers apoB but significantly reduces.Therefore, grifola frondosus
Polysaccharide F2 may reduce LDL in serum and LDL-C be horizontal, prevent artery athero- by inhibiting the expression of apoB
The occurrence and development of hardening.
4. combine the reducing blood lipid mechanism of the experiment of qPCR and 2-DE
The main source of cholesterol is itself synthesis of liver, and acetyl coenzyme A ultimately generates courage by multistep enzymatic reaction
Sterol, In this cholesterol metabolic approach, HMGCR is the rate-limiting enzyme of entire metabolic response, controls courage
The synthesis quantity of sterol, and HMGCS2 is the enzyme for catalyzing and synthesizing 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC), if
The quantity of HMGCS2 is reduced, and catalyzing and synthesizing the quantity of HMGC can also be reduced, then the substrate quantity of HMGCR just tails off therewith, most
The synthetic quantity of cholesterol is caused to reduce eventually, therefore, the synthetic quantity of cholesterol can be reduced by lowering the expressing quantity of HMGCS2.This reality
The HMGCS2 identified with MALDI-TOF-MS is tested, the protein expression amount in grifolan F2 group rat livers will be less than
Model group, this is also just explained it is observed that grifolan F2 groups Serum TC is significantly lower than model group serum TC
Reason.
Triglycerides be by aliphatic acid and glycerine with reference to and generate, acetyl coenzyme A is as starting material, in a series of enzymes
Under the action of ultimately generate triglycerides, In this triglyceride metabolic pathway, ACSL1 is that free fatty is made to be converted into acyl
The important enzyme of coacetylase, finally, three ester of acyl coenzyme A and diacylglycerol synthetic glycerine.If the expression quantity of ACSL1 is raised
Words, the synthetic quantity of acyl coenzyme A will increase, and triglycerides synthetic quantity is consequently increased.In similar research, it was also found that
Toll-like receptor agonists increase the synthesis of TG by improving the expression quantity of ACSL1, finally make mouse and people
Class macrophage takes in many TG.The ACSL1 that this experimental identification goes out, the albumen in grifolan F2 group rat livers
Matter expression quantity will be less than model group, this is also just explained it is observed that grifolan F2 group rat blood serums TG is significantly lower than
The reason of model group serum TG.
5. grifolan F2 reducing blood lipid mechanisms
Reducing blood lipid mechanism Fig. 5 the result shows that, grifolan F2 regulates and controls the metabolism of cholesterol to realize drop by 4 approach
The biosynthesis of cholesterol is reduced in the purpose of low serum TC, LDL-C, (1) by lowering the expression quantity of HMGCR；(2) under
It adjusts the expression quantity of ACAT2 and reduces the absorption of cholesterol；(3) assembling of LDL is reduced by lowering the expression quantity of apoB；(4)
Increase the conversion of cholesterol by raising the expression quantity of CYP7A1.These are the result shows that grifolan F2 is consolidated by regulating and controlling courage
The expression of alcohol key enzyme and play norcholesterol effect, therefore they are the natural functions of auxiliary treatment hypercholesterolemia
Food.
Claims (8)
1. grifolan F2 is preparing the application in treating hyperlipidemia.
2. application according to claim 1, which is characterized in that the treatment hyperlipidemia is treatment high cholesterol
Mass formed by blood stasis drug or treatment hypertriglyceridemia drug.
3. application according to claim 2, which is characterized in that the treatment hypercholesterolemicagents agents object is with inhibition
The treatment hypercholesterolemicagents agents of the mRNA expressions of HMGCR, ACAT2, apoB and the protein expression amount of downward HMGCR2
Object.
4. application according to claim 2, which is characterized in that the treatment hypertriglyceridemia drug is with suppression
The treatment hypertriglyceridemia drug of the mRNA expressions of ACC1 and FAS processed.
5. a kind of treat hyperlipidemia, which is characterized in that it contains a effective amount of grifolan F2 as active constituent.
6. applications of the grifolan F2 in preparing prevention or delaying atherosclerosis drug.
7. application according to claim 6, which is characterized in that the prevention delays atherosclerosis drug as tool
There is the pre- of the expressing quantity of the expressing quantity for raising antioxidase SOD1, Prdx-1 and HPX and downward HSP60 and CPPED1
Prevent or delay atherosclerosis drug.
8. a kind of prevention delays atherosclerosis drug, which is characterized in that it contains a effective amount of grifolan F2 and makees
For active constituent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810079130.7A CN108159069A (en) | 2018-01-26 | 2018-01-26 | Applications of the grifolan F2 in preparing treatment hyperlipidemia, preventing or delay atherosclerosis drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810079130.7A CN108159069A (en) | 2018-01-26 | 2018-01-26 | Applications of the grifolan F2 in preparing treatment hyperlipidemia, preventing or delay atherosclerosis drug |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108159069A true CN108159069A (en) | 2018-06-15 |
Family
ID=62516079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810079130.7A Pending CN108159069A (en) | 2018-01-26 | 2018-01-26 | Applications of the grifolan F2 in preparing treatment hyperlipidemia, preventing or delay atherosclerosis drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108159069A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008089581A1 (en) * | 2007-01-26 | 2008-07-31 | Merck Frosst Canada Ltd. | Fused aromatic ptp-1b inhibitors |
| CN103724445A (en) * | 2013-12-25 | 2014-04-16 | 广东省微生物研究所 | Preparation method and blood sugar lowering function of Grifola frondosa polysaccharide F2 |
-
2018
- 2018-01-26 CN CN201810079130.7A patent/CN108159069A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008089581A1 (en) * | 2007-01-26 | 2008-07-31 | Merck Frosst Canada Ltd. | Fused aromatic ptp-1b inhibitors |
| CN103724445A (en) * | 2013-12-25 | 2014-04-16 | 广东省微生物研究所 | Preparation method and blood sugar lowering function of Grifola frondosa polysaccharide F2 |
Non-Patent Citations (2)
| Title |
|---|
| CHUN XIAO等: "Hypoglycemic effects of Grifola frondosa (Maitake) polysaccharides F2 and F3 through improvement of insulin resistance in diabetic rats", 《FOOD & FUNCTION》 * |
| WEI QIU等: "Hepatic PTP-1B Expression Regulates the Assembly and Secretion of Apolipoprotein B–Containing Lipoproteins", 《DIABETES》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gruhlke et al. | The human allicin-proteome: S-thioallylation of proteins by the garlic defence substance allicin and its biological effects | |
| Bonenfant et al. | Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry | |
| Pahlich et al. | Protein arginine methylation: Cellular functions and methods of analysis | |
| Guo et al. | Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS | |
| Gueugneau et al. | Proteomics of muscle chronological ageing in post-menopausal women | |
| Tevatia et al. | The taurine biosynthetic pathway of microalgae | |
| Kizawa et al. | Specific citrullination causes assembly of a globular S100A3 homotetramer: a putative Ca2+ modulator matures human hair cuticle | |
| JPH0667827B2 (en) | Maillard reaction inhibitor | |
| Shen et al. | Response of the critically endangered Przewalski's gazelle (Procapra przewalskii) to selenium deprived environment | |
| Robinson et al. | Lysine and arginine protein post-translational modifications by enhanced DIA libraries: quantification in murine liver disease | |
| Wang et al. | Quantitative Proteomics Reveals a Novel Role of Karyopherin Alpha 2 in Cell Migration through the Regulation of Vimentin–pErk Protein Complex Levels in Lung Cancer | |
| Aminin et al. | Immunomodulatory effects of holothurian triterpene glycosides on mammalian splenocytes determined by mass spectrometric proteome analysis | |
| Nikitashina et al. | Metabolic adaptation of diatoms to hypersalinity | |
| Gaudio et al. | Chemoproteomic fishing identifies arzanol as a positive modulator of brain glycogen phosphorylase | |
| Khaleel et al. | Disturbance of Arginase Activity and Nitric Oxide Levels in Iraqi Type 2 Diabetes Mellitus | |
| Tsai et al. | Garcinia multiflora inhibits FPR1-mediated neutrophil activation and protects against acute lung injury | |
| Hepowit et al. | Enhancing lifespan of budding yeast by pharmacological lowering of amino acid pools | |
| Yang et al. | Absolute quantification of E1, ubiquitin-like proteins and Nedd8–MLN4924 adduct by mass spectrometry | |
| US20060024234A1 (en) | Protein markers for pharmaceuticals and related toxicity | |
| Lakowski et al. | Arginine methylation in yeast proteins during stationary-phase growth and heat shock | |
| Mushtaq et al. | N-acetyl cysteine inhibits endothelin-1-induced ROS dependent cardiac hypertrophy through superoxide dismutase regulation | |
| Young et al. | Identification of adenine modulating AMPK activation in NIH/3T3 cells by proteomic approach | |
| CN108159069A (en) | Applications of the grifolan F2 in preparing treatment hyperlipidemia, preventing or delay atherosclerosis drug | |
| Reed et al. | Transcriptome and metabolome changes induced by bitter melon (Momordica charantia)-intake in a high-fat diet induced obesity model | |
| Cooke et al. | Evidence for attenuated cellular 8-oxo-7, 8-dihydro-2′-deoxyguanosine removal in cancer patients |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180615 |