Use of a wheat germ extract enriched in polyamines

Abstract

The application discloses a new application of a wheat germ extract rich in polyamines, and results show that the wheat germ extract rich in polyamines can significantly increase cell migration ability, has the effects of promoting skin repair and healing, and can significantly promote skin fibroblast proliferation, has the effect of promoting cell proliferation activity; can significantly inhibit B16 cell melanin synthesis, has the effect of whitening activity; can delay skin aging, promote skin collagen production, and improve skin hyaluronic acid synthesis. It is shown that the wheat germ extract rich in polyamines prepared in the application can be prepared into health care products, food, cosmetics or medicines for skin repair, skin whitening and delaying skin aging, and a new way is provided for development and utilization of wheat germ.

Description

TECHNICAL FIELD

[0001] The present application relates to a new application of wheat germ extract rich in polyamines, in particular to the application of wheat germ extract rich in polyamines in the preparation of compositions for skin repair, skin whitening and delaying skin aging. BACKGROUND

[0002] Wheat germ accounts for about 3% of wheat kernels, and contains 15-40% high-quality protein, 10-20% oil and dietary fiber, minerals, vitamins and other ingredients beneficial to human health, and is praised by nutritionists as "human natural nutrient bank". In foreign countries, wheat germ is widely used in various nutritional products, health products, therapeutic foods. The prolamin in wheat germ protein is one of the main raw materials for preparing drug coatings, drug sustained-release agents, hair shampoo smoothing agents, food preservation coating films, etc., and is also used for preparing antioxidant peptides, high-F value oligopeptides, antihypertensive peptides and fat substitutes, etc. Wheat germ peptides and wheat germ antioxidant and anti-aging liquid contain various small molecule short peptides, amino acids, vitamins and mineral elements such as glutathione, and have very high nutritional value, and can be used as nutritional fortifiers, anti-aging, anti-fatigue, antioxidant agents, hair, skin and other nutritional repair agents in the health product, food and daily chemical industries. Therefore, in recent years, the development and utilization of wheat germ resources has become a research hotspot in the food, biochemical and nutritional industries at home and abroad.

[0003] At present, the utilization of wheat germ mainly focuses on three aspects: first, most of the wheat germ is mixed into bran and used as feed, causing great waste of resources; second, it is directly added to food, such as wheat germ baked food, soup base, leisure small food, etc.; third, wheat germ oil is extracted, and the defatted wheat germ is sold as feed at a low price. The main reason why wheat germ has not been reasonably utilized is that the research and development of wheat germ has been limited to the level of nutritional source, and the research on its health function is not deep enough. At present, there is no literature report on the application of wheat germ extract rich in polyamines in the preparation of compositions for skin repair, skin whitening and delaying skin aging. SUMMARY

[0004] In view of this, the purpose of the present application is to provide a new use of wheat germ extract rich in polyamines. It is found through research that the wheat germ extract rich in polyamines prepared by the present application has the effects of promoting skin repair and healing, whitening activity, delaying skin aging, promoting collagen production and improving hyaluronic acid synthesis, which provides a new direction for the research of wheat germ.

[0005] The present application provides the application of wheat germ extract rich in polyamines in the preparation of compositions for skin repair.

[0006] The second aspect of the present application provides use of the wheat germ extract rich in polyamines in the preparation of a composition for skin whitening.

[0007] The third aspect of the present application provides use of the wheat germ extract rich in polyamines in the preparation of a composition for delaying skin aging.

[0008] Preferably, the wheat germ extract contains spermidine and / or spermine.

[0009] The present application provides a preparation method of the wheat germ extract, comprising the following steps:

[0010] Step S1, the wheat germ powder is soaked in a 60-80% ethanol solution containing 0.6-1% NaOH, and the extraction supernatant is collected after filtration as the soaking liquid;

[0011] Step S2, the soaking liquid is combined as the extraction liquid, the pH of the extraction liquid is adjusted to 5-7, and the mixture is stirred and left to stand to obtain a solid-liquid mixture;

[0012] Step S3, the solid-liquid mixture is subjected to centrifugal separation to collect the solid phase rich in polyamines;

[0013] Step S4, the solid phase rich in polyamines is dried to obtain the wheat germ extract rich in polyamines.

[0014] Preferably, in step S1, the volume ratio of the wheat germ powder to the ethanol solution is 1:5-10; the soaking temperature is 25-40℃, and the soaking times are 1-3 times.

[0015] The above method uses an alkaline alcohol solution for extraction, which can swell the wheat germ raw material and avoid the dissolution of a large amount of starch and protein, so as to facilitate the release of polyamines and play a more effective role in the dissolution and extraction of polyamines. Through step S1, the polyamines are released into the soaking liquid; after acidification in step S2, the polyamines are precipitated from the soaking liquid in the form of salts, and then the solid-liquid separation and drying treatment are performed to obtain the wheat germ extract rich in polyamines.

[0016] The present application also provides another preparation method of the wheat germ extract, comprising the following steps:

[0017] (1) the wheat germ raw material is soaked in a pH 3-4 dilute acid solution containing 0.8-1.2wt% VC (ascorbic acid);

[0018] (2) the soaked wheat germ raw material is subjected to percolation with a pH 3-4 dilute acid solution, and the percolation liquid is collected;

[0019] (3) the percolation liquid is filtered through an organic membrane and / or an inorganic membrane to collect the clear liquid;

[0020] (4) the clear liquid is further filtered through a 2500-3500 Dal ultrafiltration membrane, and the retentate is collected;

[0021] (5) the retentate is boiled, sterilized and dried to obtain the wheat germ extract containing polyamines.

[0022] Preferably, in step (3), the organic membrane is a suction filtration bag, and the inorganic membrane is a ceramic membrane.

[0023] Preferably, in step (1), the amount of the dilute acid solution is 3-10 times the amount of the raw material.

[0024] Preferably, in step (5), the drying is spray drying, and the inlet air temperature of the spray dryer is controlled at 185-215℃, and the outlet air temperature is controlled at 90-110℃.

[0025] The method uses an acid solution containing ascorbic acid (VC) to soak the raw material, and then uses an acid solution to treat the soaked raw material, removes impurities in the obtained treatment liquid, and retains the target component to prepare the wheat germ extract containing polyamines, and the preparation process has a high extraction rate of polyamines.

[0026] The dosage form of the composition of the present application is a liquid preparation, tablet, capsule, granule, emulsion or suspension, which can be prepared by the conventional method of the present application.

[0027] Compared with the prior art, the present application has the following excellent effects:

[0028] The wheat germ extract rich in polyamines prepared by the method of the present application can be used to prepare a composition for skin repair, skin whitening and delaying skin aging. The results show that, on the one hand, the wheat germ extract rich in polyamines can significantly increase the cell migration ability, has the effect of promoting skin repair and healing, and can significantly promote the proliferation of skin fibroblasts, and has the effect of promoting cell proliferation activity; on the other hand, the wheat germ extract rich in polyamines can significantly inhibit the synthesis of B16 cell melanin, and has whitening activity; on the other hand, the wheat germ extract rich in polyamines can delay skin aging, promote the generation of skin collagen, and enhance the synthesis of skin hyaluronic acid. This shows that the wheat germ extract rich in polyamines prepared by the present application can be used in health care products, food, cosmetics or drugs for skin repair, skin whitening and delaying skin aging, and provides a new way for the development and utilization of wheat germ. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The preparation process diagram of the wheat germ extract;

[0030] Figure 2 The promotion of different concentrations of DA1 on cell healing;

[0031] Figure 3 Promotion of cell healing by different concentrations of DA2;

[0032] Figure 4 Effect of wheat germ extract DA1, DA2 on cell survival;

[0033] Figure 5 Effect of wheat germ extract DA1, DA2 on cell growth;

[0034] Figure 6 Effect of different concentrations of DA1 on cell cycle;

[0035] Figure 7 The proportion of cells in the division and proliferation phase under the influence of different concentrations of DA1;

[0036] Figure 8 RT-PCR detection of the effect of wheat germ extract DA1 on HAS1, HAS2, HAS3;

[0037] Figure 9 RT-PCR detection of the effect of wheat germ extract DA2 on HAS1, HAS2, HAS3. DETAILED DESCRIPTION

[0038] The application will be further described by specific embodiments, and the following examples are specific embodiments of the application, but the embodiments of the application are not limited by the following examples.

[0039] Raw materials: wheat germ (spermidine content 0.0339%, spermine content 0.0093%), commercially available.

[0040] Example 1: Preparation method of polyamine-rich wheat germ extract

[0041] Step S1: The wheat germ powder is passed through a 60-mesh screen, soaked in 80% ethanol solution containing 1% NaOH at 40°C, and the volume ratio of wheat germ powder to ethanol solution is 1:5. After soaking, the extraction supernatant is collected by filtration as the soaking liquid, and the soaking time is 1-3 times;

[0042] Step S2: Combine the soaking liquid as the extraction liquid, adjust the pH of the extraction liquid to 5-7, stir for 10 min, and stand for 10 min to obtain a solid-liquid mixture;

[0043] Step S3: Centrifugal separation of the solid-liquid mixture to collect the polyamine-rich solid phase;

[0044] Step S4: Hot air drying of the polyamine-rich solid phase at 40°C to obtain the polyamine-rich wheat germ extract DA1. Optimization process of the preparation process:

[0045] 1.1 The effect of NaOH addition on extraction

[0046] Table 1: The effect of NaOH addition on extraction

[0047]

[0048] From the above table, it can be seen that after adding NaOH in the ethanol solution, the content of spermine and spermidine in the extract can be significantly improved. In the case of only adding ethanol, the extraction rate of spermidine is about 20% of the case of adding NaOH. However, if only NaOH is added, the soaking solution is too thick and cannot be extracted and filtered to obtain the extract.

[0049] 1.2 The effect of different concentrations of NaOH on extraction

[0050] Adjust the concentration of NaOH, and the rest is the same as Example 1. The content of spermidine and spermine in the extract is detected by HPLC, and the specific values are shown in Table 2.

[0051] Table 2: The effect of NaOH concentration on extraction

[0052]

[0053] From the above table, it can be seen that as the content of sodium hydroxide in the alkaline alcohol solution increases, the dissolution rate of spermine and spermidine increases. However, when the content of sodium hydroxide exceeds 1%, the dissolution rate of spermidine and spermine no longer increases, and even the dissolution rate of spermidine decreases. This may be due to the excessive alkalinity leading to the decrease of the stability of spermidine, and part of the spermidine is decomposed.

[0054] 1.3 The effect of different concentrations of ethanol on extraction

[0055] Adjust the concentration of ethanol, and the rest is the same as Example 1. The content of spermidine and spermine in the extract is detected by HPLC, and the specific values are shown in Table 3.

[0056] Table 3: The effect of ethanol concentration on extraction

[0057]

[0058] From the above table, it can be seen that the concentration of ethanol in the range of 40-80% has little effect on the extraction and dissolution rate of spermidine. However, it was found in subsequent tests that too high water content in the ethanol solution would make it difficult to precipitate spermidine subsequently. Therefore, the selection of 80% ethanol concentration is a better choice.

[0059] 1.4 The effect of pH value on extraction

[0060] Take extraction group 1 as an example, citric acid is added to the extract liquid to acidify to the target pH value, and stirred for 10 min, and then standed for 10 min to obtain a solid-liquid mixture. The solid-liquid mixture is subjected to filtration treatment to obtain separated filtrate and precipitate. The precipitate is hot air dried at 40℃ to obtain the wheat germ extract rich in polyamines. The target pH values of each example are recorded in Table 4.

[0061] The contents of spermine and spermidine in the filtrate separated from the solid-liquid mixture and the dried precipitate are detected by the method specified in GB5009.208-2016, and the yield of spermine and spermidine is calculated by the weight of spermine and spermidine in the precipitate, and the calculation formula is yield = total amount of target substance after drying / total amount of target substance before precipitation, and the detection results are recorded in Table 4.

[0062] Table 4 Influence of pH value on extraction effect

[0063]

[0064] As can be seen from the above table, the lower the pH value, the higher the precipitation rate of spermine and spermidine, that is, the higher the yield of spermine and spermidine. However, the lower the pH value, the more impurities precipitated with spermine and spermidine, so the total amount of the obtained precipitate is more, resulting in lower purity of spermine and spermidine. In summary, when acidified to a pH value of 4-7, spermine and spermidine can be well precipitated. Further tests of the precipitation collection of extraction 2 and extraction group 3 under different acidification pH values show the same trend as the results shown in Table 4, and the higher the concentration of ethanol in the range of 40%-80%, the more conducive to the precipitation of spermine and spermidine. Therefore, considering the precipitation of impurities and the precipitation of target products, it is more conducive to improve the purity of the product to select a pH value between 5 and 7.

[0065] Example 2: Preparation method of wheat germ extract rich in polyamines

[0066] The process flow chart is shown in Figure 1 :

[0067] (1) The wheat germ raw material is soaked in 8 times the amount of pH 3 dilute hydrochloric acid solution containing 1wt% ascorbic acid (VC) for 1h;

[0068] (2) The soaked wheat germ raw material is percolated with 8 times the amount of pH 3 dilute hydrochloric acid solution, and the percolate is collected;

[0069] (3) The percolate is first filtered through a 1μm filter bag, and then the filtrate is transferred to a 0.1μm ceramic membrane for filtration, and purified water is supplemented twice during the process, each time supplementing 1 times the volume of the percolate, and the clear liquid is collected;

[0070] (4) The supernatant was further filtered through a 3500 Dal ultrafiltration membrane, and the retentate was collected;

[0071] (5) The retentate was sterilized by boiling and spray dried, with the inlet temperature of the spray dryer controlled at 185-215°C and the outlet temperature controlled at 90-110°C, to obtain a wheat germ extract DA2 rich in polyamines.

[0072] Optimization process of the preparation process:

[0073] 2.1 Effect of VC addition on extraction

[0074] Table 5: Effect of VC addition on extraction

[0075] Group Spermidine content % Spermidine relative yield % With 1 wt% VC 0.1198 94.6** Without VC, otherwise as in Example 2 0.0652 55.1

[0076] HPLC detection was performed according to the method specified in GB5009.208—2016. As shown in Table 5, the addition of VC helps release and stabilize spermidine during the spermidine extraction process, and the relative yield is increased from 55.1% to 94.6% compared to the case without VC addition.

[0077] 2.2 Effect of VC addition amount on extraction

[0078] The amount of VC added was adjusted, and the other steps were the same as in Example 2. The content of spermidine in the extract was detected by HPLC, and the results are shown in Table 6.

[0079] Table 6: Effect of different VC addition amounts on spermidine extraction

[0080]

[0081]

[0082] As shown in Table 6, the addition of VC in an amount of less than 0.8% has little effect on the extraction rate of spermidine, and the effect does not improve when the amount of VC added is more than 1.2%. Therefore, an amount of 0.8%-1.2% is a suitable range.

[0083] 2.3 Effect of pH value on extraction of spermidine from plants

[0084] The pH value of the material liquid was adjusted, and the other steps were the same as in Example 2. The content and yield of spermidine in the extract were detected by HPLC, and the results are shown in Table 7.

[0085] Table 7: Effect of different pH values on spermidine extraction

[0086] Feed pH Spermidine content % in dry product Relative yield % pH = 1 0.039 79.13 pH = 2 0.052 82.41 pH = 3 0.115 96.22 pH = 4 0.113 94.00 pH = 5 0.101 83.32 pH = 6 0.082 59.83 pH = 7 0.069 19.02 pH = 8 0.033 8.43 pH = 9 0.024 5.67

[0087] wherein the relative yield = (total amount of target component in final product / total amount of target component in raw material) x 100%.

[0088] From Table 7, it can be seen that the pH value during the extraction process changes from high to low, and the relative yield and content of spermidine increase first and then decrease. The pH value of 1-6 is suitable for the extraction of spermidine. When the pH value is 3 or 4, the content of spermidine in the solid and the yield of spermidine reach the highest value. Therefore, the solvent with pH = 3-4 can be preferably used for extraction.

[0089] 2.4 Selection of molecular weight cutoff of ultrafiltration membrane

[0090] The content of spermidine in the extract and the yield of spermidine were detected by HPLC using different molecular weight cutoff ultrafiltration membranes to separate the target components in the supernatant obtained in step (3). The results are shown in Table 8.

[0091] Table 8 Effect of ultrafiltration membranes with different molecular weight cutoff on spermidine extraction

[0092]

[0093]

[0094] As shown in Table 8 above:

[0095] (1) In the separation of spermidine, 100000 Dal, 50000 Dal and 10000 Dal ultrafiltration membranes have large pore sizes, and the solid and spermidine can all pass through the membranes, so there is no purification effect.

[0096] (2) In the separation process, the content of spermidine in the membrane concentrate can be increased, but some spermidine can pass through the membrane and the proportion of the passed spermidine is more than 30%, which causes the yield of the target product to be relatively low.

[0097] (3) In the separation of spermidine, the 3500 Dal and 2500 Dal ultrafiltration membranes have little spermidine passing through the membranes (within 10%), and the content of the membrane concentrate is relatively high, which has an enrichment effect. The retentate (concentrate) is used as the target product, and the yield of spermidine is high, so the nanofiltration concentration process can be omitted and the drying process can be directly performed.

[0098] (4) In the separation of spermidine, although the spermidine does not pass through the 1000 Dal and 500 Dal ultrafiltration membranes, the impurities in the solid also have little passing through the membranes, so the purification and enrichment effect is not obvious.

[0099] In summary, the optimal selection of the ultrafiltration membrane is between 2500 Dal and 3500 Dal, and the yield of spermidine can reach more than 90%.

[0100] Example 3: Efficacy test of wheat germ extract

[0101] 3.1 Wheat germ extract promotes skin repair and healing

[0102] Cell migration refers to the movement of cells after receiving a migration signal or feeling the stimulation of certain substances. Increased cell migration ability indicates a promoting effect on wound repair.

[0103] This test example evaluates the repair effect of the test substance by calculating the scratch healing rate based on the scratch healing speed of cells.

[0104] 1) Cell line: HaCat (human immortalized keratinocytes from the American Type Culture Collection (ATCC));

[0105] 2) Culture medium: DMEM high-sugar medium;

[0106] 3) Culture conditions: 37°C, 5% CO, saturated humidity;

[0107] 4) Solution and control: Control: cell culture medium; Test sample (TA): diluted with culture medium to the desired test concentration.

[0108] Cells were seeded in a 24-well plate, and the amount of cells was determined to reach confluence every other day. After 24 hours of adherent culture, a straight scratch was made on the bottom of the culture plate using a plastic pipette (20 μL) to ensure uniformity in force, angle, and thickness. The floating cells were washed away with PBS, and new culture medium (containing 1% fetal bovine serum) was added. The control group did not undergo any intervention, and the sample group was added with culture medium containing the test substance. The same part of the scratch with consistent thickness and cell density was selected, the coordinates were recorded, and photographs were taken at different time points for observation. The scratch healing rates of each group were compared, and the percentage of scratch healing area of each group was measured using ImageJ software.

[0109] Scratch healing rate / % = (0h scratch area - scratch area at each time point) / 0h scratch area x 100%.

[0110] Test results: The promotion of cell healing by DA1 and DA2 at different concentrations is shown in Table 9. Figure 1-2

[0111] Table 9: Promotion of cell healing by DA1 and DA2 at different concentrations

[0112]

[0113] ​The scratch healing experiment results show that the scratch healing rate of wheat germ extract of different processes at each test concentration is significantly higher than that of the blank control group (p<0.05), and the results show that 1.25-5 μg / mL of wheat germ extract can significantly promote cell migration.

[0114] The test object (wheat germ extract) is used on skin fibroblasts, and whether it can improve cell vitality is evaluated by detecting cell survival rate, cell growth curve and cell cycle.

[0115] Cell line: HSF (human skin fibroblast), culture medium: DMEM / F12 medium containing 10% FBS (complete culture medium), culture conditions: 37°C, 5% CO, saturated humidity, solution and control: blank control (Control): cell culture medium; test sample (TA): diluted with culture medium to the required test concentration, MTT, cell cycle and apoptosis detection kit from Biyun Tian Bio. In the test, cell survival rate was detected by MTT method, cell growth curve was drawn by cell counting method, and cell cycle was detected by flow cytometry (for related specific test methods, refer to Cell Experiment Guide and supplier's kit instruction).

[0116] Table 10 Effect of wheat germ extract DA1 and DA2 on cell survival

[0117]

[0118] MTT detection shows (as shown in Figure 4 and Table 10), compared with the blank control group, 2.5-10.0 mg / mL of wheat germ extract of different processes can significantly promote the proliferation of skin fibroblasts (p<0.01), and has the effect of promoting cell proliferation.

[0119] According to the culture time and cell counting results, the cell growth curve is obtained, as shown in Figure 5 . The initial growth of cells is slow, which is the lag phase, and after two to three days, the cells enter the logarithmic growth phase. Compared with the blank control group, 2.5-10 ug / ml of wheat extract of different processes can promote the growth of skin fibroblasts.

[0120] Based on the above results, the cell cycle of cells after adding the test object wheat germ extract DA1 was further detected, and the results are shown in Figure 6-7 and Table 11. With the increase of concentration, the cells in G2 / M+S phase significantly increased, indicating that the cell division was active and the proliferation activity was high.

[0121] Table 11 Effect of different concentrations of DA1 on cell cycle

[0122]

[0123] 3.3 Wheat germ extract melanin synthesis inhibition efficacy detection method:

[0124] Experimental principle: by determining the change of melanin synthesis after the test substance acts on melanocytes, the whitening activity of the test substance is evaluated.

[0125] Experimental reagents: cell line: B16 (mouse melanoma cells), culture medium: DMEM high glucose medium containing 10% FBS (complete culture medium), culture conditions: 37°C, 5% CO, saturated humidity, solution and control: negative control (CTRL): cell culture medium, positive control (PC): kojic acid (50 μg / mL);

[0126] Test sample (TA): diluted with culture solution to the required test concentration, kojic acid, L-DOPA purchased from Shanghai Yuan Ye Company;

[0127] Experimental method:

[0128] Cell plating: resuspend the cells after reviving culture with fresh complete culture medium, inoculate 2500 cells per well into a 6-well cell culture plate, and adhere to culture for 24 h. Discard the old culture medium, and add the prepared negative control, test sample group and positive control group solutions to the 6-well cell culture plate, and continue to culture for 48 hours.

[0129] Discard the old culture medium, and replace it with new culture medium containing negative control, test sample group and positive control group, and continue to culture for 48 hours. Repeat the above operation, and then continue to culture for 48-72 h (to allow the cell fusion rate to reach more than 90%).

[0130] Melanin content detection: wash the cells with PBS twice, add 0.25% trypsin to digest the cells, count, adjust the cell amount of each group to be consistent, centrifuge at 4000 rpm to collect the cell precipitate in a centrifuge tube. Add 200 μL of melanin extraction solution to each tube, heat at 80°C for 1 hour. After cooling, transfer the extraction solution to a 96-well plate, and detect the absorbance at 405 nm of each well using an enzyme marker.

[0131] Calculate the melanin synthesis inhibition rate according to formula (1).

[0132]

[0133] In the formula: T - absorbance of test sample well;

[0134] C - average of 3 absorbances of negative control group;

[0135] C0 - background absorbance of melanin extraction solution.

[0136] The results of the intracellular melanin synthesis determination are shown in Table 12. Compared with the negative control group, the melanin synthesis inhibition rate of the positive control group (kojic acid, 50 μg / mL) had a significant difference (p<0.05), and the C.V value was ≤20%, indicating that the experimental system was effective. Compared with the negative control group, the melanin synthesis inhibition rate of each test concentration had a significant difference (p<0.05), indicating that 0.25-1.00 mg / mL wheat germ extract could significantly inhibit the melanin synthesis of B16 cells.

[0137] Table 12 Results of intracellular melanin synthesis determination

[0138]

[0139]

[0140] 3.4 Fibroblast collagen production detection method:

[0141] Experimental principle: By determining the up-regulation rate of human skin fibroblast type I collagen content after administration of the test substance, the efficacy of the test substance in promoting collagen synthesis is evaluated.

[0142] Experimental reagents:

[0143] Cell line: HSF (human skin fibroblast);

[0144] Culture medium: DMEM / F12 medium containing 10% FBS (complete culture medium);

[0145] Solutions and controls: Blank control (Control): cell culture medium, test sample (TA): diluted with culture medium to the required test concentration, TGF-β1 purchased from PEPROTECH company, Collagen I elisa detection kit purchased from Wuhan Dr. Deki Biological Company.

[0146] After the cells were recovered and cultured, the cells were diluted with culture medium to the inoculation density (fusion degree reached 45%-60% 24 h after inoculation), inoculated into a 96-well plate, and the liquid volume in each well was 200 μL. After inoculation, the 96-well plate was placed in a CO incubator for 24 h±2 h. The culture medium in the 96-well plate was discarded, and the dosing operation was carried out. The test substance well was added with culture medium containing the test substance, the positive control well was added with culture medium containing the positive control, and the blank / solvent control well was added with normal cell culture medium, 200 μL per well. After the dosing was completed, the 96-well plate was placed in a CO incubator for 48 h±2 h. After the incubation and culture were completed, the cell culture supernatant was collected in a sterile centrifuge tube and stored in a -80℃ ultra-low temperature freezer. The type I collagen content was detected according to the instructions of the human type I collagen enzyme-linked immunoassay kit.

[0147] Statistical Analysis:

[0148] The standard curve regression equation was calculated by using CurveExpert 1.4 standard curve analysis software with the concentration of standard and OD450 value. The OD450 value of sample was substituted into the equation to calculate the collagen I content of sample. The average value of three duplicate wells of each group was taken as the final collagen I result. The up-regulation rate of collagen I was calculated according to formula (1).

[0149] Up-regulation rate (%) = (T / C-1) x 100% (1)

[0150] In the formula: T - the average value of collagen I content of test substance;

[0151] C - the average value of collagen I content of blank / solvent control.

[0152] The positive control should be set for each batch of test. Compared with the blank / solvent control, the up-regulation rate of collagen I content of positive control should be ≥20% to consider that the test system is effective. The coefficient of variation (C.V) was calculated by the standard deviation (SD) of optical density between duplicate wells of each group measured by microplate reader. The C.V value ≤20% to consider that the test parallelism is effective.

[0153] Table 13 Collagen I detection results

[0154]

[0155] The collagen I detection results are shown in Table 13. Compared with the Control group, the up-regulation rate of collagen I content of the positive control (TGF-β1) was (26.64±8.78) %≥20 %, and the test system was effective. The C.V value of the optical density between the duplicate wells of each group was ≤20 %, and the test parallelism was effective. Compared with the Control group, 2.5 μg / ml of wheat germ extract DA1 had no significant effect on the up-regulation of collagen I content of the cells (p>0.05), and the up-regulation rates of collagen I content of 5.0 μg / ml and 10.0 μg / ml of wheat germ extract DA1 were (14.68±2.35) % and (19.76±5.84) % respectively, and the statistical analysis had significant difference (p<0.05); the results showed that the wheat germ extract DA1 could promote the up-regulation of collagen I content of human skin fibroblasts. The up-regulation rates of collagen I content of 2.5 μg / ml, 5.0 μg / ml and 10.0 μg / ml of wheat germ extract DA2 were (13.48±2.51) %, (17.98±3.65) % and (21.46±6.34) % respectively, and the statistical analysis had significant difference (p<0.05); the results showed that the wheat germ extract DA2 could promote the up-regulation of collagen I content of human skin fibroblasts.

[0156] 3.5 Hyaluronic acid synthase expression detection method:

[0157] Experimental principle: Human hyaluronic acid synthase (Hyaluronan synthase, HAS) is a class of enzymes that play an important role in the synthesis of hyaluronic acid (Hyaluronic acid, HA), which can be divided into HAS1, HAS2 and HAS3. In this test, by determining the changes of hyaluronic acid synthase gene expression in human skin fibroblasts after administration of the test substance, the effect of the test substance on the synthesis of hyaluronic acid was evaluated.

[0158] Cell line: HSF (human skin fibroblasts);

[0159] Culture medium: DMEM / F12 culture medium containing 10 % FBS (complete culture medium);

[0160] Solution and control: blank control (Control): cell culture medium, test sample (TA): diluted with culture solution to the required test concentration, RNA extraction kit, cDNA reverse transcription synthesis kit purchased from Tiangeng Biochemical Company, SYBRGreen Pro Taq HS, premixed qPCR reagent kit purchased from Aikewei Biological Company;

[0161] After diluting the cells to the inoculation density with cell culture medium (fusion degree reached 45%~60% after 24h inoculation), inoculate into 12-well plates, each well with 1000μL of liquid. After inoculation, place in a CO incubator for 24h±2h. Discard the original culture medium and carry out the dosing operation. Add the culture medium containing the test substance to the test substance well, and add the normal cell culture medium to the blank / solvent control well, 1000μL per well. After dosing, place the 12-well plate in a CO incubator for 48h±2h. After incubation, wash with PBS for 1~2 times, collect cells, extract mRNA, quantify, reverse transcribe to obtain cDNA for Real-Time PCR reaction.

[0162] The relative expression amount of the gene was analyzed by 2-△△CT method. The average Ct value of each cDNA sample and each gene with 3 repeats was set as the amplification result, and the amplification amount of the GAPDH gene was used as the internal reference gene. The cycle threshold value of the gene was calculated as △Ct=Ct gene-Ct GAPDH, and the relative expression amount of the gene was calculated as △△Ct=△Ct test-△Ct blank control. The ratio of the expression amount of the test substance group to the blank control group was calculated by 2-△△Ct analysis.

[0163] Statistical analysis software GraphPad Prism 8.0 was used for comparison among multiple groups by one-way ANOVA, and p<0.05 was considered statistically significant.

[0164] The RT-PCR detection results of the effect of wheat germ extract DA1 on HAS1, HAS2 and HAS3 are shown in Figure 8-9 Compared with the blank control group, 2.5μg / mL, 5.0μg / mL and 10.0μg / mL wheat germ extract had a significant promoting effect on the expression of hyaluronic acid synthase HAS2 in human skin fibroblasts (p<0.05). The test concentrations of wheat germ extract had no obvious promoting effect on the expression of hyaluronic acid synthase HAS1 and HAS3 in human skin fibroblasts.

Claims

1. Use of a wheat germ extract enriched in polyamines for the preparation of a composition for the repair of the skin, the lightening of the skin or the delay of the ageing of the skin, characterized in that, The wheat germ extract contains spermidine and / or spermine, and the preparation method of the wheat germ extract comprises the following steps: S1, the wheat germ powder is soaked in a 60-80% ethanol solution containing 0.6-1% NaOH, and the extraction supernatant is collected after filtration as the soaking liquid; S2, the soaking liquid is combined as the extraction liquid, the pH of the extraction liquid is adjusted to 5-7, and stirring and standing are performed to obtain a solid-liquid mixture; S3, the solid-liquid mixture is subjected to centrifugal separation, and the solid phase rich in polyamines is collected; S4, the solid phase rich in polyamines is dried to obtain the wheat germ extract rich in polyamines.

2. Use according to claim 1, characterized in that, In the step S1, the volume ratio of the wheat germ powder to the ethanol solution is 1:5-10; the soaking temperature is 25℃-40℃, and the soaking times are 1-3 times.

3. Use of a wheat germ extract enriched in polyamines for the preparation of a composition for skin repair, skin lightening or delaying skin aging, characterized in that, The wheat germ extract contains spermidine and / or spermine, and the preparation method of the wheat germ extract comprises the following steps: (1) the wheat germ raw material is soaked in a pH 3-4 dilute acid solution containing 0.8-1.2wt% ascorbic acid; (2) the soaked wheat germ raw material is subjected to percolation with a pH 3-4 dilute acid solution, and the percolation liquid is collected; (3) the percolation liquid is filtered through an organic membrane and / or an inorganic membrane, and the clear liquid is collected; (4) the clear liquid is subjected to ultrafiltration through a 2500-3500Dal membrane, and the cut-off liquid is collected; (5) the cut-off liquid is boiled for sterilization and drying to obtain the wheat germ extract containing polyamines.

4. Use according to claim 3, characterized in that, In the step (3), the organic membrane is a suction filter bag, and the inorganic membrane is a ceramic membrane.

5. Use according to claim 3, characterized in that, In the step (1), the amount of the dilute acid solution is 3-10 times the amount of the raw material; in the step (5), the drying is spray drying, the inlet air temperature of the spray dryer is controlled to be 185-215℃, and the outlet air temperature is controlled to be 90-110℃.

6. The use according to any one of claims 1 or 3, characterized in that, The dosage form of the composition is a liquid preparation, a tablet, a capsule or a granule.

Images