CN113546161B - Yak I-type collagen product for repairing skin photodamage - Google Patents
Yak I-type collagen product for repairing skin photodamage Download PDFInfo
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- CN113546161B CN113546161B CN202011305340.7A CN202011305340A CN113546161B CN 113546161 B CN113546161 B CN 113546161B CN 202011305340 A CN202011305340 A CN 202011305340A CN 113546161 B CN113546161 B CN 113546161B
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Abstract
The invention belongs to the field of skin photodamage repair, and particularly relates to a yak type-I collagen product for skin photodamage repair. The invention discovers that the yak type I collagen has obvious repairing effect on the skin damaged by ultraviolet light, can improve the inflammatory symptom after the skin is damaged, promotes the growth of epidermal cells of the skin, and induces the proliferation and differentiation of the epidermal cells; meanwhile, the skin surface moisture can be kept, the repair and reconstruction of collagen fibers are promoted, and the collagen fibers can be used for preparing skin photodamage repair products. The invention also provides a skin photodamage repairing composition containing yak type I collagen, which can improve the inflammatory symptoms of skin after photodamage, promote the growth of epidermal cells, improve the content of collagen in damaged skin and have obvious skin photodamage repairing effect.
Description
Technical Field
The invention belongs to the field of skin photodamage repair, and particularly relates to a yak type-I collagen product for skin photodamage repair.
Background
Photodamage to the skin is primarily acute or chronic UV damage to the skin, including phototoxic reactions, photoallergy, photoaging, and carcinogenesis. The solar energy is inevitably irradiated by sunlight in daily life, and the damage of ultraviolet rays in the sunlight to human skin is not negligible. Most of the ultraviolet rays in sunlight are UVA with wavelengths of 320-400nm, about 50% of which penetrate into the epidermis and are deeper than UVB radiation, producing a fast but persistent tan by the photo-oxidation of melanin or direct skin pigmentation (Meirowsky effect); and is also a major cause of photoaging, photosensitivity and damage to the retina, as well as the appearance of cataracts. Sunlight contains a small part of UVB with the ultraviolet ray of 280-320nm, 90% of UVB radiation is absorbed by the epidermis, and solar erythema, immunosuppression and carcinogenesis are caused. The damage of collagen fibers and elastic fibers in the skin due to light damage, and excessive irradiation can cause acute effects on the skin, such as erythema, inflammation, papules, blisters, exfoliation, pigmentation, photosensitivity, systemic immunosuppression, etc.; and other chronic effects such as skin aging, DNA alterations, malignant melanoma, etc. Currently, skin photodamage can be treated topically by topical corticosteroid or soothing lotion without blisters, but the above methods have less effect on repairing photodamaged skin.
Collagen is the most abundant protein in human body, accounting for about 30% of the total protein in human body, and is widely distributed in various parts of human body, and is the most key protein of tissues such as skin, bone, tendon, blood vessel and the like. Collagen has a unique triple helix structure, which is the basis of the physicochemical properties and biological activity of collagen. Collagen exhibits excellent biocompatibility, biodegradability and low immunogenicity, and thus is widely used in the fields of medical devices, cosmetics, and the like.
The yaks are unique precious resources in northwest of China, and 95% of the yaks in the world live in Qinghai-Tibet plateau of China. The yak lives in a relatively closed natural environment of a plateau snow mountain, the risk of being infected by various viruses is small, the risk of heavy metal residue and drug residue is small, and the yak is a highly safe collagen raw material. The invention extracts and prepares high-quality yak type-I collagen which keeps a complete triple helical structure from yaks, and unexpectedly discovers that compared with a collagen product sold on the market, the yak type-I collagen has a remarkable repairing effect on the skin with light injury, can improve the inflammatory symptom of the injured skin, promotes the repairing and reconstruction of collagen fibers, and improves the content of the collagen in the skin with light injury.
Disclosure of Invention
The invention aims to provide application of yak type I collagen in preparation of a skin photodamage repair product.
Preferably, the skin photodamage repair product comprises a drug, a cosmetic, a food, a medical device.
Preferably, the cosmetic comprises stock solution, lotion, essence cream, toner, conditioning water, cream, mask, makeup cream, eye cream, spray, sunscreen cream, foundation, body lotion.
Preferably, the medical device comprises a stock solution, a dressing, a cold pack, a gel, a sponge, a film, a hydro-acupuncture, a milk, a cream, a paste.
Preferably, the photodamage is ultraviolet damage.
Preferably, the preparation method of the yak type I collagen comprises the following steps:
(1) Pretreating yak tendon, treating with surfactant, and/or oxidant, and/or sterilizing, and/or defatting and decalcifying to obtain raw material powder;
(2) Adding 0.01-2.0M sodium hydroxide solution into the raw material powder in the step (1), and keeping the temperature at 4-30 ℃ for 2-24h;
(3) Removing alkali solution, washing with water to neutrality, and extracting by acid dissolution enzymolysis to obtain enzymolysis solution;
(4) And (4) inactivating the enzymolysis liquid in the step (3), salting out, dialyzing, freezing and drying to obtain the yak type I collagen.
Another object of the present invention is to provide a composition for photodamage repair of skin, comprising the following components in percentage by weight: 0.1 to 50 percent of yak I type collagen, 0.1 to 30 percent of butanediol, 0.1 to 20 percent of glycerol, 0.1 to 10 percent of betaine, 0.1 to 5 percent of chamomile extract, 0.1 to 10 percent of sodium hyaluronate, 0.1 to 10 percent of caprylic/capric triglyceride, 0.1 to 0.3 percent of phenoxyethanol, 0.1 to 5 percent of glycerol stearic acid, 0.1 to 5 percent of cetearyl glucoside, 0.1 to 20 percent of polysiloxane and 0.1 to 5 percent of centella extract.
Preferably, the composition comprises the following components in percentage: 15% of yak I type collagen, 10% of butanediol, 8% of glycerol, 7% of betaine, 1% of chamomile flower extract, 1% of sodium hyaluronate, 6% of caprylic/capric triglyceride, 0.2% of phenoxyethanol, 1% of glycerol stearic acid, 3% of cetearyl glucoside, 6% of polydimethylsiloxane and 0.1% of centella extract.
Preferably, the preparation method of the yak type I collagen comprises the following steps:
(1) Pretreating yak tendon, treating with surfactant, and/or oxidant, and/or sterilizing, and/or defatting and decalcifying to obtain raw material powder;
(2) Adding 0.01-2.0M sodium hydroxide solution into the raw material powder in the step (1), and keeping the temperature at 4-30 ℃ for 2-24h;
(3) Removing alkali solution, washing to neutrality, and extracting by acid-soluble enzymolysis method to obtain enzymolysis solution;
(4) And (4) inactivating the enzymolysis liquid in the step (3), salting out, dialyzing, freezing and drying to obtain the yak type I collagen.
Another object of the present invention is to provide the use of one of the above-mentioned compositions in the preparation of a product for repairing photodamaged skin.
Preferably, the skin photodamage repair product comprises a drug, a cosmetic, a food, a medical device.
Preferably, the cosmetic comprises stock solution, lotion, essence cream, toner, conditioning water, cream, mask, makeup cream, eye cream, spray, sunscreen cream, foundation, body lotion.
Preferably, the medical device comprises a stock solution, a dressing, a cold pack, a gel, a sponge, a film, a hydro-acupuncture, a milk, a cream, a paste.
Preferably, the photodamage is ultraviolet damage.
Another object of the present invention is to provide a cosmetic composition for the repair of photodamage to skin, characterized in that it comprises the following components in percentage by weight: 1 to 50 percent of yak I type collagen, 0.1 to 30 percent of butanediol, 0.1 to 30 percent of erythritol, 0.1 to 5 percent of butyrospermum parkii extract, 0.1 to 20 percent of glycerol, 0.1 to 10 percent of nicotinamide, 0.1 to 10 percent of hyaluronic acid, 0.1 to 10 percent of bisabolol and 0.1 to 0.15 percent of methylparaben.
Preferably, the composition comprises the following composition in percentage: yak type-I collagen 10%, butanediol 10%, erythritol 8%, butyrospermum parkii extract 1%, glycerol 5%, nicotinamide 3%, hyaluronic acid 2%, bisabolol 0.5%, and methylparaben 0.14%.
Preferably, the preparation method of the yak type I collagen comprises the following steps:
(1) Pretreating yak tendon, and treating with surfactant, and/or oxidant, and/or sterilizing, and/or defatting and decalcifying to obtain raw material powder;
(2) Adding 0.01-2.0M sodium hydroxide solution into the raw material powder in the step (1), and keeping the temperature at 4-30 ℃ for 2-24h;
(3) Removing alkali solution, washing with water to neutrality, and extracting by acid dissolution enzymolysis to obtain enzymolysis solution;
(4) And (4) inactivating the enzymolysis liquid obtained in the step (3), salting out, dialyzing, freezing and drying to obtain the yak type I collagen.
Another object of the present invention is to provide the use of one of the above-mentioned compositions for the preparation of a product for the repair of photodamaged skin.
Preferably, the skin photodamage repair product comprises a drug, a cosmetic, a food, a medical device.
Preferably, the cosmetic comprises stock solution, lotion, essence cream, toner, conditioning water, cream, mask, makeup cream, eye cream, spray, sunscreen cream, foundation, body lotion.
Preferably, the medical device comprises a stock solution, a dressing, a cold pack, a gel, a sponge, a film, a hydro-acupuncture, a milk, a cream, a paste.
Preferably, the photodamage is ultraviolet damage.
The invention has the beneficial effects that:
(1) the invention discovers that the yak type I collagen has an obvious repairing effect on ultraviolet light damaged skin, can improve the inflammation symptom of the skin after being dried in the sun, promotes the growth of epidermal cells, improves the content of collagen in the damaged skin, and has a skin repairing effect after being dried in the sun which is superior to that of the existing collagen sold on the market.
(2) The invention also provides a yak type I collagen-containing skin photodamage repairing cream compound and a skin photodamage liquid compound, wherein the skin photodamage repairing cream compound and the skin photodamage liquid compound have obvious skin after-sun repairing effect, can improve inflammation symptoms of the after-sun skin, promote growth of epidermal cells and improve the content of collagen in damaged skin; the skin photodamage repairing effect is superior to that of the existing commercially available collagen; therefore, the compound can be prepared into medicines, medical instruments or cosmetics such as cream or emulsion which are easy to apply and use, and can be conveniently stored and used.
Drawings
FIG. 1 is a SDS-PAGE pattern of type I collagen from yaks prepared in example 1 of the present invention;
FIG. 2 is a circular dichroism diagram of type I collagen of yak prepared in example 1 of the present invention;
FIG. 3 shows HE staining results of skin tissues of mice in a normal blank group, a model group and an experimental group 1;
FIG. 4 shows HE staining results of skin tissues of mice in experiment group 2 and experiment group 3;
FIG. 5 shows HE staining results of skin tissues of mice in control group 1, control group 2 and control group 3;
FIG. 6 Masson staining results of skin tissues of mice in blank group, model group and experimental group 1;
FIG. 7 shows Masson staining results of skin tissues of mice in experiment group 2 and experiment group 3;
FIG. 8 Masson staining results of skin tissues of mice of control group 1, control group 2 and control group 3;
FIG. 9 measurement of hydroxyproline content in mouse skin tissue.
Detailed Description
The technical solution of the present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited to the following.
Example 1 preparation of Yak type I collagen
1. Preparation method
Cleaning yak tendon, removing foreign matter, and pulverizing into small pieces; treating the crushed small blocks with 1% of chlorhexidine and 5% of hydrogen peroxide, taking the precipitate, and washing the precipitate to be neutral; degreasing the obtained precipitate with 10% n-butanol, collecting the precipitate, and washing with water to neutrality; decalcifying the precipitate with 0.5M hydrochloric acid, collecting the precipitate, and washing with water to neutrality; soaking the tissue precipitate in 0.5M sodium hydroxide solution, stirring at 10 deg.C for 2 hr, collecting precipitate, and washing with water to neutrality; extracting collagen with 0.5M acetic acid solution containing 1g/L pepsin to obtain crude collagen extractive solution; adjusting the pH value to be neutral, and performing enzyme inactivation; and (4) dialyzing the yak type-I collagen with low endotoxin content by using an 8-14kDa dialysis bag, and after dialysis is finished, freeze-drying to finally obtain the yak type-I collagen with low endotoxin content.
2. Determination of endotoxin content
Using a ToxinSensor TM The LAL endotoxin detection kit adopting the color development method obtains a standard curve of endotoxin content according to the method described in the kit specification, wherein the standard curve is as follows: y =0.5038x +0.3387 (R) 2 = 0.9483); the method for determining the content of endotoxin after each operation step in the extraction process of the yak type I collagen is sensitive and reliable, and the determination result is shown in the following table 1 (the content of endotoxin in known medical equipment is required to be<0.5Eu/mL, and the endotoxin content requirement of the medicine is generally<0.3Eu/mL)。
TABLE 1 detection results of endotoxin content in each step of yak type I collagen preparation
As can be seen from table 1: the yak type-I collagen extracted and prepared by the method can obviously reduce the content of endotoxin in a yak type-I collagen product, so that the content of the endotoxin in the yak type-I collagen product is lower than the detection limit, and the yak type-I collagen is safe and low-toxicity.
3. Identification of collagen structure
Gel electrophoresis experiment: the structure of the low endotoxin collagen prepared in the above examples was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The gel electrophoresis pattern of the yak collagen prepared by the invention is shown in figure 1, two alpha bands, namely alpha 1 and alpha 2, are arranged near the molecular weight of 100kDa, two beta bands, namely beta 11 and beta 12, are arranged near the molecular weight of 200kDa, wherein the beta 11 and the beta 12 are dimer peptide chains formed by the two alpha chains, and no redundant band is arranged at the low molecular weight position.
Circular dichroism chromatography experiment: circular dichroism is a common method for characterizing protein structure, wherein a positive peak at 225nm and a negative peak at 205nm are characteristic peaks of the triple helix structure of collagen. Performing circular dichroism chromatogram identification on the yak collagen prepared by the invention, wherein the result is shown in figure 2a, the yak collagen prepared by the invention has a positive peak at about 225nm and a negative peak at about 205nm, and accords with a characteristic triple helical structure of the collagen; meanwhile, the heat change temperature of the yak collagen is measured, and as can be seen from fig. 2b, the heat change temperature of the yak collagen is 37 ℃, which accords with the characteristics of natural collagen.
Example 2 preparation of liquid composition for skin photodamage repair
The skin photodamage repair liquid compound is prepared from the following components in percentage by mass: yak collagen 10%, butanediol 10%, erythritol 8%, butyrospermum parkii extract 1%, glycerol 5%, nicotinamide 3%, hyaluronic acid 2%, bisabolol 0.5%, methylparaben 0.14%, and the balance of water.
The preparation process comprises the following steps: heating phase A materials (water, glycerol, erythritol, nicotinamide, and hyaluronic acid) to 85 deg.C, stirring to dissolve completely, homogenizing for 3min, and keeping the temperature for 10-15min; pre-dissolving a B-phase substance (butanediol), completely dissolving to be transparent, cooling to 70 ℃, and adding; pre-dissolving C phase substance (Butyrospermum parkii L. Extract, yak collagen), and D phase substance (methylparaben, bisabolol); cooling to 40 deg.C, adding phase D material, cooling to 35 deg.C, adding phase C material, and stirring until completely dissolved. Cooling to 30 ℃, filtering by a clean filter screen, discharging and standing. And obtaining the yak collagen liquid compound.
Example 3 preparation of a skin photodamage repair cream composition
The skin photodamage repair cream compound is prepared from the following components in percentage by mass: yak collagen 15%, butanediol 10%, glycerol 8%, betaine 7%, caprylic/capric triglyceride 6%, polysiloxane 6%, cetearyl glucoside 3%, chamomile flower extract 1%, glycerol stearic acid 1%, sodium hyaluronate 1%, phenoxyethanol 0.2%, centella asiatica extract 0.1%, and the balance water.
Stirring phase A substances (caprylic/capric triglyceride, cyclohexasiloxane, polydimethylsiloxane, glycerol stearic acid, cetostearyl glucoside) and heating to 90 deg.C, stirring phase B substances (water, glycerol, betaine, sodium hyaluronate) and heating to 90 deg.C, stirring, vacuum pumping phase A into phase B, mixing and emulsifying the two phases, homogenizing for 3-5min, stirring, and keeping the temperature for 10-20min; cooling to 45 deg.C, adding C phase material (butanediol, herba Centellae extract, flos Matricariae Chamomillae extract, phenoxyethanol), and homogenizing for 5min; cooling to 35 deg.C, adding yak collagen, mixing and stirring. Cooling to 30 ℃, filtering by a clean filter screen, discharging and standing. Obtaining the yak collagen cream compound.
Example 4 post-basking repair experiment
1. Procedure of experiment
40 healthy adult mice of SPF class, female, weighing 27 + -2 g were selected. The mice were shaved on their backs with a philips hair cutter and depilated with depilatory cream in an area of approximately 2 x 4cm. Mice were randomized into 8 groups: blank group, model group, experimental group 1 (yak type I collagen solution prepared in example 1, collagen concentration of 1.5 mg/ml), experimental group 2 (yak collagen liquid compound prepared in example 2), experimental group 3 (yak collagen cream compound prepared in example 3), control group 1 (commercially available fish collagen solution, collagen concentration of 1.5 mg/ml), control group 2 (commercially available pig collagen solution, collagen concentration of 1.5 mg/ml), control group 3 (commercially available bovine collagen solution, collagen concentration of 1.5 mg/ml). Wherein the blank group was not treated except for depilation; the model set does not undergo any repair process.
Establishing an ultraviolet injury model of hairless mice: irradiating model group and experimental group mice with continuous-wavelength UVA (wavelength: 320-440 nm) and UVB (280-320 nm) ultraviolet lamps, and irradiating with ultraviolet irradiatorThe total amount of UVA and UVB irradiation was determined to be 100mJ/cm 2 Can cause acute skin inflammation of the skin on the back of the mouse.
And (3) processing: 1h after irradiation, the corresponding experimental group and the control group were respectively treated by applying yak type I collagen solution (prepared in example 1), the skin photodamage repair dressing prepared in example 2, the skin photodamage repair cream prepared in example 2, a commercially available fish collagen solution, a commercially available pig collagen solution and a commercially available bovine collagen solution; the dose was 4ml (with a collagen concentration of 1.5 mg/ml) or 4g. On day 4 of treatment mice were sacrificed by dislocation and dorsal skin was harvested and skin tissue was frozen; the collected skin tissue was fixed with 10% neutral formaldehyde for 48h. Then taking the fixed skin tissue, dipping in wax, dehydrating, transparentizing, embedding, and carrying out paraffin section. The slice thickness was 3.5 μm.
HE staining: dewaxing and dehydrating the paraffin sections, then carrying out HE (high intensity intrinsic) staining, carrying out water immersion, staining with hematoxylin for 5-8min, and washing with running water for 1min; differentiating the differentiation liquid for 1-3s, and washing for 1min; returning the blue liquid to blue for 30-60s; soaking in 95% ethanol for alcoholization for 1min; dip-dyeing with alcohol-soluble eosin for 5-8s; soaking in 95% ethanol I and II for 2-3s respectively; soaking in 100% ethanol I and II, and dehydrating for 1min; drying by using an electric hair drier; the xylene is transparent for 5-15min; and sealing the neutral gum into a piece, drying at room temperature, and taking a picture.
Masson staining: dewaxing and dehydrating the paraffin section, performing Masson trichrome staining, staining for 5-10min by Weigert hematoxylin, and washing; differentiating the differentiation liquid for 5-15s, and washing with water; returning blue liquid to blue for 1-5min, and washing with water; dyeing the fuchsin for 5-10min; washing with weak acid for 1min; washing with phosphomolybdic acid solution for 1-2min; washing with weak acid for 1min; dyeing with aniline blue for 2min; quickly dehydrating by 95% ethanol; absolute ethyl alcohol I, II and III are respectively 5-10s; xylene I, II and III are respectively 1-2min; and (5) sealing by using neutral gum.
Hydroxyproline content determination: firstly, weighing 20-30mg of skin tissue without fat, cutting up the skin tissue as much as possible, extracting the skin tissue with 0.5M acetic acid and 10g/L pepsin, and stirring the skin tissue with magnetons for 12 hours; centrifuging to remove precipitate, taking supernatant, adding concentrated hydrochloric acid with the same volume in the supernatant, and hydrolyzing at 110 ℃ for 16h; diluting the hydrolyzed sample by a certain multiple according to a national standard hydroxyproline method, adjusting the pH to be neutral, adding a chloramine T solution, uniformly mixing, and standing at room temperature for 20min; adding color developing agent, mixing, sealing with aluminum foil, and water bath at 60 deg.C for 20min; and cooling with flowing water for at least 3min, standing at room temperature for 30 min, comparing with blank color developing agent, measuring the absorption value of each sample at 560nm, and calculating by standard curve to obtain hydroxyproline content. The same procedure was used to prepare hydroxyproline standard working solution for standard curve drawing and the experiment was repeated 3 times independently.
2. Results of the experiment
2.1 skin Damage and repair situations
2.1.1 mouse skin HE staining results
(1) Blank group
The blank mice were not irradiated with ultraviolet light and treated except for depilating, and as can be seen from fig. 3, on the 2 nd day after depilating, the skin of the blank mice was observed to undulate in a longitudinal section, wherein the epidermis grew downward to form a skin process; the skin of the back of the mouse is thin, the granular layer and the transparent layer are not obvious, and the basal layer and the spinous layer can be seen; the dermis is mainly composed of a papillary layer and a reticular layer, no obvious boundary exists between the papillary layer and the reticular layer, the fibers are compact and ordered, the shape of the cell nucleus is good, inflammatory cells are not generated, and the shape of the accessory is good. On day 4, the subcutaneous layer of the skin was not waved significantly in the cross section of the mice in the blank group, and the appendages extended from the epidermis to the dermis. The dermis layer has continuous fibers which are orderly arranged, the shape of cell nucleus is good, and inflammatory cells do not exist; subcutaneous fat and reticular fiber are in good form. As can be seen, the skin of the mice in the blank group was entirely healthy and normal on days 2 and 4.
(2) Model set
The mice of the model group after ultraviolet irradiation were not treated, and as can be seen from fig. 3, HE staining results of the skin on day 2 after ultraviolet irradiation showed that the skin of the mice had damaged the whole epidermis, edema of the local basal layer, and loss of the local structure; the blood vessels in the dermis layer are obviously dilated and hyperemic, the reticular layer is loose and edema, and inflammatory cells are infiltrated; the appendages are clearly damaged and most of the follicles are damaged (the fibrous sheath shrinks or even disappears). On day 4, the epidermal structure of the skin of the mouse is more complete than that on the next day, but the spinous layer is loosened and locally thickened; the dermal layer fibers become thicker and looser obviously, the matrix among the fibers is increased, the edema is serious, and inflammatory cells are infiltrated; the number of accessories is significantly reduced. It can be seen that, without any treatment, at day four after uv light injury, the mice continued to have increased skin edema, increased inflammatory cell infiltration, and increased injury.
(3) Experimental group 1
Mice of experimental group 1 were treated with 4ml of yak collagen solution (1.5 mg/ml of yak type I collagen prepared in example 1) at 1h after uv irradiation, and thereafter applied once daily at the same dose. As can be seen from fig. 3, when the skin of the mice treated by the yak collagen solution is on day 2, the epidermal layer of the mice is obviously damaged, the structure is incomplete, and the local basal layer is damaged; loose edema of the dermis, infiltration of inflammatory cells, damaged hair follicles (atrophy or even disappearance of the fibrous sheath). The whole epidermis is visible on day 4, and the structure is complete; the dermis has complete structure, and the number and the shape of the attachments are normal; no blood vessel hyperemia and dilatation were seen.
Compared with a model group, the yak type I collagen solution has a good effect of repairing the skin damaged by ultraviolet light, and the damaged skin of the mice is recovered in the fourth day after the damage and is basically close to the healthy form of the skin of the mice in a blank group.
(4) Experimental group 2
Mice of experimental group 2 were treated with 4ml of yak collagen liquid complex (prepared in example 2) 1h after uv irradiation, and thereafter treated once daily at the same dose. As can be seen from fig. 4, on day 2, the epidermal layer of the mouse was significantly damaged, the structure was incomplete, and the local basal layer was damaged; loose edema in the dermis, inflammatory cell infiltration, damaged hair follicles (atrophy or even disappearance of the fibrous sheath). On day 4, the epidermis structure of the mouse skin is complete, the whole layer of the epidermis is visible, and the layering is obvious; edema is not seen in the dermis layer, the fibers are compact and continuous, and no vasodilatation is seen; the hair follicle is repaired, the number of the attachments is normal, and the shape is good.
(5) Experimental group 3
Mice of experimental group 3 were treated with 4g of yak collagen cream complex (prepared in example 3) 1h after uv irradiation, and thereafter treated once daily at the same dose. From fig. 4, it can be seen that the mouse epidermis was damaged, the structure was incomplete and the basal layer was injured at day 2; loose edema of the dermis, inflammatory cell infiltration, vasodilation and congestion. On day 4, the mouse epidermis is complete in structure, the whole layer of the epidermis is visible, and layering is obvious; the local epidermis is thicker, the fibers of the dermis layer are arranged normally, the number and the shape of the attachments are normal, and edema and vasodilatation are not seen.
Therefore, the liquid and cream compound prepared from the yak collagen has a good repairing effect on the skin damaged by ultraviolet light.
(6) Control group 1
Mice of control group 1 were treated with 4ml (1.5 mg/ml) of commercially available fish collagen solution 1h after UV irradiation, and thereafter treated once daily with the same dose. From FIG. 5, it can be seen that the epidermal layer of the mouse was seriously damaged and the basal layer was damaged at day 2; the dermis is loose and edema, inflammatory cell infiltration and blood vessel hyperemia and dilatation are obvious. On day 4, the epidermis structure is complete but local basal layer is edematous, acantho stratum is loosened compared with the next day; the dermis is loose and edematous, the fiber is thickened, and the stroma is increased; the number of attachments is obviously reduced, hair follicles are seriously damaged, and inflammatory cells are infiltrated. The repairing effect is not obvious.
(7) Control group 2
Mice of control group 2 were treated with 4ml (1.5 mg/ml) of a commercially available porcine collagen solution 1h after UV irradiation, and thereafter treated once daily at the same dose. From fig. 5, it can be seen that the mouse epidermal layer is obviously damaged, the structure is incomplete, and the local basal layer is damaged on the 2 nd day; loose edema of the dermis, infiltration of inflammatory cells, damaged hair follicles (atrophy or even disappearance of the fibrous sheath). On day 4, the epidermis is intact but partially defective, damaging the basal lamina, accompanied by acantholysis; the dermis is loose and edematous, the fiber is thickened, and the stroma is increased; the number of attachments is obviously reduced, hair follicles are seriously damaged, and inflammatory cells are infiltrated. The repairing effect is not obvious.
(8) Control group 3
Mice of control group 3 were treated with 4ml (1.5 mg/ml) of a commercially available bovine collagen solution 1h after UV irradiation, and thereafter treated once daily at the same dose. From fig. 5, it can be seen that on day 2, the mouse has obvious epidermal injury, incomplete structure and partial exfoliation (continuous basal layer and local defect); edema of the dermis, thickening and breaking of the fibers, infiltration of inflammatory cells, damage of hair follicles (atrophy and even disappearance of the fibrous sheath), vasodilation and congestion. On day 4, the epidermal structure of the skin of the mouse is complete but discontinuous compared with that on the next day, and the part of the skin is broken; edema of the dermis layer, thickening and breaking of fibers are more obvious, and blood vessels are dilated and congested; the number of accessories is significantly reduced. The repairing effect is not obvious.
Therefore, the skin repair effect of the commercially available fish collagen, the commercially available pig collagen and the commercially available bovine collagen on ultraviolet injury is not obvious.
In conclusion, the yak type I collagen and the composition thereof provided by the invention have an obvious repairing effect on the skin of a mouse with ultraviolet injury, and the effect is superior to that of commercially available fish collagen, commercially available fish collagen and commercially available bovine collagen.
2.1.2 mouse skin histology Masson staining results
(1) Blank group
The blank group of mice was not exposed to uv irradiation and treatment except for hair removal. As can be seen from fig. 6, on day 2, collagen fibers in the dermal papilla layer of the skin of the blank group of mice were fine and interwoven into a web; in addition, the hair-sebaceous gland unit, the eccrine sweat gland and the apocrine sweat gland are all surrounded by the thin reticular collagen, and the blood vessels are also surrounded by the thin layer of the thin collagen fibers; the collagen fibers of the reticular layer are the largest constituent of the dermis, and the collagen fibers therebetween form a coarse fiber bundle, which is slightly wavy in longitudinal section and extends in all directions in a horizontal plane, with fibroblasts interspersed among the collagen fibers. On the 4 th day, the collagen fibers of the dermis layer of the mouse skin are compact and continuous, and the number and the shape of the accessories are good and are surrounded by the collagen fibers; the collagen fibers form relatively uniform and large fiber bundles, which are slightly wavy in longitudinal section and extend in all directions in the horizontal plane, and fibroblasts are scattered among the collagen fibers
As can be seen, the collagen fibers and the appendages in the dermis layer of the skin of the blank group of mice on the 2 nd day and the 4 th day are in good, healthy and normal states.
(2) Model set
Mice in the model group after uv irradiation were not treated. As can be seen from FIG. 6, collagen fibers in the dermis layer of the model group on day 2 of ultraviolet irradiation were disorganized, and were significantly reduced, broken, crushed, thickened, kinked and gathered into clusters; collagen fibers in the dermis are thicker, aggregated into blocks, seriously fractured and blood vessels in the dermis are congested and expanded on the 4 th day.
It can be seen that the damage to collagen fibers in the dermal layer of mouse skin continues to increase at the fourth day after the uv light damage without any treatment.
(3) Experimental group 1
Mice of experimental group 1 were treated with 4ml of yak collagen solution (1.5 mg/ml of yak type i collagen prepared in example 1) at 1h after uv irradiation by applying once daily with the same dose. As can be seen from FIG. 6, masson staining results show that collagen fibers in the dermis of the yak group are disorganized on day 2, the collagen fibers are obviously reduced, broken and curled, hair follicles are broken and broken, and blood vessels are congested and dilated; the collagen fibers of the dermis layer are obviously increased on the 4 th day, the arrangement is compact and orderly, the shape of the attachment is normal, the continuous collagen fibers are in a slight wavy shape and extend to all directions on the horizontal plane, and fibroblasts are scattered among the collagen fibers.
As seen in a comparison model group, the yak type I collagen solution has a good repairing effect on collagen fibers of the dermis layer of the skin damaged by ultraviolet light, and the collagen fibers are restored to be close to the normal shape and density of a blank group.
(4) Experimental group 2
Mice of experimental group 2 were treated with 4ml of yak collagen liquid complex (prepared in example 2) 1h after uv irradiation, and thereafter treated once daily at the same dose. From FIG. 7, it can be seen that collagen fibers in the dermis layer of the mouse skin at the 2 nd day are obviously reduced, broken and twisted to be aggregated into a mass, and inflammatory cells infiltrate; the collagen fibers of the dermis layer are obviously increased and continuous at the 4 th day, the collagen fibers are in a slight wave shape and extend to all directions on the horizontal plane, the fibroblasts are scattered among the collagen fibers, and the accessory is in a normal state.
(5) Experimental group 3
Mice of experimental group 3 were treated with 4g of yak collagen cream complex (prepared in example 2) 1h after uv irradiation, and thereafter treated once daily at the same dose. From fig. 7, it can be seen that collagen fibers in the dermis are obviously reduced, broken and aggregated at day 2, and blood vessels in the dermis are hyperemia and dilated; the collagen fibers of the dermis layer are increased obviously at day 4, and are arranged in a wave-like order around the intact appendage, and simultaneously extend to all directions on the horizontal plane, the fibroblasts are scattered among the collagen fibers, and the appendage is in a normal shape
Therefore, the liquid and cream compound mainly prepared from yak type I collagen has an obvious effect on repairing collagen fibers in the dermis.
(6) Control group 1
Mice of control group 1 were treated with 4ml (1.5 mg/ml) of commercially available fish collagen solution 1h after UV irradiation, and thereafter treated once daily with the same dose. From FIG. 8, it can be seen that collagen fibers in the dermis are significantly reduced, broken, kinked, aggregated, and dilated by blood vessel congestion at day 2; at the fourth day, the collagen fibers in the dermis are broken and aggregated into blocks, and the collagen fibers are still arranged loosely, are not increased obviously and are infiltrated by inflammatory cells. Basically has no repairing effect.
(7) Control group 2
Mice of control group 2 were treated with 4ml (1.5 mg/ml) of a commercially available porcine collagen solution 1h after UV irradiation, and thereafter treated once daily at the same dose. From fig. 8, it can be seen that at day 2, collagen fibers in the dermis layer are significantly reduced, broken, distorted and aggregated, and inflammatory cell infiltration; at the fourth day, the collagen fibers in the dermis layer are broken, aggregated and loosely arranged, and are not obviously increased. The repairing effect is not obvious.
(8) Control group 3
Mice of control group 3 were treated with 4ml (1.5 mg/ml) of commercially available bovine collagen solution 1h after UV irradiation, and thereafter treated once daily at the same dose. From FIG. 8, it can be seen that at day 2, collagen fibers in the dermis layer are significantly reduced, broken, kinked, aggregated, and blood vessels are dilated and engorged; collagen fibers in the dermis layer at day 4 are broken, aggregated into blocks, arranged loosely, hyperemia and dilated in blood vessels, and infiltrated by inflammatory cells, but the number of attachments is normal. The repair effect is less obvious.
It can be seen that commercially available fish collagen, commercially available pig collagen and commercially available bovine collagen do not contribute significantly to the restoration of dermal collagen fibers.
In conclusion, the yak type I collagen and the composition thereof provided by the invention have a remarkable repairing effect on the skin of a mouse with ultraviolet injury, can promote the recovery of collagen fibers in the dermis layer of the skin with ultraviolet injury, and have an effect superior to that of commercially available fish collagen, commercially available pig collagen and commercially available bovine collagen.
2.2 measurement of hydroxyproline content in skin
The relationship between the absorbance and the hydroxyproline content is that a standard curve is y =0.183x +0.0232, and R2=0.9983.
The hydroxyproline content in the skin of the mice in the normal blank group was set as a standard, i.e., 100% (shown in fig. 9, 1), and other groups including the model group, the experimental group, and the control group were normalized. The hydroxyproline content in the skin of the model group mice is only 60.59% of that of the normal blank group (described in figure 9, 5). Compared with the model group, the hydroxyproline content in the skin of the mice of the experimental group 1 (yak type I collagen solution) is 106.13 percent of that of the normal blank group (shown in figure 9 and 3); the hydroxyproline content in the skin of the experimental group 2 (yak collagen liquid complex prepared in example 2) mice is 106.65% of that of a normal blank group (shown in figure 9 and figure 2); experiment group 3 (yak collagen cream compound prepared in example 2) the hydroxyproline content in the skin of the mice is 96.02% of that of the normal blank group (described in fig. 9, 4); the hydroxyproline content in the skin of the mice in the experimental groups 1-3 is recovered to be between 96% and 107% of that of the normal blank group, and is close to or better than that of the normal blank group. Among commercially available collagens, the skin of the mice in the control group 1 (commercially available fish collagen) has a lower hydroxyproline content than that of the model group, and is only 42.41% of that of the blank group (shown in a commercial figure 9, 8); the content of hydroxyproline in the skin of the mice in the control group 2 (commercial pig collagen) is higher than that of the mice in the model group, but is only 62.82 percent of that of the blank group, and is obviously different from the results (96 percent to 107 percent) of the experimental group (shown in figure 9 and 6); the content of hydroxyproline in the control group 3 (commercially available bovine collagen) was close to that in the model group, and was 60.96% (shown in FIG. 9, 7). The results show that only the yak type I collagen and the composition thereof can obviously improve the content of hydroxyproline in the skin of the ultraviolet injury mouse. Among them, hydroxyproline is an amino acid specific to collagen, and the more hydroxyproline content, the more collagen content. Therefore, only the yak type I collagen and the composition thereof can obviously improve the content of collagen in the skin of the ultraviolet-damaged mouse and promote the repair and reconstruction of collagen fibers in the skin of the ultraviolet-damaged mouse.
In conclusion, the yak I type collagen and the composition thereof can obviously improve the content of collagen in the skin of a mouse damaged by ultraviolet light and promote the repair of the collagen in the skin damaged by the mouse, and the yak collagen and the compound thereof have a good repair effect on the skin damaged by the ultraviolet light. The damage of ultraviolet light to skin is mainly reflected in the damage of epidermal layer cells, the damage of dermal fibers and the like, the ultraviolet light can directly damage DNA or induce an oxidation reaction to generate free radicals and other mechanisms to act on body cells, the light-induced damage can change the space structure of the DNA, so that the DNA replication and transcription are hindered, the biological function of protein is further influenced, and meanwhile, the ultraviolet light radiation provides energy for the aggregation, crosslinking, breaking and other behaviors of dermal collagen fibers. The accumulation of UV radiation damage in skin cells can lead to corresponding pathological changes, such as skin inflammation, immunosuppression, and in severe cases, skin cancer. The photodamage in this experiment was all 100mJ/cm 2 The damage caused by ultraviolet radiation can be deduced by the inference that other types of damage caused by at least the same intensity can be repaired by yak collagen and the compound thereof, such as electromagnetic radiation from different sources, such as laser, red light, blue light and the like.
The above description is only for details of a specific exemplary embodiment of the present invention, and it is obvious to those skilled in the art that various modifications and changes may be made in the present invention in the practical application process according to specific preparation conditions, and the present invention is not limited thereto. All that comes within the spirit and principle of the invention is to be understood as being within the scope of the invention.
Claims (8)
1. The application of yak type I collagen in preparing a product for repairing skin photodamage; the preparation method of the yak type I collagen comprises the following steps:
(1) Pretreating yak tendon, treating with surfactant, and/or oxidant, and/or sterilizing, and/or defatting and decalcifying to obtain raw material powder;
(2) Adding 0.01-2.0M sodium hydroxide solution into the raw material powder in the step (1), and keeping the temperature at 4-30 ℃ for 2-24h;
(3) Removing alkali solution, washing with water to neutrality, and extracting by acid dissolution enzymolysis to obtain enzymolysis solution;
(4) And (4) inactivating the enzymolysis liquid obtained in the step (3), salting out, dialyzing, freezing and drying to obtain the yak type I collagen.
2. The use of claim 1, wherein the photodamage to skin is ultraviolet damage to skin.
3. The use of claim 1, wherein the skin photodamage repair product comprises a pharmaceutical, cosmetic, medical device.
4. A composition for photodamage repair of skin, the composition comprising the following components in percent: 0.1 to 50 percent of yak I type collagen, 0.1 to 30 percent of butanediol, 0.1 to 20 percent of glycerol, 0.1 to 10 percent of betaine, 0.1 to 5 percent of chamomile extract, 0.1 to 10 percent of sodium hyaluronate, 0.1 to 10 percent of caprylic/capric triglyceride, 0.1 to 0.3 percent of phenoxyethanol, 0.1 to 5 percent of glycerol stearic acid, 0.1 to 5 percent of cetearyl glucoside, 0.1 to 20 percent of polysiloxane and 0.1 to 5 percent of centella extract;
the preparation method of the yak type I collagen comprises the following steps:
(1) Pretreating yak tendon, and treating with surfactant, and/or oxidant, and/or sterilizing, and/or defatting and decalcifying to obtain raw material powder;
(2) Adding 0.01-2.0M sodium hydroxide solution into the raw material powder in the step (1), and keeping the temperature at 4-30 ℃ for 2-24h;
(3) Removing alkali solution, washing with water to neutrality, and extracting by acid dissolution enzymolysis to obtain enzymolysis solution;
(4) And (4) inactivating the enzymolysis liquid in the step (3), salting out, dialyzing, freezing and drying to obtain the yak type I collagen.
5. The composition of claim 4, wherein the composition comprises the following components in percent: 15% of yak type I collagen, 10% of butanediol, 8% of glycerol, 7% of betaine, 1% of chamomile flower extract, 1% of sodium hyaluronate, 6% of caprylic/capric triglyceride, 0.2% of phenoxyethanol, 1% of glycerol stearic acid, 3% of cetearyl glucoside, 6% of polydimethylsiloxane and 0.1% of centella asiatica extract.
6. A composition for photodamage repair of skin, the composition comprising the following components in percent: 1 to 50 percent of yak I type collagen, 0.1 to 30 percent of butanediol, 0.1 to 30 percent of erythritol, 0.1 to 5 percent of avocado extract, 0.1 to 20 percent of glycerol, 0.1 to 10 percent of nicotinamide, 0.1 to 10 percent of hyaluronic acid, 0.1 to 10 percent of bisabolol and 0.1 to 0.15 percent of methylparaben;
the preparation method of the yak type I collagen comprises the following steps:
(1) Pretreating yak tendon, treating with surfactant, and/or oxidant, and/or sterilizing, and/or defatting and decalcifying to obtain raw material powder;
(2) Adding 0.01-2.0M sodium hydroxide solution into the raw material powder in the step (1), and keeping the temperature at 4-30 ℃ for 2-24h;
(3) Removing alkali solution, washing with water to neutrality, and extracting by acid dissolution enzymolysis to obtain enzymolysis solution;
(4) And (4) inactivating the enzymolysis liquid in the step (3), salting out, dialyzing, freezing and drying to obtain the yak type I collagen.
7. The composition of claim 6, wherein the composition comprises the following components in percent: 10% of yak I type collagen, 10% of butanediol, 8% of erythritol, 1% of butyrospermum parkii extract, 5% of glycerol, 3% of nicotinamide, 2% of hyaluronic acid, 0.5% of bisabolol and 0.14% of methylparaben.
8. The composition of any one of claims 4-7, wherein the photodamage to the skin is ultraviolet damage to the skin.
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| CN109349419A (en) * | 2018-10-09 | 2019-02-19 | 王书敏 | A kind of compound Yak Bone collagen protein peptide powder for repairing human body cell |
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| CN107362356A (en) * | 2017-03-17 | 2017-11-21 | 中国科学院西北高原生物研究所 | A kind of Yak-skin Gelatin original protein product with hemostasia effect and preparation method thereof |
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