CN103585052A - Application of phyllanthus emblica extract in preparation of health food or cosmetics with anti-radiation and anti-aging efficacy - Google Patents
Application of phyllanthus emblica extract in preparation of health food or cosmetics with anti-radiation and anti-aging efficacy Download PDFInfo
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Abstract
The invention discloses application of a phyllanthus emblica extract in preparation of health food or cosmetics with anti-radiation, anti-inflammation and anti-aging efficacy, the phyllanthus emblica extract has anti-UVA (Ultra Violet A) radiation efficacy, can substantially reduce cell damage caused by UV radiation, simultaneously has an obvious inhibition effect on macrophage inflammatory factor expression, and can play anti-inflammation and anti-aging effects; the invention also illuminates, in cell and molecular level, that the phyllanthus emblica extract has anti-UV radiation and anti-aging effects. In addition, the invention also discloses a preferred preparation method of the phyllanthus emblica extract having the anti-radiation and anti-aging effects.
Description
Technical field
The invention belongs to Fructus Phyllanthi technical field, be specifically related to Fructus Phyllanthi extract and there is the health food of radioprotective and senile-resistant efficacy or the application in cosmetics in preparation.
Technical background
In recent years, the active component that derives from plant is more and more for the compositions of Yao Wu ﹑ Zhi Liao ﹑ cosmetic purpose.At present, plant Fructus Phyllanthi extract 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.At molecular level, show as the aging of cell, this is the progressive event accompanying with organism aging process.The old and feeble physiological function of body that not only causes is degenerated, and the immune decorum of body is also being degenerated gradually.In human aging process not only due to self physiology and immune reason, also have a lot of exopathogenic factors also can accelerate aging, the radiation of its medium ultraviolet is exactly to cause old and feeble the most common reason, and because the aging chronic inflammatory disease causing is also the sign that immune system is degenerated.
Ultraviolet radiation is to cause the topmost environmental factors of skin aging, and ultraviolet radiation makes to produce a large amount of oxygen-derived free radicals in skin histology, makes to organize anti-oxidative defense System Capacity to weaken, and finally causes skin aging.Free radical is very easily encroached on the unsaturated fatty acid in cell membrane, cause lipid peroxidation, form lipid peroxide, catabolite malonaldehyde (MDA) and the aminoacid of lipid peroxide, nucleic acid, reaction forms alicyclic diradical with free amine group for protein and phospholipid etc., and it participates in physiology and the pathological process of body widely.Under normal physiological state, in body, oxygen-derived free radicals is difficult for accumulating and peroxide injury, wherein SOD plays vital effect to the oxidative and anti-oxidative balance of body, when body is subject to ultraviolet radiation for a long time, in body the generation of ultra-oxygen anion free radical with remove just disequilibrium, oxygen-derived free radicals produces when too much, can produce toxic action 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 body weakens the adaptive capacity of environment so that loses.Old and feeble mechanism is rather complicated, relates to the structure of each system and the change of function of body, and relevant 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 Senile disease has substantial connection with the change of immunologic function in many aspects.Think that the interior most of organs of immune system and body and cell keep continuous contact, its change certainly will affect these Organ and tissue cells.With advancing age, immune function goes down, and induces thus the disease that some have a strong impact on histoorgan, has aggravated the aging course of each system of body.Stop or reverse the exhaustion of immunologic function, can slow down aging, and change the order of severity of old and feeble pathological changes histoorgan.Old and feeble immunology performance has immune organ function decline, and along with the age increases, the function of each immune organ day by day fails, and is wherein apparent that most that thymus, atrophy of thymus gland are the keys of old people's immunologic function degression.Cellular immunization and humoral immune function also change to some extent in old and feeble process, and cellular immunization is the T cells play effect that relies on thymus differentiation and maturation, and therefore along with old atrophy of thymus gland, it is undoubtedly that the quality and quantity of T cell is all affected.Immunity is in close relations with aging, improves the immunity of body come defying age and slow down aging to have great significance from immune angle.
In addition, also have close contact between aging and inflammation, inflammatory aging receives increasing concern, and inflammation aging refers to the process of a chronic inflammatory disease of body appearance with advancing age.Inflammation is the reply event of host system to the series of complex of the generations such as pathogenic infection and various tissue injurys, it is by affecting the interaction of various kinds of cell and the factor in body microenvironment, the balance trend of the regulation and control multiple physiology of body and pathological signals network, shown the dual character of height: in the ordinary course of things after proinflammatory factors is as infection or tissue injury's elimination, inflammatory reaction terminates immediately, be transformed into afterwards that a kind of height is active, the poised state of finely regulating, this inflammation is called as controllability inflammation.But under the effect of some uncertain factor, as that continue or low intensive stimulation, target tissue is when long-term or overreaction, and inflammation cannot be transformed into the state of balance and stability from infection tissue injury pattern, and what cause inflammatory reaction continues to show 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 immunoreation, and the inflammatory reaction of appropriateness is favourable to body, 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 mutual dependence for existence mutually.Relation in a dynamic equilibrium between them under normal circumstances, the pathologic inflammation in inflammatory aging be inflammatory cytokine network unbalance due to, extremely closely related with inflammatory mediator also.
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 adjustment reaction of general that body occurs when various internal and external environment factors and socio-psychological factors stimulation.Stress be a double-edged sword, favourable also harmful to body.In inflammatory senescence process, body is in the formed environment of stressor for a long time.Stressor is the reason that causes and maintain chronic pro-inflammatory state existence.Oxidation inflammation, oxidative stress, inflammatory aging and immunosenescence three are closely related.Cytokine theory, pro-inflammatory cytokine plays an important role in the inflammatory aging of body due to chronic inflammatory disease.
At present, about Fructus Phyllanthi extract is had to the health food of radioprotective and senile-resistant efficacy or the research of the application aspect in cosmetics in preparation, yet there are no open.
Summary of the invention
Technical problem to be solved by this invention is to provide Fructus Phyllanthi extract and has the health food of radioprotective and senile-resistant efficacy and the application in cosmetics in preparation.
The present invention has carried out radioprotective, antiinflammatory and the checking of antidotal effect cytobiology to Fructus Phyllanthi extract, because radiation causes inflammation, and inflammation can cause aging, so applicant studied Fructus Phyllanthi extract opposing UVA radiation effects, and at present about this type of application seldom.The Fructus Phyllanthi extract that the application provides has anti-UVA radiation effects, significantly reduce the radiation-induced hypodermal cell of UVA and epidermis cell damage, in addition, between aging and inflammation, also there is close contacting, applicant has studied Fructus Phyllanthi extract antiphlogistic effects in vitro simultaneously, finds that it can suppress macrophage inflammatory Cytokines Expression effectively.Proinflammatory cytokine energy inducing cell is old and feeble, 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 induction fibroblast, and DNA damage activates the signal paths such as NF-Κ B and produces proinflammatory cytokine (IL-1, IL-6, IL-8 etc.), thereby block cell cycle, induce and maintain cell ageing phenotype.DNA damage theory, because DNA damage accumulation makes stem cell and substrate fibroblast be divided into pro-inflammatory cytokine overexpressing cell, makes the collapse of multilamellar shape cytokine network, causes inflammatory and causes aging.
Preferably, in Fructus Phyllanthi extract of the present invention the quality percentage composition of Chebulagic acid more than 70%.
In the present invention, the above-mentioned Fructus Phyllanthi extract with radioprotective and senile-resistant efficacy, can make commercially available prod, can be also the Fructus Phyllanthi extract that adopts following preferred method provided by the invention to prepare.
The above-mentioned preparation method with the Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy provided by the invention, contains following steps:
(1) pre-treatment: raw material Fructus Phyllanthi is cleaned, pulverized;
(2) extract: after adopting the ultrasonic room temperature of methanol to extract, filter, filtrate vacuum decompression is processed and reclaimed methanol, obtain B extractum;
(3) extraction: B extractum is adopted after n-butyl alcohol ultrasonic extraction, filter, obtain C extract;
(4) loading is separated: by after C extract dilute with water, filter, be splined in two detached dowels that PIPO-02 parting material is housed that are in series, with after aqueous formic acid eluting, by two detached dowels separately, second detached dowel first washes with water, with methanol, wash again, obtain target phase D fraction;
(5) concentrate drying: after vacuum-concentrcted, vacuum drying obtains Fructus Phyllanthi extract by D fraction.
In above-mentioned steps:
After pulverizing in step of the present invention (1), be preferably screened to 30～60 orders.
The consumption of the middle methanol of step of the present invention (2) is preferably 8-10 times of Fructus Phyllanthi raw material gross weight.
In step of the present invention (2), the ultrasonic room temperature of methanol is extracted preferably 2 times, every less preferred 20-40 minute, and preferred 500-800 order filter-cloth filtering, merging filtrate, vacuum decompression reclaims methanol, obtains B extractum, and the weight of B extractum accounts for the 20-30% of Fructus Phyllanthi raw material gross weight.
In step of the present invention (3), the consumption of n-butyl alcohol and B extractum is identical in quality, and the ultrasonic extraction time is preferably 30-50 minute.
In step of the present invention (4), C extract is preferably diluted with accounting for its gross mass 15-20 water doubly.
In step of the present invention (4), preferably with the heavy eluent of 10-15 times of Fructus Phyllanthi raw material of aqueous formic acid eluting, in aqueous formic acid, the volumn concentration of formic acid is preferably 0.2%; Second detached dowel preferably washes the heavy eluent of 3-5 times of Fructus Phyllanthi raw material with water, more preferably with methanol, washes the heavy eluent of 2-3 times of Fructus Phyllanthi raw material.
In step of the present invention (5) by D fraction preferably at 40-70 ℃ after vacuum-concentrcted, the Fructus Phyllanthi extract that vacuum drying obtains, the quality percentage composition that detects Chebulagic acid through HPLC in Fructus Phyllanthi extract is more than 70%.
It is more than the preparation method of the preferred Fructus Phyllanthi extract of the present invention.
Tool of the present invention has the following advantages:
(1) Fructus Phyllanthi extract that adopts the inventive method to extract, wherein Chebulagic acid composition accounts for 70%(quality percentage composition) more than, Chebulagic acid conventionally in health product or cosmetics application quantity little, the clear and definite easily quality control of content;
(2) the Fructus Phyllanthi extract color and luster that adopts the inventive method to prepare is brief talked, and is applicable to applying in cosmetics, does not affect former coloured pool of cosmetics;
(3) adopt the extraction efficiency of the inventive method extraction Fructus Phyllanthi extract high, can make full use of resource;
(4) Fructus Phyllanthi extract manufacture cost of the present invention is low, is suitable in health product or cosmetics and applies, and has the market competitiveness;
(5) the present invention finds through test; Fructus Phyllanthi extract can significantly reduce the radiation-induced cell injury of UV; macrophage inflammatory Cytokines Expression is had to obvious inhibitory action simultaneously; thereby performance anti-aging effects, from cell and molecular level, illustrate the Fructus Phyllanthi extract product the present invention and there is anti-UV radiation, Cell protection and anti-aging effects.
Accompanying drawing explanation
Fig. 1 is the 3T3 cytoprotection of (1.2) Fructus Phyllanthi extract under UVA is irradiated in the embodiment of the present invention 4;
Fig. 2 is the experimental result of (1.3) comet electrophoresis detection DNA Damage in the embodiment of the present invention 4, wherein left figure is that 3T3 cell, the middle figure irradiating without UVA is that 3T3 cell, the right figure that has UVA to irradiate added the cell DNA of the 3T3 cell of Fructus Phyllanthi extract to produce comettail rate, wherein slogan banner is followed successively by the 3T3 cell that UVA does not irradiate, the 3T3 cell that has UVA to irradiate, has added the 3T3 cell of Fructus Phyllanthi extract; The vertical cell DNA that is designated as produces comettail;
Fig. 3 is that in the embodiment of the present invention 4, (1.4) flow cytometer detects the protective effect of Fructus Phyllanthi extract to UVA radiation 3T3 cell cycle;
Fig. 4 is that in the embodiment of the present invention 4, (1.5) mtt assay detects Fructus Phyllanthi extract to RAW264.7 cytotoxicity;
Fig. 5 is embodiment of the present invention 4(1.5) in PCR detect Fructus Phyllanthi extract and at transcriptional level, suppress or reduce the generation of inflammatory factor IL-6;
Fig. 6 is embodiment of the present invention 4(1.5) in PCR detect Fructus Phyllanthi extract and at transcriptional level, suppress or reduce the generation of inflammatory factor IL-1;
Fig. 7 is embodiment of the present invention 4(1.5) in PCR detect Fructus Phyllanthi extract and at transcriptional level, suppress or reduce the generation of inflammatory factor TNF-alpha;
Fig. 8 is embodiment of the present invention 4(1.5) in PCR detect Fructus Phyllanthi extract and at transcriptional level, suppress or reduce the generation of inflammatory factor MCP-1;
Fig. 9 is embodiment of the present invention 4(1.5) in PCR detect Fructus Phyllanthi extract and at transcriptional level, suppress or reduce the generation of inflammatory factor MCP-1.
The specific embodiment
That the present invention is further illustrated in conjunction with the embodiments below.
(1) extracting method of the preferred Fructus Phyllanthi extract of the present invention
The preferred preparation method of Fructus Phyllanthi extract provided by the invention, contains following steps:
A pre-treatment: raw material Fructus Phyllanthi is washed, pulverize, be screened to 30-60 order, weigh 1 kilogram;
B extracts: by the 8-10 kilogram of ultrasonic room temperature of methanol, extracts 2 times, and each 20-40 minute, 500-800 order filter-cloth filtering, merging filtrate, vacuum decompression reclaims methanol, obtains the B extractum of 0.2-0.3 kg feed material weight;
C extraction: the n-butyl alcohol ultrasonic extraction 30-50 minute by B extractum weight equivalent, filter, obtain 0.3-0.5 kilogram of C extract;
D loading is separated: 4.5-10 kg water dilution for C extract, filter, be splined in two pillars that PIPO-02 parting material is housed, use 0.2%(volumn concentration, lower same) formic acid water elution 10-15 kilogram, separate two detached dowels, second post washes 3-5 kilogram with water, with methanol, wash 2-3 kilogram again, obtain target phase D flow point;
E concentrate drying: vacuum decompression is at 40-70 ℃ of concentrated D flow point, and vacuum drying obtains Fructus Phyllanthi extract, with the test of HPLC Chebulagic acid reference substance, finds, wherein the quality percentage composition of Chebulagic acid, more than 70%, is weighed as 50-80 gram.
Embodiment 2
The preferred preparation method of Fructus Phyllanthi extract provided by the invention, contains following steps:
A pre-treatment: raw material Fructus Phyllanthi is washed, pulverize, be screened to 30-60 order, weigh 10 kilograms;
B extracts: by the 80-100 kilogram of ultrasonic room temperature of methanol, extracts 2 times, and each 20-40 minute, 500-800 order filter-cloth filtering, merging filtrate, vacuum decompression reclaims methanol, obtains the B extractum of 2-3 kg feed material weight;
C extraction: the n-butyl alcohol ultrasonic extraction 30-50 minute by B extractum weight equivalent, filter, obtain 3-5 kilogram of C extract;
D loading is separated: 45-100 kg water dilution for C extract, filter, be splined in two pillars that PIPO-02 parting material is housed, with 0.2% formic acid water elution 100-150 kilogram, separate two detached dowels, second post washes 30-50 kilogram with water, then washes 20-30 kilogram with methanol, obtains target phase D flow point;
E concentrate drying: vacuum decompression is at 40-70 ℃ of concentrated D flow point, and vacuum drying obtains Fructus Phyllanthi extract, with the test of HPLC Chebulagic acid reference substance, finds, wherein the quality percentage composition of Chebulagic acid, more than 70%, is weighed as 500-800 gram.
Embodiment 3
The preferred preparation method of Fructus Phyllanthi extract provided by the invention, contains following steps:
A pre-treatment: raw material Fructus Phyllanthi is washed, pulverize, be screened to 30-60 order, the double centner of weighing;
B extracts: by the 800-1000 kilogram of ultrasonic room temperature of methanol, extracts 2 times, and each 20-40 minute, 500-800 order filter-cloth filtering, merging filtrate, vacuum decompression reclaims methanol, obtains the B extractum of 20-30 kg feed material weight;
C extraction: the n-butyl alcohol ultrasonic extraction 30-50 minute by B extractum weight equivalent, filter, obtain 30-50 kilogram of C extract;
D loading is separated: 450-1000 kg water dilution for C extract, filter, be splined in two pillars that PIPO-02 parting material is housed, with 0.2% formic acid water elution 1000-1500 kilogram, separate two detached dowels, second post washes 300-500 kilogram with water, then washes 200-300 kilogram with methanol, obtains target phase D flow point;
E concentrate drying: vacuum decompression is at 40-70 ℃ of concentrated D flow point, and vacuum drying obtains Fructus Phyllanthi extract, with the test of HPLC Chebulagic acid reference substance, finds, wherein the quality percentage composition of Chebulagic acid, more than 70%, is weighed as 5-8kg.
(1) efficacy test of Fructus Phyllanthi extract
Embodiment 4 Fructus Phyllanthi extract radioprotectives and senile-resistant efficacy checking
4.1 experimental raw
Below adopt in the embodiment of the present invention 1 and prepare Fructus Phyllanthi extract, carry out radioprotective 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 Fructus Phyllanthi extract to UVA radiation 3T3 cell, method is as follows:
By mtt assay, detect cell proliferation situation and measure the protective action of compound to UVA radiation 3T3 cell.
(1) collect logarithmic (log) phase 3T3 cell, adjust 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 ℃, 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 multiple holes, every group arranges 3 blank well, and matched group does not deal with 5%CO
2, hatch 16-24 hour for 37 ℃;
(3) radiation group respectively organize cell by UVA radiation dose condition grope experiment the ultraviolet condition obtaining carry out ultraviolet radiation: exposure time 45min; Radiation dose 1.4J/cm
2, matched group does not deal with, 5%CO
2, hatch 16-24 hour for 37 ℃;
(4) every hole adds 10 μ LMTT solution, continues to cultivate 4h, stops cultivating, and carefully sucks culture fluid in hole;
(5) every hole adds 100 μ L dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, and 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 Fructus Phyllanthi extract extracting in the embodiment of the present invention 1 UVA radiation 3T3 cell to be had to the activity (>=90%cell viability) of protection.
4.3 antiinflammatory anti-aging effects effect confirmatory experiments
Adopt in the comet electrophoresis detection embodiment of the present invention 1 and be prepared into the protective effect of Fructus Phyllanthi extract to UVA radiation 3T3 cell DNA; method is as follows: DNA damage is more serious; cause DNA superhelix looser; the breakaway poing producing 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 larger.By measuring the indexs such as length, area or fluorescence intensity of comet afterbody, can carry out quantitative analysis to the degree of injury of DNA.
(1) pre-12 orifice plates of 3T3 cell, cell density is 70%;
(2) compound treatment: we adopt effective Cmax as the medicine working concentration of this experiment diluted chemical compound to the 200 μ g/mL(that above-mentioned testing sieve is selected), inhale and 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 processing, 60uW/cm
2, 37 ℃, 5%CO
2incubator cultivate 24h;
(4) trypsin digestion cell, the culture medium termination digestion containing serum, proceeds to Cell sap 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 fusing point agarose that dissolves 0.7% concentration, drip fast 70 μ L to clean microscope slide, use fast coverslip moulding, 4 ℃ of gel 20min, peel-off covers slide, mixes cell suspension and 0.8% low melting-point agarose gently, drops to fast on ground floor glue, coverslip moulding fast, 4 ℃ of gel 20min;
(6) peel off gently the coverslip on second layer glue, glue is put into alkaline bleach liquor cleavage liquid, 4 ℃ of cracking 1.5～2h;
(7) take out gently microscope slide, with single, steam washing glue 2 times;
(8) glue is put into alkaline electrophoresis liquid, standing 20min, 25V, electrophoresis 15min, the Tris-HCl neutralization of pH7.5 three times, 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 Fructus Phyllanthi extract the radiation-induced DNA damage of UVA is had to inhibitory action significantly in the embodiment of the present invention 1.
4.4 antiinflammatory anti-aging effects effect confirmatory experiments
In the flow cytometer detection of drugs embodiment of the present invention 1, be prepared into the protective effect of Fructus Phyllanthi extract to UVA radiation 3T3 cell cycle; method is as follows: DNA Damage; start cellular genome repair mechanism; regulate cell cycle and then complete genomic injury repairing, guaranteeing the complete of cellular genome.UV radiation causes fracture damage to the genomic DNA of cell, will cause cell cycle to change.Therefore utilize Flow cytometry to come detection compound whether to there is adjusting inhibitory action to the cell cycle changing.
(1) pre-12 orifice plates of 3T3 cell, overnight incubation;
(2) cell drug is processed: medicine is done to concentration dilution, add cell, UVA radiation 1h after 24h, 60 μ W/cm
2, 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 twice, and 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 ℃ of 70% ethanol of 500 μ L fixedly to spend the night;
(5) 1000rpm/min, 4 ℃, 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 cell cycle, result as shown in Figure 3, the presynthetic phase that wherein G1 being DNA, cell resting stage; S is DNA replication dna period; G2 is DNA post-synthetic phase, is the cell of proliferation period.If, after >G1 dosing, the cell proportion of S phase and G2 phase increases before G1 dosing, it is active that cell shows propagation.
Streaming result in Fig. 3 shows: in the embodiment of the present invention 1, be prepared into Fructus Phyllanthi extract and compare with UVA group data, Fructus Phyllanthi extract has protective effect.
4.5 antiinflammatory anti-aging effects Mechanism Validation experiments
Mtt assay detects in the embodiment of the present invention 1 of screening and is prepared into Fructus Phyllanthi extract to RAW264.7 cytotoxicity, and method is as follows:
(1) collect logarithmic (log) phase RAW264.7 cell, adjust 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 ℃, at the bottom of being paved with hole to cell monolayer (96 hole flat underside), be divided at random 60 groups, one group of each medicine, every group of Fructus Phyllanthi extract that adds Concentraton 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 every group arranges 3 blank well, 5%CO
2, hatch 16-48 hour for 37 ℃;
(3) every hole adds 10 μ LMTT solution, continues to cultivate 4h, stops cultivating, and carefully sucks culture fluid in hole;
(4) every hole adds 100 μ L dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, and crystal is fully dissolved, and measures the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD570nm place;
In the embodiment of the present invention 1, be prepared into Fructus Phyllanthi extract to the toxicity of RAW264.7 cell as shown in Figure 4, as can be seen from Figure 4, the maximal non-toxic concentration of Fructus Phyllanthi extract to cell nonhazardous.
4.6 antiinflammatory anti-aging effects effect confirmatory experiments
PCR detects in the embodiment of the present invention 1 and is prepared into Fructus Phyllanthi extract in the generation of transcriptional level inhibition or reduction inflammatory factor, and method is as follows: the compound Fructus Phyllanthi extract with anti-UVA radiation that screening is obtained carries out anti-inflammatory activity experiment at macrophage RAW264.7 cell model.
(1) the phase RAW264.7 cell of taking the logarithm, pre-24 orifice plates, it is 3 * 10 that bed board is adjusted cell density
5cells/mL;
(2) add the PMA of 50ng/mL, cultivate 48h, examine under a microscope cellular morphology;
(3) carefully suck culture medium in hole, with PBS, rinse after 2 times, add 1640 culture medium of serum-free, cultivate 24h;
(4) add LPS and the Fructus Phyllanthi extract of 100ng/mL, cultivate 18～24h;
(5) RNA of extracting administration group cell, use real-time PCR method to IL-6 and IL-1 (Interleukin-6, nterleukin-1, interleukin-6 interleukin 1), TNF-α (tumor necrosis factor-alpha, tumor necrosis factor), MCP-1 (monocyte chemotactic protein-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 Fructus Phyllanthi extract 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 (tumor necrosis factor-alpha, tumor necrosis factor) result as shown in Figure 7, MCP-1 (monocyte chemotactic protein-1, macrophage chemoattractant protein 1) and iNOS result are as shown in Fig. 8 and 9.
From Fig. 5-9, can find out: the Fructus Phyllanthi extract that detects preparation in the embodiment of the present invention 1 shows the different scorching effects that presses down, and in the embodiment of the present invention 1, the Fructus Phyllanthi extract of preparation is remarkable to the inhibitory action of IL-6.
In fact, the present patent application people also carries out above-mentioned test to the Fructus Phyllanthi extract in other sources as commercially available Fructus Phyllanthi extract or the Fructus Phyllanthi extract prepared with other method simultaneously, result shows also have above-mentioned test effect, has radioprotective and senile-resistant efficacy.
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 of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, be included in protection scope of the present invention.
Claims (10)
1. Fructus Phyllanthi extract has the health food of radioprotective and senile-resistant efficacy or the application in cosmetics in preparation.
2. Fructus Phyllanthi extract according to claim 1 has the health food of radioprotective and senile-resistant efficacy or the application in cosmetics in preparation, it is characterized in that: in described Fructus Phyllanthi extract, the quality percentage composition of Chebulagic acid is more than 70%.
3. a preparation method with the Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy, is characterized in that: contain following steps:
(1) pre-treatment: raw material Fructus Phyllanthi is cleaned, pulverized;
(2) extract: after adopting the ultrasonic room temperature of methanol to extract, filter, filtrate vacuum decompression is processed and reclaimed methanol, obtain B extractum;
(3) extraction: B extractum is adopted after n-butyl alcohol ultrasonic extraction, filter, obtain C extract;
(4) loading is separated: by after C extract dilute with water, filter, be splined in two detached dowels that PIPO-02 parting material is housed that are in series, with after aqueous formic acid eluting, by two detached dowels separately, second detached dowel first washes with water, with methanol, wash again, obtain target phase D fraction;
(5) concentrate drying: after vacuum-concentrcted, vacuum drying obtains Fructus Phyllanthi extract by D fraction.
4. the preparation method with the Fructus Phyllanthi extract of radioprotective 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 Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy according to claim 3, is characterized in that: the consumption of the middle methanol of step (2) is 8-10 times of Fructus Phyllanthi raw material gross weight.
6. the preparation method with the Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy according to claim 3, it is characterized in that: in step (2), the ultrasonic room temperature of methanol is extracted 2 times, each 20-40 minute, 500-800 order filter-cloth filtering, merging filtrate, vacuum decompression reclaims methanol, obtains B extractum, and the weight of B extractum accounts for the 20-30% of Fructus Phyllanthi raw material gross weight.
7. the preparation method with the Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy according to claim 3, is characterized in that: in step (3), the consumption of n-butyl alcohol and B extractum is identical in quality, and the ultrasonic extraction time is 30-50 minute.
8. the preparation method with the Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy according to claim 3, is characterized in that: in step (4), C extract is diluted with accounting for its gross mass 15-20 water doubly.
9. the preparation method with the Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy according to claim 3, it is characterized in that: in step (4), with the heavy eluent of 10-15 times of Fructus Phyllanthi raw material of aqueous formic acid eluting, in aqueous formic acid, the volumn concentration of formic acid is 0.2%; Second detached dowel washes the heavy eluent of 3-5 times of Fructus Phyllanthi raw material with water, then washes the heavy eluent of 2-3 times of Fructus Phyllanthi raw material with methanol.
10. the preparation method with the Fructus Phyllanthi extract of radioprotective and senile-resistant efficacy according to claim 3, it is characterized in that: in step (5) by D fraction at 40-70 ℃ after vacuum-concentrcted, the Fructus Phyllanthi extract that vacuum drying obtains, the quality percentage composition through HPLC detection Chebulagic acid in Fructus Phyllanthi extract is more than 70%.
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