CN107744525A - A kind of preparation method of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people's derived stem cell active factors composition - Google Patents
A kind of preparation method of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people's derived stem cell active factors composition Download PDFInfo
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- CN107744525A CN107744525A CN201711014620.0A CN201711014620A CN107744525A CN 107744525 A CN107744525 A CN 107744525A CN 201711014620 A CN201711014620 A CN 201711014620A CN 107744525 A CN107744525 A CN 107744525A
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses the preparation method of a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people's derived stem cell active factors composition, the derived mesenchymal stem cells in vitro culture of people source；After cell culture to some hours, it is degerming to centrifuge tube, filtering supernatant to collect supernatant；Add physiological saline and continue culture 12 hours, smudge cells centrifugal filtration film obtains stem cell extract solution；Raw sugar is extracted in gynostemma pentaphylla；Raw sugar purifies to obtain polysaccharide powder；By stem cell extract solution and polysaccharide powder according to mass ratio 56：1 is sufficiently mixed and produces.People source and plant source stem cell properties bioactive extracts are carried out compatibility by the present invention, are acted synergistically on the basis of displaying one's respective advantages, and are allowed to play bigger comprehensive effect, prepared composition treatment hypertension, high fat of blood, the disease such as hyperglycaemia, fatty liver.There is the effect of eliminating inflammation and expelling toxin, cough-relieving apophlegmatic, protecting liver and detoxication, lowering blood pressure and blood fat blood glucose.
Description
Technical field
The invention belongs to biomedical sector, more particularly to a kind of gynostemma pentaphylla biology with reducing blood pressure and blood fat effect
The preparation method of active material and people's derived stem cell active factors composition.
Background technology
Mescenchymal stem cell is the important member of stem cell line, from the mesoderm of mesoderm growing early stage, belongs to more competent
Cell, MSC initially have found in marrow, because it has multi-lineage potential, hematopoiesis support and promotes stem cell implantation, immune tune
Control and the concern of people is increasingly subject to the features such as self-replacation.
Under mescenchymal stem cell in vivo or in vitro specific inductive condition, can be divided into fat, bone, cartilage, muscle,
The Various Tissues cell such as tendon, ligament, nerve, liver, cardiac muscle, endothelium, still have after continuous passage culture and freezen protective multidirectional
Differentiation potential, preferable seed cell can be used as to be used for injuries of tissues and organs reparation caused by aging and lesion.
The content of the invention
It is an object of the invention to provide a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people
The preparation method of derived stem cell active factors composition,
The present invention is achieved by the following technical solutions：
A kind of system of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people's derived stem cell active factors
Preparation Method, this method comprise the following steps：
S1, the derived mesenchymal stem cells in vitro culture of people source；
After S2, cell culture to some hours, it is degerming to centrifuge tube, filtering supernatant to collect supernatant；
S3, addition physiological saline continue culture 12 hours, and smudge cells centrifugal filtration film obtains stem cell extract solution；
Raw sugar is extracted in S4, gynostemma pentaphylla；
S5, raw sugar purify to obtain polysaccharide powder；
S6, by stem cell extract solution and polysaccharide powder according to mass ratio 5-6：1 is sufficiently mixed and produces.
Further, the S1 is concretely comprised the following steps, and after taking the mescenchymal stem cell recovery of people source, is resuspended with physiological saline,
Suspension is inoculated in complete medium, is placed in 37 DEG C, 5%CO2Cultivated in incubator, change liquid for the first time after cultivating 24h and remove
Attached cell, liquid is changed once every 2d afterwards, with 1 after cell growth to 85% degree of converging：2 ratios carry out Secondary Culture.
Further, the complete medium is DF12 culture mediums, containing volume fraction is 2% tire ox blood in the culture medium
Clearly, 40%MCDB201,10 μ g/L platelet derived growth factors, 10 μ g/L basic fibroblast growth factors, 10 μ g/L tables
Skin growth factor.
Further, the S4 is concretely comprised the following steps, weigh gynostemma pentaphylla it is broken after, add after the dissolving of 95% ethanol to flow back and carry
Take, normal temperature volatilization ethanol obtains gynostemma pentaphylla powder, gynostemma pentaphylla powder is scattered in distilled water, supernatant is collected in extraction, will be upper
Clear liquid is concentrated under reduced pressure into the 1/3 of original volume, is centrifuged off precipitating, and supernatant adds ethanol solution, stands, centrifugation obtains white wadding
Shape is precipitated, and white flock precipitate is scattered in ethanol solution, centrifuged after being sufficiently stirred, and precipitation vacuum drying obtains raw sugar
Powder.
Further, concretely comprising the following steps in the S5, raw sugar powder is scattered in distilled water, 4 DEG C of standing 12-16h
After centrifuge, supernatant is added into chloroform-n-butanol mixed solution, solid-to-liquid ratio 5-6：1, wherein chloroform in the mixed solution
Volume ratio with n-butanol is 5：1, mixed solution centrifuges to obtain polysaccharide powder.
Further, a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people's derived stem cell activity
The preparation method of the factor, it is characterised in that stem cell extract solution is with polysaccharide powder according to mass ratio 5-6：1 be sufficiently mixed after
Arrive.
The invention has the advantages that：
People source and plant source stem cell properties bioactive extracts are carried out compatibility by the present invention, what is displayed one's respective advantages
On the basis of act synergistically, be allowed to play bigger comprehensive effect, prepared composition treatment hypertension, high fat of blood, hyperglycaemia,
The diseases such as fatty liver.There is the effect of eliminating inflammation and expelling toxin, cough-relieving apophlegmatic, protecting liver and detoxication, lowering blood pressure and blood fat blood glucose.
Certainly, any product for implementing the present invention it is not absolutely required to reach all the above advantage simultaneously.
Embodiment
Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only
Part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art
The all other embodiment obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
The present invention is a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people's derived stem cell activity
The preparation method of factor composition, this method comprise the following steps that：
Embodiment 1
S1, after taking the mescenchymal stem cell recovery of people source, it is resuspended with physiological saline, suspension is inoculated in complete medium, is put
In 37 DEG C, 5%CO2Cultivated in incubator, change liquid for the first time after cultivating 24h and remove attached cell, change liquid one every 2d afterwards
It is secondary, with 1 after cell growth to 85% degree of converging：2 ratios carry out Secondary Culture；
The complete medium is DF12 culture mediums, and it is 2% hyclone, 40% that volume fraction is contained in the culture medium
MCDB201,10 μ g/L platelet derived growth factors, 10 μ g/L basic fibroblast growth factors, 10 μ g/L epidermal growths
The factor；
After S2, cell culture to some hours, it is degerming to centrifuge tube, 0.22 μm of membrane filtration supernatant to collect supernatant；
S3,2 blake bottles are cleaned with physiological saline, discarded, continuously added physiological saline and be placed in T225 blake bottles and continue to train
Support 12 hours；
5min is centrifuged with 1400rpm after Ultrasonic Cell Disruptor smudge cells, collection supernatant crosses 0.22 μm of filter membrane and obtains stem cell
Extract solution；
S4, weigh gynostemma pentaphylla it is broken after, add refluxing extraction after the dissolving of 95% ethanol, solid-to-liquid ratio 1：10, normal temperature volatilization
Ethanol obtains gynostemma pentaphylla powder；
S5, gynostemma pentaphylla powder is scattered in distilled water, solid-to-liquid ratio 1：10,100 DEG C of extraction 30min, collect supernatant,
1/3, the 5000rpm that supernatant is concentrated under reduced pressure into original volume centrifuges 10min, removes precipitation；
S6, supernatant add 3 times 95% of ethanol solution, volume ratio 1：2,12h, 5000rpm are stood after stirring
Centrifugation 10min obtains white flock precipitate；
S7, white flock precipitate is scattered in ethanol solution, centrifuged after being sufficiently stirred, precipitation vacuum drying obtains
Raw sugar powder；
S8, raw sugar powder is scattered in distilled water, 4 DEG C stand 5000rpm centrifugation 10min after 12h, supernatant add to
In chloroform-n-butanol mixed solution, solid-to-liquid ratio 5：1, wherein the volume ratio of chloroform and n-butanol is 5 in the mixed solution：
1, mixed solution centrifuges to obtain polysaccharide powder；
S9, by stem cell extract solution and polysaccharide powder according to mass ratio 5：1 is sufficiently mixed and produces.
Embodiment 2
S1, after taking the mescenchymal stem cell recovery of people source, it is resuspended with physiological saline, suspension is inoculated in complete medium, is put
In 37 DEG C, 5%CO2Cultivated in incubator, change liquid for the first time after cultivating 24h and remove attached cell, change liquid one every 2d afterwards
It is secondary, with 1 after cell growth to 85% degree of converging：2 ratios carry out Secondary Culture；
The complete medium is DF12 culture mediums, and it is 2% hyclone, 40% that volume fraction is contained in the culture medium
MCDB201,10 μ g/L platelet derived growth factors, 10 μ g/L basic fibroblast growth factors, 10 μ g/L epidermal growths
The factor；
After S2, cell culture to some hours, it is degerming to centrifuge tube, 0.22 μm of membrane filtration supernatant to collect supernatant；
S3,2 blake bottles are cleaned with physiological saline, discarded, continuously added physiological saline and be placed in T225 blake bottles and continue to train
Support 12 hours；
5min is centrifuged with 1400rpm after Ultrasonic Cell Disruptor smudge cells, collection supernatant crosses 0.22 μm of filter membrane and obtains stem cell
Extract solution；
S4, weigh gynostemma pentaphylla it is broken after, add refluxing extraction after the dissolving of 95% ethanol, solid-to-liquid ratio 1：10, normal temperature volatilization
Ethanol obtains gynostemma pentaphylla powder；
S5, gynostemma pentaphylla powder is scattered in distilled water, solid-to-liquid ratio 1：10,100 DEG C of extraction 40min, collect supernatant,
1/3, the 6000rpm that supernatant is concentrated under reduced pressure into original volume centrifuges 20min, removes precipitation；
S6, supernatant add 3 times 95% of ethanol solution, volume ratio 1：3,16h, 6000rpm are stood after stirring
Centrifugation 20min obtains white flock precipitate；
S7, white flock precipitate is scattered in ethanol solution, centrifuged after being sufficiently stirred, precipitation vacuum drying obtains
Raw sugar powder；
S8, raw sugar powder is scattered in distilled water, 4 DEG C stand 6000rpm centrifugation 20min after 16h, supernatant add to
In chloroform-n-butanol mixed solution, solid-to-liquid ratio 6：1, wherein the volume ratio of chloroform and n-butanol is 5 in the mixed solution：
1, mixed solution centrifuges to obtain polysaccharide powder；
S9, by stem cell extract solution and polysaccharide powder according to mass ratio 6：1 is sufficiently mixed and produces.
Embodiment 3
S1, after taking the mescenchymal stem cell recovery of people source, it is resuspended with physiological saline, suspension is inoculated in complete medium, is put
In 37 DEG C, 5%CO2Cultivated in incubator, change liquid for the first time after cultivating 24h and remove attached cell, change liquid one every 2d afterwards
It is secondary, with 1 after cell growth to 85% degree of converging：2 ratios carry out Secondary Culture；
The complete medium is DF12 culture mediums, and it is 2% hyclone, 40% that volume fraction is contained in the culture medium
MCDB201,10 μ g/L platelet derived growth factors, 10 μ g/L basic fibroblast growth factors, 10 μ g/L epidermal growths
The factor；
After S2, cell culture to some hours, it is degerming to centrifuge tube, 0.22 μm of membrane filtration supernatant to collect supernatant；
S3,2 blake bottles are cleaned with physiological saline, discarded, continuously added physiological saline and be placed in T225 blake bottles and continue to train
Support 12 hours；
5min is centrifuged with 1400rpm after Ultrasonic Cell Disruptor smudge cells, collection supernatant crosses 0.22 μm of filter membrane and obtains stem cell
Extract solution；
S4, weigh gynostemma pentaphylla it is broken after, add refluxing extraction after the dissolving of 95% ethanol, solid-to-liquid ratio 1：10, normal temperature volatilization
Ethanol obtains gynostemma pentaphylla powder；
S5, gynostemma pentaphylla powder is scattered in distilled water, solid-to-liquid ratio 1：10,100 DEG C of extraction 35min, collect supernatant,
1/3, the 5500rpm that supernatant is concentrated under reduced pressure into original volume centrifuges 15min, removes precipitation；
S6, supernatant add 3 times 95% of ethanol solution, volume ratio 1：2.5,14h is stood after stirring,
5500rpm centrifugations 15min obtains white flock precipitate；
S7, white flock precipitate is scattered in ethanol solution, centrifuged after being sufficiently stirred, precipitation vacuum drying obtains
Raw sugar powder；
S8, raw sugar powder is scattered in distilled water, 4 DEG C stand 5500rpm centrifugation 15min after 14h, supernatant add to
In chloroform-n-butanol mixed solution, solid-to-liquid ratio 5.5：1, wherein the volume ratio of chloroform and n-butanol is in the mixed solution
5：1, mixed solution centrifuges to obtain polysaccharide powder；
S9, by stem cell extract solution and polysaccharide powder according to mass ratio 5.5：1 is sufficiently mixed and produces.
Above content is only citing made for the present invention and explanation, and affiliated those skilled in the art are to being retouched
The specific embodiment stated is made various modifications or supplement or substituted using similar mode, without departing from invention or super
More scope defined in the claims, protection scope of the present invention all should be belonged to.
Claims (6)
- A kind of 1. preparation of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect and people's derived stem cell active factors Method, it is characterised in that this method comprises the following steps：S1, the derived mesenchymal stem cells in vitro culture of people source；After S2, cell culture to some hours, it is degerming to centrifuge tube, filtering supernatant to collect supernatant；S3, addition physiological saline continue culture 12 hours, and smudge cells centrifugal filtration film obtains stem cell extract solution；Raw sugar is extracted in S4, gynostemma pentaphylla；S5, raw sugar purify to obtain polysaccharide powder；S6, by stem cell extract solution and polysaccharide powder according to mass ratio 5-6：1 is sufficiently mixed and produces.
- 2. a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect according to claim 1 is done with people source The preparation method of cell active factor, it is characterised in that：The S1's concretely comprises the following steps, and takes the mescenchymal stem cell recovery of people source Afterwards, it is resuspended with physiological saline, suspension is inoculated in complete medium, is placed in 37 DEG C, 5%CO2Cultivated in incubator, cultivate 24h Change liquid for the first time afterwards and remove attached cell, change liquid once every 2d afterwards, with 1 after cell growth to 85% degree of converging：2 ratios Example carries out Secondary Culture.
- 3. a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect according to claim 2 is done with people source The preparation method of cell active factor, it is characterised in that：The complete medium is DF12 culture mediums, contains body in the culture medium Fraction is 2% hyclone, 40%MCDB201,10 μ g/L platelet derived growth factors, 10 μ g/L basic fibroblasts Growth factor, 10 μ g/L EGFs.
- 4. a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect according to claim 1 is done with people source The preparation method of cell active factor, it is characterised in that：The S4's concretely comprises the following steps, weigh gynostemma pentaphylla it is broken after, add Refluxing extraction after the dissolving of 95% ethanol, normal temperature volatilization ethanol obtain gynostemma pentaphylla powder, gynostemma pentaphylla powder are scattered in distilled water, Supernatant is collected in extraction, and supernatant is concentrated under reduced pressure into the 1/3 of original volume, is centrifuged off precipitating, and supernatant adds ethanol solution, Standing, centrifugation obtain white flock precipitate, and white flock precipitate is scattered in ethanol solution, centrifuged after being sufficiently stirred, Precipitation vacuum drying obtains raw sugar powder.
- 5. a kind of gynostemma pentaphylla bioactive substance with reducing blood pressure and blood fat effect according to claim 1 is done with people source The preparation method of cell active factor, it is characterised in that：Concretely comprising the following steps in the S5, raw sugar powder is scattered in distilled water In, centrifuged after 4 DEG C of standing 12-16h, supernatant is added into chloroform-n-butanol mixed solution, solid-to-liquid ratio 5-6：1, wherein institute It is 5 to state the volume ratio of chloroform and n-butanol in mixed solution：1, mixed solution centrifuges to obtain polysaccharide powder.
- 6. a kind of gynostemma pentaphylla bioactive substance according to claim 1 with reducing blood pressure and blood fat effect is done with people source The preparation method of cell active factor, it is characterised in that stem cell extract solution is with polysaccharide powder according to mass ratio 5-6：1 is fully mixed Obtained after conjunction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113730439A (en) * | 2021-09-09 | 2021-12-03 | 陕西中鸿瑞康健康管理有限公司 | Stem cell factor freeze-dried powder capable of reducing triglyceride and preparation method and application thereof |
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| CN105902598A (en) * | 2016-05-31 | 2016-08-31 | 安徽惠恩生物科技股份有限公司 | Human-derived stem cell and gynostemma pentaphyllum bioactive substance composition and preparation method thereof |
| CN106344492A (en) * | 2016-09-12 | 2017-01-25 | 博雅干细胞科技有限公司 | Stem cell active factor and lyophilized powder thereof |
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- 2017-10-25 CN CN201711014620.0A patent/CN107744525A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105902598A (en) * | 2016-05-31 | 2016-08-31 | 安徽惠恩生物科技股份有限公司 | Human-derived stem cell and gynostemma pentaphyllum bioactive substance composition and preparation method thereof |
| CN106344492A (en) * | 2016-09-12 | 2017-01-25 | 博雅干细胞科技有限公司 | Stem cell active factor and lyophilized powder thereof |
Non-Patent Citations (2)
| Title |
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| 周宝珍等: "绞股蓝多糖脱蛋白工艺及相对分子质量的测定", 《陕西师范大学学报（自然科学版）》 * |
| 景永帅等: "绞股蓝提取物抗氧化活性评价", 《山地农业生物学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113730439A (en) * | 2021-09-09 | 2021-12-03 | 陕西中鸿瑞康健康管理有限公司 | Stem cell factor freeze-dried powder capable of reducing triglyceride and preparation method and application thereof |
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