CN110448563A - Application of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose in preparation prevention and treatment osteoporosis drug - Google Patents
Application of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose in preparation prevention and treatment osteoporosis drug Download PDFInfo
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- CN110448563A CN110448563A CN201910653874.XA CN201910653874A CN110448563A CN 110448563 A CN110448563 A CN 110448563A CN 201910653874 A CN201910653874 A CN 201910653874A CN 110448563 A CN110448563 A CN 110448563A
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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
- A61K31/7024—Esters of saccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physical Education & Sports Medicine (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Rheumatology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses application of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose in preparation prevention and treatment osteoporosis drug.The survival rate of osteoblast under oxidative stress status can be improved in 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, restores mitochondria dysfunction, inhibits Apoptosis and then generates protective effect to osteoblast.I.e. 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses can be finally reached the purpose for the treatment of osteoporosis by inhibiting TNF-a Induced Apoptosis in Osteoblasts.
Description
Technical field
The invention belongs to osteoporosis Prevention Technique fields, and in particular to 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose exists
Application in preparation prevention and treatment osteoporosis drug.
Background technique
Osteoporosis (Osteoporosis, OP) is the most common skeletal diseases, is that one kind is low with bone amount, bone tissue is micro-
Structural failure causes bone brittleness to increase, the systemic osteopathy that fracture is characterized easily occurs.OP not only causes patient and society huge
Big financial burden, and middle-aged and the old's quality of life is seriously reduced, jeopardize the health of the middle-aged and the old.Bone metabolism is mainly with bone weight
The mode built carries out.Bone remoulding includes mediating bon e formation and origin derived from marrow hemopoiesis by osteoblast (Osteoblast, OB)
The osteoclast (Osteoclast, OC) of system mediates bone resorption.It has been more than the bon e formation of OB when the bone resorption ability enhancing of OC
It is horizontal, it will to induce OP.
Clinically the therapeutic agent of OP can be divided into following a few classes according to its mechanism of action: (1) antagonism bone resorption drug: female
Hormone, calcitonin (CT) and diphosphonate etc.；(2) Bone formation drug: vitamin K and fluoride etc.；(3) bone mine class drug:
Calcium agent, vitamin D and boron preparation etc..These drugs are there are different degrees of toxic side effect, and that there are therapeutic effects is low for prolonged application
The problems such as lower and skeletonization poor quality, therefore finding new OP therapeutic agent is global difficult point and hot spot.Chinese medicine is " whole
Under idea " and the guidance of the theory of medicine of diagnosis and treatment based on an overall analysis of the illness and the patient's condition, to unique advantage is highlighted in the treatment and prevention of OP, cause both at home and abroad
The extensive concern of scholar.
1,2,3,4,6- Penta-O-galloyl-D-glucopyranose is that one kind has no toxic side effect tannin class native compound, is contained in Chinese gall
Measure highest, followed by cortex moutan and Chinese herbaceous peony.There are no about 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose in currently available technology
In the report of prevention and treatment osteoporosis.
Summary of the invention
The technical issues of solution: the purpose of the present invention is in 1,2,3,4, the 6- existing pharmacological action of Penta-O-galloyl-D-glucopyranose
On the basis of, it is furtherd investigate by many experiments and develops its pharmacological effect, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses are provided and are existed
The new application of anti-curing osteoporosis, further to promote its medical value.
Technical solution: application of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose in preparation prevention and treatment osteoporosis drug.
The osteoporosis is a kind of with bone amount reduction, bone micro-structure destruction, and bone brittleness is caused to increase, easily fracture
The systemic osteopathy being characterized.
Further, the osteoporosis includes primary osteoporosis and secondary osteoporosis.
Further, the primary osteoporosis is that female postmenopausal osteoporosis or old degenerative sclerotin are dredged
Loose disease.
Further, the secondary osteoporosis is by incretion metabolism disease, connective tissue disease, kidney disease
Disease, disease of digestive tract and/or drug-induced osteoporosis.
Further, the application is that auxiliary material system will be subjected in 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose and pharmacy
At pill, tablet, capsule, granule or freeze dried powder.
1,2,3,4,6- Penta-O-galloyl-D-glucopyranose is preparing the application in promoting bone growing drug.
The drug of anti-curing osteoporosis, effective component include 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses.
In the drug of the anti-curing osteoporosis, the minimum effective concentration of 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose
It is 10-9M。
The utility model has the advantages that 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose can be by anti-oxidation stress, so that anti-osteoblast withers
It dies, is finally reached the purpose for the treatment of osteoporosis.To which 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose can be used for preparing prevention and treatment
The drug of osteoporosis.
Detailed description of the invention
Fig. 1 is that 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses dredge zebra fish sclerotin caused by prednisolone in embodiment 1
The influence of pine, wherein a is zebra fish skull in-seam images, b is zebra fish vertebra stock area statistics figure.
Fig. 2 is influence of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranoses to osteoblast differentiation in embodiment 2, and wherein a is
MC3T3-E1 breaks up the 7th, 14,21 day Alizarin red staining figure, b is that BMSC breaks up the 14th day Alizarin red staining figure.
Fig. 3 is 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses in embodiment 3, embodiment 4 and embodiment 5 to oxidative stress shape
The influence of the anti-thin apoptosis of MC3T3-E1 under state.Wherein a be MTT detect PGG to the survival rate of MC3T3-E1 under oxidative stress,
B is the anti-MC3T3-E1 Apoptosis figure of flow cytometer detection PGG, c is apoptosis rate statistical chart, d is WB detection apoptosis-related protein Bcl2
It is Bcl2/Bax ratio with Bax figure, e.
Fig. 4 be embodiment 6, embodiment 7, in embodiment 81,2,3,4,6- Penta-O-galloyl-D-glucopyranoses to oxidative stress shape
The influence of MC3T3-E1 mitochondrial function is as a result, wherein a is flow cytometer detection mitochondrial membrane potential figure, b is mitochondria under state
Film potential statistical chart, c are ATP content detection, d is that ROS level, e are ROS level statistic figure in flow cytometer detection MC3T3-E1.
Specific embodiment
Technical solution of the present invention is described further combined with specific embodiments below.
Of the present invention 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, structural formula is as shown in formula I:
1,2,3,4,6- Penta-O-galloyl-D-glucopyranose at present can be by the conventional Chinese medicine containing the ingredient, such as Chinese gall, male
It extracts and obtains in the plants such as the root bark of tree peony, Radix Paeoniae Alba, radix paeoniae rubra, since 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose is in many natural plants
All exist in object, therefore in an embodiment of the present invention, 1 used as effective component, 2,3,4,6- five nutgall acyl grapes
Sugar, by using source it is more abundant it is naturally occurring in the form of 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses advantageously.Below
1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses used by embodiment are purchase commercial product, and CAS NO:14937-32-7 is produced
Product article No.: lw-00008, brand: good Wei, Chinese name: 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose, English name: 1,2,3,4,6-
penta-O-galloyl-β-d-glucose。
Embodiment 1
Influence of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose to zebra fish osteoporosis caused by prednisolone.
Experimental animal: zebra fish AB strain is purchased from triumphant base biology Co., Ltd；Circadian rhythm: daytime: night=14h:10h；Temperature
Degree: (28.5 ± 0.5) DEG C.
Reagent and drug: prednisolone is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd；Disodium etidronate is purchased from Shanghai
Yuan Ye Biotechnology Co., Ltd.
Instrument: Leica M205FA body formula fluorescence microscope etc..
Grouping and administration: it is divided into control group control (0.2%DMSO), model group model (25 μM of prednisolones), sun
Property medicine group DE (15 μ g/mL etidronate disodium), 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses (10-10、10-9With 10-8M)
Experimental method: starting at after fertilization 3 days (dpf), is handled zebra fish juvenile fish continuous 6 days with 25 μM of prednisolones.In
Disodium etidronate (DE is used after modeling 2 days；15 μ g/mL) it is used as positive drug, or 1 is given, 2,3,4,6- five nutgall acyl Portugals
Grape sugar (10-10、10-9With 10-8M it) treats.In order to detect the doped calcium in zebra fish juvenile fish, by live fish in 0.2% calcein
It is middle to be incubated for 1 hour, juvenile fish is anaesthetized with 0.02% tricaine, and be imaged with Leica M205FA body formula fluorescence microscope.Entire
Identical magnifying power, time for exposure, gain and contrast are used in experiment, obtain image.
Fig. 1 is bone amount imaging in zebra fish skull.The results show that 25 μM of prednisolone groups are compared to the blank group, zebra fish
Bone amount, which generates, in skull is reduced.With 25 μM of prednisolone group ratios, etidronate disodium (15 μ g/mL) is organized bone amount in skull and is increased,
10-10、10-9With 10-81,2,3,4,6- Penta-O-galloyl-D-glucopyranose of M is similar to the therapeutic effect of etidronate disodium.The result
Show that 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses can treat the zebra fish bon e formation inhibition of prednisolone induction.
Embodiment 2
Influence of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose to osteoblast differentiation.
Cell: preosteoblast system MC3T3-E1 is purchased from upper marine academy of sciences's cell bank, between the marrow in rat femur source
Mesenchymal stem cells (BMSCs) self-carry.
Reagent and drug: beta-glycerophosphate is purchased from Sigma；Dexamethasone is purchased from Sigma；Ascorbic acid is purchased from Sigma.
Instrument: Leica Stereo microscope etc..
Grouping and administration: it is divided into blank group (0.1%DMSO), 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose groups (10,50 and
100μM)。
Experimental method: the α-MEM culture solution of FBS is inactivated with 10% heat is contained, while pressing the thinner ratio of 1:100 in culture solution
Add it is dual anti-, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivated.Every two days replacement culture mediums.When cell is long extremely
When 90% or so, with trypsin digestion cell into centrifuge tube.3min is centrifuged with 1000rpm.α-MEM culture solution is used after cell centrifugation
It is resuspended and counts.Osteoblast is seeded in 12 orifice plates (1 × 105A cells/well) in, it is trained with the α-MEM containing 10% heat inactivation FBS
Nutrient solution adds 10mM beta-glycerophosphate, 10nM dexamethasone, 50mg/mL ascorbic acid, while existing by the thinner ratio of 1:100
Dual anti-, configuration skeletonization induction liquid, in 37 DEG C, 5%CO is added in culture solution2, saturated humidity incubator in cultivated.
Blank group (0.1%DMSO) and 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose groups (10,50 and 100 μM) induce liquid with Osteoblast Differentiation
Culture 7~21 days, Alizarin red staining observe the Mineral nodules that osteoblast differentiation is formed, obtain coloration result.
Alizarin red staining detects calcium scoring: by cell after the taking-up of experimental period point, carefully cleaning 2 times with PBS, every time
1min, when cleaning, are careful not to wash out cell calcium scoring.After cell cleaning, 4% paraformaldehyde of 3mL is added to every hole,
It is placed in draught cupboard and fixes 15min.Fixer is discarded, the PBS that 3mL is added in every hole is cleaned 2 times, each 1min.Alizarin red is contaminated
Cell culture well is added in liquid, then every hole 1mL is stored at room temperature 15min, carries out Alizarin red staining.Alizarin is discarded after dyeing
Red dye liquor, and extra dye liquor is rinsed out with tri-distilled water, tissue culture plate is then put into 30min in 60 DEG C of insulating boxs and is dried.Carefully
White calcium scoring in born of the same parents' plate will be reacted with alizarin red dyes Chinese red.The dyeing of each group calcium scoring is observed under Stereo microscope
As a result, and preservation of taking pictures.
As shown in Fig. 2, when the induction of MC3T3-E1 cell is broken up 7 days, without finding that apparent calcium scoring generates；Differentiation
At 14 days, blank group generates a small amount of calcium scoring, and 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose group is promoted in a manner of concentration dependant
Into the generation of calcium scoring；When breaking up 21 days, since the calcium scoring that blank group Osteoblast Differentiation generates is more, do not have with dosing group
Significant difference；At BMSCs induction differentiation 14 days, blank group starts to break up, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose group and sky
White group is compared, and promotes the generation of calcium scoring in a manner of concentration dependant.Should the result shows that, 1,2,3,4,6- five nutgall acyl Portugal
Grape sugar can effectively facilitate osteoblast differentiation maturation.
Embodiment 3
Influence of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose to MC3T3-E1 cell survival rate under oxidative stress status.
Cell: preosteoblast system MC3T3-E1 is purchased from upper marine academy of sciences's cell bank.
Instrument: Bio-Tek microplate reader.
Grouping and administration: it is divided into blank control group control (0.1%DMSO), model group model (0.4mM H2O2), 1,
2,3,4,6- Penta-O-galloyl-D-glucopyranose groups (10-12M~10-5M)。
Experimental method: the α-MEM culture solution of FBS is inactivated with 10% heat is contained, while pressing the thinner ratio of 1:100 in culture solution
Add it is dual anti-, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivated.Every two days replacement culture mediums.When cell is long extremely
When 90% or so, with trypsin digestion cell into centrifuge tube.3min is centrifuged with 1000rpm.α-MEM culture solution is used after cell centrifugation
It is resuspended and counts, with every hole containing 200 μ L of culture solution, 1 × 104A/hole is inoculated in 96 porocyte culture plates, cell adherent growth
After for 24 hours, H containing 0.4mM is added2O21,2,3,4,6- Penta-O-galloyl-D-glucopyranoses (10 are added after damage 4h-12M~10-5M) continue
Culture is for 24 hours.Mtt assay detects cell viability, and 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses of observation are living to cell after peroxide injury
The influence of power.
Mtt assay: culture solution in reject plate after incubation, MTT the solution 10 μ L and 100 μ L that 5mg/mL is added in every hole are free of
α-MEM the culture medium of serum.Culture supernatant is discarded after 4h, every hole is added, 150mL DMSO is added.Tissue culture plate is put into vibration
It swings and shakes 10min on device.Microplate reader wavelength 490nm is set, detects and records optical density (OD value).
Statistical analysis: experimental result is indicated using mean ± standard error (mean ± SEM), uses Graphpad Prism 7
Difference in software between the analysis method comparison each group of one-way ANOVA is represented statistically significant with P < 0.05.
A is MC3T3-E1 cell viability statistical result in Fig. 3.Compared to the blank group, 0.4mM H2O2It is thin to damage MC3T3-E1
Cell viability reduces by 20% or so after born of the same parents 4h, at this point, compared with model group, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose group cell
10-9M~10-5Cell viability can be significantly improved within the scope of M, illustrate that 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses can significantly change
Cell survival rate under kind oxidative stress status.
Embodiment 4
Influence of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose to MC3T3-E1 Apoptosis under oxidative stress status.
Cell: preosteoblast system MC3T3-E1.
Instrument: BECKMAN flow cytometer.
Grouping and administration: it is divided into blank control group control (0.1%DMSO), model group model (0.4mM H2O2), 1,
2,3,4,6- Penta-O-galloyl-D-glucopyranose groups (10-10M~10-7M)。
Experimental method: the α-MEM culture solution of FBS is inactivated with 10% heat is contained, while pressing the thinner ratio of 1:100 in culture solution
Add it is dual anti-, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivated.Every two days replacement culture mediums.When cell is long extremely
When 90% or so, with trypsin digestion cell into centrifuge tube.3min is centrifuged with 1000rpm.α-MEM culture solution is used after cell centrifugation
It is resuspended and counts.With every hole 1.5mL containing culture solution, 3 × 105A/hole is inoculated in 6 porocyte culture plates, and cell adherent growth is for 24 hours
H containing 0.4mM is added afterwards2O21,2,3,4,6- Penta-O-galloyl-D-glucopyranoses are added after damage 4h to co-culture for 24 hours.With Annexin V-
FITC/PI marks cell, flow cytomery Apoptosis.(flow cytomery Apoptosis: the film marked with FITC
Join the apoptosis of albumen-V (Annexin V FITC) detection osteoblast.Propidium iodide (PI) is for determining meronecrosis.Sudden and violent
After being exposed to various experiment conditions, cell is used into 0.25% trypsin digestion without EDTA and at 37 DEG C with fluorescent dye mark
Note, then carries out cytofluorometric analysis with flow cytometer.)
Statistical analysis: experimental result is indicated using mean ± standard error (mean ± SEM), uses Graphpad Prism 7
Difference in software between the analysis method comparison each group of one-way ANOVA is represented statistically significant with P < 0.05.
As shown in b, c in Fig. 3, compared with blank control group, 0.4mM H2O2It can be caused after damage MC3T3-E1 cell 4h bright
Aobvious Apoptosis, meanwhile, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses can resist TNF-a Induced Apoptosis in Osteoblasts compared with model group, say
Bright 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses can Apoptosis under significant anti-oxidation stress state.
Embodiment 5
1,2,3,4,6- Penta-O-galloyl-D-glucopyranose is to MC3T3-E1 cell death related protein under oxidative stress status
It influences.
Specific experiment process the following steps are included:
1, protein extraction: tissue culture plate is placed on ice, cell conditioned medium is removed, after every hole adds 2mL ice PBS to clean twice
100 μ L cell pyrolysis liquids (RIPA:PMSF=100:1), cell scraper scraping cells lysate to 1.5mL centrifuge tube is added in every hole
In, in cracking 30min, 4 DEG C of centrifugation 15min of 12000rpm on ice, take out spare.
2, protein quantification: prepare the detachable ELISA Plate in 96 holes, protein standard hole be added 0.5mg/mL BSA standard items (0,2,
4,6,8,12,16,20 μ L), and 20 μ L are complemented to PBS, sample 5 μ L and 20 μ LPBS (dilution 4 is added in the every hole in sample detection hole
Times).200 μ L BCA albumen working solutions (BCA reagent: Cu reagent=50:1) are added in every hole afterwards, and 37 DEG C are protected from light incubation 30min, In
Absorbance is detected at wavelength 562nm.According to absorbance, standard curve and Sample Dilution multiple, protein concentration is calculated.
3, albuminous degeneration: 80 μ L sample cell lysate supernatants are drawn into 1.5mL centrifuge tube, 20 μ L albumen loadings are added
Buffer (5 ×), heating 10min denaturation in 100 DEG C of dry-type thermostats, room temperature is cooling, -20 DEG C save it is stand-by.
4, glue:
The formula glue of according to the form below:
5, loading and electrophoresis: press 20 μ g albumen loadings, after electrophoresis is run under 60v voltage, after sample fully enters separation gel
Electrophoresis is run under 120V voltage.
6, transferring film: the transferring film 90min under 100V voltage.
7, primary antibody: after transferring film, TBST washes 5 × 5min of film, with 5% skim milk be incubated at room temperature 1h, TBST wash film 5 ×
5min is added primary antibody (dilution ratio 1:1000), and 4 DEG C are incubated overnight.
8, secondary antibody: absorbing primary antibody, and TBST washes 5 × 5min of film, and secondary antibody is added and is incubated at room temperature 1h, TBST washes 5 × 5min of film, adds
Enter and exposes liquid, digitlization gel imaging work station exposure.
9, Image lab gray value statisticallys analyze.
As shown in d in Fig. 3, compared to the blank group, H2O2Group reduces the expression of anti-apoptotic proteins Bcl-2, increases apoptotic proteins
The expression of Bax；With H2O2Group is compared, and PGG can significantly increase the influence of Bcl-2 albumen, reduces the expression of apoptotic protein Bax.Figure
E is the ratio statistical chart of Bcl-2/Bax in 3, and PGG can be with MC3T3-E1 apoptosis under anti-oxidation stress as the result is shown.
Embodiment 6
1,2,3,4,6- Penta-O-galloyl-D-glucopyranose is to MC3T3-E1 mitochondrial membrane potential in anoxic shadow under oxidative stress status
It rings.
Cell: preosteoblast system MC3T3-E1.
Instrument: BD-C6 flow cytometer.
Grouping and administration: it is divided into blank control group control (0.1%DMSO), model group model (0.4mM H2O2), 1,
2,3,4,6- Penta-O-galloyl-D-glucopyranose groups (10-10M~10-7M)。
Experimental method: the α-MEM culture solution of FBS is inactivated with 10% heat is contained, while pressing the thinner ratio of 1:100 in culture solution
Add it is dual anti-, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivated.Every two days replacement culture mediums.When cell is long extremely
When 90% or so, with trypsin digestion cell into centrifuge tube.3min is centrifuged with 1000rpm.α-MEM culture solution is used after cell centrifugation
It is resuspended and counts.With every ware (60mm × 15mm) 3mL containing culture solution, 3 × 105A cell inoculation is in Tissue Culture Dish, cell patch
H containing 0.4mM is added in wall growth afterwards for 24 hours2O21,2,3,4,6- Penta-O-galloyl-D-glucopyranoses are added after damage 4h to co-culture for 24 hours.It is right
0.5mL JC-1 dyeing working fluid is needed in every ten thousand cell of 50-100 of cell suspension.Appropriate JC-1 (200X) is taken, according to every 50 μ L
The dilution proportion JC-1 of JC-1 (200X) addition 8mL ultrapure water.Violent Vortex sufficiently dissolves and mixes JC-1.Then it adds
2mL JC-1 dye solution (5X) is JC-1 dyeing working fluid after mixing.Culture solution is absorbed, according to specific experiments if any must
It is primary to want can be washed cell with the appropriate solution of PBS or other, 1mL cell culture fluid is added.It can contain in cell culture fluid
Serum and phenol red.
Culture solution is absorbed, it is primary to wash cell with the appropriate solution of PBS or other if necessary according to specific experiments, adds
Enter 1mL cell culture fluid.It can be containing serum and phenol red in cell culture fluid.The complete training dyeing of 0.5mL JC-1 and 0.5mL is added
Working solution mixes well, and is incubated for 20 minutes for 37 DEG C in cell incubator.After 37 DEG C are incubated for, ice bath, flow cytometer is waited for
Machine.It is observed under fluorescence microscope or laser confocal microscope.
Statistical analysis: experimental result is indicated using mean ± standard error (mean ± SEM), uses Graphpad Prism 7
Difference in software between the analysis method comparison each group of one-way ANOVA is represented statistically significant with P < 0.05.
As shown in a and b in Fig. 4, compared with blank control group, 0.4mM H2O2It can be caused after damage MC3T3-E1 cell 4h
Apparent mitochondrial membrane potential in anoxic decline, meanwhile, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses can rise compared with model group
High osteoblast mitochondrial membrane potential.
Embodiment 7
1,2,3,4,6- Penta-O-galloyl-D-glucopyranose is to MC3T3-E1 cell mitochondrial ATP production capacity under oxidative stress status
It influences.
Cell: preosteoblast system MC3T3-E1.
Instrument: GloMax luminometer.
Grouping and administration: it is divided into blank control group control (0.1%DMSO), model group model (0.4mM H2O2), 1,
2,3,4,6- Penta-O-galloyl-D-glucopyranose groups (10-9M)。
Experimental method: the α-MEM culture solution of FBS is inactivated with 10% heat is contained, while pressing the thinner ratio of 1:100 in culture solution
Add it is dual anti-, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivated.Every two days replacement culture mediums.When cell is long extremely
When 90% or so, with trypsin digestion cell into centrifuge tube.3min is centrifuged with 1000rpm.α-MEM culture solution is used after cell centrifugation
It is resuspended and counts.With every hole 1.5mL containing culture solution, 3 × 105A/hole is inoculated in 6 porocyte culture plates, and cell adherent growth is for 24 hours
H containing 0.4mM is added afterwards2O21,2,3,4,6- Penta-O-galloyl-D-glucopyranoses are added after damage 4h to co-culture for 24 hours.Culture solution is absorbed,
Lysate is added according to the ratio (that is, 1/10 of cell culture liquid measure 2mL) that 200 μ L lysates are added in the every hole of 6 orifice plates,
Lytic cell.In order to crack sufficiently when lytic cell, pipettor can be used being blown and beaten or shaken repeatedly culture plate makes to crack
Liquid comes into full contact with and lytic cell.Usual cell can crack immediately after contact cracking liquid.4 DEG C of 12000g are centrifuged 5 points after cracking
Clock takes supernatant, is used for subsequent measurement.
Specific continuous mode the following steps are included:
1, ATP detects the preparation of working solution: the ratio of 100 μ L ATP detection working solution is needed according to each sample or standard items
The ATP for preparing appropriate amount detects working solution.Stand-by reagent is melted on ice bath.Suitable ATP detection reagent is taken, according to 1:9
Ratio ATP detection reagent diluted ATP detection reagent.Such as 100 μ L ATP detection reagent be added 900 μ L ATP inspection
Test agent diluent preparing detects working solution at 1mL ATP.ATP detection reagent after dilution is the ATP for being used for subsequent experimental
Detect working solution.ATP detection working solution can temporarily save on ice bath.
2, the measurement of ATP concentration: a. adds in 100 μ L ATP detection working solution to detection hole or detection pipe.It is placed at room temperature for 3-5
Minute, so that the ATP of background is all consumed, to reduce background.It can be disposably 10-20 detection hole or detection
Pipe adds 100 μ L ATP detection working solution respectively, to save the time.
3,20 μ L samples or standard items are added in detection hole or detection pipe, are mixed with rifle (micropipettor) rapidly, until
After being spaced 2 seconds less, with Chemiluminescence Apparatus (luminometer) or liquid scintillation instrument measurement RLU value or CPM.(note: the volume of sample can
Voluntarily to be adjusted within the scope of 10-100 μ L.100 μ L samples can be added if the ATP concentration in sample is relatively low, if
ATP concentration is relatively high in sample, the sample of smaller size smaller can be added, while standard items are also required to using identical volume.Such as
The concentration of ATP is especially high in fruit sample, measures again after can detecting lysate dilute sample with ATP.10- is being added in this kit
When 100 μ L standard items, substantially there is good linear relationship in the concentration range of 1nM~10 μM.
Statistical analysis: experimental result is indicated using mean ± standard error (mean ± SEM), uses Graphpad Prism 7
Difference in software between the analysis method comparison each group of one-way ANOVA is represented statistically significant with P < 0.05.
As shown in c in Fig. 4, compared with blank control group, 0.4mM H2O2It can be caused after damage MC3T3-E1 cell 4h obvious
Cell mitochondrial ATP production capacity decline, meanwhile, 10 compared with model group-91,2,3,4,6- Penta-O-galloyl-D-glucopyranose of M can be with
Increase osteoblast mitochondrial ATP production capacity.
Embodiment 8
Influence of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose to MC3T3-E1 cell ROS level under oxidative stress status.
Cell: preosteoblast system MC3T3-E1.
Instrument: BECKMAN flow cytometer.
Grouping and administration: it is divided into blank control group control (0.1%DMSO), model group model (0.4mM H2O2), 1,
2,3,4,6- Penta-O-galloyl-D-glucopyranose groups (10-10M~10-7M)。
Experimental method: the α-MEM culture solution of FBS is inactivated with 10% heat is contained, while pressing the thinner ratio of 1:100 in culture solution
Add it is dual anti-, in 37 DEG C, 5%CO2, cultivated in the incubator of saturated humidity.Every two days replacement culture mediums.When cell is long extremely
When 90% or so, with trypsin digestion cell into centrifuge tube.3min is centrifuged with 1000rpm.α-MEM culture solution is used after cell centrifugation
It is resuspended and counts.With every ware (60mm × 15mm) 3mL containing culture solution, 3 × 105A cell inoculation is in Tissue Culture Dish, cell patch
H containing 0.4mM is added in wall growth afterwards for 24 hours2O21,2,3,4,6- Penta-O-galloyl-D-glucopyranoses are added after damage 4h to co-culture for 24 hours.It presses
DCFH-DA is diluted according to 1:1000 serum-free medium, makes final concentration of 10 μM/l.Cell culture fluid is removed, appropriate bulk is added
The DCFH-DA that product has diluted.The DCFH-DA 1mL diluted is added.It is incubated for 20 minutes in 37 DEG C of cell incubators.Use serum-free
Cell culture fluid washs cell three times, intracellular DCFH-DA is not entered with abundant removal, with flow cytomery and fluorescence
Microscope is taken pictures observation.
Statistical analysis: experimental result is indicated using mean ± standard error (mean ± SEM), uses Graphpad Prism 7
Difference in software between the analysis method comparison each group of one-way ANOVA is represented statistically significant with P < 0.05.
As shown in d and e in Fig. 4, compared with blank control group, 0.4mM H2O2It can be caused after damage MC3T3-E1 cell 4h
Apparent intracellular ROS level rises, meanwhile, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses can be reduced into compared with model group
ROS is horizontal in osteocyte.
Osteoblast under oxidative stress status can be improved in above-mentioned description of test, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose
Survival rate, restore mitochondria dysfunction, inhibit Apoptosis, and then to osteoblast generate protective effect.Prove 1,2,
3,4,6- Penta-O-galloyl-D-glucopyranoses may be by the damage to anti-oxidation stress to osteoblast, to reduce osteoblast
Apoptosis is finally reached the purpose for the treatment of osteoporosis, thus, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose can be applied to system
The drug of standby treatment osteoporosis.
Claims (8)
1. application of 1,2,3,4, the 6- Penta-O-galloyl-D-glucopyranose in preparation prevention and treatment osteoporosis drug.
2. application according to claim 1, it is characterised in that: the osteoporosis include primary osteoporosis and
Secondary osteoporosis.
3. application according to claim 2, it is characterised in that: the primary osteoporosis is women post menopausal sclerotin
Osteoporosis or old involutional osteoporosis.
4. application according to claim 2, it is characterised in that: the secondary osteoporosis is by endocrine metabolism disease
Disease, connective tissue disease, kidney trouble, disease of digestive tract and/or drug-induced osteoporosis.
5. application according to claim 1, it is characterised in that: the application is by 1,2,3,4,6- five nutgall acyl grape
It is subjected to auxiliary material on sugar and pharmacy, pill, tablet, capsule, granule or freeze dried powder is made.
6. 1,2,3,4,6- Penta-O-galloyl-D-glucopyranose is preparing the application in promoting bone growing drug.
7. the drug of anti-curing osteoporosis, effective component includes 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses.
8. the drug of anti-curing osteoporosis according to claim 7, it is characterised in that: 1,2,3,4,6- five galla turcica
The minimum effective concentration of acyl glucose is 10-9 M。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113181195A (en) * | 2021-04-07 | 2021-07-30 | 中山大学 | Application of glucose polyphenol acid ester derivatives in preparation of isoleucyl tRNA synthetase inhibitor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000025787A1 (en) * | 1998-10-30 | 2000-05-11 | Guilford Pharmaceuticals Inc. | Pharmaceutical compositions containing poly(adp-ribose) glycohydrolase inhibitors and methods of using the same |
| WO2008131047A2 (en) * | 2007-04-16 | 2008-10-30 | The Regents Of The University Of Michigan | Plasminogen activator inhibitor-1 inhibitors and methods of use thereof to modulate lipid metabolism |
| US20150174184A1 (en) * | 2013-12-20 | 2015-06-25 | Natreon, Inc. | Use of terminalia chebula extract for treatment of osteoarthritis |
| US20190060337A1 (en) * | 2017-08-31 | 2019-02-28 | Muhammed Majeed | Compositions comprising beta-glucogallin and therapeutic applications thereof in controlled kinetics of carbohydrate breakdown and monosaccharide absorption |
-
2019
- 2019-07-19 CN CN201910653874.XA patent/CN110448563A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000025787A1 (en) * | 1998-10-30 | 2000-05-11 | Guilford Pharmaceuticals Inc. | Pharmaceutical compositions containing poly(adp-ribose) glycohydrolase inhibitors and methods of using the same |
| WO2008131047A2 (en) * | 2007-04-16 | 2008-10-30 | The Regents Of The University Of Michigan | Plasminogen activator inhibitor-1 inhibitors and methods of use thereof to modulate lipid metabolism |
| US20150174184A1 (en) * | 2013-12-20 | 2015-06-25 | Natreon, Inc. | Use of terminalia chebula extract for treatment of osteoarthritis |
| US20190060337A1 (en) * | 2017-08-31 | 2019-02-28 | Muhammed Majeed | Compositions comprising beta-glucogallin and therapeutic applications thereof in controlled kinetics of carbohydrate breakdown and monosaccharide absorption |
Non-Patent Citations (4)
| Title |
|---|
| ADRIANA ARRUDA MATOS等: "An extract from Myracrodruon urundeuva inhibits matrix mineralization in human osteoblasts", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
| YUKIKO HOSOYAMA等: "Effects of gallotannin on osteoclastogenesis and the p38 MAP kinase pathway", 《SCIENCEDIRECT》 * |
| 王新峦: "骨碎补抗骨质疏松活性成分和心叶淫羊藿抗骨质疏松作用机制研究", 《沈阳药科大学博士学位论文 万方数据库》 * |
| 顾伟伟: "应用成骨细胞生物色谱法研究牡丹皮促进骨折愈合的效应物质", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113181195A (en) * | 2021-04-07 | 2021-07-30 | 中山大学 | Application of glucose polyphenol acid ester derivatives in preparation of isoleucyl tRNA synthetase inhibitor |
| CN113181195B (en) * | 2021-04-07 | 2023-02-07 | 中山大学 | Application of glucose polyphenol acid ester derivatives in preparation of isoleucyl tRNA synthetase inhibitor |
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