CN110003358A - A kind of pectin and its it is used for anti-aging purposes - Google Patents
A kind of pectin and its it is used for anti-aging purposes Download PDFInfo
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- CN110003358A CN110003358A CN201910365802.5A CN201910365802A CN110003358A CN 110003358 A CN110003358 A CN 110003358A CN 201910365802 A CN201910365802 A CN 201910365802A CN 110003358 A CN110003358 A CN 110003358A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
- C08B37/0048—Processes of extraction from organic materials
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- Gerontology & Geriatric Medicine (AREA)
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- Cosmetics (AREA)
Abstract
The invention discloses the external agings that the pectin of specific structure involved in right can inhibit people's primary fibroblast BJ cell of active oxygen induction；The specific structure pectin can inhibit the accumulation of the activity of beta-galactosidase and aging marker protein DEC-1 in people's primary fibroblast BJ cell.It is expected to develop the anti-aging health-oriented products for being prepared into the different dosage forms such as solid beverage, oral solution, oral tablet in the form of alone or in combination with the specific structure pectin.
Description
Technical field
The present invention relates to pectin can inhibit H2O2The aging of people's primary fibroblast BJ cell of induction, and inhibit DEC-1
The expression of albumen reaches inhibition anti-aging effects.
Background technique
Polysaccharide (polysaccharide) is by multiple monosaccharide molecules are condensed, dehydration forms, and is that structure is complicated for molecule
And huge glucide.All carbohydrate for meeting high-molecular compound concept and its derivative are referred to as polysaccharide.Polysaccharide
It is extremely wide in distributed in nature, it is also critically important.Has plenty of the constituent for constituting animal and plant cells wall, such as peptide glycan and cellulose；
Has plenty of the nutrient as animals and plants storage, such as glycogen and starch；Some has special bioactivity, as the heparin in human body
There is blood coagulation resisting function, the polysaccharide in pneumococcal cell walls has antigenic action.The structural units of polysaccharide is monosaccharide, and polysaccharide is opposite to be divided
Protonatomic mass is ten million from tens of thousands of to several.It is connected between structural units with glycosidic bond, common glycosidic bond has α-Isosorbide-5-Nitrae -, β-Isosorbide-5-Nitrae-and α -1,
6- glycosidic bond.Structural units can be linked to be straight chain, can also form branch, straight chain generally with α-Isosorbide-5-Nitrae-glycosidic bond (such as starch) and β -1,
4- glycosidic bond 9 such as cellulose) it is linked to be；The tie point of chain and chain is often α -1,6- glycosidic bond in branch.It is more and more studied in foreign countries
The unique function of polysaccharide is focused on application.Honda Yasuki etc. obtains one from Common Primrose category (Polyanthus) plant
Kind has the effects that good moisturizing, crease-resistant acid heteroglycan.Sawai Yasuko etc. is from rhizoma acori graminei (Acorusgram/
Neus isolated polysaccharide can inhibit the generation of melanin in rhizome), has anti-inflammatory, antioxidation, can be used for blacking
The treatment of disease, and because it is with good moisture-keeping function, therefore but also as the effective component of cosmetics.Shimomura K~nji
Deng obtaining a kind of acidic polysaccharose with beauty functions from the meat catabolite of shellfish.It is demonstrated experimentally that this is acid
Polysaccharide can inhibit the decomposition of the hyaluronic acid of anti-aging, reduce skin lines and dry and cracked, thus can be used as beauty food and change
The effective component of cosmetic.Technological development background of the invention is namely based on the polysaccharide of specific structure, utilizes free radical H2O2Induction
Primary fibroblast aging model develops the polysaccharide with important anti-aging effects.
Summary of the invention
The object of the present invention is to provide the structural characterizations such as the composition of arabinose involved by right and molecular weight；Another mesh of the invention
The pectin that right is related to that is to provide inhibit H2O2The aging marker beta- of people's primary fibroblast BJ cell of induction
The activity of galactosidase；Further object of the invention is to provide under pectin involved by right primary fibroblast of transferring person
The expression of BJ cell ageing mark DEC-1；
Detailed description of the invention
Sugared composition analysis, the nmr spectrum (H of pectin involved by Fig. 1 right1- NMR, C13- NMR) map；Figure 1A is
Pectin purity and molecular weight determination figure involved by right；Figure 1B is the sugared composition analysis map of pectin involved by right；Fig. 1 C is right
Involved pectin H1NMR spectra；Fig. 1 D is pectin C involved by right13NMR spectra (dept135 spectrogram)；Fig. 1 E is involved by right
Pectin C13NMR spectra (BB spectrogram)；
Fig. 2 is the activity knot that pectin inhibits people's primary fibroblast BJ cell ageing marker beta-galactosidase
Fruit figure；
Fig. 3 is the expression figure that pectin inhibits aging mark DEC-1 in people's primary fibroblast BJ cell；
Specific embodiment
Embodiment 1: the measurement of the characterization of pectin structure involved by right
1) molecular weight and purity testing
Instrument is 1260 high performance liquid chromatography of Agilent, configuration series connection waters gel chromatographic columns
UltrahydrogelTM2000 and UltrahydrogelTMThe cm of 500(25 cm × 0.75) chromatographic column.Mobile phase is 0.1
M NaNO3, flow velocity is 0.5 ml/min, and chromatographic column operating temperature is 25 DEG C, is detected with RID detector.Purity test shows this
Pectin is made of two components.Its molecular weight is respectively 8.13 × 105 D and 6.86 × 102 D.As shown in Figure 1A；
Arabinose composition analysis involved by right:
It is now ready for mixing monosaccharide standard.Weigh each monosaccharide standard (L-Fuc；L-Rha；L-Ara；L-Xyl；D-Man；D-
Gal；D-Glc；D-GalA；D-GlcA it) is dissolved in deionized water, is made into 1 mg/ml with to be analyzed；
The hydrolysis of pectin polysaccharide: weighing 2-4 mg pectin sample in chicken heart bottle (10 ml), and 1 ml H is added2O is pre-dissolved, then plus
Enter the TFA of 1 ml, 4 M, adhesive plaster seals a bottle, and sets in 110 DEG C of baking ovens and hydrolyzes 4 h；After being cooled to room temperature, add methanol multiple
Extra TFA is removed to no tart flavour (4 times)；The polysaccharide sample of hydrolysis is redissolved in 200 μ L H2O；
The PMP derivatization of monosaccharide.50 μ L hydrolyzed pectin liquid or 50 μ L are taken to mix monosaccharide standard solution in 2 ml EP pipes,
The NaOH solution with 0.6 mol/L of 50 μ L is sufficiently mixed respectively, and the PMP(0.2613 g/ of 100 μ L, 0.5 mol/L is added
3ml) methanol solution stoppers after plug is tamping, and vortex oscillation instrument mixes；100 min are reacted in 70 DEG C of water-baths, take out cooling
To room temperature.Be added 100 μ L, 0.3 mol/L HCl neutralize, then plus 700 μ l deionized waters and 1 ml chloroform extraction, whirlpool
Oscillation stands 1-2 h.900 μ l of upper strata aqueous phase is drawn, the chloroform extraction for adding 900 μ l is primary, stands 1 h, then go to upper layer
800 μ l of water phase, the chloroform extraction for adding 800 μ l is primary, or replaces stewing process with low-speed centrifugal.With with 0.22 μm
After the syringe filtering of miillpore filter, it is fitted into liquid phase bottle that sample introduction is analyzed to HPLC；
HPLC analysis sugar composition: it is realized using 1260 highly effective liquid phase chromatographic system of Agilent.Chromatographic column used is ZORBAX
Eclipse XDB-C18(250 mm × 4.6 mm, 5 μm)；Mobile phase used is+85% phosphate of Buffer A:15% acetonitrile
Buffer；+ 60% phosphate buffer of Buffer B:40% acetonitrile；Phosphate buffer: 0.05 M potassium phosphate buffer
(pH 6.7)； KH2PO4In 2 L water of 13.6 g and 1.8 g solution of NaOH；
Mobile phase is realized according to below table sequence overloads:
In test, column temperature is 20 DEG C；Ultraviolet detection wavelength is 254 nm；Flow velocity is 1 ml/min；Sampling volume is 20 μ l；
Experimental result: being made up of PMP deriveding analysis arabinose, we are by area normalization method, in conjunction with the school of each monosaccharide
Positive divisor learns that pectin monosaccharide group becomes Rha:GalA:Glc:Gal:Ara=1.6:50:38.6:7.4:2.4.It follows that this
Polysaccharide may be by equal polygalacturonic acid, RG1 type pectin and glucan composition.Sugar composition map is as shown in Figure 1B；
3) pectin nmr spectrum (H involved by right1- NMR, C13- NMR)
Pectin D2After O exchange freeze-drying, its H is analyzed with 500 MHz cryogenic probe Nuclear Magnetic Resonance1- NMR, C13-NMR；H1-NMR
Spectrogram it is as shown in Figure 1 C；Chemical shift is D at 4.85 ppm2The peak of O is acetone peak at 2.29 ppm；Chemical shift exists
It is the signal peak of C6 on Rha at 1.0-1.5；Chemical shift is at the peak that 3.5-4.5 is hydrogen all in addition to anomer hydrogen in saccharide ring；Change
Displacement study is the signal peak of anomer hydrogen at 4.5-5.5；C13As shown in figure iD, upper figure is that dept 135 is composed to NMR spectra in Fig. 1 D
Figure, Fig. 1 E is BB spectrogram.It is internal standard acetone signal peak in dept135 figure, at chemical shift 31.5.Chemical shift is in 60-65
Negative peak between ppm is the peak of the C6 of Glc or Gal.It is the signal peak of non-anomeric carbon between 65-90 ppm.90-110 ppm is
The signal peak of anomeric carbon.The signal of BB spectrum is as dept135 signal.216 ppm chemical shifts having more are the signals of acetone
Peak.
Embodiment 2: pectin inhibits people's primary fibroblast BJ cell to pass through H2O2The aging marker beta- of induction
The activity of galactosidase
1) fibroblastic culture and aging model induction:
Application on human skin primary cell BJ cell line is bought from ATCC(59899913).BJ cell use containing 10% (v/v) fetal calf serum,
The DMEM high glucose medium culture of the penicillin/streptomycin of 1% (v/v).At 37 degree, contain 5% carbon dioxide and 3% oxygen
Sterile culture in incubator.Cell initial passage number for assessment was lower than for the 5th generation (P5)；
The aging H of BJ cell2O2To simulate active Oxdative stress.With the H of 1200 μM of concentration2O2It is thin to make an addition to medium treatment BJ
Born of the same parents 2 hours, carry out the aging of induced activity oxygen induction.Experiment starting, with 40000 cell culture in 2 ml complete mediums, paving
Plate is cultured in 6 orifice plates.Experiment initial stage is defined as the 0th day.Experiment amounts to 5 groups of parallel groups of setting.First group is set as blank pair
According to group, always with standard culture procedures culture, a cell culture fluid is changed within every 24 hours；Second group, third group be for the 4th group
With the 5th group of H2O2The culture solution of replacement is provided after processing；4th group is only to use H2O2Handle the group that induced activity Oxdative stress generates
Not；5th group is 1200 μM of H2O2The group being jointly processed by with pectin.Second group and third group after replacing culture medium just no longer
Participate in experiment.It should be noted that pectin adds always in the whole process；The ultimate density of pectin is 1mg/ml.It is cultivating
When by the 4th day, is taken pictures with inverted microscope, randomly choose 9 regions, each region is taken pictures 9, counted；
2) the 1 anti-H of mg/ml pectin2O2The aging result of induction:
At the 1st day of experiment, with 1200 μM of H2O2After processing BJ fibroblast 2 hours, continue culture 72 hours, induction makes
The activity for obtaining the beta-galactosidase of BJ cell increases, in x-gal dyeing, so that 18.1% BJ cell presentation declines
Old state.And in x-gal dyeing, always with 1200 μM of H of the BJ cell of pectin culture2O2After processing 2 hours, continue culture 72
Hour is dyed without apparent cell ageing, the cells ratio 0.98% of positive staining is presented, does not have with 1.04% ratio compareed
Statistical difference.
Embodiment 3: under pectin in mediator's primary fibroblast BJ cell anti-aging aging mark Dec-1 expression
Result figure
Fig. 3 pectin inhibits H2O2The rising of the senile cell DEC-1 albumen of induction
1) cell culture and cell protein extract
Application on human skin primary cell BJ cell line is bought from ATCC(59899913).BJ cell use containing 10% (v/v) fetal calf serum,
The DMEM high glucose medium culture of the penicillin/streptomycin of 1% (v/v).At 37 degree, contain 5% carbon dioxide and 3% oxygen
Sterile culture in incubator.Cell initial passage number for assessment was lower than for the 5th generation (P5)；
The aging H of BJ cell2O2To simulate active Oxdative stress.With the H of 1200 μM of concentration2O2It is thin to make an addition to medium treatment BJ
Born of the same parents 2 hours, carry out the aging of induced activity oxygen induction.Experiment starting, with 40000 cell culture in 2 ml complete mediums, paving
Plate is cultured in 6 orifice plates.Experiment initial stage is defined as the 0th day.Experiment amounts to 5 groups of parallel groups of setting.First group is set as blank pair
According to group, always with standard culture procedures culture, a cell culture fluid is changed within every 24 hours；Second group, third group be for the 4th group
With the 5th group of H2O2The culture solution of replacement is provided after processing；4th group is only to use H2O2Handle the group that induced activity Oxdative stress generates
Not；5th group is 1200 μM of H2O2The group being jointly processed by with pectin.Second group and third group after replacing culture medium just no longer
Participate in experiment.It should be noted that pectin adds always in the whole process；The ultimate density of pectin is 1 mg/ml.It is cultivating
When by the 4th day, used with the protein for extracting cell with the expression of western blot method detection aging marker DEC-1
Beta-actin is as internal reference albumen；
2) 1 mg/ml pectin inhibits H2O2The accumulation of aging marker protein in the BJ cell of processing
At the 1st day of experiment, with 1200 μM of H2O2After processing BJ fibroblast 2 hours, continue culture 72 hours, collection
Cell protein compares the expression of cell ageing protein marker DEC-1 with immunoblotting, that is, western blot method；
The result shows that 1200 μM of H2O2After processing BJ fibroblast 2 hours, after continuing culture 72 hours, BJ is significantly had accumulated
The expression of aging marker protein DEC-1 in fibroblast.And the addition of pectin inhibits 1200 μM of H2O2This of induction declines
The accumulation of old marker protein DEC-1.
Claims (4)
1. it is white powdery solids that right, which is related to pectin, purity test shows that the pectin is made of two components, molecular weight
Respectively 8.13 × 105D and 6.86 × 102D；The pectin of claim is that molecular weight ranges are 2 × 102 -2×106D's
Pectin, the pectin become Rha:GalA:Glc:Gal by equal polygalacturonic acid, RG1 type pectin and glucan composition, monosaccharide group:
Ara=1.6:50:38.6:7.4:2.4。
2. a kind of functional application of pectin as described in claim 1；Its application is to be used to the pectin inhibit into fiber finer
Born of the same parents, the polysaccharide of aging and free radical resisting accumulation including people's primary fibroblast BJ cell.
3. a kind of functional application of pectin as described in claim 1；Its application is former using the pectin as people is able to suppress
For the activity of anti-aging marker beta-galactosidase in fibroblast BJ cell, inhibit the accumulation of Dec1 albumen,
Have the function that inhibit cell ageing.
4. a kind of functional application of pectin as described in claim 1；Will application of the pectin for the products such as anti-aging, packet
It includes and individually or in combination perhaps cosmetics or individually or is in combination used to prepare into solid for skin care item
Beverage, oral solution, oral tablet, etc. different dosage forms.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115068498A (en) * | 2022-07-06 | 2022-09-20 | 东北师范大学 | Application of AG type pectin in prolonging life and resisting aging |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115068498A (en) * | 2022-07-06 | 2022-09-20 | 东北师范大学 | Application of AG type pectin in prolonging life and resisting aging |
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Application publication date: 20190712 |