CN103584093B - Radix paeoniae alba extraction has the application in health food or the cosmetics of radioresistance and senile-resistant efficacy in preparation - Google Patents
Radix paeoniae alba extraction has the application in health food or the cosmetics of radioresistance and senile-resistant efficacy in preparation Download PDFInfo
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- CN103584093B CN103584093B CN201310521821.5A CN201310521821A CN103584093B CN 103584093 B CN103584093 B CN 103584093B CN 201310521821 A CN201310521821 A CN 201310521821A CN 103584093 B CN103584093 B CN 103584093B
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Dermatology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses radix paeoniae alba extraction and there is the application in health food or the cosmetics of radioresistance and senile-resistant efficacy in preparation, radix paeoniae alba extraction in the present invention has anti-UVA radiation effects, can significantly reduce the radiation-induced cellular damage of UV, macrophage inflammatory Cytokines Expression is had to obvious inhibitory action simultaneously, can bring into play anti-inflammatory anti-aging effects; The present invention has also illustrated radix paeoniae alba extraction from cell and molecular level and has had anti-UV radiation and anti-aging effects. In addition, the invention also discloses the preferred preparation method of one of the above-mentioned radix paeoniae alba extraction with radioresistance and senile-resistant efficacy.
Description
Technical field
The invention belongs to root of herbaceous peony technical field, be specifically related to radix paeoniae alba extraction and have in preparation the health care of radioresistance and senile-resistant efficacyApplication in food or cosmetics.
Technical background
In recent years, the active component that derives from plant is more and more for the composition of Yao Wu ﹑ Zhi Liao ﹑ cosmetic purpose. OrderBefore, plant radix paeoniae alba extraction is used to various medicine and health care aspect.
Aging is a kind of physiological phenomenon, and on physiology, performance is exactly the deterioration of each organ, causes the generation of various ageing disorders.And evidence is the aging of skin the most intuitively. Show as the aging of cell at molecular level, this is one that accompanies with organism aging processProgressive event. The old and feeble physiological function that not only causes body is degenerated, and the immune decorum of body is also being degenerated gradually. Decline at human bodyPhysiology and the immune reason due to self not only in old process, also has a lot of external causes also can accelerate aging, its medium ultravioletRadiation is exactly to cause old and feeble the most common reason, and because the aging chronic inflammation causing is also one that immune system is degeneratedCharacterize.
Ultraviolet ray irradiation is to cause the topmost environmental factor of skin senescence, and ultraviolet ray is irradiated and made to produce a large amount of oxygen certainly in skin histologyBy base, make to organize anti-oxidative defense System Capacity to weaken, finally cause skin senescence. Free radical is very easily encroached in cell membrane notSaturated fatty acid, causes peroxidatic reaction of lipid, forms lipid peroxide, the catabolite MDA (MDA) of MDAWith amino acid, nucleic acid, the reaction of protein and phosphatide etc. and free amine group forms alicyclic diradical, and it participates in the physiology of body widelyWith pathologic process. Under normal physiological state, in body, oxygen radical is difficult for accumulating and peroxide injury, wherein SOD coupleThe oxidative and anti-oxidative balance of body plays vital effect, when body is subject to ultraviolet radiation for a long time, and superoxide anion in bodyThe generation of free radical with remove just disequilibrium, oxygen radical produces when too much, can produce toxic action to body. And ultravioletThe radiation of line can also cause the damage of DNA.
Old and feeble or aging is a necessary stage in organism metabolism process, and main manifestations is that body weakens the adaptive capacity of environmentSo that lose. Old and feeble mechanism is rather complicated, relates to the structure of each system and the change of function of body, relevant agingHypothesis is a lot of. Mainly comprise neuroendocrine theory, free radical theory, immunological theory, somatic mutation theory, theory of stressDeng. Along with immunologic development in modern times, people start to recognize that old and feeble and geriatric disease is in many aspects with immunologic functionChange and have substantial connection. Think that the interior most of organs of immune system and body and cell keep continuous contact, its change certainly will affectThese Organ and tissue cells. With advancing age, immunity function goes down, and induces thus some and has a strong impact on histoorganDisease, aggravated the aging course of the each system of body. Stop or reverse the exhaustion of immunologic function, can delay senility, and changeBecome the order of severity of old and feeble pathology histoorgan. Old and feeble immunology performance has immune organ function decline, along with the age increases,The function of each immune organ day by day fails, and is wherein apparent that most that thymus gland, atrophy of thymus gland are the keys that the elderly's immunologic function declines.Cellular immunity and humoral immune function also change to some extent in old and feeble process, and cellular immunity is that the T of dependence thymus gland differentiation and maturation is thinBorn of the same parents play a role, and therefore along with old atrophy of thymus gland, it is undoubtedly that the quality and quantity of T cell is all affected. Immune and old and feebleIn close relations, the immunity that improves body from immune angle is come anti-ageing and is delayed senility and have great significance.
In addition, also have close contact between aging and inflammation, inflammatory aging receives increasing concern, and inflammation aging isRefer to the process of a chronic inflammation of body appearance with advancing age. Inflammation is host system to pathogenic infection and various groupsKnit the event of replying of the series of complex of the generations such as damage, it is by affecting the mutual work of various kinds of cell and the factor in body microenvironmentWith, the balance trend of the regulation and control multiple physiology of body and pathological signals network, the dual character that has shown height: in the ordinary course of thingsAfter proinflammatory factors is as infection or tissue damage elimination, inflammatory reaction terminates immediately, is transformed into afterwards one highly active, smartThe poised state of fine tuning control, this inflammation is called as controllability inflammation. But under the effect of some uncertain factor, as continuedOr low intensive stimulation, target tissue is in the time of long-term or overreaction, inflammation cannot change from anti-infective tissue damage patternBecome the state of balance and stability, what cause inflammatory reaction continues to show as non-controllability inflammatory conditions. The inflammation of inflammatory agingThere is the feature of non-controlled inflammation. Inflammation is the normal physiological function of body as immune response, the inflammatory reaction pair of appropriatenessBody is favourable, otherwise harmful. Inflammatory cytokine network comprises pro-inflammatory cytokine network and anti-inflammatory cytokine network.These two had not only been opposed but also the direction of the favourable or illeffects of inflammation is being controlled in the variation of the sub-network of interdependence mutually. Positive reasonRelation in a dynamic equilibrium between them under condition, the pathologic inflammation in inflammatory aging is that inflammatory cytokine network is unbalanceDue to, also extremely closely related with inflammatory mediator.
Immunosenescence and inflammatory aging are gone hand in hand, and the mechanism of inflammatory aging can be generalized into as follows: theory of stress stress be machineThe non-specific adaptive response of general that body occurs in the time of various internal and external environment factors and socio-psychological factor stimulation. Stress be oneDouble-edged sword, favourable also harmful to body. In inflammatory senescence process, body is in the environment that stressor forms for a long time. StressFormer is the reason that causes and maintain chronic pro-inflammatory state existence. Oxidation inflammation, oxidative stress, inflammatory old and feeble with exempt fromOld and feeble three is closely related for epidemic disease. Cell factor theory, pro-inflammatory cytokine is sent out in the inflammatory aging of body due to chronic inflammationWaving important effect.
At present, about radix paeoniae alba extraction is had to the application in health food or the cosmetics of radioresistance and senile-resistant efficacy in preparationThe research of aspect, yet there are no open.
Summary of the invention
Technical problem to be solved by this invention is to provide radix paeoniae alba extraction and has in preparation the health care food of radioresistance and senile-resistant efficacyApplication in product and cosmetics.
The present invention has carried out radioresistance, anti-inflammatory and the checking of antidotal effect cell biology to radix paeoniae alba extraction, because radiation is drawnPlay inflammation, and inflammation can cause aging, therefore applicant has studied radix paeoniae alba extraction opposing UVA radiation effects, and at presentApplication about this type of is little. The radix paeoniae alba extraction that the application provides has anti-UVA radiation effects, significantly reduces UVA radiationThe dermal cell causing and epidermal cell damage, in addition, also have close contacting between aging and inflammation, applicant studies simultaneouslyRadix paeoniae alba extraction antiphlogistic effects in vitro, find that it can suppress macrophage inflammatory Cytokines Expression effectively. Proinflammatory cell because ofThe aging of son energy inducing cell, proinflammatory cytokine (as TNF-α, IFN-γ, IFN-β) etc. are by producing active oxygen and activationThe aging of ATM/P53/P21 signal path induction epithelial cell, chemokine receptors CXCR2 induces into fiber by P53 pathCell ageing, DNA damage activates the signal paths such as NF-Κ B and produces proinflammatory cytokine (IL-1, IL-6, IL-8 etc.), thus resistanceThe stagnant cell cycle, induce and maintain cell ageing phenotype. DNA damage theory, due to DNA damage accumulation make stem cell andMatrix fibroblast is divided into pro-inflammatory cytokine overexpressing cell, makes the collapse of multilayer shape cytokine network, causes inflammatoryAnd cause aging.
In the present invention, the above-mentioned radix paeoniae alba extraction with radioresistance and senile-resistant efficacy, can adopt commercially available prod, also can be preferredThe radix paeoniae alba extraction that adopts following method provided by the invention to prepare.
The preparation method of the above-mentioned radix paeoniae alba extraction with radioresistance and senile-resistant efficacy provided by the invention, contains following steps:
(1) pre-treatment: the raw material root of herbaceous peony is cleaned, pulverized, weigh;
(2) ultrasonic wave extracts: with after the ultrasonic extraction of ethanol water room temperature, filter, merging filtrate, by filtrate vacuum decompressionConcentrate to obtain concentrate, in concentrate, add water, filter, obtain B extract;
(3) macroporous absorption post separate: by B extract to first PIPO-02 macroreticular resin splitter, adopt methyl alcoholWater and formic acid water are as mobile phase, and after wash-out, the methanol-water of employing variable concentrations and formic acid water, as mobile phase, carry out for the first timeAfter wash-out, carry out for the second time removal of impurities, the second PIPO-02 macropore of then connecting under first PIPO-02 macroreticular resin splitterResin separating column, adopts ethanol elution to obtain the C cut section that contains target substance;
(4) concentrate drying: vacuum-concentrcted C cut section, and vacuum drying obtains radix paeoniae alba extraction.
Grinding and sieving to 30～60 order in step of the present invention (1).
In step of the present invention (2), the volumn concentration of ethanol water is 60～80%, crosses 500～800 object filter clothes when filtration.
In step of the present invention (2) with the heavy ultrasonic extraction of ethanol water room temperature of 8～10 times of raw materials 2～3 times, each 20～40 pointsClock; Concentrate by filtrate vacuum-concentrcted to 3～5 times of weights of raw material adds water in concentrate, and the consumption of water is that concentrate is total2～4 times of weight.
In step of the present invention (3) for the first time when wash-out in methanol-water methyl alcohol volumn concentration be 3%, the body of formic acid in formic acid waterLong-pending percentage composition is 0.2%, for the first time when wash-out the quality of eluent be root of herbaceous peony raw material gross weight 3-5 doubly; For the second time when wash-outIn methanol-water, methyl alcohol volumn concentration is 5%, and in formic acid water, the volumn concentration of formic acid is 0.2%, washes when wash-out for the second timeThe quality of de-liquid is 8～12 times of root of herbaceous peony raw material gross weight.
Described in step of the present invention (3), first PIPO-02 macroreticular resin splitter separates with second PIPO-02 macroreticular resinPost is identical.
In step of the present invention (3), the volumn concentration of ethanol is more than 80%; Preferably, the volume hundred of ethanol of the present inventionDividing content is 80～95%
In step of the present invention (4), 40～70 DEG C of vacuum-concentrcted C cut sections, in radix paeoniae alba extraction, detect the root of herbaceous peony through HPLCThe quality percentage composition of glycosides is more than 70%.
Tool of the present invention has the following advantages:
(1) radix paeoniae alba extraction that adopts the inventive method to extract, wherein albiflorin composition accounts for 70%(quality percentage composition) withUpper, albiflorin conventionally in health products or cosmetics application quantity little, the clear and definite easily quality control of content;
(2) the radix paeoniae alba extraction color and luster that adopts the inventive method to prepare is brief talked, and is applicable to applying in cosmetics, does not affect cosmeticOriginal color and luster of product;
(3) adopt the extraction efficiency of the inventive method extraction radix paeoniae alba extraction high, can make full use of resource;
(4) radix paeoniae alba extraction manufacture cost of the present invention is low, is suitable in health products or cosmetics and applies, and has market competingStrive power;
(5) the present invention finds through test, and radix paeoniae alba extraction can significantly reduce the radiation-induced cellular damage of UV, bites huge simultaneouslyCellular inflammation factor expression has obvious inhibitory action, thereby performance anti-aging effects, has illustrated this from cell and molecular levelRadix paeoniae alba extraction product in invention has anti-UV radiation, Cell protection and anti-aging effects.
Brief description of the drawings
Fig. 1 be in the embodiment of the present invention 4 (1.2) radix paeoniae alba extraction to UVA irradiate under 3T3 cytoprotection;
Fig. 2 is the experimental result of (1.3) comet electrophoresis detection DNA Damage in the embodiment of the present invention 4, and wherein left figure is3T3 cell, the middle figure irradiating without UVA is that the 3T3 cell, the right figure that have UVA to irradiate are the 3T3 that has added radix paeoniae alba extractionThe cell DNA of cell produces comettail rate;
Fig. 3 is that in the embodiment of the present invention 4, (1.4) flow cytometer detects radix paeoniae alba extraction to the UVA radiation 3T3 cell cycleProtective effect, wherein A figure is that 3T3 cell, the B figure irradiating without UVA has 3T3 cell, the C figure of UVA irradiation to beAdd the flow cytometer detection result of the 3T3 cell of radix paeoniae alba extraction;
Fig. 4 is that in the embodiment of the present invention 4, (1.5) mtt assay detects radix paeoniae alba extraction to RAW264.7 cytotoxicity;
Fig. 5 is embodiment of the present invention 4(1.5) middle PCR detection radix paeoniae alba extraction is in transcriptional level inhibition or reduce inflammatory factor IL-6Generation;
Fig. 6 is embodiment of the present invention 4(1.5) middle PCR detection radix paeoniae alba extraction is in transcriptional level inhibition or reduce inflammatory factor IL-1Generation;
Fig. 7 is embodiment of the present invention 4(1.5) middle PCR detection radix paeoniae alba extraction is in transcriptional level inhibition or reduce inflammatory factorThe generation of TNF-alpha;
Fig. 8 is embodiment of the present invention 4(1.5) middle PCR detection radix paeoniae alba extraction is in transcriptional level inhibition or reduce inflammatory factorThe generation of MCP-1;
Fig. 9 is embodiment of the present invention 4(1.5) middle PCR detection radix paeoniae alba extraction is in transcriptional level inhibition or reduce inflammatory factorThe generation of MCP-1.
Detailed description of the invention
That the present invention is further illustrated in conjunction with the embodiments below.
(1) extracting method of the preferred radix paeoniae alba extraction of the present invention
Embodiment 1
The radix paeoniae alba extraction that the present embodiment provides, it prepares by the following method:
A: pre-treatment: the raw material root of herbaceous peony is washed, pulverize, be screened to 30～60 orders, weigh 1 kilogram;
B: extract: the ultrasonic room temperature of the ethanol water that is 60% with 8～10 kilograms of ethanol volumn concentrations is extracted 2 times, everyInferior 20～40 minutes, 500～800 order filter-cloth filterings, merging filtrate, obtains concentrate by filtrate vacuum-concentrcted to 3～5 kilogram,In concentrate, add 6～8 kg water, filter, obtain B extract;
C: macroporous absorption post separate: by B extract to first PIPO-02 macroreticular resin splitter, first adopt methyl alcoholVolumn concentration be 3% methanol-water and the formic acid volumn concentration formic acid water that is 0.2% as mobile phase, wash-out 3～5kg,Adopt again methyl alcohol volumn concentration be 5% methanol-water and the formic acid volumn concentration formic acid water that is 0.2% as mobile phase,Wash-out 8～12kg, carries out removal of impurities, the second PIPO-02 macropore of then connecting under first PIPO-02 macroreticular resin splitterResin separating column, adopts the ethanol elution that volumn concentration is 80% to obtain the C cut section that contains target substance;
D: concentrate drying: 40～70 DEG C of vacuum-concentrcted C cut sections, obtain radix paeoniae alba extraction through vacuum drying, weighBe 50～80 grams, find with the test of HPLC albiflorin reference substance, albiflorin content exceedes 70%.
Embodiment 2
The radix paeoniae alba extraction that the present embodiment provides, it prepares by the following method:
A: pre-treatment: the raw material root of herbaceous peony is washed, pulverize, be screened to 30～60 orders, weigh 10 kilograms;
B: extract: the ultrasonic room temperature of the ethanol water that is 80% with 80～double centner ethanol volumn concentration is extracted 2 times,Each 20～40 minutes, 500～800 order filter-cloth filterings, merging filtrate, obtains dense by filtrate vacuum-concentrcted to 30～50 kilogramContracting liquid adds 60～80 kg water in concentrate, filters, and obtains B extract;
C: macroporous absorption post separate: by B extract to first PIPO-02 macroreticular resin splitter, first adopt methyl alcoholVolumn concentration be 3% methanol-water and the formic acid volumn concentration formic acid water that is 0.2% as mobile phase, wash-out 30～50kgAdopt again methyl alcohol volumn concentration be 5% methanol-water and the formic acid volumn concentration formic acid water that is 0.2% as mobile phase,Wash-out 80～120kg carries out removal of impurities, the second PIPO-02 macropore of then connecting under first PIPO-02 macroreticular resin splitterResin separating column, adopts the ethanol elution that volumn concentration is 85% to obtain the C cut section that contains target substance;
D: concentrate drying: 40～70 DEG C of vacuum-concentrcted C cut sections, obtain radix paeoniae alba extraction through vacuum drying, weighBe 500～800 grams, find with the test of HPLC albiflorin reference substance, albiflorin content exceedes 70%.
Embodiment 3
A kind of radix paeoniae alba extraction, it prepares by the following method:
A: pre-treatment: the raw material root of herbaceous peony is washed, pulverize, be screened to 30～60 orders, the double centner of weighing;
B: extract: the ultrasonic room temperature of the ethanol water that is 75% with 800～1000 kilograms of ethanol volumn concentrations is extracted 3 times,Each 20～40 minutes, 500～800 order filter-cloth filterings, merging filtrate, obtains filtrate vacuum-concentrcted to 300～500 kilogramConcentrate adds 600～800 kg water in concentrate, filters, and obtains B extract;
C: macroporous absorption post separate: by B extract to first PIPO-02 macroreticular resin splitter, first adopt methyl alcoholVolumn concentration be 3% methanol-water and the formic acid volumn concentration formic acid water that is 0.2% as mobile phase, wash-out300～500kg, then to adopt methyl alcohol volumn concentration be that the formic acid water that 5% methanol-water and formic acid volumn concentration are 0.2% is doneFor mobile phase, wash-out 800～1200kg, carries out removal of impurities, then under first PIPO-02 macroreticular resin splitter, connects secondRoot PIPO-02 macroreticular resin splitter, the C that the ethanol elution that employing volumn concentration is 95% obtains containing target substance heats up in a steamerSegmentation;
D: concentrate drying: 40～70 DEG C of vacuum-concentrcted C cut sections, obtain radix paeoniae alba extraction through vacuum drying, weighBe 5000～8000 grams, find with the test of HPLC albiflorin reference substance, albiflorin content exceedes 70%.
(2) effect of radix paeoniae alba extraction checking
Embodiment 4 radix paeoniae alba extraction radioresistances and senile-resistant efficacy checking
4.1 experimental raw
Below adopt in the embodiment of the present invention 1 and prepare radix paeoniae alba extraction, carry out radioresistance and senile-resistant efficacy checking.
4.2 anti-radiation effect confirmatory experiments
Adopt in the embodiment of the present invention 1 and be prepared into the protective action of radix paeoniae alba extraction to UVA radiation 3T3 cell, method is as follows:
Detect cell proliferation situation by mtt assay and measure the protective action of compound to UVA radiation 3T3 cell.
(1) collect logarithmic phase 3T3 cell, adjust concentration of cell suspension, every hole adds 100 μ L, and it is close that bed board is adjusted cell to be measuredSpend to 1000-10000 hole;
（2）2.5%CO2, hatch for 37 DEG C, at the bottom of being paved with hole to cell monolayer (96 hole flat underside), gradient concentration is followed successively by:200 μ g/mL ﹑ 100 μ g/mL ﹑ 50 μ g/mL ﹑ 25 μ g/mL ﹑ 12.5 μ g/mL ﹑ 6.25 μ g/mL, each gradient arranges 3 againHole, every group arranges 3 blank well, and control group does not deal with 5%CO2, hatch 16-24 hour for 37 DEG C;
(3) radiation group respectively organize cell by UVA dose of radiation condition grope experiment the ultraviolet condition obtaining carry out ultra-violet radiation:Exposure time 45min; Dose of radiation 1.4J/cm2, control group does not deal with, 5%CO2, hatch 16-24 hour for 37 DEG C;
(4) every hole adds 10 μ LMTT solution, continues to cultivate 4h, stops cultivating, and carefully sucks nutrient solution in hole;
(5) every hole adds 100 μ L dimethyl sulfoxide (DMSO)s, puts low-speed oscillation 10min on shaking table, crystal is fully dissolved, at enzymeThe light absorption value in each hole is measured at connection immune detection instrument OD570nm place;
As shown in fig. 1, result of the test shows result, adopts the radix paeoniae alba extraction extracting in the embodiment of the present invention 1 to UVA spokePenetrate 3T3 cell and there is the activity (>=90%cellviability) of protection.
4.3 anti-inflammatory anti-aging effects effect confirmatory experiments
Adopt in the comet electrophoresis detection embodiment of the present invention 1 and be prepared into the guarantor of radix paeoniae alba extraction to UVA radiation 3T3 cell DNAProtect effect, method is as follows: DNA damage is more serious, causes DNA superhelix looser, and the breakaway poing of generation is more,DNA fragmentation is less, thereby the DNA part occurring at comet afterbody is more, and the length of comets tail, area and fluorescence intensity are moreGreatly. By measuring the index such as length, area or fluorescence intensity of comet afterbody, can carry out quantitatively the degree of injury of DNAAnalyze.
(1) pre-12 orifice plates of 3T3 cell, cell density is 70%;
(2) compound treatment: diluted chemical compound to the 200 μ g/mL(that above-mentioned testing sieve is selected we adopt effective maximum denseDegree is as the medicine working concentration of this experiment), inhale and abandon culture medium, add the diluted chemical compound liquid of dilution, every hole 1mL, 37 degree,5%CO2Incubator cultivate 24h;
(3) by the cell UVA irradiation 1h processing, 60 μ W/cm2，37℃，5%CO2Incubator cultivate 24h;
(4) trypsin digestion cell, stops digestion containing the culture medium of serum, cell liquid proceeded in 15mL centrifuge tube,1000rpm/min, centrifugal 5min, abandons supernatant, adds appropriate PBS re-suspended cell, and the same centrifugal 5min, abandons PBS, addsAppropriate PBS re-suspended cell, adjusts cell concentration to 106cells/mL；
(5) glue: dissolve the normal fusing point agarose of 0.7% concentration, drip fast 70 μ L to clean slide, use fastCover glass moulding, 4 DEG C of gel 20min, peel-off covers slide gently, mixes cell suspension and 0.8% low melting-point agarose, fastDrop on ground floor glue cover glass moulding fast, 4 DEG C of gel 20min;
(6) peel off gently the cover glass on second layer glue, glue is put into alkaline bleach liquor cleavage liquid, 4 DEG C of cracking 1.5～2h;
(7) take out gently slide, steam washing glue 2 times with single;
(8) glue is put into alkaline electrophoresis liquid, left standstill 20min, 25V, electrophoresis 15min, the Tris-HCl neutralization three of pH7.5Inferior, each 5min;
(9) PI dyeing microscopic examination, takes pictures.
The experimental result of comet electrophoresis detection DNA Damage as shown in Figure 2, is prepared into the root of herbaceous peony in the embodiment of the present invention 1Extract has inhibitory action significantly to the radiation-induced DNA damage of UVA.
4.4 anti-inflammatory anti-aging effects effect confirmatory experiments
In the flow cytometer detection of drugs embodiment of the present invention 1, be prepared into radix paeoniae alba extraction to the UVA radiation guarantor of 3T3 cell cycleProtect effect, method is as follows: DNA Damage, start cellular genome repair mechanism, and regulate the cell cycle and then complete baseBecause of the wound repair of group, ensure the complete of cellular genome. UV radiation causes fracture damage to the genomic DNA of cell, mustTo cause the cell cycle to change. Therefore utilize Flow cytometry to carry out detection compound whether the cell cycle changing is had to tuneJoint inhibitory action.
(1) pre-12 orifice plates of 3T3 cell, overnight incubation;
(2) cell drug processing: medicine is done to concentration dilution, add cell, UVA radiation 1h after 24h, 60 μ W/cm2，Cell culture incubator is received sample after cultivating 24h;
(3) trypsin digestion cell, goes to 1.5mL centrifuge tube, and the centrifugal 5min of 1000rpm/min, abandons supernatant, and PBS washes twoTime, centrifugal condition is the same, abandons PBS, adds 70% ethanol 4 degree of 500 μ L fixedly to spend the night;
(4) abandon PBS, add 4 DEG C of 70% ethanol of 500 μ L fixedly to spend the night;
(5) 1000rpm/min, 4 DEG C, centrifugal 5min, abandons ethanol, adds PBS resuspended, the same centrifugal;
(6) abandon PBS, add in the PBS that contains DNA enzyme I and PI dyestuff 200 object screen filtrations;
(7) upper Flow cytometry is surveyed the cycle;
Flow cytometer detects the cell cycle, result as shown in Figure 3, the pre-synthesis phase that wherein G1 being DNA, cell resting stage;S is DNA replication dna period; G2 is DNA post-synthesis phase, is the cell of proliferation period. If before G1 dosing > after G1 dosing,The cell proportion of S phase and G2 phase increases, and it is active that cell shows propagation.
Streaming result in Fig. 3 shows: in the embodiment of the present invention 1, be prepared into radix paeoniae alba extraction compared with UVA group data, the root of herbaceous peonyExtract has protective effect.
4.5 anti-inflammatory anti-aging effects Mechanism Validation experiments
Mtt assay detect screening the embodiment of the present invention 1 in be prepared into radix paeoniae alba extraction to RAW264.7 cytotoxicity, method asUnder:
(1) collect logarithmic phase RAW264.7 cell, adjust concentration of cell suspension, every hole adds 100 μ L, and bed board makes to be measured thinBorn of the same parents adjust density to 1000-10000 hole;
（2）2.5%CO2, hatch for 37 DEG C, at the bottom of being paved with hole to cell monolayer, (96 hole flat underside), is divided into 60 groups at random, eachOne group of medicine, every group adds the radix paeoniae alba extraction of concentration gradient, and gradient concentration is followed successively by: 200 μ g/mL ﹑ 100 μ g/mL ﹑50 μ g/mL ﹑ 25 μ g/mL ﹑ 12.5 μ g/mL ﹑ 6.25 μ g/mL, each gradient arranges 3 multiple holes, and every group arranges 3 blank well,5%CO2, hatch 16-48 hour for 37 DEG C;
(3) every hole adds 10 μ LMTT solution, continues to cultivate 4h, stops cultivating, and carefully sucks nutrient solution in hole;
(4) every hole adds 100 μ L dimethyl sulfoxide (DMSO)s, puts low-speed oscillation 10min on shaking table, crystal is fully dissolved, at enzymeThe light absorption value in each hole is measured at connection immune detection instrument OD570nm place;
In the embodiment of the present invention 1, be prepared into radix paeoniae alba extraction to the toxicity of RAW264.7 cell as shown in Figure 4, from Fig. 4Can find out the maximal non-toxic concentration of radix paeoniae alba extraction to cell nonhazardous.
4.6 anti-inflammatory anti-aging effects effect confirmatory experiments
In the PCR detection embodiment of the present invention 1, be prepared into radix paeoniae alba extraction suppresses or reduce inflammatory factor generation at transcriptional level,Method is as follows: the compound radix paeoniae alba extraction with anti-UVA radiation that screening is obtained is at macrophage RAW264.7 cell membraneType carries out anti-inflammatory activity experiment.
(1) the phase RAW264.7 cell of taking the logarithm, pre-24 orifice plates, it is 3 × 10 that bed board is adjusted cell density5cells/mL；
(2) add the PMA of 50ng/mL, cultivate 48h, examine under a microscope cellular morphology;
(3) carefully suck culture medium in hole, with after PBS flushing 2 times, add 1640 culture mediums of serum-free, cultivate 24h;
(4) add LPS and the radix paeoniae alba extraction of 100ng/mL, cultivate 18～24h;
(5) RNA of extracting administration group cell, use real-timePCR method to IL-6 and IL-1 (Interleukin-6,Nterleukin-1, interleukin-6 interleukin 1), TNF-α (tumornecrosisfactor-alpha, tumor necrosis factorSon), MCP-1 (monocytechemotacticprotein-1, macrophage chemoattractant protein 1) and iNOS carry out qualitative, quantitativeAnalyze, observe the impact that active matter is expressed inflammatory factor, in the embodiment of the present invention 1, be prepared into radix paeoniae alba extraction to inflammatory factorThe inhibitory action of expression.
The result of IL-6 and IL-1 (Interleukin-6, nterleukin-1, interleukin-6 interleukin 1) is as in Fig. 5 and 6Shown in, the result of TNF-α (tumornecrosisfactor-alpha, TNF) as shown in Figure 7, MCP-1(monocytechemotacticprotein-1, macrophage chemoattractant protein 1) and iNOS result as shown in Fig. 8 and 9,
From Fig. 5-9, can find out: the radix paeoniae alba extraction that detects preparation in the embodiment of the present invention 1 shows the different scorching effects that presses down,In the embodiment of the present invention 1, the radix paeoniae alba extraction of preparation is remarkable to the inhibitory action of MCP-1 and iNos.
In fact, the present patent application people also simultaneously to the radix paeoniae alba extraction in other sources as commercially available radix paeoniae alba extraction or additive methodThe radix paeoniae alba extraction of preparation carries out above-mentioned test, and result shows also have above-mentioned radioresistance and antidotal effect.
Above-described embodiment is preferably embodiment of the present invention, but embodiments of the present invention are not restricted to the described embodiments,Other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify, all shouldFor equivalent substitute mode, be included in protection scope of the present invention.
Claims (5)
1. radix paeoniae alba extraction has the application in health food or the cosmetics of radioresistance and senile-resistant efficacy as unique active component with radioresistance and senile-resistant efficacy in preparation;
This radix paeoniae alba extraction prepares by the method comprising the following steps:
(1) pre-treatment: the raw material root of herbaceous peony is cleaned, pulverized, weigh;
(2) ultrasonic wave extracts: with after the ultrasonic extraction of ethanol water room temperature, filter, merging filtrate, obtains concentrate by filtrate vacuum-concentrcted, in concentrate, adds water, and filters, and obtains B extract;
(3) macroporous absorption post separate: by B extract to first PIPO-02 macroreticular resin splitter, adopt methanol-water and formic acid water as mobile phase, for the first time after wash-out, the methanol-water of employing variable concentrations and formic acid water are as mobile phase, carry out after wash-out, carrying out removal of impurities for the second time, then the second PIPO-02 macroreticular resin splitter of connecting under first PIPO-02 macroreticular resin splitter, adopts ethanol elution to obtain the C cut section that contains target substance;
(4) concentrate drying: vacuum-concentrcted C cut section, and vacuum drying obtains radix paeoniae alba extraction;
In step (4), 40 ~ 70 DEG C of vacuum-concentrcted C cut sections, the quality percentage composition that detects albiflorin through HPLC in radix paeoniae alba extraction is more than 70%;
In step (2), the volumn concentration of ethanol water is 60 ~ 80%, crosses 500 ~ 800 object filter clothes when filtration;
In step (2) with the heavy ultrasonic extraction of ethanol water room temperature of 8 ~ 10 times of raw materials 2 ~ 3 times, each 20 ~ 40 minutes; Concentrate by filtrate vacuum-concentrcted to 3 ~ 5 times of weights of raw material adds water in concentrate, and the consumption of water is 2 ~ 4 times of concentrate gross weight;
In step (3) for the first time when wash-out in methanol-water methyl alcohol volumn concentration be 3%, in formic acid water, the volumn concentration of formic acid is 0.2%, for the first time when wash-out the quality of eluent be root of herbaceous peony raw material gross weight 3-5 doubly; In methanol-water, methyl alcohol volumn concentration is 5% when wash-out for the second time, and in formic acid water, the volumn concentration of formic acid is 0.2%, and the quality of eluent is 8 ~ 12 times of the root of herbaceous peony raw material gross weight when wash-out for the second time.
2. a preparation method with the radix paeoniae alba extraction of radioresistance and senile-resistant efficacy, is characterized in that: contain following steps:
(1) pre-treatment: the raw material root of herbaceous peony is cleaned, pulverized, weigh;
(2) ultrasonic wave extracts: with after the ultrasonic extraction of ethanol water room temperature, filter, merging filtrate, obtains concentrate by filtrate vacuum-concentrcted, in concentrate, adds water, and filters, and obtains B extract;
(3) macroporous absorption post separate: by B extract to first PIPO-02 macroreticular resin splitter, adopt methanol-water and formic acid water as mobile phase, for the first time after wash-out, the methanol-water of employing variable concentrations and formic acid water are as mobile phase, carry out after wash-out, carrying out removal of impurities for the second time, then the second PIPO-02 macroreticular resin splitter of connecting under first PIPO-02 macroreticular resin splitter, adopts ethanol elution to obtain the C cut section that contains target substance;
(4) concentrate drying: vacuum-concentrcted C cut section, and vacuum drying obtains radix paeoniae alba extraction;
In step (4), 40 ~ 70 DEG C of vacuum-concentrcted C cut sections, the quality percentage composition that detects albiflorin through HPLC in radix paeoniae alba extraction is more than 70%;
In step (2), the volumn concentration of ethanol water is 60 ~ 80%, crosses 500 ~ 800 object filter clothes when filtration;
In step (2) with the heavy ultrasonic extraction of ethanol water room temperature of 8 ~ 10 times of raw materials 2 ~ 3 times, each 20 ~ 40 minutes; Concentrate by filtrate vacuum-concentrcted to 3 ~ 5 times of weights of raw material adds water in concentrate, and the consumption of water is 2 ~ 4 times of concentrate gross weight;
In step (3) for the first time when wash-out in methanol-water methyl alcohol volumn concentration be 3%, in formic acid water, the volumn concentration of formic acid is 0.2%, for the first time when wash-out the quality of eluent be root of herbaceous peony raw material gross weight 3-5 doubly; In methanol-water, methyl alcohol volumn concentration is 5% when wash-out for the second time, and in formic acid water, the volumn concentration of formic acid is 0.2%, and the quality of eluent is 8 ~ 12 times of the root of herbaceous peony raw material gross weight when wash-out for the second time.
3. the preparation method of the radix paeoniae alba extraction with radioresistance and senile-resistant efficacy according to claim 2, is characterized in that: grinding and sieving to 30 ~ 60 order in step (1).
4. the preparation method of the radix paeoniae alba extraction with radioresistance and senile-resistant efficacy according to claim 2, is characterized in that: described in step (3), first PIPO-02 macroreticular resin splitter is identical with second PIPO-02 macroreticular resin splitter.
5. the preparation method of the radix paeoniae alba extraction with radioresistance and senile-resistant efficacy according to claim 2, is characterized in that: in step (3), the volumn concentration of ethanol is more than 80%.
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| KR102418101B1 (en) | 2017-11-30 | 2022-07-08 | (주)아모레퍼시픽 | Composition for preventing or alleviating intrinsic aging comprising paeoniflorin or albiflorin |
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| CN1899064A (en) * | 2006-07-18 | 2007-01-24 | 吴煌发 | Anti-radiation tea |
| CN1984637A (en) * | 2004-05-10 | 2007-06-20 | 株式会社Lg生活健康 | Skin aging treatment comprising paeoniflorin |
| CN101167901A (en) * | 2006-10-26 | 2008-04-30 | 中国人民解放军军事医学科学院毒物药物研究所 | Chinese angelica and peony formula extraction, preparation and medical use thereof |
| CN102018651A (en) * | 2010-10-19 | 2011-04-20 | 广东雅倩化妆品有限公司 | Aloe extract |
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| CN1984637A (en) * | 2004-05-10 | 2007-06-20 | 株式会社Lg生活健康 | Skin aging treatment comprising paeoniflorin |
| CN1899064A (en) * | 2006-07-18 | 2007-01-24 | 吴煌发 | Anti-radiation tea |
| CN101167901A (en) * | 2006-10-26 | 2008-04-30 | 中国人民解放军军事医学科学院毒物药物研究所 | Chinese angelica and peony formula extraction, preparation and medical use thereof |
| CN102018651A (en) * | 2010-10-19 | 2011-04-20 | 广东雅倩化妆品有限公司 | Aloe extract |
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