CN104666518A - Composition containing extracts of cichorium intybus and poncirus trifoliata for strengthening skin barrier - Google Patents
Composition containing extracts of cichorium intybus and poncirus trifoliata for strengthening skin barrier Download PDFInfo
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- CN104666518A CN104666518A CN201310636398.3A CN201310636398A CN104666518A CN 104666518 A CN104666518 A CN 104666518A CN 201310636398 A CN201310636398 A CN 201310636398A CN 104666518 A CN104666518 A CN 104666518A
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Abstract
The invention relates to a composition containing extracts of cichorium intybus and poncirus trifoliata for strengthening a skin barrier. In particular, compared with individual extracts of cichorium intybus (cichorium intybus) and poncirus trifoliate (poncirus trifoliate RAFIN), by virtue of the mixed extracts of the cichorium intybus and the poncirus trifoliate, cell growth of sebocyte is increased; the amount of lipid synthesized from the sebocyte is obviously increased; and the amount of hyaluronan as an important moisture factor in the skin is obviously increased. Therefore, the composition can be effectively used as a composition for strengthening skin barriers of infants with insufficient lipid biosynthesis capability and a composition for preventing and treating atopic dermatitis.
Description
Technical field
The present invention relates to the Prevention and Curation compositions of skin barrier strengthening compositions, atopic dermatitis.
Background technology
Skin is made up of epidermis and corium, the outermost horny layer being positioned at skin be protection live body by the impact of the physics of external environment condition, chemical stimulation and microorganism etc., and suitably regulate the evaporation of moisture, thus keep the stability skin barrier of live body.In order to the skin condition of keeping fit, horny layer needs containing a certain amount of moisture, horn cell needs to be formed balanced with the nature moisturizing factor such as lipid components and aminoacid, carbamide, organic acid and sebum, if and the equilibrium of such key element is broken or lacks, then the water quantities of keratodermatitis declines, skin barrier function generation obstacle, causes xerosis cutis.
Child less than three years old is called baby, is called child by from full three years old to the child before primary school enters school, they are referred to as infant.With regard to the skin of infant; the activity of sebaceous sebaceous gland is faint; and cause the secretion power of antiperspirant more weak because of sweat gland immaturity; therefore protect the formation of the natural sebum film of skin faint once on the outermost surface of skin; epidermis and corium thin, therefore water tariff collection and skin protective ability decline, thus cause xerosis cutis; responsive for endo reaction, and be easily upset and damage.
When xerosis cutis, scratching because scratching where it itches, therefore causing the damage of skin, finally causing atopic dermatitis.In fact, according to the anaphylactic disease investigation that disease control our department delivers, the ratio suffering from the child of atopic dermatitis, every year in increase, it is said that 75% in the middle of patient was fallen ill before completely one-year-old.The disease major part that the chronic eczema and pityriasis etc. that comprise atopic dermatitis occur at the skin of drying demonstrates chronic process, therefore constantly to skin supply moisture and oil content and to recover skin barrier function be very important, and as the auxiliary therapeutical agent that life-time service is also free from side effects, skin moisture-keeping product must be used.
In order to improve skin barrier function, the major function of the skin preparations for extenal use used at present is the water tariff collection amount keeping skin surface by smearing this skin preparations for extenal use, and the composition wherein showing moistening effect is divided into as the wetting agent of basis, sealant and as the natural extract of medicine component, vitamin and ceramide.Wetting agent is as the more material of moisture, what have according to composition itself shows moistening effect containing wet ability, sealant forms diaphragm seal as maintenance composition at skin surface, thus prevent the evaporation of moisture and go out moistening effect in cutaneous manifestations, or utilize containing wet ability and moistening effect being provided of maintenance composition agent unsaturated fatty acid.Natural goods and ceramide can improve moistening effect further, in order to recover cuticular skin barrier function, use the various moisturizing ingredient from natural goods at present.In addition, as the known material possessing sebum secretion facilitation, also have plumule essence, pantetheine and fatty acid ester etc., but be difficult to because of the character of material manufacture dosage form.
Although, using various material to improve skin barrier function, owing to using chemical raw material, causing skin irritation, allergy etc. on the contrary, in fact bad influence is produced to skin, in order to solve such problem, being badly in need of the natural component of exploitation from the Nature.
Herba Cichorii (Cichorium intybus) is the frigid zone high mountain herbaceous plant belonging to Flos Chrysanthemi section, and original producton location is near Northern Europe universe, the Central Asia, Siberian Baikal, and also grows the northwestward in China, and it is the plant that tolerance to cold is strong.Inulin (chicory inulin) from Herba Cichorii has the effect of the absorption promoting enteral mineral, and the bacillus bifidus (bifidobacteria) greatly increased wittingly as enteral probiotics and the breeding of lactobacillus (lactobacilli), and suppress the growth of intestinal toxic antibacterial, the combination of intestinal microbial group is improved to useful direction, therefore just in probiotic bacteria (probiotics) field, has carried out various research in the early time.Further, Herba Cichorii extract also has Prevention and Curation effect for hyperlipemia and the such chronic degenerative disease of diabetes, more specifically, has confirmed the numerical value of blood glucose and the numerical quantities of total glycolated hemoglobin in the body that Herba Cichorii extract can reduce blood glucose.Moreover, report is had to point out, also there is antioxidation, antiinflammatory, antiageing effect, and indicated for the factor played an important role in the expression of gene and PPAR-a(Peroxisome proliferators activated receptorsΥ-a by nearest result of study) upregulation (up-regulation) also generation effect.
Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) belongs to Rutaceae (Rutaceae) in Plant Taxonomy, is collectively referred to as Citrus.The feature of Poncirus trifoliata Raf. is that tolerance to cold is strong, has unique fragrance, is called as Poncirus trifoliata Raf., is widely used as aromatic bitter stomachic in Chinese medicine.Particularly, be used for treating fullness in the chest, chest pain, abdominal distention, stomachache and inflammation.The main component of Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) is flavonoid (flavonoid), Hesperidin (hesperidin), neohesperidin (neohesperidin), naringin (naringin) and coumalin (cumalin) class etc., and has also carried out much research about the dependency of these compositions and live body function.From ancient times, not only medicinal, but also be extensively used in beauty treatment, there is report to claim for anti-inflammatory, agaist allergic symptoms, anticancer etc. also effective in cure recently.More specifically, the activation generation effect for the transcription factor and SREBPs playing material impact in the synthesis of lipid is noted.
Skin barrier is present in the outermost horny layer being positioned at epidermis in the structure forming skin, prevents moisture from evaporating and invading from the impurity of outside, thus protection human body.The suitable moisture of skin maintains and is realized by lipid, nature moisturizing factor and sebum etc. between horn cell, and it has close relationship with suppressing the disease such as skin aging and atopic dermatitis, pityriasis, chronic eczema, chronic renal failure disease, diabetes.Therefore, secreted in the sebocyte cell of skin lipid plays important effect for maintenance skin barrier function.The synthesis (lipogenesis) of lipid is by cholesterol regulation element conjugated protein (sterol regulatory element-binding proteins (SREBPs)) and Peroxisome proliferators activated receptorsΥ s(peroxisome proliferator-activated receptors (PPARs)) etc. transcription factor regulate.PPAR is as the one of intracellular nucleic hormone receptor, activated with the combination with specific ligand, and promote the transcription factor (transcription factor) of the expression of subject proteins, have that report claims the growth and differ entiation of the cell in PPAR and skin, lipid metabolism regulates, Wound healing and bone regeneration, inflammatory reaction, sebum produce and B16 cell etc. has relation.In PPAR, there is the obform body (isoform) of these three kinds of structures of a, β and γ, wherein the differentiation of PPAR-γ reconciliation statement chrotoplast and lipid metabolism, guide the formation of normal epidermal area, thus maintain the stability of epidermis.
To this, the present inventor plays an important role for skin barrier to invent, and can fundamentally improve the synthesis of lipid from natural external preparation, utilize and probably the mixed extract of the virtuous Herba Cichorii of this tool and Japanese raisintree fruit is studied and the result of making great efforts, confirm following content: this mixed extract not only strengthens the growth of sebocyte cell (sebocyte) and the GCMS computer of lipid, but also the generation promoted as the hyaluronic acid (hyaluronan) of main Moisture factor, therefore as the skin barrier strengthening external preparation and very applicable of crowd's particularly infant of Lipid Secretion under-activity.
Summary of the invention
Therefore, main purpose of the present invention is to provide following skin barrier strengthening compositions: its mixed extract containing Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) and as effective ingredient, this mixed extract not only promotes the growth of the sebocyte cell (sebocyte) generating lipid and guides the GCMS computer of the lipid of the major function realizing skin barrier, but also has the effect of the expression promoting lipid to generate and to promote Moisture factor.
Another object of the present invention is to provide the Dermatologic preparation composition that make use of skin barrier strengthening compositions, this skin barrier strengthening compositions contains the mixed extract of described Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), as effective ingredient.
Another object again of the present invention is to provide the cosmetic combination that make use of skin barrier strengthening compositions, this skin barrier strengthening compositions contains the mixed extract of described Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), as effective ingredient.
According to a mode of the present invention, the invention provides skin barrier strengthening compositions, it contains the mixed extract of Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), as effective ingredient.
Baby is after birth before 3 months, because of the hormone that brings from parent during fetus and androgen, sebum secretion is similar with adult, but from being born latter 3 months to adolescence, drop to adult's 1/3, easily be infected by bacterial, and owing to being directly exposed to surrounding, skin is easily in drying regime.Further, when infant, although the moisture amount in skin is high, because skin is thin, cannot prevent the evaporation of moisture, its result skin, easily by chronic eczema, causes reacting outside environmentally sensitive area thus.Therefore, need the GCMS computer carrying out sebum and moisture energetically, and that in this GCMS computer, play important effect is PPAR-γ.Although do not indicate mechanism accurately, activated, express the factor needed for GCMS computer of lipid from sebocyte cell (sebocyte) by PPAR-γ, its result forms lipid and performs the effect of barrier cell.
Herba Cichorii (Cichorium intybus) is as the perennial herb of dicotyledon Canterburybells order Flos Chrysanthemi section, there is the usefulness about facilitating digestion, blood circulation, anticancer, calculus, hepatic disease, diabetes, and because the amount of inulin (inulin) is high, therefore promote the growth of enteral beneficial bacteria and improve enteral environment, and being the plant as fat-reducing material and food additives with very high value.
The broad leaved and deciduous broad leaved arbor of Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) as Rutaceae and the fruit of Japanese raisintree fruit tree, from ancient times, be not only used for the treatment of acute bronchus inflammation, asthma, the dirty disease of stomach, and be utilized as beauty treatment material.Poncirus trifoliata Raf. comprises flavonoid (flavonoids) and alkaloid (alkaloids), specifies by various research the effect that it has anti-oxidation function, antiinflammatory and whitening.
The invention is characterized in, guide the growth of sebocyte cell (sebocyte), promotion lipid GCMS computer by the mixed extract of described two kinds of materials and promote the expression of Moisture factor, thus demonstrating skin barrier strengthening effect.
According to embodiments of the invention, Growth of Cells guiding function is confirmed by MTT experiment (assay), and by chilli oil O dyeing (oilred O staining) test and total cholesterol level determination test, demonstrate the GCMS computer effectively realizing lipid, thus confirm the strengthening that can guide skin barrier.
According to embodiments of the invention, the expression confirmed by RT-PCR as a kind of PPAR-γ of intracellular nucleic hormone receptor is increased, this represents that the mixed extract of Herba Cichorii of the present invention and Poncirus trifoliata Raf. increases the expression of PPAR-γ, fundamentally can guide the GCMS computer of lipid.
According to embodiments of the invention, test is expressed by hyaluronic acid (hyaluronan), the amount confirming the hyaluronic acid (hyaluronan) of the effect playing main Moisture factor in skin increases, and this represents that the present invention can realize the effect of the water tariff collection amount increased in skin.
In the present invention, described extract extracts by known any Extraction solvent, more than one solvent such as by selecting from following group extracts: the mixed solvent of ethanol, methanol, butanols, ether, ethyl acetate, chloroform or their organic solvent and water, preferably uses water, ethanol or butanediol to extract.
About the Extracting temperature of described solvent, in the case of water, be preferably 18 DEG C ~ 80 DEG C, when 70% ethanol and butanediol, be preferably 18 DEG C ~ 40 DEG C, extraction time is preferably 6 hours ~ 168 hours.If depart from described Extracting temperature and extraction time, then may cause the change of extraction efficiency decline or composition.In foregoing, after obtaining extract utilizing solvent, by general method well known in the art, merceration, heating and filtration at normal temperatures and obtain fraction, or also can further solvent be evaporated, spraying dry or freeze-dried.
In the present invention, described extract extracts by known any extracting method, extract preferably by following method: the Herba Cichorii (Cichorium intybus) of drying and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) are put in solvent, carry out circulation extraction and make it deposition, afterwards, filter deposition thing and obtain filtrate, carries out concentrating under reduced pressure to filtrate, extracts thus.
The mixed extract of Herba Cichorii used in the present invention (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) not only comprises and leaching Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), decoct and the leachate that obtains, but also comprise and the concentrate that obtain or the preserved material that described concentrate after drying produced again concentrated to part or all leachate, decoct, tincture, fluid extract and be contained in the chemical substance and the plant itself that play main effect in Herba Cichorii and Poncirus trifoliata Raf..In addition, in the present invention, the extract gone out from all extracting section of Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) can be used, be not limited to the extract of a certain specific part.
About the manufacture method of the mixed extract of Herba Cichorii (Cichorium intybus) used in the present invention and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), known method can be used.Such as can obtain mixed extract like this: the Herba Cichorii (Cichorium intybus) of invention and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture are cleaned and after drying, put into water or organic solvent, after circulation extracts and deposits, by carrying out filter-cloth filtering and centrifugalize and isolating residue and filtrate, concentrating under reduced pressure is carried out to isolated filtrate and obtains extract.
According to another way of the present invention, the invention provides skin barrier strengthening Dermatologic preparation composition, it is containing Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixed extract, as effective ingredient.
The invention is characterized in, described Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixed extract occupy 0.001 % by weight ~ 50 % by weight in the gross weight of compositions.Because when the content of described Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixed extract is less than 0.001 % by weight, according to usefulness, low effort that described extract obtains, when more than 50 % by weight, there is the problem in cutaneous safety or dosage form.
Compositions of the present invention can be used as infant skin barrier strengthening preparation composition for external use and uses, the present invention considers the characteristic of the infant of the secretion under-activity of lipid, fundamentally guide growth and the lipid GCMS computer of sebaceous sebocyte cell (sebocyte), improve the Be very effective of the secretion of the lipid played an important role as skin barrier.And then, keep the skin intrinsic factor of moisture and the GCMS computer of hyaluronic acid (hyaluronan) because guide, therefore can be used as strengthening skin barrier, prevention and improve atopic dermatitis, prevention or improve the Dermatologic preparation composition of xeroderma, chronic eczema etc. and effectively use.
According to another way of the present invention, provide skin barrier strengthening cosmetic combination, it is containing Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixed extract, as effective ingredient.
The invention is characterized in, described Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) extract occupy 0.001 % by weight ~ 50 % by weight in composition total weight.
As mentioned above, Herba Cichorii of the present invention (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixed extract display skin barrier strengthening effect and moistening effect, therefore can be used as the skin barrier of strengthening infant, prevention and improve atopic dermatitis, prevention or improve the external preparation of xeroderma, chronic eczema etc. or cosmetic combination and effectively carry out commercialization.
Accompanying drawing explanation
Fig. 1 is the figure measuring the Growth of Cells usefulness of extract in sebocyte cell (sebocyte) people's sebocyte cell and fibroblast (fibroblast) people fibrocyte and illustrate with figure.
Fig. 2 measures the lipid GCMS computer usefulness of extract in sebocyte cell (sebocyte) people sebocyte cell and the figure illustrated with figure by chilli oil O colouring method.
Fig. 3 measures the T-CHOL GCMS computer usefulness of extract in sebocyte cell (sebocyte) people sebocyte cell and the figure illustrated with figure by total cholesterol level determination test method.
Fig. 4 be by RT-PCR method measure the PPAR-γ of extract in sebocyte cell (sebocyte) people sebocyte cell expression usefulness and with the figure that illustrates of form of band.
Fig. 5 measures the expression usefulness of the PPAR-γ of extract in sebocyte cell (sebocyte) people sebocyte cell and the figure illustrated with figure by RT-PCR method.
Fig. 6 measures the Moisture factor of extract in CCD986SK people's fibrocyte to generate usefulness and the figure that illustrates with figure by hyaluronic acid test (Hyaluronan assay) method.
Detailed description of the invention
Below, by embodiment, in further detail the present invention is described.These embodiments are only illustrate the present invention, and therefore scope of the present invention should not be only limitted to these embodiments.
Embodiment 1: the manufacture of Herba Cichorii (Cichorium intybus), Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixed extract
Utilize pulverizer, after the Herba Cichorii (Cichorium intybus) (large light Pharmaceutical) of the 50g of drying and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) (large light Pharmaceutical) are fabricated to powder, with 70% ethanol 1L, extract 3 hours at normal temperatures, then filter with Whatman NO.4 filter paper and Advantec NO.5C filter paper.After utilization decompression rotary evaporator carrys out concentrated filtrate, Herba Cichorii obtains the earning rate of 21.76%, and Japanese raisintree fruit obtains the earning rate of 14.06%.For Herba Cichorii+Poncirus trifoliata Raf. mixture, two natural goods powder are mixed with the ratio of 1:1, and has manufactured extract by said method.
Experimental example 1: cell culture
[sebocyte cell (sebocyte), SZ95]
Use with the addition of 10% hyclone (Fetal bovine serum (FBS)), 5ng/ml hEGF (human epidermal growth factor), 1mM CaCl
2dMEM (Dulbecco's modified eagle medium (DMEM))/F12 (1:1) and at CO
2incubator (incubator) (37 DEG C, 5%CO
2) middle cultivation SZ95 cell, with 2 ~ 3 days for the cycle has changed culture medium.
[fibroblast (fibroblast), CCD-986SK]
Use with the addition of 10% hyclone (FBS) DMEM (DMEM) and at CO
2incubator (incubator) (37 DEG C, 5%CO
2) in cultivate CCD-986 or cell, with 2 ~ 3 days for the cycle has changed culture medium.
Experimental example 2: Cytotoxic evaluation (MTT assay)
In order to evaluate manufacture in described embodiment 1 Herba Cichorii (Cichorium intybus), Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture extract cytotoxicity, perform MTT assay and confirm.
SZ95 and CCD-986SK cell is separately planted to 96-orifice plate (well plate), at 37 DEG C, 5%CO with the concentration of 1 × 105 cell/ml
224 hours have been cultivated in incubator (incubator).After cultivation, remove all culture medium, process utilizes the culture medium that do not comprise serum and after diluting the test portion 100ul for debita spissitudo (25ug/ml ~ 200ug/ml), has cultivated 24 hours in each hole (well).After cultivation 24 hours, utilize PBS and produce the MTT reagent of the concentration being dissolved as 5mg/ml, and having put into 20ul at every turn and cultivated 4 hours.Culture medium containing MTT reagent and test portion is all removed, the sour isopropyl alcohol (acid isopropanol) of 100ul is added in each hole (well), and stir 10 ~ 15 minutes in agitator (shaker), utilize microplate reader (ELISA reader), under 570nm, determine light absorption value.
[table 1]
As described in shown in table 1, measurement result shows, and extract does not find toxicity under all concentration, in this concentration selected maximum concentration and 100ug/ml and 200ug/ml concentration and carried out following experiment.
Experimental example 3: lipid GCMS computer evaluates (chilli oil O dyes)
In order to evaluate in described embodiment 1 the lipid GCMS computer function of manufactured Herba Cichorii (Cichorium intybus), Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture extract, performing chilli oil O dyeing and confirming.
By SZ95 cell, separately plant 12-orifice plate (well plate), at 37 DEG C, 5%CO with the concentration of 3 × 104 cells/ml
224 hours have been cultivated in incubator (incubator).After culturing, remove all culture medium, after each hole (well) middle process utilizes the culture medium containing serum and dilutes the test portion 1ml for suitable concentration (100ug/ml ~ 200ug/ml), cultivate 48 hours.After 48 hours, remove all culture medium, after PBS cleaning (washing), intactly remove PBS.10% formalin (formalin) is put into 2ml at every turn, cultivates at normal temperatures and (incubation) 10 minutes.Again put into 10% new formalin and cultivate and (incubation) more than 1 hour or 1 hour.Remove 10% formalin, utilize D.W and clean and (washing) 2 times.After cleaning (washing), put into 60% isopropyl alcohol (isopropanol), after cultivating (incubation) 5 minutes at normal temperatures, remove isopropyl alcohol (isopropanol), drying has been carried out to orifice plate (well plate).Make after chilli oil O working solution (Oil Red O working solution) reacts 10 minutes, (washing) 4 times with D.W cleaning at once.Now, capable of taking pictures.Put into 100% isopropyl alcohol (isopropanol) after taking pictures and cultivate (incubation) 10 minutes, and determine light absorption value under 500nm.
[table 2]
As described in shown in table 2, in whole test portion, lipid GCMS computer function and concentration proportionally increase, and wherein in the mixture of Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture extract, usefulness is the most outstanding.
Experimental example 4: total cholesterol level evaluation (Total cholesterol assay)
In order to evaluate manufacture in described embodiment 1 Herba Cichorii (Cichorium intybus), Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture extract total cholesterol level, perform total cholesterol level evaluation and confirm.
By SZ95 cell, with in the concentration of 3 × 104 cells/ml separately plantation to 12-orifice plate (well plate), and at 37 DEG C, 5%CO
224 hours have been cultivated in incubator (incubator).After cultivation, remove all culture medium, process utilizes the culture medium that do not include serum and after diluting the test portion 1ml for debita spissitudo (100ug/ml ~ 200ug/ml), has cultivated 48 hours in each hole (well).After cultivation 48 hours, remove all culture medium, after PBS cleaning (washing), remove PBS completely.Use RIPA buffer (buffer) (150mM NaCl, 1.0%
0.5% NaTDC (sodium deoxycholate), 0.1%SDS, 50mM Tris, pH8.0) and carry out lysis (cell lysis), and carried out quantification of protein by BCA method.After carrying out quantitatively, in cytolysate, add chloroform: methanol (Chloroform:methaol) (2:1) solution, and fully mix.The centrifugalize (centrifuge) of 15 minutes is carried out under 2000rpm, take out subnatant and after removing, in remaining solution, add chloroform: methanol (Chloroform:methaol) (2:1) solution points for 2 times, use the same method to be separated subnatant and obtain lipid after removing.When the lipid of test portion is concentrated, puts into ethanol (ethanol) 300ul and after dissolving, utilize T-CHOL box (total cholesterol kit (AM202-K)) and measure.In the enzyme reagent of 3ml, add the test portion of 20ul and mix, and after cultivating 5 minutes at 37 DEG C, under the wavelength of 500nm, utilize microplate reader (ELISA reader) and determine absorbance.About the cholesterol (cholesterol) of titer, put in the enzyme reagent of 3ml 20ul titer and as 3mg/ml value.
[table 3]
As described in shown in table 3, in Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) and Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture group, confirm the total cholesterol level more outstanding than positive controls, and confirming Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture group, to organize usefulness than Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) more outstanding.
Experimental example 5: lipid GCMS computer correlation factor confirms to evaluate (RT-PCR [PPAR-γ])
In order to confirm Herba Cichorii (Cichorium intybus) manufactured in described embodiment 1, Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) the lipid GCMS computer correlation factor of mixture extract and the expression of PPAR-γ, performing RT-PCR and confirming.
1) total serum IgE is separated
By SZ95 cell, with in the concentration of 1.5 × 105 cells/ml separately plantation to 6-orifice plate (well plate), and at 37 DEG C, 5%CO
224 hours have been cultivated in incubator (incubator).After culturing, remove all culture medium, process utilizes the culture medium that do not comprise serum and after diluting the test portion 2ml for suitable concentration (100ug/ml ~ 200ug/ml), has cultivated 48 hours in each hole (well).Remove all culture medium, put into the QIAzol of 1ml in each sample (sample) after, shake (shaking) 5 minutes at normal temperatures, to make to isolate nucleoprotein.Knee-action (up & down) is carried out with dropper (pipette), cell (cell) is reclaimed in miniature tube (micro tube), and put into chloroform (Chloroform) 0.2ml and rock after about 50 times, cultivate (incubation) 2 ~ 3 minutes at normal temperatures, and in 4 DEG C 12,000g, carried out the centrifugalize of 20 minutes.Upper liquid 300 ~ the 400ul of the pipe (tube) completing centrifugalize is moved on in the pipe (tube) that isopropyl alcohol (isopropanol) is housed, then its eddy current (vortexing) is made, after cultivation (incubation) at normal temperatures 10 minutes, the centrifugalize of 15 minutes has been carried out under 4 DEG C 12,000g.Note granule (pellet) is not fallen down, remove upper liquid, and after adding 75% ethanol (ethanol) (in0.1%DEPC) 1ml and making its eddy current (vortexing), at 4 DEG C 12, in 000g, carried out the centrifugalize of 10 minutes.Remove upper liquid, miniature tube (micro tube) is stood upside down and after carrying out drying, puts into the tip-tap (tapping) without RNA enzyme water (RNAase Free Water) and carried out necking in spindown (spin down) afterwards.
2) RNA is quantitative
In miniature tube (micro tube), separately plant 0.1%DEPC998ul, the RNA of separation is put into 2ul at every turn and carries out mixing (mixing).Utilize quartz test tube, in 260nm, 0.1%DEPC is determined absorbance as OA.Calculated by [2ug/{ absorbance × 40 (specific absorbance) × 500 (extension rate) }] × 1000 (unit conversions), obtained the amount of RNA and nuclease free water (Nuclease free water).
3) cDNA synthesis
5X reaction buffer (Reaction buffer) 4ul and Maxima enzyme (Enzyme) 2ul of heat (Thermo) is put in PCR pipe (tube), according to the amount quantitatively obtained with RNA, put into RNA and nuclease free water (Nuclease free water), carry out RT-PCR with 25 DEG C of 10 minutes-50 DEG C of 15 minutes-85 DEG C of 5 minutes-4 DEG C of conditions of 8 minutes.
4) PCR confirms
Utilize the GoTaq Green Master Mix of Promega and perform PCR.Manufacture 1.5%agarose gel and carry out surge, and confirming group.
[table 4]
As described in shown in table 4, in Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) extract, confirm the expression of more than 150%, under the concentration of 100 μ g/ml of Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture group, confirm the expression of 200% nearly.
Experimental example 6: moisturizing efficiency evaluation (hyaluronic acid evaluation)
In order to evaluate manufacture in described embodiment 1 Herba Cichorii (Cichorium intybus), Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN), Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture extract moisturizing usefulness, perform hyaluronic acid evaluation and confirm.
By HaCaT and CCD-986SK cell, separately plant 6-orifice plate (well plate) with each 2ml ground of the concentration of 1 × 105 cell/ml, and at 37 DEG C, 5%CO
224 hours have been cultivated in incubator (incubator).After cultivation, remove all culture medium, after each hole (well) middle process utilizes the culture medium containing serum and dilutes the test portion 2ml for suitable concentration (100ug/ml ~ 200ug/ml), cultivate 48 hours.In each hole (well), reclaim culture medium 1.2ml respectively and move on in miniature tube (micro tube), and under 4 DEG C, 12,000rpm, carrying out the centrifugalize of 10 points of kinds, and the 1ml in upper liquid having been moved on in new miniature tube (micro tube).By test case (assay kit) (echelon, K-1200) sample (standard) 100ul and sample (sample) 100ul of recovery is put into yellow plate (yellow plate) respectively, and put into the working detector of 50ul respectively and seal (sealing), then cultivate at 37 DEG C and (incubation) 1 hour.After cultivation (incubation), in institute porose (well), get 100ul respectively and move on to detection plate (detector plate) and seal (sealing) tip-tap (tapping) after, and 30 points of kinds (incubation) in cultivation at 4 DEG C.Remove all solution (solution) in the detection plate (detection plate), (washing) 3 times with 1X concentrated solution (wash concentrate) cleaning of being correlated with.In each hole (well), put into work enzyme (working enzyme) and seal (sealing), cultivating at 37 DEG C and (incubation) 30 points of kinds.Again clean (washing) 3 times, and after putting into matrix solution (substrate solution), under the dark condition of room temperature, make it reaction.After reacting, in units of 15 points of kinds, under 405nm, determine absorbance.
[table 5]
As described in shown in table 5, confirm compared with all test portions at this, in Herba Cichorii (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixture extract, the amount of hyaluronic acid contents (Hyaluronan content) increases at most.
As mentioned above, Herba Cichorii of the present invention (Cichorium intybus) and Poncirus trifoliata Raf. (Poncirus trifoliata RAFIN) mixed extract strengthen the growth of sebocyte cell (sebocyte), and promote the GCMS computer of lipid and promote the GCMS computer of Moisture factor, be not form barrier cell artificially from outside, but fundamentally generate lipid and Moisture factor, thus barrier cell can be formed voluntarily, therefore effectively can be used in infant barrier cell and strengthen in external preparation, the exploitation of cosmetic preparation and production.
Claims (8)
1. a skin barrier strengthening compositions, it contains the mixed extract of Herba Cichorii and Poncirus trifoliata Raf., as effective ingredient.
2. skin barrier strengthening compositions according to claim 1, is characterized in that,
Described extract not only guides lipid GCMS computer, also has moistening effect.
3. skin barrier strengthening compositions according to claim 1, is characterized in that,
Described extract uses alcohol solvent and extracts.
4. skin barrier strengthening compositions according to claim 1, is characterized in that,
Described extract extracts by the following method: the mixture of Herba Cichorii and Poncirus trifoliata Raf. is put into solvent, and carry out circulation extraction and make it deposition, then filter deposition thing, obtains filtrate thus, carries out concentrating under reduced pressure and extract this filtrate.
5. a skin barrier strengthening Dermatologic preparation composition, it contains the mixed extract of Herba Cichorii and Poncirus trifoliata Raf., as effective ingredient.
6. skin barrier strengthening Dermatologic preparation composition according to claim 5, is characterized in that,
The mixed extract of described Herba Cichorii and Poncirus trifoliata Raf. occupies 0.001 % by weight ~ 50 % by weight in composition total weight.
7. a skin barrier strengthening cosmetic combination, it contains the mixed extract of Herba Cichorii and Poncirus trifoliata Raf., as effective ingredient.
8. skin barrier strengthening cosmetic combination according to claim 7, is characterized in that,
The mixed extract of described Herba Cichorii and Poncirus trifoliata Raf. occupies 0.001 % by weight ~ 50 % by weight in composition total weight.
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| CN108354857A (en) * | 2018-04-08 | 2018-08-03 | 大连军门保健食品有限公司 | Water beautifying liquid and preparation method thereof is refined after a kind of extraction of witloof polysaccharide |
| CN109771350A (en) * | 2019-03-19 | 2019-05-21 | 广州睿森生物科技有限公司 | A kind of skin barrier remediation composition, Essence and preparation method thereof |
| CN113544259A (en) * | 2020-01-31 | 2021-10-22 | 瑞帝安有限公司 | Method for differentiating human adipose-derived mesenchymal stem cells into hair papilla cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108096089A (en) * | 2017-12-11 | 2018-06-01 | 百朗德生物化学(海门)有限公司 | A kind of cosmetic composition of Gotu Kola P.E with anti-inflammatory anti-oxidant complex function and its preparation method and application |
| CN108354857A (en) * | 2018-04-08 | 2018-08-03 | 大连军门保健食品有限公司 | Water beautifying liquid and preparation method thereof is refined after a kind of extraction of witloof polysaccharide |
| CN109771350A (en) * | 2019-03-19 | 2019-05-21 | 广州睿森生物科技有限公司 | A kind of skin barrier remediation composition, Essence and preparation method thereof |
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| CN113544259A (en) * | 2020-01-31 | 2021-10-22 | 瑞帝安有限公司 | Method for differentiating human adipose-derived mesenchymal stem cells into hair papilla cells |
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