Preparation method, composition and application of yak collagen hydrolysate with anti-aging effect

Abstract

The invention belongs to the field of skin medicine, and particularly relates to a preparation method of a yak collagen hydrolysate with an anti-aging effect, a composition and application of the yak collagen hydrolysate, the yak collagen hydrolysate prepared through the method and an oral liquid composition containing the yak collagen hydrolysate. Good biocompatibility and safety are realized; the density of a corium layer can be increased, the water content can be recovered, the TEWL value can be recovered, the pathological change of light-aged skin tissue can be improved, the regeneration of collagen in the light-aged skin tissue can be promoted, the activity of SOD in the light-aged skin tissue can be improved, and the content of MDA in the light-aged skin tissue can be reduced, so that the oxidative stress reaction caused by ultraviolet radiation can be reduced; and the composition can be applied to anti-aging of the skin.

Description

Technical Field

[0001] The invention belongs to the field of dermatology, and specifically relates to a preparation method, a composition and an application of a yak collagen hydrolyzate with anti-aging effects. Background Art

[0002] Skin aging is one of the most intuitive manifestations of body aging and is an inevitable physiological process. According to the speed of aging, skin aging can be divided into two types: acute aging and subacute aging. Subacute skin aging is a common type of skin aging, usually caused by factors such as long-term exposure to ultraviolet radiation, continuous intake of excessive sugar, and long-term exposure to heavily polluted environments. Long-term exposure to ultraviolet rays is one of the most common factors leading to skin aging, called photoaged skin. The characteristics of photoaged skin include deepening and coarsening of wrinkles, rough skin, loss of elasticity, and may be accompanied by localized excessive pigmentation or capillary dilation, and may even cause skin cancer, seriously threatening people's life and health.

[0003] Collagen, as the main component of the dermis of the skin, is widely used in anti-aging products. Collagen hydrolysate is the product of further hydrolysis of collagen, and has many properties such as good biocompatibility and antioxidant properties. Collagen hydrolysate has a wide range of sources and types, and there are significant differences in quality and efficacy. How to prepare collagen hydrolysate with good anti-aging function is still a huge challenge. Small molecular weight collagen hydrolysate is widely considered to have the most application prospects because it is easy to absorb. Niu Jinge found that under the same concentration conditions, the Tibetan sheepskin collagen hydrolysate has the highest OH free radical scavenging ability, DPPH free radical scavenging ability, ABTS free radical scavenging ability and reducing power activity <1k Da. [1] .

[0004] Yaks are a precious resource unique to the Qinghai-Tibet Plateau in my country. Their high-altitude, oxygen-deficient, green and pollution-free environment endows yak collagen with unique properties. Invention patent CN112569347B discloses the use of yak collagen peptides in hypoglycemic drugs or hypoglycemic health foods. The yak collagen peptides prepared by this method have a wide range of molecular weights. The connective tissue components of yaks, such as skin, are complex, and how to prepare yak collagen hydrolysates with precise molecular weight and high activity is still difficult.

[0005] In view of the above technical problems, the present invention provides a method for preparing a yak collagen hydrolyzate with anti-aging efficacy. The preparation method obtains a yak collagen hydrolyzate with an accurate molecular weight distribution. The yak collagen hydrolyzates with different molecular weight distributions have different anti-aging effects, especially the medium molecular weight yak collagen hydrolyzate (MYCH) has the best anti-aging effect, and has good water solubility, biosafety and biological activity. The oral product prepared by using the same has significant effects in reducing skin wrinkles, increasing skin water content, increasing skin density and alleviating skin oxidative stress, and has broad market prospects in the fields of nutritional health products and medical cosmetology.

[0006] References:

[0007] [1] Niu Jinge. Extraction and functional properties of Tibetan sheepskin collagen peptides[D]. Qinghai University, 2023. DOI: 10.27740 / d.cnki.gqhdx.2023.000735. Summary of the invention

[0008] In order to solve the above technical problems existing in the prior art, the object of the present invention is to provide a method for preparing yak collagen hydrolysate with anti-aging effect, comprising the following steps:

[0009] (1) Yak leather pretreatment

[0010] The hair and subcutaneous tissue on the yak skin were removed, the skin was cut into small pieces, 10% n-butanol was used for degreasing, 1M NaOH was added for reaction to remove the non-collagen components in the yak skin, and the skin was washed with water until the pH value was 7 to obtain the pretreated yak skin;

[0011] (2) Preparation of yak collagen filtrate

[0012] The pretreated yak skin obtained in step (1) is added to a 0.5 M citric acid solution at a solid-liquid ratio of 1:10, pepsin is added to react under low temperature conditions, filtered, and the pH value is adjusted to neutral to obtain a yak collagen filtrate;

[0013] (3) Preparation of yak collagen hydrolysate

[0014] Add 8-120 U / mL of papain to the yak collagen filtrate (50%-100%) obtained in step (2), and hydrolyze at 50° C. for 3-6 hours to obtain yak collagen hydrolyzate;

[0015] (4) Enzyme inactivation

[0016] The yak collagen hydrolysate obtained in step (3) is placed in boiling water;

[0017] (5) Purification of yak collagen hydrolysate

[0018] Take 100 mL of the yak collagen hydrolyzate obtained in step (4) and elute it in the chromatography column. The absorbance of the eluate is measured at 220 nm, and the elution curve is drawn. The molecular weight distribution of the eluate is determined by size exclusion chromatography. According to the measurement results, the target product is collected, freeze-dried, and stored at -20°C for future use.

[0019] Preferably, the concentration of papain in step (3) is 16-120 U / mL.

[0020] Preferably, the concentration of papain in step (3) is 80-120 U / mL.

[0021] Preferably, the hydrolysis time in step (3) is 4 to 6 hours.

[0022] Preferably, the hydrolysis time in step (3) is 5 to 6 hours.

[0023] The second object of the present invention is to provide yak collagen hydrolysate prepared by the preparation method.

[0024] The third object of the present invention is to provide the use of the yak collagen hydrolyzate in the preparation of skin repair materials / products.

[0025] The fourth object of the present invention is to provide the use of the yak collagen hydrolyzate in the preparation of anti-aging and anti-oxidation health products and / or cosmetics.

[0026] The fifth object of the present invention is to provide an oral liquid composition with anti-aging efficacy, wherein the oral liquid composition comprises the following raw materials in percentage by mass: 6% yak collagen hydrolysate, 0.2% bilberry extract, 0.6% vitamin C, 0.04% hyaluronic acid, 0.02% black wolfberry powder, 0.01% salidroside, and the balance is water.

[0027] Preferably, the yak collagen hydrolysate is a medium molecular weight yak collagen hydrolysate.

[0028] Technical effects and advantages of the present invention:

[0029] (1) The present invention provides a method for preparing a yak collagen hydrolysate with anti-aging effects, wherein the preparation method can prepare a collagen hydrolysate with a specific molecular weight, and the preparation method is simple, easy to scale up, and low in cost;

[0030] (2) The yak collagen hydrolyzate prepared by the method has good biocompatibility, no damage to the immune organs of mice, and good safety; it can increase the density of the dermis, restore the water content, restore the TEWL value, improve the pathological changes of photoaged skin tissue, accelerate the expression of collagen fibers in photoaged skin tissue, promote the regeneration of collagen in photoaged skin tissue, increase the activity of SOD in photoaged skin tissue, and reduce the content of MDA in photoaged skin tissue, thereby alleviating the oxidative stress response caused by ultraviolet irradiation and repairing the photoaged skin of mice.

[0031] (3) An oral liquid composition was prepared using the collagen hydrolyzate, comprising the following raw materials in percentage by mass: 6% yak collagen hydrolyzate, 0.2% bilberry extract, 0.6% vitamin C, 0.04% hyaluronic acid, 0.02% black wolfberry powder, 0.01% salidroside, and the remainder being water.

[0032] (4) The oral liquid composition can increase the density of the dermis, restore the water content, restore the TEWL value, improve the pathological changes of photoaged skin tissue, accelerate the expression of collagen fibers in photoaged skin tissue, promote the regeneration of collagen in photoaged skin tissue, enhance the activity of SOD in photoaged skin tissue, and reduce the content of MDA in photoaged skin tissue, thereby alleviating the oxidative stress response caused by ultraviolet irradiation and repairing photoaged skin, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Qualitative evaluation of the performance of yak skin collagen hydrolysate in repairing photoaged skin. Note: a. Dermatoscope; b. Ultrasound image.

[0034] Figure 2 Quantitative evaluation of the performance of yak skin collagen hydrolysate in repairing photoaged skin. Note: ad. Skin density of mouse skin tissue; eh. Skin water content; il. Transepidermal water loss.

[0035] Figure 3 H&E staining was used to evaluate the repairing effect of yak collagen hydrolysate on photoaged skin.

[0036] Figure 4 Masson staining was used to evaluate the repairing effect of yak collagen hydrolysate on photoaged skin.

[0037] Figure 5 Histological analysis to evaluate the skin repair ability of yak skin collagen hydrolysate.

[0038] Note: a. Quantification of collagen fibers by Image J software; b. Relative content of hydroxyproline; c. Superoxide dismutase activity; d. Malondialdehyde content.

[0039] Figure 6 To qualitatively evaluate the performance of different doses of medium molecular weight yak skin collagen hydrolysate and its oral solution in repairing subacute aged skin.

[0040] Note: a. Dermatoscope; b. Ultrasound image.

[0041] Figure 7 Quantitative evaluation of the performance of different doses of medium molecular weight yak skin collagen hydrolysate and its oral solution in repairing subacute aged skin.

[0042] Note: ad. Skin density of mouse skin tissue; eh. Skin water content; il. Transepidermal water loss.

[0043] Figure 8 H&E staining was used to evaluate the repairing effects of different doses of medium molecular weight yak skin collagen hydrolysate and its oral solution on subacute aging skin.

[0044] Fig. 9 Masson staining was used to evaluate the repairing effect of different doses of medium molecular weight yak skin collagen hydrolysate and its oral solution on subacute aging skin.

[0045] Fig.10 Histological analysis was used to evaluate the repair ability of different doses of medium molecular weight yak skin collagen hydrolysate and its oral solution on subacute aged skin.

[0046] Note: a. Quantification of collagen fibers by Image J software; b. Relative content of hydroxyproline; c. Superoxide dismutase activity; d. Malondialdehyde content. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following.

[0048] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by ordinary technicians in the field to which the method belongs. Although any method similar or equivalent to that described herein can also be used for the precise preparation of yak collagen hydrolysate, it is not limited to the representative exemplary methods described in the following examples. It should also be understood that the terms used in the present invention are only for the purpose of describing specific embodiments and do not limit the scope of protection of the present invention and the appended claims.

[0049] In the following examples, the methods used are conventional methods unless otherwise specified, and the reagents used are conventional reagents that can be obtained from commercial sources.

[0050] Example 1. Preparation of yak collagen hydrolysate

[0051] The yak collagen hydrolysates are all prepared by a green method of molecular weight-directed enzymatic hydrolysis-chromatography, and the molecular weight-directed enzymatic hydrolysis-chromatography method is specifically as follows:

[0052] 1. Yak leather pretreatment

[0053] The yak skin was thawed in tap water at room temperature, the hair and subcutaneous tissue on the yak skin were removed, and it was cut into small pieces for later use. 10% n-butanol was used for degreasing for 12 hours, and then 1M NaOH was added for reaction for 11 hours to remove the non-collagen components in the yak skin. The yak skin treated with alkali was rinsed with water until pH = 7 to obtain pretreated yak skin.

[0054] 2. Preparation of Yak Collagen Filtrate

[0055] The pretreated yak skin was added to a 0.5M citric acid solution with a solid-liquid ratio of 1:10. Pepsin was added to react for 36 hours under low temperature conditions to extract collagen from the yak skin tissue. After the reaction was completed, the filtrate was filtered and the pH value of the filtrate was adjusted to neutral for later use.

[0056] 3. Preparation of Yak Collagen Hydrolysate

[0057] By adding 8U / mL of papain to the filtrate, the conditions for preparing ultra-high molecular weight yak collagen hydrolysate (UYCH) were explored at 50°C for different hydrolysis times. The results of SDS-PAGE gel electrophoresis showed that the product was yak collagen at 0h, most of the yak collagen in the hydrolyzate was not hydrolyzed at 1h and 2h, most of the yak collagen was hydrolyzed at 3h, and the yak collagen was hydrolyzed more thoroughly at 4h, 5h and 6h. In summary, considering factors such as production cost, the hydrolysis time was determined to be 3h.

[0058] Table 1 Preparation of ultra-high molecular weight yak collagen hydrolysate (UYCH)

[0059]

[0060] The optimal conditions for preparing high molecular weight yak collagen hydrolysate (HYCH) were explored by adding different concentrations of papain to the filtrate and hydrolyzing it at 50°C for a certain period of time. According to the liquid phase results, the optimal conditions for preparing high molecular weight yak collagen hydrolysate (HYCH) were 50% filtrate concentration, 16U / mL papain was added and hydrolyzed at 50°C for 4 hours.

[0061] Table 2 Preparation of high molecular weight yak collagen hydrolysate (HYCH)

[0062]

[0063] The optimal conditions for preparing medium molecular weight yak collagen hydrolysate (MYCH) were explored by adding different concentrations of papain to the filtrate and hydrolyzing it at 50°C for a certain period of time. According to the liquid phase results, the optimal conditions for preparing medium molecular weight yak collagen hydrolysate (MYCH) were 75% filtrate concentration, 80U / mL papain was added and hydrolyzed at 50°C for 5 hours.

[0064] Table 3 Preparation of yak collagen hydrolysate (MYCH) with medium molecular weight

[0065]

[0066] The optimal conditions for preparing low molecular weight yak collagen hydrolysate (LYCH) were explored by adding different concentrations of papain to the filtrate and hydrolyzing it at 50°C for a certain period of time. According to the liquid phase results, the optimal conditions for preparing low molecular weight yak collagen hydrolysate (LYCH) were 50% filtrate concentration, 120U / mL papain was added and hydrolyzed at 50°C for 6 hours.

[0067] Table 4 Preparation of low molecular weight yak collagen hydrolysate (LYCH)

[0068]

[0069] 4. Inactivate enzymes

[0070] After the hydrolysis, the reaction system was placed in boiling water for 10 minutes to stop the enzymatic reaction.

[0071] 5. Purification of Yak Collagen Hydrolysate

[0072] After pretreatment of Sephadex G25 gel, it was loaded into a prepared polytetrafluoro sand core glass chromatography column to ensure the column was balanced and stable. A certain volume of the enzymolysis solution was loaded onto the column, and distilled water was used as the eluent to control the elution flow rate. During the elution process, every 5 mL of the eluate was taken as one tube. The absorbance of each tube of eluate at 220 nm was measured with an ultraviolet spectrophotometer, and an elution curve of absorbance versus the number of tubes was plotted. Size exclusion chromatography was used to determine the molecular weight distribution of the eluate in each tube, and the target product was collected according to the measurement results for freeze-drying and stored at -20 °C for standby.

[0073] Example 2. Characterization of the properties of yak collagen hydrolysate

[0074] According to the method provided by the national standard (GB 31648-2018), high performance liquid chromatography (HPLC) equipped with a TSK gel G2000 SW XL chromatographic column was used to determine the molecular weight distribution of yak collagen hydrolysate (Table 5). The molecular weight of low molecular weight yak collagen hydrolysate (LYCH) mainly concentrated below 1000 Da, and the proportion of M < 1000 Da could reach 75.56%. The molecular weight of medium molecular weight yak collagen hydrolysate (MYCH) mainly concentrated between 1000 Da and 3000 Da, and the proportion of 1000 Da < M < 3000 Da could reach 57.81%. The molecular weight of high molecular weight yak collagen hydrolysate (HYCH) mainly concentrated between 3000 Da and 10000 Da, and the proportion of 3000 Da < M < 10000 Da could reach 52.38%. The molecular weight distribution of ultra-high molecular weight yak collagen hydrolysate (UYCH) was characterized by SDS-PAGE gel electrophoresis, and the results showed that it mainly concentrated between 35 kDa and 180 kDa.

[0075] Table 5 Molecular weight distribution table of yak collagen hydrolysate

[0076]

[0077] Example 3. Photodamaged skin repair ability of yak collagen hydrolysate

[0078] 1. Establishment and repair of ultraviolet photoaged skin

[0079] Female Kunming mice aged 3 months were randomly divided into 6 groups, including normal group (Normal), model group (Model), LYCH group, MYCH group, HYCH group and UYCH group, with 24 mice in each group. Mice in the normal group and model group were orally administered with 0.4 mL of normal saline daily. The LYCH group, MYCH group, HYCH group and UYCH group were orally administered with the same volume of LYCH, MYCH, HYCH and UYCH solutions prepared in Example 1 by gavage, respectively, at a dose of 200 mg / kg BW.

[0080] The construction of the mouse photoaging model was first carried out by shaving the hair on the back skin of the mice with an electric shaver, and then further removing the hair with a commercially available depilatory cream. The mice in the normal group did not receive any treatment, and the back skin of the mice in the other groups was placed under a UV lamp for irradiation. The irradiation was performed 3 times a week for 8 consecutive weeks, with each irradiation lasting 20 minutes. The radiation was quantified using an ultraviolet irradiator. The total doses of UVA and UVB irradiation were 20.4 J / cm2 and 1.44 J / cm2, respectively.

[0081] 2. Toxicological effects of yak collagen hydrolysate

[0082] We weighed and recorded the weight of mice in each group at weeks 1, 2, 4, 6, and 8 to evaluate whether yak collagen hydrolysate had a significant adverse effect on the growth of mice. During the entire experimental period, the weight of mice in the normal group, model group, and experimental groups showed a trend of gradual increase, and there was no statistical significance between the groups in the same week (Table 2-4). At week 8, the weights of mice in the model group, LYCH, MYCH, HYCH, and UYCH groups were 41.91±1.84g, 42.50±0.90g, 41.72±1.55g, 41.63±2.09g, and 41.97±1.76g, respectively, which was not statistically significant compared with the weight of mice in the normal group (42.07±0.98g). The above experimental results show that oral administration of yak collagen hydrolysate at a dose of 200mg / kg BW per day has no significant adverse effect on the growth of mice.

[0083] We weighed and recorded the spleen and thymus of each group of mice at week 8 to evaluate whether yak collagen hydrolysate produced significant toxicological effects on mice (Table 6). The spleen and thymus are two key immune organs. The spleen is rich in lymphocytes and macrophages, while the thymus is where T cells differentiate and mature. The spleen index (SI) and thymus index (TI) are considered important indicators of immune function. The average values ​​of SI and TI of mice in the normal group were 3.76±0.33 mg / g and 2.23±0.19 mg / g, respectively. The average values ​​of SI and TI of mice in the model group, LYCH, MYCH, HYCH and UYCH groups were 3.89±0.37mg / g and 2.19±0.23mg / g, 3.86±0.31mg / g and 2.21±0.20mg / g, 3.70±0.23mg / g and 2.23±0.24mg / g, 3.70±0.31mg / g and 2.26±0.40mg / g, 3.92±0.33mg / g and 2.30±0.34mg / g, respectively. There was no significant difference between the SI and TI values ​​of the above groups and those of mice in the normal group. Therefore, the results show that yak collagen hydrolysate has good biocompatibility, does not damage the immune organs of mice, and does not produce obvious toxicological effects on mice.

[0084] Table 6 Toxicological evaluation of yak collagen hydrolysate: mouse body weight, spleen index (SI) and thymus index (TI)

[0085]

[0086]

[0087] 3. Dermoscopy and Combo Ultrasound Images of Mouse Skin Tissue

[0088] Combo and dermatoscope were used to evaluate the ability of YCH to repair photoaged skin. Figure 1 As shown in a: During the entire experimental period, the back skin of the mice in the normal group was smooth. In the second week, the back skin of the mice in the model group was dry and fine lines appeared, and fine lines also appeared on the back skin of the mice in each experimental group. In the fourth week, the back skin of the model and UYCH groups became dry, the fine lines deepened and the number increased, while the fine lines of the LYCH and MYCH skin became shallower. In the sixth and eighth weeks, the skin of the model and UYCH groups became drier and rougher, the wrinkles deepened, and the fine lines of the HYCH group decreased. The skin condition of the LYCH and MYCH groups returned to normal. The above results show that the YCH prepared in the present application can repair the apparent state of photoaged skin, which is specifically manifested in reducing skin wrinkles and improving skin roughness. LYCH and MYCH have the best effects, followed by HYCH, and UYCH has almost no effect.

[0089] Combo high-frequency ultrasound probe was used to detect dermal density and epidermal thickness at 2, 4, 6, and 8 weeks to evaluate the ability of yak collagen hydrolyzate (YCH) to repair photoaged skin. The relationship between the color of ultrasound images and the density and thickness of the skin is black < green < blue < red < yellow < white. The ultrasound images of mouse skin tissue are shown in Figure 2. Figure 1 As shown in Figure b: During the entire experiment, the back skin of the normal group mice always maintained a skin state with high dermis density and thin epidermis. In contrast, the back skin of the model group mice showed a trend of gradually decreasing dermis density, indicating that long-term UV exposure will lead to a decrease in skin dermis density. In the second week, compared with the model group, the dermis density of the LYCH and MYCH groups increased, but was lower than that of the normal group. In the fourth week, compared with the model group, except for the UYCH group, the dermis density of the other groups of mice increased, among which the dermis density of the LYCH and MYCH groups was close to that of the normal group. In the sixth and eighth weeks, compared with the model group, except for the UYCH group, the dermis density of the other groups of mice increased to varying degrees, among which the dermis density of the LYCH and MYCH groups was equivalent to that of the normal group. The above results show that the YCH prepared in this application can repair the photoaged skin of mice, specifically by increasing the density of its dermis, among which LYCH and MYCH have the best effect.

[0090] 4.Combo evaluation of skin density, moisture and transepidermal water loss in mouse skin tissue

[0091] The density of the dermis layer of the skin was measured using a Combo high-frequency ultrasound probe to evaluate the ability of yak collagen hydrolysate (YCH) to repair photoaged skin. The better the skin condition, the higher the value obtained by the instrument. Figure 2As shown in ad: During the entire experimental period, the dermal density of the skin in the normal group remained at a high level. In contrast, the dermal density of the skin in the model group gradually decreased to 42.81±1.68. This is because UV irradiation destroyed the structure of collagen in the skin, causing it to gradually degrade and lose. At week 2, the dermal density of the skin in the LYCH, MYCH, HYCH, and UYCH groups was lower than that in the normal group (92.29±1.89), decreasing to 81.47±1.83, 84.03±1.85, 77.31±1.58, and 71.66±1.68, respectively. At weeks 4 and 6, the dermal density of the skin in the LYCH, MYCH, and HYCH groups increased slowly. At week 8, the dermal density of the skin in the LYCH, MYCH, and HYCH groups increased to 86.80±1.29, 90.42±1.53, and 81.91±1.73, respectively. It is worth noting that from the 6th week, there was no significant difference in the dermis density of the MYCH group compared with the normal group. The above results show that the YCH prepared in the present application helps to restore the dermis density of photoaged skin, and the effect is MYCH>LYCH>HYCH, while UYCH has almost no effect.

[0092] The moisture probe detects the moisture content of the skin to study the ability of yak collagen hydrolyzate (YCH) to repair the moisture content of photo-aged skin. When the skin is in good condition, the moisture content of its stratum corneum is usually high, and the value detected by the instrument is higher. The experimental results are as follows Figure 2As shown in eh: At week 2, the water content of the skin in the model, LYCH, MYCH, HYCH and UYCH groups was lower than that in the normal group (365.33±16.05μS), decreasing to 152.46±13.22μS, 234.18±17.00μS, 256.77±16.79μS, 184.58±14.68μS and 162.06±11.72μS, respectively. At week 4, the water content of the skin in the LYCH, MYCH and HYCH groups increased to 257.50±16.16μS, 301.51±18.31μS and 204.69±17.51μS, respectively, while the water content in the model and UYCH groups decreased to 117.32±13.53μS and 122.47±18.04μS, respectively. At week 6, the water content of the skin in the LYCH, MYCH and HYCH groups further increased to 277.60±17.24μS, 338.67±12.25μS and 209.40±17.05μS. At week 8, the water content of the skin in the LYCH, MYCH and HYCH groups increased to 282.22±14.87μS, 343.40±12.92μS and 216.48±14.87μS, with no significant difference compared with week 6. The water content of the skin in the UYCH group gradually decreased to 57.11±11.60μS, with no significant difference compared with the model group (51.10±9.90μS). The above results show that the YCH prepared in this application helps to restore the water content of photoaged skin, and the effects are MYCH>LYCH>HYCH, while the effect of UYCH is almost negligible.

[0093] Combo's TEWL probe evaluates transepidermal water loss on the back of mice. TEWL refers to the rate at which water in the dermis evaporates through the epidermis. The higher the value, the more water is lost from the skin and the worse the skin barrier function. Figure 2 As shown in il: At the second week, the 2 *h) compared with the TEWL value of the model group (46.74±1.45g / m 2 *h) increased significantly, which is because UV irradiation destroyed the skin barrier. The TEWL values ​​of the skin of mice in the LYCH, MYCH, HYCH and UYCH groups were between the normal group and the model group. At the 4th and 6th weeks, the TEWL values ​​of the skin of mice in the LYCH, MYCH and HYCH groups slowly decreased, indicating that oral YCH can repair the skin barrier of photoaged skin, thereby restoring its TEWL. At the 8th week, the TEWL value of the MYCH group (18.90±1.82g / m 2 *h) and the 6th week (19.00±1.92g / m 2*h) had no significant difference, and had no significant difference with the normal group. The above results show that the YCH prepared in the present application helps to restore the TEWL value of photoaged skin, and the effect is in the order of MYCH>LYCH>HYCH, while UYCH has almost no effect.

[0094] 5. H&E Staining of Mouse Skin Tissue

[0095] The repair ability of yak collagen hydrolysate (YCH) on inflammatory response in photoaged skin was studied at the tissue level by H&E staining. Figure 3 As shown in the figure: During the whole experiment, the back skin of the mice in the normal group remained healthy, the thickness of the epidermis was uniform and thin, the structure of the dermal-epidermal junction (EDJ) area was wavy, and there was no inflammatory cell infiltration. However, the back skin condition of the mice in the model group gradually deteriorated, the epidermis gradually thickened, the EDJ area structure was flat, and the dermis was infiltrated with inflammatory cells and accompanied by vascular dilation. At the second week, the thickness of the epidermis of the back skin of the mice in each experimental group was uneven, the EDJ area structure was flat, and the dermis was infiltrated with inflammatory cells. At the fourth and sixth weeks, the number of dermal papillae in the skin of the LYCH, MYCH and HYCH groups increased, and the inflammatory reaction in the dermis gradually disappeared. At the eighth week, the dermis of the skin of the UYCH and model groups was significantly thickened, and the EDJ area structure of the skin of the LYCH, MYCH and HYCH groups began to appear wavy; among them, the thickness of the epidermis of the mice in the LYCH and MYCH groups was similar to that of the normal group. The above experimental results show that the YCH prepared in the present application can improve the pathological changes of photoaged skin tissue, LYCH and MYCH have the best effects, HYCH is second, and UYCH has almost no effect.

[0096] 6. Masson staining of mouse skin tissue

[0097] Masson staining was used to study the ability of yak collagen hydrolysate (YCH) to repair and regenerate collagen fibers in photoaged skin tissue. Figure 4As shown in the figure: During the whole experiment, the collagen fibers in the dermis of the skin of the mice in the normal group were arranged in an orderly and wavy shape. At the second week, the collagen fibers in the dermis of the back skin of the mice in the model and experimental groups were broken to varying degrees, and the fiber bundles were arranged loosely. Compared with other experimental groups, the collagen fibers in the model and UYCH groups were severely broken. At the fourth and sixth weeks, compared with the mice in the model and UYCH groups, the broken collagen fibers in the dermis of the LYCH, MYCH and HYCH groups were gradually repaired and arranged neatly. At the eighth week, the collagen fiber bundles in the dermis of the back skin of the mice in the model group were severely broken, while the collagen fiber bundles in the dermis of the back skin of the mice in the UYCH group were scarce and arranged in a disordered manner. The collagen fibers in the dermis of the back skin of the mice in the LYCH and MYCH groups were full and arranged neatly. The epidermis of the skin of the HYCH group was consistent with that of the model group, but the collagen fiber bundles in its dermis were full and arranged neatly. The above experimental results show that the YCH prepared in the present application can improve the pathological changes of photoaged skin tissue, and the effects are in the order of MYCH>LYCH>HYCH, while UYCH has almost no promoting effect.

[0098] 7. Quantitative evaluation of the ability of yak collagen hydrolysate to promote collagen regeneration

[0099] Image J software was used to quantify the collagen fiber content in the back skin tissue of mice at 2, 4, 6, and 8 weeks to evaluate the ability of yak collagen hydrolysate (YCH) to repair photoaged skin. Figure 5 As shown in a: At the second week, the collagen fiber content in the skin tissue of mice in the model, LYCH, MYCH, HYCH and UYCH groups was lower than that in the normal group (32.60±0.71%), and decreased to 24.65±0.79%, 25.98±0.81%, 26.30±0.74%, 25.23±0.63% and 24.33±0.55%, respectively. At the fourth and sixth weeks, the collagen fiber content in the LYCH, MYCH and HYCH groups gradually increased, while that in the model and UYCH groups gradually decreased. At the eighth week, the collagen fiber content in the LYCH, MYCH and HYCH groups increased to 28.60±0.63%, 29.82±0.57% and 27.29±0.63%, respectively, among which there was no significant difference between the LYCH and MYCH groups and the normal group (28.50±0.67%). The above results show that the YCH prepared in the present application can accelerate the expression of collagen fibers in photoaged skin tissue, LYCH and MYCH have the best effects, HYCH is second, and UYCH has almost no effect.

[0100] The chloramine T method was used to further quantitatively analyze the relative content of hydroxyproline (Hyp) in the back skin tissue of each group of mice at different time periods to evaluate the ability of yak collagen hydrolysate (YCH) to repair photoaged skin. Figure 5 As shown in b: The Hyp content of the normal group at week 2 was used as the benchmark. At week 2, the Hyp content of the model, LYCH, MYCH, HYCH and UYCH groups decreased to 86.85±3.61%, 89.88±2.32%, 90.72±3.73%, 88.59±3.15% and 87.13±3.70%, respectively. At week 4, the Hyp content of the LYCH, MYCH and HYCH groups increased to 91.39±2.42%, 94.59±4.29% and 91.09±2.12%, respectively, and the Hyp content of the model and UYCH groups decreased to 78.41±4.07% and 78.96±4.04%, respectively. At week 6, the Hyp contents in the LYCH, MYCH and HYCH groups increased to 92.13±3.14%, 99.15±2.67% and 87.27±3.21%, respectively, among which there was no significant difference between the MYCH group and the normal group (99.73±2.40%). At week 8, the Hyp contents in the skin tissues of the LYCH, MYCH and HYCH groups were 92.83±2.85%, 100.48±2.56% and 86.47±3.04%, respectively, which were not significantly different from those in week 6. The above results show that the YCH prepared in the present application can promote the regeneration of collagen in photoaged skin tissues, and the effects are MYCH>LYCH>HYCH, while UYCH has almost no effect.

[0101] 8. Analysis of the antioxidant activity of yak collagen hydrolysate in vivo

[0102] We used a superoxide dismutase (SOD) activity detection kit to measure the SOD activity in the back skin tissue of mice to evaluate the in vivo antioxidant activity of yak collagen hydrolysate (YCH). Ultraviolet radiation can lead to a decrease in SOD activity in skin tissue. SOD plays a key antioxidant role in the skin. It can help scavenge free radicals to reduce oxidative stress and thus protect skin cells from oxidative damage. The better the skin condition, the higher the SOD value detected. The experimental results are as follows: Figure 5As shown in Figure c: During the entire experimental period, the skin of the normal group of mice remained in a healthy state, and its SOD value remained at a high level. In contrast, the SOD value of the model group mice gradually decreased to 81.84±12.87U / g. The SOD activity of the LYCH, MYCH and HYCH groups gradually increased, increasing to 187.55±13.87U / g, 219.08±9.33U / g and 173.12±14.97U / g, respectively, in the 8th week, among which there was no significant difference between the MYCH group and the normal group (224.64±9.23U / g). The SOD value of the UYCH group showed a gradual downward trend, decreasing to 85.36±11.95U / g in the 8th week. The above results show that the YCH prepared in this application can enhance the activity of SOD in photoaged skin tissue, thereby reducing the oxidative stress response caused by ultraviolet irradiation, and the effects are MYCH>LYCH>HYCH, and the effect of UYCH can be ignored.

[0103] Malondialdehyde (MDA) content detection kit was used to further evaluate the in vivo antioxidant activity of yak collagen hydrolysate (YCH). MDA is a lipid oxidation product, and its increased content is usually associated with oxidative stress and cell damage. Ultraviolet radiation can lead to an increase in MDA content in skin tissue. The experimental results are as follows Figure 5d shows: At week 2, the MDA contents in the model, LYCH, MYCH, HYCH and UYCH groups were higher than those in the normal group (2.51±0.25nmol / g), increasing to 4.05±0.22nmol / g, 3.73±0.19nmol / g, 3.48±0.32nmol / g, 3.94±0.19nmol / g and 3.98±0.19nmol / g, respectively. At week 4, the MDA contents in the LYCH, MYCH and HYCH groups decreased to 3.69±0.20nmol / g, 3.30±0.37nmol / g and 3.84±0.19nmol / g, respectively, and increased to 4.16±0.22nmol / g and 4.01±0.14nmol / g in the model and UYCH groups, respectively. At week 6, the MDA content of LYCH, MYCH and HYCH groups decreased to 3.15±0.25nmol / g, 2.54±0.24nmol / g and 3.44±0.16nmol / g, respectively, and there was no significant difference between the MYCH group and the normal group (2.54±0.14nmol / g). At week 8, the MDA content of LYCH, MYCH and HYCH groups was 3.15±0.19nmol / g, 2.40±0.08nmol / g and 3.33±0.19nmol / g, respectively, which was not significantly different from that of week 6. The MDA content of the model and UYCH groups decreased to 6.38±0.42nmol / g and 6.06±0.35nmol / g, respectively, with no significant difference between the two groups. The above results show that the YCH prepared in the present application can reduce the content of MDA in photoaged skin tissue, thereby alleviating the oxidative stress response caused by ultraviolet irradiation, and the effects are MYCH>LYCH>HYCH, while UYCH has almost no effect.

[0104] Example 4: Preparation of yak collagen hydrolysate oral product

[0105] The invention discloses an oral product of yak collagen hydrolyzate for repairing skin aging. The oral product comprises the following components in percentage by mass: 6% of MYCH, 0.2% of bilberry extract, 0.6% of vitamin C, 0.04% of hyaluronic acid, 0.02% of black wolfberry powder, 0.01% of salidroside and water.

[0106] Preparation process: (1) accurately weigh each component and mix well; (2) add a certain volume of drinking water to homogenize and dissolve; (3) sterilize; (4) clean and disinfect the glass bottle; (5) fill.

[0107] Example 5: Skin aging repair ability of yak collagen hydrolysate oral product

[0108] 1. Subacute aging skin modeling and treatment

[0109] Three-month-old female Kunming mice were randomly divided into 7 groups, including normal group (Normal), model group (Model), low-dose medium molecular weight yak collagen hydrolysate group (MYCH-200), medium-dose medium molecular weight yak collagen hydrolysate group (MYCH-400), high-dose medium molecular weight yak collagen hydrolysate group (MYCH-800), commercial product group (Commercial product) and homemade oral solution group (Oral solution), with 24 mice in each group. The mice in the normal group did not receive any treatment, while the mice in the subacute aging model group were orally administered with D-galactose solution at a dose of 500 mg / kg BW every day. After 2 months, compared with the normal group, the moisture and density of the back skin of the mice administered with D-galactose significantly decreased, while the transepidermal water loss significantly increased, indicating that the subacute skin aging model induced by D-galactose was successfully established. After the model was established, the mice in the normal group and the model group were orally administered with 0.4 mL of normal saline every day. The mice in the MYCH-200, MYCH-400 and MYCH-800 groups were orally administered with the same volume of MYCH solution by gavage, with the doses being 200 mg / kg BW, 400 mg / kg BW and 800 mg / kg BW, respectively. The mice in the commercial oral liquid group were orally administered with 0.4 mL of commercial collagen product solution (Fancl collagen oral liquid drink, FANCL Co., Ltd.), and the mice in the homemade oral liquid group were orally administered with 0.4 mL of the yak collagen hydrolyzate oral product prepared in Example 1.

[0110] 2. Dermoscopy and Skin Ultrasound of Mouse Skin Tissue

[0111] The Combo dermoscopy probe was used to evaluate the ability of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution to repair subacute aged skin. Figure 6 As shown in a: During the entire experimental period, the back skin of the mice in the normal group remained smooth, while the back skin of the mice in the model group was relatively dry, rough and had obvious wrinkles. In the second week, the wrinkles on the back skin of the mice in each experimental group were slightly reduced compared with the model group. In the fourth and sixth weeks, the wrinkles on the back skin of each experimental group were further reduced. In particular, the skin condition of the homemade oral liquid group was close to that of the normal group. In the eighth week, the skin of the MYCH-200 group was slightly wrinkled, the skin of the MYCH-400 and commercially available product groups was slightly fine, and the skin condition of the MYCH-800 and homemade oral liquid groups was comparable to that of the normal group. The above experimental results show that the different doses of MYCH and its oral liquid described in this application can repair the apparent state of subacute aging skin, which is specifically manifested in reducing skin wrinkles. As the dose of MYCH increases, its repair effect is better. In addition, the oral product prepared using MYCH is better than commercially available products.

[0112] Combo high-frequency ultrasound probe was used to detect dermal density and epidermal thickness at 2, 4, 6, and 8 weeks to evaluate the ability of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution to repair subacute aging skin. The relationship between the color of ultrasound images and the density and thickness of the skin is black < green < blue < red < yellow < white. The experimental results are as follows Figure 6 As shown in b: During the entire experimental period, the back skin of the mice in the normal group always maintained a skin state with a high dermis density. In contrast, the back skin of the mice in the model group showed a skin state with a low dermis density, indicating that oral administration of D-galactose would lead to a decrease in the dermis density of the skin. Compared with the model group, the dermis density of the skin in each experimental group showed a gradual increasing trend. In the 8th week, the dermis density of the skin in the homemade oral liquid group was close to that of the normal group. The above results show that the different doses of MYCH and its oral liquid described in this application can repair the subacutely aged skin of mice, which is specifically manifested in an increase in the density of the dermis. Among them, the homemade oral liquid has the most significant effect, followed by MYCH-800 and commercially available products.

[0113] 3. Skin density, moisture, and transepidermal water loss (TEWL) of mouse skin tissue

[0114] Combo high-frequency ultrasound probe quantitatively measures the density of the dermis layer of the skin to evaluate the ability of different doses of medium molecular weight collagen hydrolysate (MYCH) and its oral solution to repair subacute aging skin. The better the skin condition, the higher the value obtained by the instrument test. The experimental results are as follows Figure 7As shown in ad: During the entire experimental period, the dermis density of the skin in the normal group remained at a high level, while the dermis density of the skin in the model group was relatively low. In the second week, the dermis density of the skin in the MYCH-200, MYCH-400, MYCH-800, commercial product group, and homemade oral liquid group was lower than that in the normal group (85.75±2.00), but higher than that in the model group (51.94±1.91), which were 59.18±1.76, 62.94±1.94, 69.14±2.19, 66.63±1.81, and 71.33±1.93, respectively. In the fourth and sixth weeks, the dermis density of the skin in each experimental group continued to increase. At week 8, the dermal density of the skin in the MYCH-200, MYCH-400, MYCH-800, commercial product group, and homemade oral liquid group increased to 72.07±1.78, 77.02±1.85, 82.16±1.72, 81.09±1.83, and 85.33±1.86, respectively. It is worth noting that compared with the normal group, the dermal density of the skin in the homemade oral liquid group had no significant difference from the normal group since week 6. The above results show that different doses of MYCH and its oral liquid are helpful in restoring the dermal density of subacute aging skin. As the dose of MYCH increases, the repair effect gradually increases. In addition, the repair effect of MYCH-800 is better than that of the commercial product group and slightly lower than that of the homemade oral liquid group.

[0115] The Combo moisture probe detects the moisture content of the skin to study the effects of different doses of medium molecular weight collagen hydrolysate (MYCH) and its oral solution on repairing subacute aged skin. Generally, when the skin is in good condition, the moisture content of its stratum corneum is high, and the value detected by the instrument is high. The experimental results are as follows Figure 7As shown in eh: In the second week, the water content of the skin in the model, MYCH-200, MYCH-400, MYCH-800, commercial products and homemade oral solution groups was lower than that in the normal group (346.11±21.35μS), which were 166.28±21.60μS, 175.48±18.46μS, 183.80±19.84μS, 204.83±18.25μS, 195.39±19.67μS and 220.38±18.90μS, respectively. In the fourth week, the water content of the skin in the MYCH-200, MYCH-400 and MYCH-800 groups increased to 210.27±18.32μS, 230.55±19.46μS and 254.18±19.63μS, respectively. The water content of the skin in the commercial product and homemade oral liquid groups increased to 246.08±18.36μS and 272.85±18.14μS, respectively, and there was a significant difference between the two. At the 6th and 8th weeks, the moisture content of the back skin of mice in each experimental group remained basically unchanged. At the 8th week, the water content of the skin in the MYCH-200, MYCH-400, MYCH-800, commercial product and homemade oral liquid groups increased to 246.20±16.58μS, 285.32±19.29μS, 305.76±18.71μS, 296.50±19.45μS and 326.10±19.02μS, respectively, and the homemade oral liquid group was significantly better than the commercial product group. The above results show that the different doses of medium molecular weight MYCH and its oral liquid described in this application are helpful in restoring the water content of subacute aging skin.

[0116] Combo's TEWL probe evaluates transepidermal water loss. TEWL refers to the rate at which water in the dermis evaporates through the epidermis. The higher the value, the more water is lost from the skin, indicating that the skin barrier function is poorer. The experimental results are as follows Figure 7 il: During the entire experimental period, the TEWL value of the skin in the normal group remained at a low level, while the TEWL value of the skin in the model group gradually increased. At the 2nd, 4th, and 6th weeks, the TEWL values ​​of the skin in each experimental group showed a gradual downward trend. At the 8th week, the TEWL values ​​of the skin in the MYCH-200, MYCH-400, MYCH-800, commercially available products, and homemade oral liquid groups were significantly lower than those in the model group (67.31±0.95g / m 2 *h), 44.54±1.79g / m 2 *h, 42.54±1.76g / m 2 *h, 39.88±2.01g / m 2 *h, 41.35±1.98g / m 2 *h and 38.33±1.57g / m 2*h. The TEWL of the skin in the homemade oral solution group was significantly better than that in the commercial product group. It is worth noting that the TEWL of the skin in the homemade oral solution group was significantly lower than that in the normal group (38.77±1.66g / m 2 *h) No significant difference. The above results show that different doses of MYCH and its oral solution described in the present application can repair the skin barrier of subacute aged skin, thereby restoring its TEWL. At the same time, the repair effect of MYCH is positively correlated with its dose.

[0117] 4. H&E Staining of Mouse Skin Tissue

[0118] The ability of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution to repair subacute aging skin was studied at the tissue level by H&E staining. Figure 8 As shown: Throughout the experiment, the skin of the mice in the normal group was always in a healthy state, the thickness of the epidermis was uniform and thin, the dermal-epidermal junction (EDJ) area was wavy, and there was no inflammatory cell infiltration. The epidermis of the mice in the model group had different thicknesses, the EDJ area was flat, the dermal papilla was not obvious, and inflammatory cell infiltration and vasodilation occurred. At the second week, the inflammatory cell infiltration in the dermis of the skin of each experimental group was slightly reduced. In the second and fourth weeks, the inflammatory reaction and vasodilation of the dermis of the skin of each experimental group gradually disappeared, and the number of dermal papillae increased. At the eighth week, the EDJ area of ​​the skin of each experimental group was wavy again, and the skin of the homemade oral liquid group returned to the level of the normal group. The above experimental results show that different doses of MYCH and its oral solution described in this application can improve the pathological changes of photoaging skin tissue. As the dose of MYCH increases, its repair effect is better. In addition, the oral product prepared using MYCH is better than commercially available products.

[0119] 5. Masson staining of mouse skin tissue

[0120] Masson staining was used to study the ability of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution to promote the repair and regeneration of collagen fibers in subacute aging skin tissue. Fig. 9As shown: During the entire experiment, the collagen fibers in the dermis of the skin of the normal group of mice were arranged tightly and orderly, in a wavy shape. The collagen fibers in the dermis of the skin of the model group of mice were significantly reduced, and there were phenomena of breakage and aggregation. In the second week, new collagen fibers began to be generated at the broken and fractured parts of the collagen fibers in the dermis of the skin of each experimental group, and the arrangement was relatively loose and disordered. In the fourth and sixth weeks, the broken collagen fibers in the dermis of the skin of each experimental group tended to be neat. In the eighth week, the collagen fibers in the dermis of the skin of each experimental group were repaired and arranged in a wavy shape, especially the collagen fibers in the dermis of the skin of the homemade oral liquid group were basically restored to a normal state. The above results show that the different doses of MYCH and its oral liquid described in this application can promote the regeneration and repair of collagen fibers in the dermis of subacute aging skin. As the dose of MYCH increases, its effect is better. In addition, the oral product prepared using MYCH is better than the commercially available product.

[0121] 6. Quantitative evaluation of the ability of different doses of medium molecular weight yak collagen hydrolysate and its oral solution to promote collagen regeneration in subacute aging skin

[0122] Image J software was used to quantify the collagen fiber content in the skin tissue of mice at 2, 4, 6, and 8 weeks to evaluate the ability of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution to repair subacute aging skin. The experimental results are shown in 10a: At week 2, the collagen fiber content in the skin tissue of mice in the MYCH-200, MYCH-400, MYCH-800, commercial products, and homemade oral solution groups was lower than that in the normal group (31.44±1.64%), but higher than that in the model group (19.07±1.67%), which were 21.05±1.71%, 22.60±1.48%, 23.50±1.03%, 22.94±1.15%, and 24.82±1.66%, respectively. At weeks 4 and 6, the collagen fiber content in the skin tissue of each experimental group gradually increased. At the 6th week, there was no significant difference in the collagen fiber content in the skin tissue of the homemade oral liquid group (31.92±1.43%) and the normal group (31.07±1.79%). At the 8th week, the collagen fiber content in the skin tissue of the MYCH-200, MYCH-400, MYCH-800, commercially available products and homemade oral liquid groups increased to 25.73±1.71%, 27.90±1.62%, 29.73±1.21%, 27.13±1.41% and 32.01±1.61%, respectively. The collagen fiber content in the skin tissue of the homemade oral liquid group was significantly higher than that of the commercially available product group. According to the existing experimental results, the effect of MYCH-800 described in this application on promoting the regeneration of collagen fibers in subacute skin is worse than that of the commercially available product group, but not as good as the effect of the homemade oral liquid.

[0123] The chloramine T method was used to further quantitatively analyze the relative content of hydroxyproline (Hyp) in the back skin tissue of each group of mice at different time periods to evaluate the ability of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution to promote collagen regeneration. Fig.10 As shown in b: The Hyp content of the normal group at week 2 was used as the benchmark. At week 2, the Hyp content of the model, MYCH-200, MYCH-400, MYCH-800, commercially available products, and homemade oral liquid groups decreased to 72.90±3.60%, 74.26±3.10%, 75.12±2.26%, 76.00±2.88%, 75.70±2.77%, and 77.99±2.93%, respectively. At weeks 4 and 6, the Hyp content of each experimental group gradually increased. At week 6, there was no significant difference in the Hyp content of the homemade oral liquid group (96.40±1.98%) and the normal group (99.66±2.35%). At week 8, the Hyp contents of MYCH-200, MYCH-400, MYCH-800, commercially available products and homemade oral liquid groups were 83.00±3.64%, 88.92±2.98%, 95.14±1.91%, 90.41±2.94% and 100.78±2.33%, respectively. Among them, the Hyp content of the homemade oral liquid group was significantly better than that of the commercially available product group. The above results show that different doses of MYCH and its oral liquid described in this patent can promote the regeneration of collagen in subacute aging skin tissue. As the dose of MYCH increases, its ability to promote collagen regeneration in skin tissue becomes stronger.

[0124] 7. Analysis of the in vivo antioxidant activity of different doses of medium molecular weight yak collagen hydrolysate and its oral solution

[0125] We used a superoxide dismutase (SOD) activity detection kit to measure the SOD activity in the back skin tissue of mice to evaluate the in vivo antioxidant activity of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution. D-galactose will lead to a decrease in SOD activity in skin tissue. SOD plays a key antioxidant role in the skin. It can help scavenge free radicals to reduce oxidative stress and thus protect skin cells from oxidative damage. The better the skin condition, the higher the SOD value detected. The experimental results are as follows: Fig.10c: During the entire experiment, the skin of the mice in the normal group remained healthy, and its SOD value remained at a high level, while the SOD value of the skin of the mice in the model group remained at a low level. The SOD activity of the skin of mice in each experimental group showed a gradual upward trend. In the 8th week, the SOD activity of the skin of the MYCH-200, MYCH-400, MYCH-800, commercial products and homemade oral liquid groups increased to 139.58±9.78U / g, 162.23±13.90U / g, 175.98±11.34U / g, 166.38±12.10U / g and 192.73±10.11U / g, respectively. There was no significant difference in the SOD activity of the homemade oral liquid group and the normal group (197.97±9.12U / g). In addition, the SOD value of the homemade oral liquid group was significantly higher than that of the commercial product group. The above results show that different doses of MYCH and its oral solution described in the present application can enhance the activity of SOD in subacute aging skin tissue, thereby reducing oxidative stress. As the dose of MYCH increases, its antioxidant effect becomes stronger. In addition, the antioxidant effect of MYCH-800 is worse than that of the commercially available product group, but not as good as that of the homemade oral solution group.

[0126] Malondialdehyde (MDA) content detection kit was used to further evaluate the in vivo antioxidant activity of different doses of medium molecular weight yak collagen hydrolysate (MYCH) and its oral solution. MDA is a lipid oxidation product, and its increased content is usually associated with oxidative stress and cell damage. D-galactose can lead to an increase in MDA content in skin tissue. The experimental results are as follows Fig.10d shows: At week 2, the MDA content of the model, MYCH-200, MYCH-400, MYCH-800, commercial products and homemade oral solution groups were higher than that of the normal group (2.23±0.19nmol / g), increasing to 5.46±0.29nmol / g, 5.17±0.29nmol / g, 4.88±0.15nmol / g, 4.74±0.19nmol / g, 4.85±0.27nmol / g and 4.74±0.22nmol / g, respectively. At weeks 4 and 6, the MDA content of each experimental group was gradually decreasing. At the 8th week, the MDA contents of MYCH-200, MYCH-400, MYCH-800, commercially available products and homemade oral liquid groups were 3.09±0.13nmol / g, 2.95±0.24nmol / g, 2.66±0.21nmol / g, 2.91±0.09nmol / g and 2.52±0.22nmol / g, respectively. There was no significant difference between the MDA content of the homemade oral liquid group and the normal group (2.45±0.19nmol / g). The above results show that the different doses of MYCH and its oral liquid described in this application can reduce the content of MDA in subacute aging skin tissue, thereby alleviating oxidative stress. As the MYCH dosage increases, its repair effect is better. In addition, the oral product prepared using MYCH is better than the commercially available product.

[0127] In summary, a yak collagen hydrolyzate with skin aging effect can significantly improve photoaged skin, especially the medium molecular weight yak collagen hydrolyzate (MYCH). The oral product prepared using MYCH can accelerate the density, chromaticity, moisture and transepidermal water loss of subacute aged skin to restore to healthy skin levels, while effectively promoting the restoration of the original structure and function of each layer of aged skin and reducing oxidative stress.

[0128] The above description is only the details of individual exemplary implementation cases of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to specific preparation conditions during actual application, and is not intended to limit the present invention. Anything within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing yak collagen hydrolysate with anti-aging effect, characterized in that: The steps include: (1) Yak leather pretreatment The hair and subcutaneous tissue on the yak skin were removed, the skin was cut into small pieces, 10% n-butanol was used for degreasing, 1M NaOH was added for reaction to remove the non-collagen components in the yak skin, and the skin was washed with water until the pH value was 7 to obtain the pretreated yak skin; (2) Preparation of yak collagen filtrate The pretreated yak skin obtained in step (1) is added to a 0.5 M citric acid solution at a solid-liquid ratio of 1:10, pepsin is added to react under low temperature conditions, filtered, and the pH value is adjusted to neutral to obtain a yak collagen filtrate; (3) Preparation of yak collagen hydrolysate Add 8-120 U / mL of papain to the 50%-100% yak collagen filtrate obtained in step (2), and hydrolyze at 50° C. for 3-6 hours to obtain yak collagen hydrolyzate; (4) Enzyme inactivation The yak collagen hydrolysate obtained in step (3) is placed in boiling water; (5) Purification of yak collagen hydrolysate Take 100 mL of the yak collagen hydrolyzate obtained in step (4) and elute it in the chromatography column. The absorbance of the eluate is measured at 220 nm, and the elution curve is drawn. The molecular weight distribution of the eluate is determined by size exclusion chromatography. According to the measurement results, the target product is collected, freeze-dried, and stored at -20°C for future use.

2. The preparation method according to claim 1, characterized in that The concentration of papain in step (3) is 16-120 U / mL.

3. The preparation method according to claim 2, characterized in that: The concentration of papain in step (3) is 80-120 U / mL.

4. The preparation method according to claim 1, characterized in that: The hydrolysis time in step (3) is 4 to 6 hours.

5. The preparation method according to claim 4, characterized in that: The hydrolysis time in step (3) is 5 to 6 hours.

6. Yak collagen hydrolysate prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the yak collagen hydrolyzate as claimed in claim 6 in the preparation of skin repair materials / products.

8. Use of the yak collagen hydrolyzate according to claim 6 in the preparation of anti-aging and anti-oxidation health products and / or cosmetics.

9. An oral liquid composition with anti-aging effect, characterized in that: The oral liquid composition comprises the following raw materials in percentage by mass: 6% yak collagen hydrolysate, 0.2% bilberry extract, 0.6% vitamin C, 0.04% hyaluronic acid, 0.02% black wolfberry powder, 0.01% salidroside, and the balance is water.

10. The oral liquid composition according to claim 9, characterized in that The yak collagen hydrolysate is a yak collagen hydrolysate with a medium molecular weight.

Images