WO2023030041A1 - I型胶原蛋白产生促进剂 - Google Patents

I型胶原蛋白产生促进剂 Download PDF

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Publication number
WO2023030041A1
WO2023030041A1 PCT/CN2022/113589 CN2022113589W WO2023030041A1 WO 2023030041 A1 WO2023030041 A1 WO 2023030041A1 CN 2022113589 W CN2022113589 W CN 2022113589W WO 2023030041 A1 WO2023030041 A1 WO 2023030041A1
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Prior art keywords
extract
polygonatum
concentration
motherwort
collagen
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PCT/CN2022/113589
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English (en)
French (fr)
Inventor
李念哲
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株式会社资生堂
李念哲
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Publication of WO2023030041A1 publication Critical patent/WO2023030041A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—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
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • 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
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Definitions

  • the present invention relates to novel type I collagen production promoters, the use of these new type I collagen production promoters in cosmetics, and cosmetics containing these new type I collagen production promoters.
  • Beauty, beauty and anti-aging are the goals that people have been pursuing, and our company has been committed to finding products with beauty, beauty and anti-aging effects.
  • Collagen present in the dermis is the main factor affecting skin aging. If the collagen content in the dermis is reduced, the skin will show signs of aging, lose its elasticity, gradually loosen, and form wrinkles. If the collagen content in the dermis is rich, the skin will look full of elasticity, young and shiny, so if you can find Products that promote the increase of collagen content in the dermis can realize the dream of beauty and anti-aging.
  • the methods of supplementing collagen in the prior art include, 1. Eat more foods rich in collagen: animal skin (pig skin, fish skin, etc.), tendon (pig tendon, beef tendon, trotter, etc.) of daily food ), cartilage (flesh and bone, brittle bone) are rich in collagen. However, after collagen is digested by the gastrointestinal tract, it is decomposed into small molecular amino acids and polypeptides by protease, and the form and structure of collagen no longer exist. Oral administration of collagen has little effect on improving skin quality. 2.
  • Collagen smearing There are many smearing collagen products on the market, such as facial masks, etc., but due to the large molecular weight of collagen, it is difficult to penetrate the tight skin barrier and be absorbed by the skin. Smearing collagen does not increase skin collagen . 3. Injection water light needle: through vacuum negative pressure technology, inject the solution based on hyaluronic acid directly into the dermis, so that the skin can absorb 100% of the nutrients. Achieve the effect of smoothing wrinkles, diluting melanin, whitening and rejuvenating skin. However, the short-term effect of injection by breaking the skin, the risks should not be underestimated, so we need to be very cautious. 4. Taking collagen health products, there are two kinds of oral liquid and powder; some are called collagen, some are called “collagen peptide”, and some are called “hydrolyzed collagen”.
  • the inventor of the present invention believes that the above-mentioned method for increasing collagen in the prior art cannot fully meet people's needs.
  • the above-mentioned method may be effective, but the effect is relatively slow. If can directly add the composition that can promote collagen production in cosmetics, make this composition infiltrate in the dermis layer of skin and directly promote collagen production in dermis layer while applying cosmetics, it will be better than directly supplementing collagen in the prior art. effective method. For this reason, the present inventor has carried out repeated and in-depth research to find products that can promote the production of collagen in the dermis.
  • Motherwort the name of traditional Chinese medicine. It is the fresh or dried aerial part of Leonurus japonicus Houtt. Fresh products are harvested from seedling stage in spring to early flowering stage in early summer; dry products are harvested when stems and leaves are lush in summer, flowers are not bloomed or just bloomed, and dried in the sun, or cut into sections and dried in the sun. Widely distributed throughout the country.
  • the efficacy and role of motherwort include: 1.
  • Postpartum diseases such as postpartum hemorrhage or lochia, abdominal pain, less bleeding, or mixed with blood clots, caused by weak uterine contraction, often combined with angelica, wine white peony root, mugwort leaf, Chuanxiong, Jiaohawthorn, colder Adding canned ginger, Taiwan black medicine, etc., the effect is more ideal.
  • motherwort is compatible with other medicines to achieve the purpose of treating kidney stones, acute nephritis edema and hematuria and other kidney diseases.
  • motherwort contains a variety of trace elements.
  • Selenium can enhance the vitality of immune cells, alleviate the occurrence of atherosclerosis and improve the function of the body's disease defense system; manganese can resist oxidation, anti-aging, anti-fatigue and inhibit the proliferation of cancer cells. 8. Improving eyesight and benefiting essence, motherwort has the effect of treating dizziness and name, and is used for dizziness and blurred vision caused by body deficiency, water and dampness.
  • CN108670943A discloses a plant extract composition for moisturizing, whitening and lightening spots, which contains various plant extracts in the water phase, including motherwort extract.
  • CN12516046A discloses a motherwort extract acne cream and a preparation method thereof.
  • CN109758393A discloses a skin care product with whitening, moisturizing and anti-aging effects and its preparation method and application.
  • the skin care product contains 5-10 parts by weight of ginseng extract, 1-5 parts by weight of gentian extract, firewood 5-10 parts by weight of the thorn extract, 1-5 parts by weight of the motherwort extract and 3-5 parts by weight of the Polygonatum odoratum extract.
  • Motherwort has the functions of promoting blood circulation and regulating menstruation, diuresis and swelling, clearing away heat and detoxifying.
  • Polygonatum Polygonatum is known as the top grade in "Compendium of Materia Medica", and it has extremely high nutritional value and pharmacological effects.
  • the efficacy and functions of Polygonatum Polygonatum include: 1. Nourishing yin and moistening dryness, promoting body fluid and quenching thirst, health care and anti-aging.
  • the polysaccharides, vitamin A and niacin in Yuzhu can enhance the body's resistance to disease and delay aging. 2. Calming nerves and strengthening the heart.
  • Polygonatum odoratum contains cardiac glycosides, alkaloids, vitamin A substances, and mucus polysaccharides—polysaccharides from Polygonatum odoratum, Polygonatum fructan, etc., which can improve myocardial hypoxia and adrenal cortex. Hormone-like effects.
  • the steroidal glycosides contained in Polygonatum Polygonatum have a similar effect on the myocardium as Lily of the Valley preparations.
  • Polygonatum glycoside has a cardiotonic effect on the isolated frog heart, and the effect of Polygonatum odoratum decoction is similar to that of Polygonatum glycoside.
  • Yuzhu has a certain effect on heart palpitations, angina pectoris, rheumatic heart disease, coronary atherosclerotic heart disease, and heart failure caused by pulmonary heart disease. 3. Lowering blood fat and blood sugar. Modern pharmacological research shows that there are more than 40 kinds of traditional Chinese medicines that have the effect of lowering blood lipids.
  • the methanol extract of Polygonatum Polygonatum can significantly reduce the blood sugar level of mice with hyperglycemia induced by adrenaline. And showed a tendency to improve glucose tolerance function.
  • One of its mechanisms of action is to inhibit the liver glycolysis system.
  • gavage rats with Polygonatum odoratum has inhibitory effect on the increase of blood sugar in rats caused by glucose and alloxan.
  • Polygonatum Polygonatum has the effect of lowering low-density lipoprotein, and it also has the effect of lowering blood sugar, so as to effectively prevent the occurrence of diabetes. 4.
  • Treatment of internal fire, rising internal fire can cause oral diseases.
  • Traditional Chinese medicine reminds us to control emotions, reduce tension, and reduce worries and troubles. In particular, reduce thinking about those annoying things that are delayed, complicated, and involve many interpersonal relationships, so as not to get angry and induce heart and brain diseases.
  • the sweet soup cooked with Yuzhu has the effect of clearing heart, nourishing yin and reducing heart fire. 5.
  • Polygonatum odoratum is sweet and fatty, soft and moist, and it is a good medicine for nourishing yin and promoting body fluid.
  • Polygonatum Polygonatum is rich in vitamin A substances and mucus. Vitamin A can improve dry, rough skin and make it soft and lubricated. So as to play the role of beauty and skin care. It has been confirmed by modern medical research that it also has the effect of moisturizing the skin, dissipating chronic inflammation of the skin and treating bruises and sprains. 6.
  • Polygonatum Polygonatum is the main ingredient, decocted together with mint, ginger, and honey, which can cure dark eyes and dark eyes. 7. Improve sleep.
  • CN109758393A discloses a skin care product with whitening, moisturizing and anti-aging effects and its preparation method and application.
  • the skin care product contains 5-10 parts by weight of ginseng extract, 1-5 parts by weight of gentian extract, firewood 5-10 parts by weight of the thorn extract, 1-5 parts by weight of the motherwort extract and 3-5 parts by weight of the Polygonatum odoratum extract.
  • the dried rhizome of Polygonatum Polygonatum derived from Liliaceae mainly contains steroidal saponins, polysaccharides, chelidic acid and other active ingredients, and the polysaccharides contained in Polygonatum Polygonatum have good Hygroscopicity, can effectively increase the moisture content of the skin, and can remove free radicals in the body, inhibit the formation of melanin, and play the role of moisturizing and removing freckles.
  • the above-mentioned method for increasing collagen in the prior art cannot fully satisfy people's needs.
  • the above-mentioned method may be effective, but the effect is relatively slow. If can directly add the composition that can promote collagen production in cosmetics, make this composition infiltrate in the dermis layer of skin and directly promote collagen production in dermis layer while applying cosmetics, it will be better than directly supplementing collagen in the prior art. effective method. For this reason, the present inventor has carried out repeated and in-depth research to find products that can promote the production of collagen in the dermis.
  • motherwort extracts and Polygonatum odoratum extracts have the effect of promoting the production of type I collagen. If these extracts are added to cosmetics and coated on the skin, they can promote the production of type I collagen in the dermis. The production of collagen increases skin elasticity and realizes the anti-aging effect, thereby completing the present invention.
  • the present invention specifically relates to the following aspects:
  • the present invention provides, as a first aspect, a type I collagen production promoter characterized by being at least one selected from Leonurus japonicus extract and Polygonatum odoratum extract.
  • the present invention provides, as a second aspect, the type I collagen production promoter according to the first aspect, wherein the active ingredient content of the motherwort extract is 0.0015% by weight or more.
  • the present invention provides the type I collagen production promoter according to the second aspect, wherein the active ingredient content of the motherwort extract is 0.00185 wt % or more.
  • the present invention provides, as a fourth aspect, the type I collagen production accelerator according to the first aspect, wherein the active ingredient content of the Polygonatum odoratum extract is 0.00025 wt% or more.
  • the present invention provides the type I collagen production accelerator according to the fourth aspect, wherein the active ingredient content of the Polygonatum odoratum extract is 0.0004 wt% or more.
  • the present invention provides the type I collagen production accelerator according to the first aspect, wherein the motherwort extract is extracted from a mixture of motherwort powder with water and a lower alcohol having 1 to 6 carbon atoms. Afterwards, the obtained extract is further extracted with a mixed solution of water and a polyhydric alcohol having 3 to 6 carbon atoms.
  • the present invention provides the type I collagen production accelerator according to the sixth aspect, wherein the motherwort extract is obtained by ultrasonically stirring the motherwort powder, ethanol and deionized water, heating, extracting, and concentrating The extract obtained is dried and treated with water and butanediol to decolorize and deodorize.
  • the present invention provides, as an eighth aspect, the type I collagen production accelerator according to the first aspect, wherein the Polygonatum Polygonatum extract is a mixture of Polygonatum Polygonatum powdered water and a lower alcohol having 1 to 6 carbon atoms. After liquid extraction, the obtained extract is further extracted with a mixed solution of water and a polyhydric alcohol having 3 to 6 carbon atoms.
  • the Polygonatum Polygonatum extract is a mixture of Polygonatum Polygonatum powdered water and a lower alcohol having 1 to 6 carbon atoms.
  • the obtained extract is further extracted with a mixed solution of water and a polyhydric alcohol having 3 to 6 carbon atoms.
  • the present invention provides the type I collagen production accelerator described in the eighth aspect, wherein the polygonatum polygonatum extract is obtained by ultrasonically stirring Polygonatum odoratum powder, ethanol and deionized water, and extracting by heating. , concentrated and dried, and then decolorized and deodorized with water and butanediol to obtain the extract.
  • the present invention provides the use of the type I collagen production accelerator described in the first viewpoint for the preparation of cosmetics, characterized in that the type I collagen production accelerator is selected from Leonurus japonicus ) extract, Polygonatum odoratum (Polygonatum odoratum) extract at least one.
  • the motherwort extract in the present invention is the extract obtained by extracting the motherwort powder with a mixture of water and alcohol. More preferably, it is an extract obtained by extracting with a mixed solution of water and a lower alcohol having 1 to 6 carbon atoms, and then further extracting with a mixed solution of water and a lower polyhydric alcohol having 3 to 6 carbon atoms. More preferably, it is an extract obtained by extracting with a mixed solution of water and a lower alcohol having 1 to 3 carbon atoms, and then further extracting with a mixed solution of water and a lower polyhydric alcohol having 3 to 5 carbon atoms. Most preferably, after extraction with a mixed solution of water and ethanol, the extract obtained is further extracted with a mixed solution of water and butanediol.
  • the lower alcohols having 1 to 6 carbon atoms include, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, n-pentanol, 2-pentanol, tert-butanol, Pentanol, n-hexanol, 2-hexanol, 3-hexanol, etc.
  • the lower polyols having 3 to 6 carbon atoms include, for example, propylene glycol, glycerin, butanediol, butylene glycol, butylene glycol, pentylene glycol, pentaerythritol, pentaerythritol, hexylene glycol, hexylene glycol, Triol, hexanediol, etc.
  • the mixing ratio of the mixed solution of water and lower alcohol used in the first step of extraction is not particularly limited, preferably a mixing ratio of 3 to 7:7 to 3, more preferably a mixing ratio of 4 to 6:6 to 4, most preferably 1:1 the mixing ratio.
  • the mixing ratio of the water used in the second step extraction and the mixed solution of lower polyols is not particularly limited, preferably a mixing ratio of 2 to 6:8 to 4, more preferably a mixing ratio of 3 to 6:7 to 4, most preferably 6: 4 mix ratio.
  • the heating temperature is not particularly limited, but the heating temperature is preferably 70-90°C, more preferably 75-80°C.
  • the heating time is not particularly limited, but is preferably 5 to 10 hours, more preferably 5 to 8 hours.
  • the Polygonatum Polygonatum Extract in the present invention is the extract obtained by extracting Polygonatum Polygonatum Powder with a mixed solution of water and alcohol. More preferably, it is an extract obtained by extracting with a mixed solution of water and a lower alcohol having 1 to 6 carbon atoms, and then further extracting with a mixed solution of water and a lower polyhydric alcohol having 3 to 6 carbon atoms. More preferably, it is an extract obtained by extracting with a mixed solution of water and a lower alcohol having 1 to 3 carbon atoms, and then further extracting with a mixed solution of water and a lower polyhydric alcohol having 3 to 5 carbon atoms. Most preferably, after extraction with a mixed solution of water and ethanol, the extract obtained is further extracted with a mixed solution of water and butanediol.
  • the lower alcohols having 1 to 6 carbon atoms include, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, n-pentanol, 2-pentanol, tert-butanol, Pentanol, n-hexanol, 2-hexanol, 3-hexanol, etc.
  • the lower polyols having 3 to 6 carbon atoms include, for example, propylene glycol, glycerin, butanediol, butylene glycol, butylene glycol, pentylene glycol, pentaerythritol, pentaerythritol, hexylene glycol, hexylene glycol, Triol, hexanediol, etc.
  • the mixing ratio of the mixed solution of water and lower alcohol used in the first step of extraction is not particularly limited, preferably a mixing ratio of 3 to 7:7 to 3, more preferably a mixing ratio of 4 to 6:6 to 4, most preferably 1:1 the mixing ratio.
  • the mixing ratio of the water used in the second step extraction and the mixed solution of lower polyols is not particularly limited, preferably a mixing ratio of 5 to 10:5 to 1, more preferably a mixing ratio of 7 to 10:3 to 1, most preferably 10: 1 mixing ratio.
  • the heating temperature is not particularly limited, but the heating temperature is preferably 70-90°C, more preferably 75-80°C.
  • the heating time is not particularly limited, but is preferably 5 to 10 hours, more preferably 5 to 8 hours.
  • the reaction vessel According to the size of the reaction vessel, add 5 to 10 parts by weight of a 1:1 mixture of deionized water and ethanol to 1 part by weight of motherwort powder, and carry out ultrasonic stirring under the condition that the motherwort powder is completely immersed in the solvent, and then mix it at 75-80 °C for heating and extraction for 5-8 hours, and then concentrated and dried to obtain Motherwort extract.
  • the obtained motherwort extract is a brownish yellow to brownish red transparent liquid with a density of 0.95-1.05g/cm 3 , a refractive index of 1.340-1.400 and an active ingredient concentration of 3wt%-5wt%.
  • Col I Human type I collagen (Col I) Collagen test principle: Coat the microwell plate with purified antibody to make a solid phase carrier, add specimen or standard substance, biotinylation to the microwell coated with anti-Col I antibody The anti-ColI antibody and HRP-labeled avidin were thoroughly washed and then developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with Col I in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader, and calculate the sample concentration.
  • OD value absorbance
  • HFF-1 fibroblasts
  • HaCaT human skin keratinocytes
  • DMEM medium GIBCO, 500ml
  • fetal bovine serum GIBCO, 500ml
  • trypsin- EDTA GIBCO, 100ml
  • phosphate buffer solution CORNING, 500ml
  • active oxygen detection kit Biyuntian
  • human type I collagen Col I
  • ELISA kit Wild Huamei
  • RTCA instrument RTCA instrument
  • cell detection plate E-plate16
  • inverted microscope OYMPUS IX73
  • multifunctional microplate reader TECAN, Infinite M200 Pro
  • incubator Thermoscientific, DIRECT HEAT CO 2 Incubator
  • centrifuge eppendorf, Centrifuge 5810 R
  • T25 cell culture flask COSTAR, 430168
  • cell culture plate 96 wells) (Costar, 3599); cell counter (Life, Countess II FL).
  • Test implementation conditions sterile, ultra-clean, cells need to maintain 5% CO2, and culture at 37 degrees.
  • HFF-1 in the logarithmic growth phase was selected, digested with 0.25% trypsin, subcultured in DMEM medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO 2 .
  • the concentration of the motherwort extract when the concentration of the motherwort extract is 0.10%, that is, when the concentration of the motherwort active ingredient is 0.003wt%, the collagen content in the skin cell model is 0.6652 ⁇ g/ml, and the motherwort extract
  • the concentration is 0.05%, that is, when the active ingredient concentration of Motherwort is 0.0015wt%, the collagen content is 0.5455 ⁇ g/ml;
  • the concentration of the motherwort extract is 0.01%, that is, when the active ingredient concentration of Motherwort is 0.0003wt%, the collagen content is 0.2822 ⁇ g /ml, the collagen content of the negative control DMEM was 0.5083 ⁇ g/mL.
  • the motherwort extract concentration is 0.01% motherwort extract (when the motherwort active ingredient concentration is 0.0003wt%), the ability to promote collagen synthesis is not obvious, and the motherwort extract concentration is 0.1% and 0.05% motherwort extract (that is, the motherwort activity Concentration of ingredients 0.003wt% and 0.0015wt%), the production of type I collagen in skin cells has a promoting effect, and the results show that the motherwort extract concentration is 0.05% and 0.1% of the motherwort extract (that is, the motherwort active ingredient concentration is 0.003wt% and 0.0015wt%) has a certain anti-aging ability to human skin cells.
  • Col I Human type I collagen (Col I) Collagen test principle: Coat the microwell plate with purified antibody to make a solid phase carrier, add specimen or standard substance, biotinylation to the microwell coated with anti-Col I antibody The anti-ColI antibody and HRP-labeled avidin were thoroughly washed and then developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with Col I in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader, and calculate the sample concentration.
  • OD value absorbance
  • HFF-1 fibroblasts
  • HaCaT human skin keratinocytes
  • DMEM medium GIBCO, 500ml
  • fetal bovine serum GIBCO, 500ml
  • trypsin- EDTA GIBCO, 100ml
  • phosphate buffer solution CORNING, 500ml
  • active oxygen detection kit Biyuntian
  • human type I collagen Col I
  • ELISA kit Wild Huamei
  • RTCA instrument RTCA instrument
  • cell detection plate E-plate16
  • inverted microscope OYMPUS IX73
  • multifunctional microplate reader TECAN, Infinite M200 Pro
  • incubator Thermoscientific, DIRECT HEAT CO 2 Incubator
  • centrifuge eppendorf, Centrifuge 5810 R
  • T25 cell culture flask COSTAR, 430168
  • cell culture plate 96 wells) (Costar, 3599); cell counter (Life, Countess II FL).
  • Test implementation conditions sterile, ultra-clean, cells need to maintain 5% CO 2 , and culture at 37 degrees.
  • HFF-1 in the logarithmic growth phase was selected, digested with 0.25% trypsin, subcultured in DMEM medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO 2 .
  • the motherwort extract concentration is 0.01% motherwort extract (when the motherwort active ingredient concentration is 0.00037wt%), the ability to promote collagen synthesis is not obvious, and the motherwort extract concentration is 0.1% and 0.05% motherwort extract (that is, the motherwort activity Concentrations of 0.0037wt% and 0.00185wt%) have a promoting effect on skin cell type I collagen production, and the results show that the motherwort extract concentration is 0.05% and 0.1% of the motherwort extract (that is, the motherwort active ingredient concentration is 0.0037wt% and 0.00185wt%) has a certain anti-aging ability to human skin cells. in conclusion:
  • Col I Human type I collagen (Col I) Collagen test principle: Coat the microwell plate with purified antibody to make a solid phase carrier, add specimen or standard substance, biotinylation to the microwell coated with anti-Col I antibody The anti-Col I antibody and HRP-labeled avidin were thoroughly washed and then developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with Col I in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader, and calculate the sample concentration.
  • OD value absorbance
  • HFF-1 fibroblasts
  • HaCaT human skin keratinocytes
  • DMEM medium GIBCO, 500ml
  • fetal bovine serum GIBCO, 500ml
  • trypsin- EDTA GIBCO, 100ml
  • phosphate buffer solution CORNING, 500ml
  • active oxygen detection kit Biyuntian
  • human type I collagen Col I
  • ELISA kit Wild Huamei
  • RTCA instrument RTCA instrument
  • cell detection plate E-plate16
  • inverted microscope OYMPUS IX73
  • multifunctional microplate reader TECAN, Infinite M200 Pro
  • incubator Thermoscientific, DIRECT HEAT CO 2 Incubator
  • centrifuge eppendorf, Centrifuge 5810 R
  • T25 cell culture flask COSTAR, 430168
  • cell culture plate 96 wells) (Costar, 3599); cell counter (Life, Countess II FL).
  • Test implementation conditions sterile, ultra-clean, cells need to maintain 5% CO 2 , and culture at 37 degrees.
  • HFF-1 in the logarithmic growth phase was selected, digested with 0.25% trypsin, subcultured in DMEM medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO 2 .
  • the concentration of the motherwort extract when the concentration of the motherwort extract is 0.10%, that is, when the concentration of the motherwort active ingredient is 0.005wt%, the collagen content in the skin cell model is 0.7888 ⁇ g/ml, and the motherwort extract
  • the concentration is 0.05%, that is, when the active ingredient concentration of Motherwort is 0.0025wt%, the collagen content is 0.6122 ⁇ g/ml; when the concentration of the motherwort extract is 0.01%, that is, when the active ingredient concentration of Motherwort is 0.0005wt%, the collagen content is 0.2745 ⁇ g /ml, the collagen content of the negative control DMEM was 0.5083 ⁇ g/mL.
  • the motherwort extract concentration is 0.01% motherwort extract (when the motherwort active ingredient concentration is 0.0005wt%), the ability to promote collagen synthesis is not obvious, and the motherwort extract concentration is 0.1% and 0.05% motherwort extract (that is, the motherwort active ingredient 0.005wt% and 0.0025wt% of the component concentration), it has a promoting effect on the production of type I collagen in skin cells, and the results show that the motherwort extract concentration is 0.05% and 0.1% of the motherwort extract (that is, the motherwort active ingredient concentration is 0.005wt% and 0.0025wt%) has certain anti-aging ability to human skin cells.
  • reaction vessel According to the size of the reaction vessel, add 5 to 10 parts by weight of a 1:1 mixture of deionized water and ethanol to 1 part by weight of Polygonatum odoratum, and perform ultrasonic waves under the condition that Polygonatum odoratum is completely immersed in the solvent. stirring, heating and extracting at 75-80° C. for 5-8 hours, and then concentrating and drying to obtain Polygonatum odoratum extract.
  • the semi-finished product is tested in the quality control department, wherein the product with Polygonatum polysaccharide content greater than or equal to 0.2wt% is a qualified product, and the finished product of Polygonatum polysaccharide extract is obtained through GMP packaging.
  • the obtained Polygonatum odoratum extract is a light yellow to deep yellow transparent liquid with a density of 1.00-1.05g/cm 3 , a refractive index of 1.310-1.390 and an active component concentration of 0.5wt%-0.9wt%.
  • Col I Human type I collagen (Col I) Collagen test principle: Coat the microwell plate with purified antibody to make a solid phase carrier, add specimen or standard substance, biotinylation to the microwell coated with anti-Col I antibody The anti-Col I antibody and HRP-labeled avidin were thoroughly washed and then developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with Col I in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader, and calculate the sample concentration.
  • OD value absorbance
  • HFF-1 fibroblasts
  • HaCaT human skin keratinocytes
  • DMEM medium GIBCO, 500ml
  • fetal bovine serum GIBCO, 500ml
  • trypsin- EDTA GIBCO, 100ml
  • phosphate buffer solution CORNING, 500ml
  • active oxygen detection kit Biyuntian
  • human type I collagen Col I
  • ELISA kit Wild Huamei
  • RTCA instrument RTCA instrument
  • cell detection plate E-plate16
  • inverted microscope OYMPUS IX73
  • multifunctional microplate reader TECAN, Infinite M200 Pro
  • incubator Thermoscientific, DIRECT HEAT CO 2 Incubator
  • centrifuge eppendorf, Centrifuge 5810 R
  • T25 cell culture flask COSTAR, 430168
  • cell culture plate 96 wells) (Costar, 3599); cell counter (Life, Countess II FL).
  • Test implementation conditions sterile, ultra-clean, cells need to maintain 5% CO 2 , and culture at 37 degrees.
  • HFF-1 in the logarithmic growth phase was selected, digested with 0.25% trypsin, subcultured in DMEM medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO 2 .
  • the collagen content was 0.6424 ⁇ g/ml, when the concentration of Polygonatum odoratum extract was 0.05%, that is, when the active ingredient concentration of Polygonatum odoratum was 0.00025wt%, the collagen content was 0.6055 ⁇ g/ml, and when the concentration of Polygonatum odoratum extract was 0.01% That is, when the active ingredient concentration of Polygonatum odoratum is 0.00005 wt%, the collagen content is 0.2987 ⁇ g/ml, and the collagen content of the negative control DMEM is 0.5083 ⁇ g/mL.
  • Polygonatum odoratum extract concentration is 0.01%, that is, the Polygonatum odoratum active ingredient concentration is 0.00005wt%
  • Polygonatum polygonatum extract has no obvious ability to promote collagen
  • Polygonatum odoratum extract concentration is 0.05%, that is, Polygonatum odoratum active ingredient concentration is 0.00025wt%
  • Both the bamboo extract and the Polygonatum odoratum extract with a concentration of 0.1%, that is, the Polygonatum odoratum active ingredient concentration of 0.0005wt% have a very good effect of promoting the production of type I collagen, indicating that the concentration of Polygonatum odoratum extract is 0.05%.
  • Polygonatum odoratum extract When the active ingredient concentration of Polygonatum odoratum extract reaches above 0.00025wt%, it can promote the production of type I collagen in the human skin fibroblast model, and play an excellent anti-aging effect.
  • Col I Human type I collagen (Col I) Collagen test principle: Coat the microwell plate with purified antibody to make a solid phase carrier, add specimen or standard substance, biotinylation to the microwell coated with anti-Col I antibody The anti-Col I antibody and HRP-labeled avidin were thoroughly washed and then developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with Col I in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader, and calculate the sample concentration.
  • OD value absorbance
  • HFF-1 fibroblasts
  • HaCaT human skin keratinocytes
  • DMEM medium GIBCO, 500ml
  • fetal bovine serum GIBCO, 500ml
  • trypsin- EDTA GIBCO, 100ml
  • phosphate buffer solution CORNING, 500ml
  • active oxygen detection kit Biyuntian
  • human type I collagen Col I
  • ELISA kit Wild Huamei
  • RTCA instrument RTCA instrument
  • cell detection plate E-plate16
  • inverted microscope OYMPUS IX73
  • multifunctional microplate reader TECAN, Infinite M200 Pro
  • incubator Thermoscientific, DIRECT HEAT CO 2 Incubator
  • centrifuge eppendorf, Centrifuge 5810 R
  • T25 cell culture flask COSTAR, 430168
  • cell culture plate 96 wells) (Costar, 3599); cell counter (Life, Countess II FL).
  • Test implementation conditions sterile, ultra-clean, cells need to maintain 5% CO 2 , and culture at 37 degrees.
  • HFF-1 in the logarithmic growth phase was selected, digested with 0.25% trypsin, subcultured in DMEM medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO 2 .
  • the collagen content was 0.7015 ⁇ g/ml, and when the concentration of Polygonatum odoratum extract was 0.05%, that is, when the active ingredient concentration of Polygonatum odoratum was 0.0004wt%, the collagen content was 0.8638 ⁇ g/ml, and when the concentration of Polygonatum odoratum extract was 0.01% That is, when the active ingredient concentration of Polygonatum odoratum is 0.00008wt%, the collagen content is 0.3275 ⁇ g/ml, and the collagen content of the negative control DMEM is 0.5083 ⁇ g/mL.
  • Polygonatum odoratum extract concentration is 0.01%, that is, Polygonatum odoratum active ingredient concentration is 0.00008wt% Polygonatum odoratum extract collagen-promoting ability is not obvious, Polygonatum odoratum extract concentration is 0.05%, that is Polygonatum odoratum active ingredient concentration is 0.0004wt% Both the bamboo extract and the Polygonatum odoratum extract with a concentration of 0.1%, that is, the Polygonatum odoratum active ingredient concentration of 0.0008wt%, have a very good effect of promoting the production of type I collagen, indicating that the concentration of Polygonatum odoratum extract is 0.05%.
  • Polygonatum odoratum extract When the active ingredient concentration of Polygonatum odoratum extract reaches 0.0004wt% or more, it can promote the production of type I collagen in the human skin fibroblast model, and play an excellent anti-aging effect.
  • Col I Human type I collagen (Col I) Collagen test principle: Coat the microwell plate with purified antibody to make a solid phase carrier, add specimen or standard substance, biotinylation to the microwell coated with anti-Col I antibody The anti-Col I antibody and HRP-labeled avidin were thoroughly washed and then developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with Col I in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader, and calculate the sample concentration.
  • OD value absorbance
  • HFF-1 fibroblasts
  • HaCaT human skin keratinocytes
  • DMEM medium GIBCO, 500ml
  • fetal bovine serum GIBCO, 500ml
  • trypsin- EDTA GIBCO, 100ml
  • phosphate buffer solution CORNING, 500ml
  • active oxygen detection kit Biyuntian
  • human type I collagen Col I
  • ELISA kit Wild Huamei
  • RTCA instrument RTCA instrument
  • cell detection plate E-plate16
  • inverted microscope OYMPUS IX73
  • multifunctional microplate reader TECAN, Infinite M200 Pro
  • incubator Thermoscientific, DIRECT HEAT CO 2 Incubator
  • centrifuge eppendorf, Centrifuge 5810 R
  • T25 cell culture flask COSTAR, 430168
  • cell culture plate 96 wells) (Costar, 3599); cell counter (Life, Countess II FL).
  • Test implementation conditions sterile, ultra-clean, cells need to maintain 5% CO 2 , and culture at 37 degrees.
  • HFF-1 in the logarithmic growth phase was selected, digested with 0.25% trypsin, subcultured in DMEM medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO 2 .
  • the collagen content was 0.7308 ⁇ g/ml, when the concentration of Polygonatum odoratum extract was 0.05%, that is, when the active ingredient concentration of Polygonatum odoratum was 0.00045wt%, the collagen content was 0.6812 ⁇ g/ml, and when the concentration of Polygonatum odoratum extract was 0.01% That is, when the active ingredient concentration of Polygonatum odoratum is 0.00009 wt%, the collagen content is 0.3321 ⁇ g/ml, and the collagen content of the negative control DMEM is 0.5083 ⁇ g/mL.
  • Polygonatum odoratum extract concentration is 0.01%, that is, the Polygonatum odoratum active ingredient concentration is 0.00009wt%
  • Polygonatum polygonatum extract has no obvious ability to promote collagen
  • Polygonatum odoratum extract concentration is 0.05%, that is, Polygonatum odoratum active ingredient concentration is 0.00045wt%
  • Both the bamboo extract and the Polygonatum odoratum extract with a concentration of 0.1%, that is, the Polygonatum odoratum active ingredient concentration of 0.0009wt% have a very good effect of promoting the production of type I collagen, indicating that the concentration of Polygonatum odoratum extract is 0.05%.
  • Polygonatum odoratum extract When the active ingredient concentration of Polygonatum odoratum extract reaches 0.00045wt% or more, it can promote the production of type I collagen in the human skin fibroblast model, and play an excellent anti-aging effect.

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Abstract

本发明提供一种I型胶原蛋白产生促进剂，其特征在于，是选自益母草(Leonurus japonicus)提取物、玉竹(Polygonatum odoratum)提取物中的至少一种。

Description

I型胶原蛋白产生促进剂 技术领域
本发明涉及新的I型胶原蛋白产生促进剂，这些新的I型胶原蛋白产生促进剂在化妆品中的应用，以及含有这些新的I型胶原蛋白产生促进剂的化妆品。
背景技术
美容养颜抗衰老是人们一直追求的目标，本公司也一直在致力于寻找具有美容养颜抗衰老功效的产品。真皮层中存在的胶原蛋白是影响皮肤衰老的主要因素。真皮层中胶原蛋白含量如果减少，则皮肤就会出现老化迹象，失去弹性，日渐松弛，形成皱纹，如果真皮层中胶原蛋白含量丰富，则皮肤看起来充满弹性，年轻有光泽，因此如果能找到促进真皮层中胶原蛋白含量增加的产品，则可以实现美容养颜抗衰老的梦想。
现有技术中补充胶原蛋白的方法包括，1.多吃富含胶原的食物：日常食物的动物皮肤(猪皮、鱼皮、之类的)、筋腱(猪蹄筋、牛蹄筋、猪蹄等)、软骨(骨肉相连、脆骨)中含有丰富的胶原蛋白。但胶原蛋白经胃肠消化后，被蛋白酶分解成小分子氨基酸和多肽，胶原形态和结构已不复存在，口服胶原蛋白对改善肤质几乎没有效果。2.涂抹胶原蛋白：市面上有很多涂抹类胶原蛋白产品，如面膜等，但由于胶原蛋白分子量大，很难穿透紧密的肌肤屏障被皮肤吸收，涂抹胶原蛋白，并不能使皮肤胶原蛋白增多。3.注射水光针：通过真空负压技术，把以玻尿酸为基底的溶液直接注射进真皮层，让肌肤100％吸收养分。达到抚平皱纹、淡化黑色素、美白嫩肤的效果。然而，通过破皮注射方式效果短暂，存在的风险也是不可小觑，需要非常谨慎。4.服用胶原蛋白保健品，有口服液和粉剂两种；有的叫胶原蛋白，有的叫“胶原蛋白肽”，还有的叫“水解胶原蛋白”。
本发明者认为，上述现有技术的增加胶原蛋白的方法并不能充分满足人们的需要，上述方法可能有效，但是见效比较缓慢。如果能在化妆品中 直接添加能促进胶原蛋白产生的成分，在涂抹化妆品的同时使得该成分渗入皮肤真皮层中直接在真皮层内促进胶原蛋白产生，会是比现有技术中直接补充胶原蛋白更有效的方法。为此，本发明人进行了反复深入的研究，寻找能够促进真皮层中胶原蛋白产生的产品。
益母草，中药名。为唇形科植物益母草Leonurus japonicus Houtt.的新鲜或干燥地上部分。鲜品春季幼苗期至初夏花前期采割；干品夏季茎叶茂盛、花未开或初开时采割，晒干，或切段晒干。广泛分布于全国各地。益母草的功效与作用包括：1.对心血管的作用，益母草对离体豚鼠心脏，用异丙肾上腺素造成心肌缺血模型,能显著增加冠脉流量及相当显著地减慢心率，静注益母草制剂使麻醉犬明显增加冠脉流量，降低冠脉阻力，减慢心率及减少输出量和左心室作功的作用。2.对子宫的好处，益母草浸膏，及煎剂对子宫有强而持久的兴奋作用，不但能增强其收缩，同时能提高其紧张度和收缩率。对外伤内有瘀血者也可用，如《外台秘要》记载的益母草膏。3.淡化色斑，若是坚持使用加了益母草的美白祛斑膏按摩洗脸，加上一星期食用2至3次益母草汤的话，内调外治，多数色斑都能有效地消退，不少严重的色斑也能淡化。4.缓解痛经，治气滞血瘀引起的痛经，常与延胡索、当归、白芍、香附、川牛膝等补血养血，行气止痛药物组合成方，益母草剂量要大一些，常用量30克左右，大多有明显效果。5.产后病，如产后出血或恶露不绝，腹部胀痛，出血量少，或夹杂血块，由子宫收缩无力引起者，常配合当归、酒白芍、艾叶、川芎、焦山楂，偏寒者再加炮姜、台乌药等，效果较为理想。6.治疗肾病，益母草与其它药物配伍，可以达到治疗肾结石、急性肾炎水肿以及血尿等肾病的目的。7.抑制癌症发生，益母草含有多种微量元素。硒具有增强免疫细胞活力、缓和动脉粥样硬化之发生以及提高肌体防御疾病功能体系之作用；锰能抗氧化、防衰老、抗疲劳及抑制癌细胞的增生。8.明目益精，益母草有治疗头晕，名目的作用，用于体虚水湿上泛之头晕、视物不清。
CN108670943A中公开了一种保湿、美白、淡斑植物提取物组合物，在水相部分中包含多种植物提取物，其中含有益母草提取物。
CN12516046A中公开了一种益母草提取物祛痘膏及其制备方法。
CN109758393A中公开了一种具有美白、保湿、抗衰老作用的护肤品及其制备方法和应用，所述护肤品含有人参提取物5-10重量份、龙胆草提取物1-5重量份、火棘提取物5-10重量份、益母草提取物1-5重量份以及玉竹提取物3-5重量份。其中，说明书0039段中记载了益母草的作用是，活血调经、利尿消肿、清热解毒。
玉竹在《本草纲目》中被称为上品，它有极高的营养价值和药理作用。玉竹的功效与作用包括：1.养阴润燥，生津止渴，保健抗衰。玉竹里的多糖、维他命A与烟酸，能够增强人体抗病能力，推迟衰老。2.镇静神经、强心，现代医学认为它含强心苷、生物碱和维生素A类物质及粘液多糖———玉竹粘多糖、玉竹果聚糖等,有改善心肌缺氧和肾上腺皮质激素样作用。玉竹含有的甾甙，对心肌的作用与铃兰制剂类似。玉竹配糖体对离体蛙心有强心作用，玉竹煎剂的作用与玉竹配糖体类似。同时玉竹对心悸、心绞痛，风湿性心脏病、冠状动脉粥样硬化性心脏病、肺原性心脏病引起的心力衰竭有一定疗效。3.降血脂、血糖，现代药理研究表明，具有调脂作用的中药有40多种，按中药药性分为补益降脂药、活血降脂药、化痰利湿降脂药等类别。玉竹甲醇提取物对肾上腺素所致高血糖小鼠的血糖值具有明显降低作用。并显示有改善耐糖功能的倾向。其作用机理之一是抑制了肝脏糖酵解系统。亦有报道，用玉竹灌胃大鼠对葡萄糖和四氧嘧啶引起的大鼠血糖升高有抑制作用。其中玉竹药有降低低密度脂蛋白作用，其还有降血糖作用，从而有效预防糖尿病的发生。4.治疗上火，心火上升可引起口腔疾病。中医提醒需要控制情绪，减少紧张，少生心事烦事。尤其是减少思虑那些迟延不决、处理繁杂、涉及众多人际关系的烦心事，以免心火气盛，诱发心脑疾病。用玉竹煮的甜汤，具有清心养阴，降心火的作用。5.美容护肤，玉竹味甘多脂，质柔而润，是一味养阴生津的良药。玉竹富含维生素A类物质和粘液质，维生素A有改善干裂、粗糙的皮肤状况，使之柔软润滑。从而起到美容护肤的作用。经现代医学研究证实，还有润泽皮肤，消散皮肤慢性炎症和治疗跌伤扭伤的功效。6.预防老眼昏花，玉竹 为主，配薄荷、生姜、蜜共煎服，可以治目视黑花昏暗。7.改善睡眠，早在明代，李时珍就把这两种中药视为上品，玉竹与沙参同煮，则有养阴润燥、生津止渴之功效，两味合用滋补养阴作用极大。虽然玉竹沙参属中药，但是煲出来没什么药味，却能安神入眠，对睡眠特别有帮助。8.增强身体免疫力，玉竹中有抗氧化作用的成分，可调节人体免疫力，抑制肿瘤的生长。常服玉竹可提高身体素质，远离各类疾病。
CN109758393A中公开了一种具有美白、保湿、抗衰老作用的护肤品及其制备方法和应用，所述护肤品含有人参提取物5-10重量份、龙胆草提取物1-5重量份、火棘提取物5-10重量份、益母草提取物1-5重量份以及玉竹提取物3-5重量份。其中，说明书0039段中记载了来源于百合科的植物玉竹的干燥根茎，主要含甾体皂苷类，多糖类，白屈菜酸等活性成分，其中所含玉竹黏多糖具有很好的吸湿性，能够有效提高皮肤的水分含量，并能清除体内自由基，抑制黑色素的形成，发挥润肤祛斑的作用。
如上所述，现有技术中关于益母草、玉竹的功效和作用有很多记载，但是从未公开或暗示益母草提取物、玉竹提取物具有I型胶原蛋白产生促进作用。本发明者经过反复深入的研究首次发现，益母草提取物、玉竹提取物、具有I型胶原蛋白产生促进效果，如果将这些提取物加入化妆品，涂布在皮肤上可以促进皮肤真皮层中I型胶原蛋白的产生，增加皮肤弹性，实现抗衰老的功效，从而完成了本发明。
发明内容
发明要解决的技术课题
上述现有技术的增加胶原蛋白的方法并不能充分满足人们的需要，上述方法可能有效，但是见效比较缓慢。如果能在化妆品中直接添加能促进胶原蛋白产生的成分，在涂抹化妆品的同时使得该成分渗入皮肤真皮层中直接在真皮层内促进胶原蛋白产生，会是比现有技术中直接补充胶原蛋白更有效的方法。为此，本发明人进行了反复深入的研究，寻找能够促进真皮层中胶原蛋白产生的产品。
解决课题的技术手段
本发明者经过反复深入的研究首次发现，益母草提取物、玉竹提取物、具有I型胶原蛋白产生促进效果，如果将这些提取物加入化妆品，涂布在皮肤上可以促进皮肤真皮层中I型胶原蛋白的产生，增加皮肤弹性，实现抗衰老的功效，从而完成了本发明。
本发明具体涉及以下方面：
本发明，作为第1观点，提供一种I型胶原蛋白产生促进剂，其特征在于，是选自益母草(Leonurus japonicus)提取物、玉竹(Polygonatum odoratum)提取物中的至少一种。
本发明，作为第2观点，提供第1观点所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物的活性成分含量在0.0015wt％以上。
本发明，作为第3观点，提供第2观点所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物的活性成分含量在0.00185wt％以上。
本发明，作为第4观点，提供第1观点所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物的活性成分含量在0.00025wt％以上。
本发明，作为第5观点，提供第4观点所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物的活性成分含量在0.0004wt％以上。
本发明，作为第6观点，提供第1观点所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物是益母草粉末用水和碳原子数1～6的低级醇的混合液提取后，用水和碳原子数3～6的多元醇的混合液进一步提取得到的提取物。
本发明，作为第7观点，提供第6观点所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物是将益母草粉末与乙醇和去离子水一起超声搅拌，加热提取，浓缩干燥，再用水和丁二醇脱色脱味处理后得到的提取物。
本发明，作为第8观点，提供第1观点所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物是玉竹粉末用水和碳原子数1～6的低级醇的混合液提取后，用水和碳原子数3～6的多元醇的混合液进一步提取得 到的提取物。
本发明，作为第9观点，提供第8观点所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物是将玉竹粉末与乙醇和去离子水一起超声搅拌，加热提取，浓缩干燥，再用水和丁二醇脱色脱味处理后得到的提取物。
本发明，作为第10观点，提供第1观点所述的I型胶原蛋白产生促进剂用于制备化妆品的用途，其特征在于，所述的I型胶原蛋白产生促进剂是选自益母草(Leonurus japonicus)提取物、玉竹(Polygonatum odoratum)提取物中的至少一种。
具体实施方式
本发明中的益母草提取物，是益母草粉末用水和醇的混合液提取得到的提取物。更优选是用水和碳原子数1～6的低级醇的混合液提取后，用水和碳原子数3～6的低级多元醇的混合液进一步提取得到的提取物。进一步优选是用水和碳原子数1～3的低级醇的混合液提取后，用水和碳原子数3～5的低级多元醇的混合液进一步提取得到的提取物。最优选是用水和乙醇的混合液提取后，用水和丁二醇的混合液进一步提取得到的提取物。
所述碳原子数1～6的低级醇，可列举例如，甲醇、乙醇、正丙醇、异丙醇、正丁醇、仲丁醇、叔丁醇、正戊醇、2-戊醇、叔戊醇、正己醇、2-己醇、3-己醇等。
所述碳原子数3～6的低级多元醇，可列举例如，丙二醇、丙三醇、丁二醇、丁三醇、丁四醇、戊二醇、戊三醇、季戊四醇、己二醇、己三醇、己四醇等。
第一步提取所用的水和低级醇的混合液的混合比例没有特别限制，优选3～7:7～3的混合比，进一步优选4～6:6～4的混合比，最优选1:1的混合比。
第二步提取所用的水和低级多元醇的混合液的混合比例没有特别限制，优选2～6:8～4的混合比，进一步优选3～6:7～4的混合比，最优选6:4 的混合比。
用水和低级醇的混合液进行提取时，要进行加热提取，加热温度没有特别的限制，优选加热温度为70～90℃，更优选75～80℃。加热时间也没有特别限制，优选为5～10小时，更优选为5～8小时。
本发明中的玉竹提取物，是玉竹粉末用水和醇的混合液提取得到的提取物。更优选是用水和碳原子数1～6的低级醇的混合液提取后，用水和碳原子数3～6的低级多元醇的混合液进一步提取得到的提取物。进一步优选是用水和碳原子数1～3的低级醇的混合液提取后，用水和碳原子数3～5的低级多元醇的混合液进一步提取得到的提取物。最优选是用水和乙醇的混合液提取后，用水和丁二醇的混合液进一步提取得到的提取物。
所述碳原子数1～6的低级醇，可列举例如，甲醇、乙醇、正丙醇、异丙醇、正丁醇、仲丁醇、叔丁醇、正戊醇、2-戊醇、叔戊醇、正己醇、2-己醇、3-己醇等。
所述碳原子数3～6的低级多元醇，可列举例如，丙二醇、丙三醇、丁二醇、丁三醇、丁四醇、戊二醇、戊三醇、季戊四醇、己二醇、己三醇、己四醇等。
第一步提取所用的水和低级醇的混合液的混合比例没有特别限制，优选3～7:7～3的混合比，进一步优选4～6:6～4的混合比，最优选1:1的混合比。
第二步提取所用的水和低级多元醇的混合液的混合比例没有特别限制，优选5～10:5～1的混合比，进一步优选7～10:3～1的混合比，最优选10:1的混合比。
用水和低级醇的混合液进行提取时，要进行加热提取，加热温度没有特别的限制，优选加热温度为70～90℃，更优选75～80℃。加热时间也没有特别限制，优选为5～10小时，更优选为5～8小时。
实施例
下面，通过实施例对本发明进行更具体的说明，但是本发明并不限于这些实施例。
制备例1：益母草(Leonurus japonicus)提取物的制备
根据反应容器大小，在1重量份益母草粉末中加入去离子水和乙醇的1:1混合液5～10重量份，在益母草粉末完全浸在溶剂中的条件下进行超声波搅拌，然后在75-80℃进行加热提取5～8小时，之后浓缩干燥，获得益母草提取物。
在1重量份上述获得的益母草提取物中加入水和丁二醇的6:4混合液19重量份，溶解，进行脱色脱味处理，处理后在6-8℃进行低温静置48小时，然后过滤除去不溶物，加入防腐剂1,2-己二醇和对羟基苯乙酮，使它们的浓度分别达到0.4wt％～0.6wt％，由此获得益母草提取液半成品，然后在质控部检测，检测其中水苏碱含量为0.1％以上的产品为合格产品，GMP包装获得益母草提取液成品。所获得的益母草提取液成品，为棕黄色至棕红色透明液体，密度为0.95～1.05g/cm 3，折光率为1.340～1.400，活性成分浓度为3wt％～5wt％。
实施例1.益母草提取物的抗老化功效测试实验
(1)测试方法原理的叙述：
人Ⅰ型胶原(Col Ⅰ)型胶原测试原理：用纯化的抗体包被微孔板，制成固相载体，往包被抗Col Ⅰ抗体的微孔中依次加入标本或标准品、生物素化的抗ColⅠ抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样本中的Col Ⅰ呈正相关。用酶标仪在450nm波长下测定吸光度(OD值)，计算样本浓度。
(2)试验药品、材料、仪器及准备：成纤维细胞(HFF-1)；人皮肤角质细胞(HaCaT)；DMEM培养基(GIBCO，500ml)；胎牛血清(GIBCO，500ml)；胰蛋白酶-EDTA(GIBCO，100ml)；磷酸盐缓冲溶液(CORNING，500ml)，活性氧检测试剂盒(碧云天)；人Ⅰ型胶原(Col Ⅰ)酶联免疫试剂盒(武汉华美)、RTCA仪器(艾森)；细胞检测板(E-plate16)；倒置显 微镜(OLYMPUS IX73)；多功能酶标仪(TECAN，Infinite M200 Pro)；培养箱(Thermoscientific，DIRECT HEAT CO 2Incubator)；离心机(eppendorf，Centrifuge 5810 R)；T25细胞培养瓶(COSTAR，430168)；细胞培养板(96孔)(Costar，3599)；细胞计数仪(Life，Countess II FL)。
(3)试验实施条件：无菌、超净、细胞需要保持5％CO2、37度下培养。
(4)试验实施
1.将按照上述制备例获得的益母草提取液(Leonurus Japonicus extract使用活性成分浓度为3wt％的提取液)室温储存备用。临用时将样品取出摇匀，用无血清培养基梯度稀释。
2.细胞培养。选择对数生长期的HFF-1，经0.25％胰蛋白酶消化，用含有10％胎牛血清的DMEM培养基传代，置于培养箱37℃，5％CO 2环境中进行培养。
3.选择生长密度90％左右的HFF-1进行实验，铺至E-plate16板中培养，24h后按照规定浓度，使用DMEM培养基对益母草提取液进行稀释使得益母草提取液浓度分别达到0.10％，0.05％，0.01％(即益母草提取物活性成分浓度分别达到0.003wt％，0.0015wt％，0.0003wt％)并加至E-plate16板中继续培养24h后观察，并选出最佳浓度进行后续实验。
(5)试验结果与讨论：
促胶原蛋白Ⅰ生成
表1
Figure PCTCN2022113589-appb-000001
从上述表格中的数据可知，在预设浓度范围内，益母草提取液浓度为0.10％时即益母草活性成分浓度为0.003wt％时，皮肤细胞模型中胶原蛋白含量为0.6652μg/ml，益母草提取液浓度为0.05％时即益母草活性成分浓度 为0.0015wt％时，胶原蛋白含量为0.5455μg/ml，益母草提取液浓度为0.01％时即益母草活性成分浓度为0.0003wt％时，胶原蛋白含量为0.2822μg/ml，阴性对照DMEM的胶原蛋白含量为0.5083μg/mL。说明益母草提取液浓度为0.01％的益母草提取物(即益母草活性成分浓度0.0003wt％时)，促胶原蛋白合成能力不明显，益母草提取液浓度为0.1％和0.05％的益母草提取物(即益母草活性成分浓度0.003wt％和0.0015wt％时)，对皮肤细胞Ⅰ型胶原蛋白生成具有促进效果，结果表明益母草提取液浓度为0.05％和0.1％的益母草提取物(即益母草活性成分浓度0.003wt％和0.0015wt％时)对人皮肤细胞具有一定的抗老化能力。
结论：
益母草提取物活性成分浓度达到0.0015wt％以上时，可促进人皮肤成纤维细胞模型中Ⅰ型胶原蛋白生成，起到优秀的抗衰老功效。
实施例2.益母草提取物的抗老化功效测试实验
(1)测试方法原理的叙述：
人Ⅰ型胶原(Col Ⅰ)型胶原测试原理：用纯化的抗体包被微孔板，制成固相载体，往包被抗Col Ⅰ抗体的微孔中依次加入标本或标准品、生物素化的抗ColⅠ抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样本中的Col Ⅰ呈正相关。用酶标仪在450nm波长下测定吸光度(OD值)，计算样本浓度。
(2)试验药品、材料、仪器及准备：成纤维细胞(HFF-1)；人皮肤角质细胞(HaCaT)；DMEM培养基(GIBCO，500ml)；胎牛血清(GIBCO，500ml)；胰蛋白酶-EDTA(GIBCO，100ml)；磷酸盐缓冲溶液(CORNING，500ml)，活性氧检测试剂盒(碧云天)；人Ⅰ型胶原(Col Ⅰ)酶联免疫试剂盒(武汉华美)、RTCA仪器(艾森)；细胞检测板(E-plate16)；倒置显微镜(OLYMPUS IX73)；多功能酶标仪(TECAN，Infinite M200 Pro)；培养箱(Thermoscientific，DIRECT HEAT CO 2Incubator)；离心机 (eppendorf，Centrifuge 5810 R)；T25细胞培养瓶(COSTAR，430168)；细胞培养板(96孔)(Costar，3599)；细胞计数仪(Life，Countess II FL)。
(3)试验实施条件：无菌、超净、细胞需要保持5％CO 2、37度下培养。
(4)试验实施
1.将按照上述制备例获得的益母草提取液(Leonurus Japonicus extract使用活性成分浓度为3.7wt％的提取液)室温储存备用。临用时将样品取出摇匀，用无血清培养基梯度稀释。
2.细胞培养。选择对数生长期的HFF-1，经0.25％胰蛋白酶消化，用含有10％胎牛血清的DMEM培养基传代，置于培养箱37℃，5％CO 2环境中进行培养。
3.选择生长密度90％左右的HFF-1进行实验，铺至E-plate16板中培养，24h后按照规定浓度，使用DMEM培养基对益母草提取液进行稀释使得益母草提取液浓度分别达到0.10％，0.05％，0.01％(即益母草提取物活性成分浓度分别达到0.0037％，0.00185％，0.00037％)并加至E-plate16板中继续培养24h后观察，并选出最佳浓度进行后续实验。
(5)试验结果与讨论：
促胶原蛋白Ⅰ生成
表2
Figure PCTCN2022113589-appb-000002
从上述表格中的数据可知，在预设浓度范围内，益母草提取液浓度为0.10％时即益母草活性成分浓度为0.0037wt％时，皮肤细胞模型中胶原蛋白含量为0.6669μg/ml，益母草提取液浓度为0.05％时即益母草活性成分浓 度为0.00185wt％时，胶原蛋白含量为0.5757μg/ml，益母草提取液浓度为0.01％时即益母草活性成分浓度为0.00037wt％时，胶原蛋白含量为0.2848μg/ml，阴性对照DMEM的胶原蛋白含量为0.5083μg/mL。说明益母草提取液浓度为0.01％的益母草提取物(即益母草活性成分浓度0.00037wt％时)，促胶原蛋白合成能力不明显，益母草提取液浓度为0.1％和0.05％的益母草提取物(即益母草活性成分浓度0.0037wt％和0.00185wt％时)，对皮肤细胞Ⅰ型胶原蛋白生成具有促进效果，结果表明益母草提取液浓度为0.05％和0.1％的益母草提取物(即益母草活性成分浓度0.0037wt％和0.00185wt％时)对人皮肤细胞具有一定的抗老化能力。结论：
益母草提取物活性成分浓度达到0.00185wt％以上时，可促进人皮肤成纤维细胞模型中Ⅰ型胶原蛋白生成，起到优秀的抗衰老功效。
实施例3.益母草提取物的抗老化功效测试实验
(1)测试方法原理的叙述：
人Ⅰ型胶原(Col Ⅰ)型胶原测试原理：用纯化的抗体包被微孔板，制成固相载体，往包被抗Col Ⅰ抗体的微孔中依次加入标本或标准品、生物素化的抗Col Ⅰ抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样本中的Col Ⅰ呈正相关。用酶标仪在450nm波长下测定吸光度(OD值)，计算样本浓度。
(2)试验药品、材料、仪器及准备：成纤维细胞(HFF-1)；人皮肤角质细胞(HaCaT)；DMEM培养基(GIBCO，500ml)；胎牛血清(GIBCO，500ml)；胰蛋白酶-EDTA(GIBCO，100ml)；磷酸盐缓冲溶液(CORNING，500ml)，活性氧检测试剂盒(碧云天)；人Ⅰ型胶原(Col Ⅰ)酶联免疫试剂盒(武汉华美)、RTCA仪器(艾森)；细胞检测板(E-plate16)；倒置显微镜(OLYMPUS IX73)；多功能酶标仪(TECAN，Infinite M200 Pro)；培养箱(Thermoscientific，DIRECT HEAT CO 2Incubator)；离心机 (eppendorf，Centrifuge 5810 R)；T25细胞培养瓶(COSTAR，430168)；细胞培养板(96孔)(Costar，3599)；细胞计数仪(Life，Countess II FL)。
(3)试验实施条件：无菌、超净、细胞需要保持5％CO 2、37度下培养。
(4)试验实施
1.将按照上述制备例获得的益母草提取液(Leonurus Japonicus extract使用活性成分浓度为5.0wt％的提取液)室温储存备用。临用时将样品取出摇匀，用无血清培养基梯度稀释。
2.细胞培养。选择对数生长期的HFF-1，经0.25％胰蛋白酶消化，用含有10％胎牛血清的DMEM培养基传代，置于培养箱37℃，5％CO 2环境中进行培养。
3.选择生长密度90％左右的HFF-1进行实验，铺至E-plate16板中培养，24h后按照规定浓度，使用DMEM培养基对益母草提取液进行稀释使得益母草提取液浓度分别达到0.10％，0.05％，0.01％(即益母草提取物活性成分浓度分别达到0.005wt％，0.0025wt％，0.0005％)并加至E-plate16板中继续培养24h后观察，并选出最佳浓度进行后续实验。
(5)试验结果与讨论：
促胶原蛋白Ⅰ生成
表3
Figure PCTCN2022113589-appb-000003
从上述表格中的数据可知，在预设浓度范围内，益母草提取液浓度为0.10％时即益母草活性成分浓度为0.005wt％时，皮肤细胞模型中胶原蛋白含量为0.7888μg/ml，益母草提取液浓度为0.05％时即益母草活性成分浓度 为0.0025wt％时，胶原蛋白含量为0.6122μg/ml，益母草提取液浓度为0.01％时即益母草活性成分浓度为0.0005wt％时，胶原蛋白含量为0.2745μg/ml，阴性对照DMEM的胶原蛋白含量为0.5083μg/mL。说明益母草提取液浓度为0.01％的益母草提取物(即益母草活性成分浓度0.0005wt％时)，促胶原蛋白合成能力不明显，益母草提取液浓度为0.1％和0.05％的益母草提取物(即益母草活性成分浓度0.005wt％和0.0025wt％时)，对皮肤细胞Ⅰ型胶原蛋白生成具有促进效果，结果表明益母草提取液浓度为0.05％和0.1％的益母草提取物(即益母草活性成分浓度0.005wt％和0.0025wt％时)对人皮肤细胞具有一定的抗老化能力。
结论：
益母草提取物活性成分浓度达到0.0025wt％以上时，可促进人皮肤成纤维细胞模型中Ⅰ型胶原蛋白生成，起到优秀的抗衰老功效。
制备例2.玉竹提取物的制备
根据反应容器的大小，在1重量份玉竹粉末(Polygonatum odoratum)中加入去离子水和乙醇的1:1混合液5～10重量份，在玉竹粉末完全浸在溶剂中的条件下进行超声波搅拌，在75～80℃加热提取5～8小时，然后浓缩干燥，获得玉竹提取物。
在上述获得的玉竹提取物1重量份中加入水和丁二醇的10:1的混合液99重量份，溶解，进行脱色脱味处理，处理后在6-8℃进行低温静置48小时，然后过滤除去不溶物，加入防腐剂1,2-己二醇和对羟基苯乙酮，使它们的浓度分别达到0.3～0.7wt％，获得玉竹提取液半成品。将半成品在质控部检测，其中玉竹多糖含量大于等于0.2wt％的产品为合格产品，GMP包装获得玉竹提取液成品。所获得的玉竹提取液成品，为淡黄色至深黄色透明液体，密度为1.00～1.05g/cm 3，折光率为1.310～1.390，活性成分浓度为0.5wt％～0.9wt％。
实施例4.玉竹提取物的抗老化功效测试报告
(1)测试方法原理的叙述：
人Ⅰ型胶原(Col Ⅰ)型胶原测试原理：用纯化的抗体包被微孔板，制成固相载体，往包被抗Col Ⅰ抗体的微孔中依次加入标本或标准品、生物素化的抗Col Ⅰ抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样本中的Col Ⅰ呈正相关。用酶标仪在450nm波长下测定吸光度(OD值)，计算样本浓度。
(2)试验药品、材料、仪器及准备：成纤维细胞(HFF-1)；人皮肤角质细胞(HaCaT)；DMEM培养基(GIBCO，500ml)；胎牛血清(GIBCO，500ml)；胰蛋白酶-EDTA(GIBCO，100ml)；磷酸盐缓冲溶液(CORNING，500ml)，活性氧检测试剂盒(碧云天)；人Ⅰ型胶原(Col Ⅰ)酶联免疫试剂盒(武汉华美)、RTCA仪器(艾森)；细胞检测板(E-plate16)；倒置显微镜(OLYMPUS IX73)；多功能酶标仪(TECAN，Infinite M200 Pro)；培养箱(Thermoscientific，DIRECT HEAT CO 2Incubator)；离心机(eppendorf，Centrifuge 5810 R)；T25细胞培养瓶(COSTAR，430168)；细胞培养板(96孔)(Costar，3599)；细胞计数仪(Life，Countess II FL)。
(3)试验实施条件：无菌、超净、细胞需要保持5％CO 2、37度下培养。
(4)试验实施：
1.将上述制备例得到的玉竹提取液(POLYGONATUM ODORATUM EXTRACT，使用活性成分浓度为0.5wt％的提取液)室温储存备用。临用时将样品取出摇匀，用无血清培养基梯度稀释。
2.细胞培养。选择对数生长期的HFF-1，经0.25％胰蛋白酶消化，用含有10％胎牛血清的DMEM培养基传代，置于培养箱37℃，5％CO 2环境中进行培养。
3.选择生长密度90％左右的HFF-1进行实验，铺至E-plate16板中培养，24h后按照规定浓度，使用DMEM培养基对玉竹提取液进行稀释使得玉竹提取液浓度分别达到0.10％，0.05％，0.01％(即玉竹提取物活性成 分浓度分别达到0.0005wt％，0.00025wt％，0.00005wt％)并加至E-plate16板中继续培养24h后观察，并选出最佳浓度进行后续实验。
(5)试验结果与讨论：
促胶原蛋白Ⅰ生成
表4
Figure PCTCN2022113589-appb-000004
从上述表格中的数据可知，在预设浓度范围内，玉竹提取物的促Ⅰ型胶原蛋白生成测试结果中，当玉竹提取液浓度为0.10％时即玉竹活性成分浓度为0.0005wt％时，胶原蛋白含量为0.6424μg/ml，玉竹提取液浓度为0.05％时即玉竹活性成分浓度为0.00025wt％时，胶原蛋白含量为0.6055μg/ml，玉竹提取液浓度为0.01％时即玉竹活性成分浓度为0.00005wt％时，胶原蛋白含量为0.2987μg/ml，阴性对照DMEM的胶原蛋白含量为0.5083μg/mL。玉竹提取液浓度为0.01％即玉竹活性成分浓度为0.00005wt％的玉竹提取物促胶原蛋白能力不明显，玉竹提取液浓度为0.05％即玉竹活性成分浓度为0.00025wt％的玉竹提取物和玉竹提取液浓度为0.1％即玉竹活性成分浓度为0.0005wt％的玉竹提取物均有非常好的促进Ⅰ型胶原蛋白生成效果，说明玉竹提取液浓度为0.05％即玉竹活性成分浓度为0.00025wt％的玉竹提取物和玉竹提取液浓度为0.1％即玉竹活性成分浓度为0.0005wt％的玉竹提取物有抗老化功效。
结论：
玉竹提取物活性成分浓度达到0.00025wt％以上时，可促进人皮肤成纤维细胞模型中Ⅰ型胶原蛋白生成，起到优秀的抗抗衰老功效。
实施例5.玉竹提取物的抗老化功效测试报告
(1)测试方法原理的叙述：
人Ⅰ型胶原(Col Ⅰ)型胶原测试原理：用纯化的抗体包被微孔板，制成固相载体，往包被抗Col Ⅰ抗体的微孔中依次加入标本或标准品、生物素化的抗Col Ⅰ抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样本中的Col Ⅰ呈正相关。用酶标仪在450nm波长下测定吸光度(OD值)，计算样本浓度。
(2)试验药品、材料、仪器及准备：成纤维细胞(HFF-1)；人皮肤角质细胞(HaCaT)；DMEM培养基(GIBCO，500ml)；胎牛血清(GIBCO，500ml)；胰蛋白酶-EDTA(GIBCO，100ml)；磷酸盐缓冲溶液(CORNING，500ml)，活性氧检测试剂盒(碧云天)；人Ⅰ型胶原(Col Ⅰ)酶联免疫试剂盒(武汉华美)、RTCA仪器(艾森)；细胞检测板(E-plate16)；倒置显微镜(OLYMPUS IX73)；多功能酶标仪(TECAN，Infinite M200 Pro)；培养箱(Thermoscientific，DIRECT HEAT CO 2Incubator)；离心机(eppendorf，Centrifuge 5810 R)；T25细胞培养瓶(COSTAR，430168)；细胞培养板(96孔)(Costar，3599)；细胞计数仪(Life，Countess II FL)。
(3)试验实施条件：无菌、超净、细胞需要保持5％CO 2、37度下培养。
(4)试验实施：
1.将上述制备例得到的玉竹提取液(POLYGONATUM ODORATUM EXTRACT，使用活性成分浓度为0.8wt％的提取液)室温储存备用。临用时将样品取出摇匀，用无血清培养基梯度稀释。
2.细胞培养。选择对数生长期的HFF-1，经0.25％胰蛋白酶消化，用含有10％胎牛血清的DMEM培养基传代，置于培养箱37℃，5％CO 2环境中进行培养。
3.选择生长密度90％左右的HFF-1进行实验，铺至E-plate16板中培养，24h后按照规定浓度，使用DMEM培养基对玉竹提取液进行稀释使得玉竹提取液浓度分别达到0.10％，0.05％，0.01％(即玉竹提取物活性成 分浓度分别达到0.0008wt％，0.0004wt％，0.00008wt％)并加至E-plate16板中继续培养24h后观察，并选出最佳浓度进行后续实验。
(5)试验结果与讨论：
促胶原蛋白Ⅰ生成
表5
Figure PCTCN2022113589-appb-000005
从上述表格中的数据可知，在预设浓度范围内，玉竹提取物的促Ⅰ型胶原蛋白生成测试结果中，当玉竹提取液浓度为0.10％时即玉竹活性成分浓度为0.0008wt％时，胶原蛋白含量为0.7015μg/ml，玉竹提取液浓度为0.05％时即玉竹活性成分浓度为0.0004wt％时，胶原蛋白含量为0.8638μg/ml，玉竹提取液浓度为0.01％时即玉竹活性成分浓度为0.00008wt％时，胶原蛋白含量为0.3275μg/ml，阴性对照DMEM的胶原蛋白含量为0.5083μg/mL。玉竹提取液浓度为0.01％即玉竹活性成分浓度为0.00008wt％的玉竹提取物促胶原蛋白能力不明显，玉竹提取液浓度为0.05％即玉竹活性成分浓度为0.0004wt％的玉竹提取物和玉竹提取液浓度为0.1％即玉竹活性成分浓度为0.0008wt％的玉竹提取物均有非常好的促进Ⅰ型胶原蛋白生成效果，说明玉竹提取液浓度为0.05％即玉竹活性成分浓度为0.0004wt％的玉竹提取物和玉竹提取液浓度为0.1％即玉竹活性成分浓度为0.0008wt％的玉竹提取物有抗老化功效。
结论：
玉竹提取物活性成分浓度达到0.0004wt％以上时，可促进人皮肤成纤维细胞模型中Ⅰ型胶原蛋白生成，起到优秀的抗衰老功效。
实施例6.玉竹提取物的抗老化功效测试报告
(1)测试方法原理的叙述：
人Ⅰ型胶原(Col Ⅰ)型胶原测试原理：用纯化的抗体包被微孔板，制成固相载体，往包被抗Col Ⅰ抗体的微孔中依次加入标本或标准品、生物素化的抗Col Ⅰ抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样本中的Col Ⅰ呈正相关。用酶标仪在450nm波长下测定吸光度(OD值)，计算样本浓度。
(2)试验药品、材料、仪器及准备：成纤维细胞(HFF-1)；人皮肤角质细胞(HaCaT)；DMEM培养基(GIBCO，500ml)；胎牛血清(GIBCO，500ml)；胰蛋白酶-EDTA(GIBCO，100ml)；磷酸盐缓冲溶液(CORNING，500ml)，活性氧检测试剂盒(碧云天)；人Ⅰ型胶原(Col Ⅰ)酶联免疫试剂盒(武汉华美)、RTCA仪器(艾森)；细胞检测板(E-plate16)；倒置显微镜(OLYMPUS IX73)；多功能酶标仪(TECAN，Infinite M200 Pro)；培养箱(Thermoscientific，DIRECT HEAT CO 2Incubator)；离心机(eppendorf，Centrifuge 5810 R)；T25细胞培养瓶(COSTAR，430168)；细胞培养板(96孔)(Costar，3599)；细胞计数仪(Life，Countess II FL)。
(3)试验实施条件：无菌、超净、细胞需要保持5％CO 2、37度下培养。
(4)试验实施：
1.将上述制备例得到的玉竹提取液(POLYGONATUM ODORATUM EXTRACT，使用活性成分浓度为0.9wt％的提取液)室温储存备用。临用时将样品取出摇匀，用无血清培养基梯度稀释。
2.细胞培养。选择对数生长期的HFF-1，经0.25％胰蛋白酶消化，用含有10％胎牛血清的DMEM培养基传代，置于培养箱37℃，5％CO 2环境中进行培养。
3.选择生长密度90％左右的HFF-1进行实验，铺至E-plate16板中培养，24h后按照规定浓度，使用DMEM培养基对玉竹提取液进行稀释使得玉竹提取液浓度分别达到0.10％，0.05％，0.01％(即玉竹提取物活性成 分浓度分别达到0.0009wt％，0.00045wt％，0.00009wt％)并加至E-plate16板中继续培养24h后观察，并选出最佳浓度进行后续实验。
(5)试验结果与讨论：
表6
Figure PCTCN2022113589-appb-000006
从上述表格中的数据可知，在预设浓度范围内，玉竹提取物的促Ⅰ型胶原蛋白生成测试结果中，当玉竹提取液浓度为0.10％时即玉竹活性成分浓度为0.0009wt％时，胶原蛋白含量为0.7308μg/ml，玉竹提取液浓度为0.05％时即玉竹活性成分浓度为0.00045wt％时，胶原蛋白含量为0.6812μg/ml，玉竹提取液浓度为0.01％时即玉竹活性成分浓度为0.00009wt％时，胶原蛋白含量为0.3321μg/ml，阴性对照DMEM的胶原蛋白含量为0.5083μg/mL。玉竹提取液浓度为0.01％即玉竹活性成分浓度为0.00009wt％的玉竹提取物促胶原蛋白能力不明显，玉竹提取液浓度为0.05％即玉竹活性成分浓度为0.00045wt％的玉竹提取物和玉竹提取液浓度为0.1％即玉竹活性成分浓度为0.0009wt％的玉竹提取物均有非常好的促进Ⅰ型胶原蛋白生成效果，说明玉竹提取液浓度为0.05％即玉竹活性成分浓度为0.00045wt％的玉竹提取物和玉竹提取液浓度为0.1％即玉竹活性成分浓度为0.0009wt％的玉竹提取物有抗老化功效。
结论：
玉竹提取物活性成分浓度达到0.00045wt％以上时，可促进人皮肤成纤维细胞模型中Ⅰ型胶原蛋白生成，起到优秀的抗抗衰老功效。

Claims (10)

  1. 一种I型胶原蛋白产生促进剂，其特征在于，是选自益母草(Leonurus japonicus)提取物、玉竹(Polygonatum odoratum)提取物中的至少一种。
  2. 如权利要求1所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物活性成分的浓度在0.0015wt％以上。
  3. 如权利要求2所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物活性成分的浓度在0.00185wt％以上。
  4. 如权利要求1所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物活性成分的浓度在0.00025wt％以上。
  5. 如权利要求4所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物活性成分的浓度在0.0004wt％以上。
  6. 如权利要求1所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物是益母草粉末用水和碳原子数1～6的低级醇的混合液提取后，用水和碳原子数3～6的多元醇的混合液进一步提取得到的提取物。
  7. 如权利要求6所述的I型胶原蛋白产生促进剂，其特征在于，所述益母草提取物是将益母草粉末与乙醇和去离子水一起超声搅拌，加热提取，浓缩干燥，再用水和丁二醇脱色脱味处理后得到的提取物。
  8. 如权利要求1所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物是玉竹粉末用水和碳原子数1～6的低级醇的混合液提取后，用水和碳原子数3～6的多元醇的混合液进一步提取得到的提取物。
  9. 如权利要求7所述的I型胶原蛋白产生促进剂，其特征在于，所述玉竹提取物是将玉竹粉末与乙醇和去离子水一起超声搅拌，加热提取，浓缩干燥，再用水和丁二醇脱色脱味处理后得到的提取物。
  10. 权利要求1所述的I型胶原蛋白产生促进剂用于制备化妆品的用途，其特征在于，所述的I型胶原蛋白产生促进剂是选自益母草(Leonurus  japonicus)提取物、玉竹(Polygonatum odoratum)提取物中的至少一种。
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