CN103564503B - Forsythia suspense extraction is preparing the application in the health food or cosmetics with uv-resistant A radiation and senile-resistant efficacy - Google Patents
Forsythia suspense extraction is preparing the application in the health food or cosmetics with uv-resistant A radiation and senile-resistant efficacy Download PDFInfo
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Classifications
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- 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
- 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
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses forsythia suspense extraction and prepare the application in the health food or cosmetics with radioresistance and senile-resistant efficacy, forsythia suspense extraction in the present invention has uv-resistant A radiation effects, significantly can reduce the radiation-induced cellular damage of UV, macrophage inflammatory factor is expressed simultaneously and there is obvious inhibitory action, anti-inflammatory anti-aging effects can be played; The present invention also illustrates forsythia suspense extraction from cell and molecular level and has uv-resistant radiation and anti-aging effects.In addition, the invention also discloses the above-mentioned preferred preparation method of one with the forsythia suspense extraction of radioresistance and senile-resistant efficacy.
Description
Technical field
The invention belongs to capsule of weeping forsythia technical field, be specifically related to forsythia suspense extraction and preparing the application in the health food or cosmetics with uv-resistant A radiation and senile-resistant efficacy.
Technical background
In recent years, the active component deriving from plant is increasing secondary for the composition for the purpose of Yao Wu ﹑ treatment and beauty treatment etc.
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 and skin the most intuitively is aging.Show as the aging of cell at molecular level, this is the progressive event of of accompanying with organism aging process.Aging not only causes the physiological function of body to be degenerated, and the immune decorum of body is also being degenerated gradually.Physiology and immune reason not only due to self in human aging process, also have a lot of external cause also can accelerate aging, the radiation of its medium ultraviolet is exactly cause old and feeble reason the most common, and due to the aging chronic inflammation caused also be the sign that immune system is degenerated.
Ultraviolet radiation causes the topmost environmental factor of skin senescence, and Ultraviolet radiation makes to produce a large amount of oxygen radical in skin histology, makes to organize anti-oxidative defense System Capacity to weaken, finally causes skin senescence.Free radical very easily encroaches on the unrighted acid in cell membrane, cause peroxidatic reaction of lipid, form lipid peroxide, the catabolite MDA (MDA) of MDA and amino acid, nucleic acid, protein and phosphatide etc. form alicyclic diradical with free amine group reaction, and it participates in physiology and the pathologic process of body widely.Under normal physiological condition, in body not easily there is accumulation and peroxide injury in oxygen radical, wherein the oxidative and anti-oxidative balance of SOD to body plays vital effect, when body is for a long time by ultraviolet radiation, in body ultra-oxygen anion free radical generation with remove just disequilibrium, when oxygen radical produces too much, toxic action can be produced to body.And ultraviolet radiation 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 the adaptive capacity of body to environment weakens so that lose.Old and feeble mechanism is rather complicated, relates to the change of the structure and fuction of each system of body, about aging hypothesis is a lot of.Mainly comprise neuroendocrine theory, free radical theory, immunological theory, somatic mutation theory, theory of stress etc.Along with immunologic development in modern times, people start to recognize that old and feeble and geriatric disease has substantial connection with the change of immunologic function in many aspects.Think that most of organ and cell keep continuous contact in immune system and body, its change certainly will affect these Organ and tissue cells.With advancing age, immunity function goes down, and induces the disease that some have a strong impact on histoorgan thus, exacerbates the aging course of each system of body.Stop or reverse the exhaustion of immunologic function, can delay senility, and change the order of severity of old and feeble pathology histoorgan.Old and feeble immunology performance has immune organ function to fail, and along with the age increases, the function of each immune organ day by day fails, and is wherein apparent that most thymus gland, and atrophy of thymus gland is the key that the elderly's immunologic function declines.Cellular immunity and humoral immune function also change to some extent in the process of aging, and cellular immunity is that the T cell relying on thymus gland differentiation and maturation plays a role, and therefore along with aged thymus atrophy, it is undoubtedly that the quality and quantity of T cell is all affected.Immune and old and feeble is in close relations, has great significance from the immunity of the angle raising body of immunity is next anti-ageing with delaying senility.
In addition, also have close contact between aging and inflammation, inflammatory aging receives increasing concern, and inflammation aging refers to that the process of a chronic inflammation appears in body with advancing age.Inflammation is the response event of host system to the series of complex that pathogenic infection and various tissue damages etc. produce, it is by affecting the interaction of various kinds of cell and the factor in body microenvironment, the balance of the regulation and control multiple physiology of body and pathological signals network is moved towards, show the dual character of height: in the ordinary course of things after proinflammatory factors is as infection or tissue damage elimination, inflammatory reaction terminates immediately, be transformed into the poised state of a kind of high activity, finely regulating afterwards, this inflammation is called as controllability inflammation.But under the effect of some uncertain factor, as that continue or low intensive stimulation, when target tissue is in long-term or overreaction, inflammation cannot be transformed into the state of balance and stability from anti-infective tissue damage pattern, cause inflammatory reaction continue carry out showing as non-controllability inflammatory conditions.The inflammation of inflammatory aging has the feature of non-controlled inflammation.Inflammation is the normal physiological function of body as immune response, and the inflammatory reaction of appropriateness is favourable to body, otherwise is then harmful to.Inflammatory cytokine network comprises pro-inflammatory cytokine network and anti-inflammatory cytokine network.These two had not only been opposed but also the change of the sub-network of interdependence controls the direction of the favourable of inflammation or illeffects mutually.Be in the relation of a dynamic equilibrium under normal circumstances between them, the pathological inflammatory in inflammatory aging be inflammatory cytokine network unbalance caused by, also closely related with the exception of 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 the non-specific adaptive responses of general that body occurs when various internal and external environment factor and socio-psychological factor stimulate.Stress be a double-edged sword, also be harmful to body is favourable.In inflammatory senescence process, body is in the environment that stressor formed for a long time.Stressor is the reason causing and maintain the existence of chronic pro-inflammatory reactiveness.Oxidation inflammation, oxidative stress, inflammatory are old and feeble closely related with immunosenescence three.Cell factor theory, pro-inflammatory cytokine plays an important role in the inflammatory aging of body caused by chronic inflammation.
Because radiation causes inflammation, and inflammation can cause aging, and at present, plant forsythia suspense extraction is used to various medicine and health care aspect, but forsythia suspense extraction is used for radioresistance, and the research of anti-inflammatory and anti-ageing aspect does not also find temporarily.
Summary of the invention
Technical problem to be solved by this invention is to provide forsythia suspense extraction and is preparing the application in the health food or cosmetics with radioresistance and senile-resistant efficacy.
The present invention has carried out radioresistance and the checking of antidotal effect cell biology to forsythia suspense extraction, because radiation causes inflammation, and inflammation can cause aging, therefore applicants studied forsythia suspense extraction opposing UVA radiation effects, and little about this type of application at present.The forsythia suspense extraction that the application provides has uv-resistant A radiation effects, the radiation-induced dermal cell of remarkable reduction UVA and epidermal cell damage, in addition, also close contacting is had between aging and inflammation, applicant have studied forsythia suspense extraction antiphlogistic effects in vitro simultaneously, finds that it can suppress macrophage inflammatory factor to be expressed effectively.Proinflammatory cytokine energy Induction of Cellular senescence, proinflammatory cytokine is (as TNF-α, IFN-γ, IFN-β) etc. old and feeble by producing active oxygen and activating ATM/P53/P21 signal path induction epithelial cell, chemokine receptors CXCR2 is old and feeble by P53 path induced fibroblast, and DNA damage activates the signal paths such as NF-Κ B and produces proinflammatory cytokine (IL-1, IL-6, IL-8 etc.), thus arresting cell cycle, induction and maintenance cell ageing phenotype.DNA damage theory, because DNA damage accumulation makes stem cell and stromal fibroblast cells be divided into pro-inflammatory cytokine overexpressing cell, makes multilayer shape cytokine network collapse, causes inflammatory and cause aging.
In the present invention, the above-mentioned forsythia suspense extraction with radioresistance and senile-resistant efficacy, can adopt commercially available product, also preferably can adopt forsythia suspense extraction prepared by following preferred method provided by the invention.
As a preferred embodiment of the present invention, the forsythia suspense extraction described in the present invention, it prepares by following method:
(1), after the raw material capsule of weeping forsythia cleans by pre-treatment, pulverize;
(2) after the ultrasonic extraction of ultrasonic wave process ethanol water, filter, concentrate to obtain concentrate, after being diluted with water in concentrate, filtering, obtain B filtered fluid;
(3) macroporous resin column loading: by B filtered fluid on PIPO-02 macroreticular resin splitter;
(4) be separated: with ethanol water wash-out to remove the impurity in macroreticular resin, again use ethanol water wash-out, collect eluent, obtain D parting liquid;
(5) concentrate drying: vacuum-concentrcted D parting liquid, and vacuum drying obtains forsythia suspense extraction.
In above-mentioned steps:
30-60 order is preferably screened to after pulverizing in step of the present invention (1).
In step of the present invention (2) during ultrasonic extractions of ethanol water, the consumption of ethanol water is preferably 8-10 times of raw material gross weight, and in ethanol water, the volumn concentration of ethanol is preferably 40-80%, ultrasonic extraction twice, every less preferred 20-40 minute.
The concentrated preferred concentrate obtaining 3-5 times of raw material and weigh in step of the present invention (2), when being diluted with water in concentrate, the consumption of water is preferably the 2-4 of concentrate gross weight doubly.
In step of the present invention (3), PIPIO-02 macroreticular resin splitter is preferably two, and is in series; With double-column series pattern, first wash-out polar impurity, the forsythin composition in the concentrated as far as possible capsule of weeping forsythia.
During impurity in step of the present invention (4) in removing macroreticular resin, in ethanol water, the volumn concentration of ethanol is preferably 5-12%, and its consumption is preferably the heavy 0.8-4.8 of capsule of weeping forsythia raw material doubly; Be 25-45% with the volumn concentration of ethanol in ethanol water during ethanol water wash-out again, its consumption is preferably the heavy 0.4-2.4 of capsule of weeping forsythia raw material doubly.
Preferably at 40-70 DEG C of vacuum-concentrcted D parting liquid in step of the present invention (5).
The mass percentage of forsythin is detected more than 70% through HPLC in forsythia suspense extraction in step of the present invention (5).
Tool of the present invention has the following advantages:
(1) in the forsythia suspense extraction that extracts of the inventive method, the content of forsythin is higher than 70%, and in health food or cosmetics, application quantity is little, the clear and definite easily quality control of content;
(2) forsythia suspense extraction color and luster of the present invention is brief talked, and is applicable to applying in cosmetics, does not affect original color and luster of cosmetics;
(3) extraction efficiency of the present invention is high, can make full use of resource;
(4) forsythia suspense extraction manufacture cost of the present invention is low, is suitable for and applies in health food or cosmetics, have the market competitiveness.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention 4 (1.2) forsythia suspense extraction to UVA irradiate under 3T3 cytoprotection;
Fig. 2 is the experimental result that in the embodiment of the present invention 4, (1.3) comet electrophoresis detects DNA Damage, and wherein left figure is for adding forsythia suspense extraction, and middle figure is the 3T3 cell having UVA to irradiate, and right figure is the DNA generation comettail rate of the 3T3 cell without UVA irradiation;
Fig. 3 be in the embodiment of the present invention 4 (1.4) flow cytomery forsythia suspense extraction to the protective effect of UVA radiation 3T3 cell cycle, wherein A figure is the 3T3 cell irradiated without UVA, B figure is the 3T3 cell having UVA to irradiate, and C figure is the fluidic cell result figure of the 3T3 cell adding forsythia suspense extraction;
Fig. 4 is that in the embodiment of the present invention 4, (1.5) mtt assay detects forsythia suspense extraction to RAW264.7 cytotoxicity;
Fig. 5 is embodiment of the present invention 4(1.5) in PCR detect forsythia suspense extraction and suppress at transcriptional level or reduce the generation of inflammatory factor IL-6;
Fig. 6 is embodiment of the present invention 4(1.5) in PCR detect forsythia suspense extraction and suppress at transcriptional level or reduce the generation of inflammatory factor IL-1;
Fig. 7 is embodiment of the present invention 4(1.5) in PCR detect forsythia suspense extraction and suppress at transcriptional level or reduce the generation of inflammatory factor TNF-alpha;
Fig. 8 is embodiment of the present invention 4(1.5) in PCR detect forsythia suspense extraction and suppress at transcriptional level or reduce the generation of inflammatory factor MCP-1;
Fig. 9 is embodiment of the present invention 4(1.5) in PCR detect forsythia suspense extraction and suppress at transcriptional level or reduce the generation of inflammatory factor MCP-1.
Detailed description of the invention
Here is that the present invention is further illustrated in conjunction with the embodiments.
(1) extracting method of the preferred forsythia suspense extraction of the present invention
Embodiment 1
The preferred preparation method of the forsythia suspense extraction that the present embodiment provides, containing following steps:
A: pre-treatment: washed by the raw material capsule of weeping forsythia, pulverizes, is screened to 30-60 order, weighs 1 kilogram;
B: extract: the ultrasonic extraction of ethanol water 2 times with 8-10 kilogram of ethanol contend percentage composition being 40-80%, each 20-40 minute, filter, merge extract, 3-5 kilogram of concentrate is obtained by concentrated for extract, in concentrate, add 6-20 kg water dilution concentrate, filter, obtain 9-25 kilogram of B filtered fluid;
C: loading: by B filtered fluid to the heavy 200-600 gram of the PIPO-02(parting material of 200-600 milliliter column volume) macroreticular resin splitter, same volume and be equipped with in the series connection of twin columns joint;
D: being separated, is the ethanol water wash-out 800-4800 milliliter of 5-12% with volumn concentration, assorted before removing, be the ethanol water wash-out of 25-45%% with volumn concentration, collect 400-2400 milliliter, obtain D parting liquid;
E: concentrate drying: at 40-70 DEG C of vacuum-concentrcted D parting liquid, and vacuum drying obtains forsythia suspense extraction, find with the test of HPLC forsythin reference substance, in forsythia suspense extraction, the content of forsythin is more than 70%, is weighed as 60-120 gram.
Embodiment 2
The preferred preparation method of the forsythia suspense extraction that the present embodiment provides, containing following steps:
A: pre-treatment: washed by the raw material capsule of weeping forsythia, pulverizes, is screened to 30-60 order, weighs 10 kilograms;
B: extract: the ultrasonic extraction of ethanol water 2 times with 80-100 kilogram of ethanol contend percentage composition being 40-80%, each 20-40 minute, filter, merge extract, concentrate and obtain 30-50 kilogram of concentrate, in concentrate, add 60-200 kg water dilution concentrate liquid, filter, obtain 90-250 kilogram of B filtered fluid;
C: loading: by B filtered fluid to the heavy 2000-6000 gram of the PIPO-02(parting material of 2000-6000 milliliter column volume) splitter, same volume and be equipped with in the series connection of twin columns joint;
D: being separated, is the ethanol water wash-out 8000-48000 milliliter of 5-12% with ethanol contend percentage composition, assorted before removing, be the ethanol water water elution of 25-45%% with ethanol contend percentage composition, collect 4000-24000 milliliter, obtain D parting liquid;
E: concentrate drying: at 40-70 DEG C of vacuum-concentrcted D parting liquid, and vacuum drying obtains forsythia suspense extraction, find with the test of HPLC forsythin reference substance, wherein the mass percentage of forsythin is more than 70%, is weighed as 600-1200 gram.
Embodiment 3
The preferred preparation method of the forsythia suspense extraction that the present embodiment provides, containing following steps:
A: pre-treatment: washed by the raw material capsule of weeping forsythia, pulverizes, is screened to 30-60 order, double centner of weighing;
B: extract: extract 2 times with 800-1000 kilogram of 40-80% EtOH Sonicate, each 20-40 minute, filters, and merges, and concentrates and obtains 300-500 kilogram of extract, adds 600-2000 kg water dilution extract, filters, obtain 900-2500 kilogram of B filtered fluid;
C: loading: by B filtered fluid to the heavy 2000-6000 gram of the PIPO-02(parting material of 2000-6000 milliliter column volume) splitter, same volume and be equipped with in the series connection of twin columns joint;
D: be separated, with 5-12% ethanol elution 80-480 liter, assorted before removing, with 25-45%% ethanol water elution, collect 40-240 liter, obtain D parting liquid;
E: concentrate drying: vacuum decompression concentrates D parting liquid at 40-70 degree, and vacuum drying obtains forsythia suspense extraction, find with the test of HPLC forsythin reference substance, wherein the mass percentage of forsythin is more than 70%, is weighed as the 6-12 kilogram of raw material weight.
(2) efficacy validation of forsythia suspense extraction
Embodiment 4 forsythia suspense extraction radioresistance and senile-resistant efficacy checking
4.1 experimental raw
Below adopt in the embodiment of the present invention 1 and prepare forsythia suspense 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 forsythia suspense extraction to UVA radiation 3T3 cell, method is as follows:
Detect cell proliferative conditions by mtt assay and measure the protective action of compound to UVA radiation 3T3 cell.
(1) collect logarithmic phase 3T3 cell, adjustment concentration of cell suspension, every hole adds 100 μ L, and bed board makes cell to be measured adjust density to 1000-10000 hole;
(2) 2.5%CO
2hatch for 37 DEG C, (96 hole flat underside) at the bottom of hole is paved with to cell monolayer, 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, often group arranges 3 blank well, and control group does not deal with 5%CO
2, hatch 16-24 hour for 37 DEG C;
(3) radiation group respectively organize cell by UVA dose of radiation condition grope test the ultraviolet condition obtained carry out ultra-violet radiation: exposure time 45min; Dose of radiation 1.4J/cm
2, control group does not deal with, 5%CO
2, hatch 16-24 hour for 37 DEG C;
(4) every hole adds 10 μ LMTT solution, continues to cultivate 4h, stops cultivating, 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, and measures the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD570nm place;
As shown in fig. 1, result of the test shows result, adopts the forsythia suspense extraction extracted in the embodiment of the present invention 1 UVA radiation 3T3 cell to be had to the activity (>=90%cellviability) of protection.
4.3 anti-inflammatory anti-aging effects efficacy validation experiments
Adopt comet electrophoresis to detect in the embodiment of the present invention 1 and be prepared into the protective effect of forsythia suspense extraction to UVA radiation 3T3 cell DNA; method is as follows: DNA damage is more serious; cause DNA superhelix looser; the breakaway poing produced is more; DNA fragmentation is less; thus the DNA part occurred at comet afterbody is more, then the length of comets tail, area and fluorescence intensity are larger.By measuring the indexs such as the length of comet afterbody, area or fluorescence intensity, quantitative analysis can be carried out to the degree of injury of DNA.
(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 Cmax as the medicine working concentration of this experiment), inhale abandon culture medium, add the diluted chemical compound liquid of dilution, every hole 1mL, 37 degree, 5%CO
2incubator cultivate 24h;
(3) by the cell UVA irradiation 1h of process, 60uW/cm
2, 37 DEG C, 5%CO
2incubator cultivate 24h;
(4) trypsin digestion cell, the culture medium containing serum stops digestion, is proceeded to by cell liquid in 15mL centrifuge tube, 1000rpm/min, centrifugal 5min, abandons supernatant, adds appropriate PBS re-suspended cell, the same centrifugal 5min, abandon PBS, add appropriate PBS re-suspended cell, adjust cell concentration to 10
6cells/mL;
(5) glue: the normal melting point agarose dissolving 0.7% concentration, fast drop 70 μ L is to clean slide, quick cover glass moulding, 4 DEG C of gel 20min, peel-off covers slide gently, by cell suspension and the mixing of 0.8% low melting-point agarose, fast drop is on ground floor glue, quick cover glass moulding, 4 DEG C of gel 20min;
(6) peel off the cover glass on second layer glue gently, glue is put into alkaline bleach liquor cleavage liquid, 4 DEG C of cracking 1.5 ~ 2h;
(7) take out slide gently, steam washing glue 2 times with single;
(8) glue is put into alkaline electrophoresis liquid, leave standstill 20min, 25V, the Tris-HCl of electrophoresis 15min, pH7.5 neutralizes three times, each 5min;
(9) PI dyeing microscopic examination, takes pictures.
Comet electrophoresis detects the experimental result of DNA Damage as shown in Figure 2, is prepared into forsythia suspense extraction and has inhibitory action significantly to the radiation-induced DNA damage of UVA in the embodiment of the present invention 1.
4.4 anti-inflammatory anti-aging effects efficacy validation experiments
The protective effect of forsythia suspense extraction to the UVA radiation 3T3 cell cycle is prepared in the flow cytomery medicine embodiment of the present invention 1; method is as follows: DNA Damage; start cellular genome repair mechanism; regulate the cell cycle and then complete genomic injury repair, ensureing the complete of cellular genome.The genomic DNA of UV radiation to cell causes fracture damage, and the cell cycle will be caused to change.Therefore utilize Flow cytometry to carry out detection compound, to the cell cycle changed, whether there is adjustment inhibitory action.
(1) pre-12 orifice plates of 3T3 cell, overnight incubation;
(2) cell drug process: medicine is done concentration dilution, adds cell, UVA radiation 1h after 24h, 60 μ W/cm
2, cell culture incubator receives sample after cultivating 24h;
(3) trypsin digestion cell, goes to 1.5mL centrifuge tube, the centrifugal 5min of 1000rpm/min, and abandon supernatant, PBS washes twice, and centrifugal condition is the same, abandons PBS, and 70% ethanol 4 degree adding 500 μ L fixedly spends the night;
(4) abandon PBS, 70% ethanol 4 DEG C adding 500 μ L fixedly spends the night;
(5) 1000rpm/min, 4 DEG C, centrifugal 5min, abandons ethanol, and it is resuspended, the same centrifugal to add PBS;
(6) abandon PBS, add in the PBS containing DNA enzymatic I and PI dyestuff, 200 object screen filtrations;
(7) upper Flow cytometry surveys the cycle;
The flow cytomery cell cycle, result as shown in Figure 3, the pre-synthesis phase that wherein G1 being DNA, the cells quiescent phase; 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 then shows propagation.
Streaming result in Fig. 3 shows: be prepared into forsythia suspense extraction in the embodiment of the present invention 1 compared with UVA group data, forsythia suspense extraction has protective effect.
4.5 anti-inflammatory anti-aging effects Mechanism Validation experiments
Mtt assay detects in the embodiment of the present invention 1 of screening and is prepared into forsythia suspense extraction to RAW264.7 cytotoxicity, and method is as follows:
(1) collect logarithmic phase RAW264.7 cell, adjustment concentration of cell suspension, every hole adds 100 μ L, and bed board makes cell to be measured adjust density to 1000-10000 hole;
(2) 2.5%CO
2hatch for 37 DEG C, (96 hole flat underside) at the bottom of hole is paved with to cell monolayer, be divided into 60 groups at random, each medicine one group, often group adds the forsythia suspense extraction of concentration gradient, 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 often group arranges 3 blank well, 5%CO
2, hatch 16-48 hour for 37 DEG C;
(3) every hole adds 10 μ LMTT solution, continues to cultivate 4h, stops cultivating, 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, and measures the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD570nm place;
Be prepared into forsythia suspense extraction in the embodiment of the present invention 1 to the toxicity of RAW264.7 cell as shown in Figure 4, as can be seen from Figure 4, forsythia suspense extraction is to the maximal non-toxic concentration of cytotoxic evil.
4.6 anti-inflammatory anti-aging effects efficacy validation experiments
PCR detects in the embodiment of the present invention 1 and is prepared into forsythia suspense extraction in transcriptional level suppression or the generation reducing inflammatory factor, and method is as follows: carry out anti-inflammatory activity experiment to screening the compound forsythia suspense extraction with uv-resistant A radiation obtained at macrophage RAW264.7 cell model.
(1) take the logarithm phase RAW264.7 cell, pre-24 orifice plates, bed board adjustment cell density is 3 × 10
5cells/mL;
(2) add the PMA of 50ng/mL, cultivate 48h, examine under a microscope cellular morphology;
(3) carefully suck culture medium in hole, after rinsing 2 times with PBS, add 1640 culture mediums of serum-free, cultivate 24h;
(4) add LPS and the forsythia suspense 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, TNF), MCP-1 (monocytechemotacticprotein-1, macrophage chemoattractant protein 1) and iNOS carry out qualitative and quantitative analysis, observe the impact that active matter is expressed inflammatory factor, in the embodiment of the present invention 1, be prepared into the inhibitory action of forsythia suspense extraction to the expression of inflammatory factor.
IL-6 and IL-1 (Interleukin-6, nterleukin-1, interleukin-6 interleukin 1) result as shown in Figures 5 and 6, TNF-α (tumornecrosisfactor-alpha, TNF) result as shown in Figure 7, MCP-1 (monocytechemotacticprotein-1, macrophage chemoattractant protein 1) and iNOS result as shown in figs
As can be seen from Fig. 5-9: the forsythia suspense extraction detecting preparation in the embodiment of the present invention 1 shows the different inflammation that presses down and acts on, and in the embodiment of the present invention 1, the inhibitory action of forsythia suspense extraction to IL-1, IL-6 and TNF-a of preparation is remarkable.
In fact, present invention applicant also carries out above-mentioned test to the forsythia suspense extraction in other sources as commercially available forsythia suspense extraction or additive method prepare forsythia suspense extraction simultaneously, and result shows, also has above-mentioned radioresistance and senile-resistant efficacy.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (8)
1. forsythia suspense extraction is preparing the application in the health food or cosmetics with uv-resistant A radiation and senile-resistant efficacy as the active component uniquely with uv-resistant A radiation and anti-aging effects;
Wherein forsythia suspense extraction prepares by the following method:
(1), after the raw material capsule of weeping forsythia cleans by pre-treatment, pulverize;
(2) after the ultrasonic extraction of ultrasonic wave process ethanol water, filter, concentrate to obtain concentrate, after being diluted with water in concentrate, filtering, obtain B filtered fluid;
(3) macroporous resin column loading: by B filtered fluid on PIPO-02 macroreticular resin splitter;
(4) be separated: with ethanol water wash-out to remove the impurity in macroreticular resin, again use ethanol water wash-out, collect eluent, obtain D parting liquid;
(5) concentrate drying: vacuum-concentrcted D parting liquid, and vacuum drying obtains forsythia suspense extraction;
In step (2) during ultrasonic extractions of ethanol water, the consumption of ethanol water is 8-10 times of raw material gross weight, and in ethanol water, the volumn concentration of ethanol is 40-80%, ultrasonic extraction twice, each 20-40 minute;
During impurity in step (4) in removing macroreticular resin, in ethanol water, the volumn concentration of ethanol is 5-12%, its consumption be 0.8-4.8 that capsule of weeping forsythia raw material is heavy doubly; Be 25-45% with the volumn concentration of ethanol in ethanol water during ethanol water wash-out again, its consumption be 0.4-2.4 that capsule of weeping forsythia raw material is heavy doubly.
2. forsythia suspense extraction according to claim 1 is as uniquely having the active component of uv-resistant A radiation and anti-aging effects preparing the application in the health food or cosmetics with uv-resistant A radiation and senile-resistant efficacy, it is characterized in that: in described forsythia suspense extraction, the mass percentage of forsythin is more than 70%.
3. there is a preparation method for the forsythia suspense extraction of uv-resistant A radiation and senile-resistant efficacy, it is characterized in that containing following steps:
(1), after the raw material capsule of weeping forsythia cleans by pre-treatment, pulverize;
(2) after the ultrasonic extraction of ultrasonic wave process ethanol water, filter, concentrate to obtain concentrate, after being diluted with water in concentrate, filtering, obtain B filtered fluid;
(3) macroporous resin column loading: by B filtered fluid on PIPO-02 macroreticular resin splitter;
(4) be separated: with ethanol water wash-out to remove the impurity in macroreticular resin, again use ethanol water wash-out, collect eluent, obtain D parting liquid;
(5) concentrate drying: vacuum-concentrcted D parting liquid, and vacuum drying obtains forsythia suspense extraction;
In step (2) during ultrasonic extractions of ethanol water, the consumption of ethanol water is 8-10 times of raw material gross weight, and in ethanol water, the volumn concentration of ethanol is 40-80%, ultrasonic extraction twice, each 20-40 minute;
During impurity in step (4) in removing macroreticular resin, in ethanol water, the volumn concentration of ethanol is 5-12%, its consumption be 0.8-4.8 that capsule of weeping forsythia raw material is heavy doubly; Be 25-45% with the volumn concentration of ethanol in ethanol water during ethanol water wash-out again, its consumption be 0.4-2.4 that capsule of weeping forsythia raw material is heavy doubly.
4. the preparation method with the forsythia suspense extraction of uv-resistant A radiation and senile-resistant efficacy according to claim 3, is characterized in that: in step (1), grinding and sieving is to 30-60 order.
5. the preparation method with the forsythia suspense extraction of uv-resistant A radiation and senile-resistant efficacy according to claim 3, it is characterized in that: in step (2), concentrate to obtain 3-5 times of raw material concentrate heavily, when being diluted with water in concentrate, the consumption of water is 2-4 times of concentrate gross weight.
6. the preparation method with the forsythia suspense extraction of uv-resistant A radiation and senile-resistant efficacy according to claim 3, is characterized in that: in step (3), PIPIO-02 macroreticular resin splitter is two, and is in series.
7. the preparation method with the forsythia suspense extraction of uv-resistant A radiation and senile-resistant efficacy according to claim 3, is characterized in that: at 40-70 DEG C of vacuum-concentrcted D parting liquid in step (5).
8. the preparation method with the forsythia suspense extraction of uv-resistant A radiation and senile-resistant efficacy according to claim 3, is characterized in that: detect the mass percentage of forsythin more than 70% through HPLC in forsythia suspense extraction in step (5).
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| Title |
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| 连翘乙醇提取物清除脂自由基和氧自由基的效果;涂秋云等;《湖南农业大学学报(自然科学版)》;20081231;第34卷(第6期);第728-731页 * |
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