Composition for treating skin decline problem as well as preparation method and application of composition

Abstract

The invention discloses a composition for treating the problem of skin decline, a preparation method and application, and belongs to the technical field of health care and skincare, and the composition is mainly prepared from the following raw materials: cactus flower extract, abalone peptide, melibiose and the like. After the cactus flower extract, the melibiose and the abalone peptide are combined, the postpartum anti-aging effect is achieved through a new mechanism, and the anti-aging effect is obviously improved and is superior to that of any single component. Experimental research proves that the composition has anti-aging activity by activating calreticulin of fibroblasts in postpartum skin, enhancing the antigen presentation capacity of dendritic cells, activating a non-classical NF-kappa B signal channel, promoting lymphocyte homing and prolonging T cell telomere, does not have the mechanism when any component is independently used, and has no toxic or side effect. The composition is innovative in the anti-aging aspect.

Description

Technical Field

[0001] The present invention relates to the technical field of health care and skin care, and in particular to a pharmaceutical composition for treating skin atrophy and the like caused by postpartum hormones and its application. Background Art

[0002] Cactus flower is the flower of perennial shrub cactus of Cactaceae. It is yellow and can be dried and used as medicine. It has the effects of clearing heat and moistening lungs, calming nerves and calming mind. It can treat tuberculosis, bronchitis, cervical lymphadenopathy, palpitation and other diseases. The main active ingredients of cactus flower are polysaccharides, flavonoids, sterols, alkaloids, etc., which have multiple biological activities and pharmacological effects such as anti-free radicals, antioxidants, antibacterial and antiviral, anti-inflammatory, and anti-cancer. It is known that external application of cactus can make the drug power directly reach the lesion to play a role, and can also make the drug properties enter the inside through the skin and fur, promote the regulation of meridians, and is often used to treat acute mastitis after childbirth. It is further discovered that cold compress of sliced ​​cactus on the breast can also reduce the skin temperature of breast swelling, keep it moist, and beautify the local skin of the breast. With the progress of the times and the improvement of living standards, cactus flower has become a popular health direction in the world as a dual-purpose product for medicine and food. After reviewing relevant literature, it was found that there are not many studies on cactus flowers at home and abroad, and there are even fewer in-depth studies on the physiological activities of cactus flowers. There is a lack of in-depth research on skin problems and molecular mechanisms caused by postpartum immune function and hormone levels.

[0003] Melibiose is a right-handed disaccharide, which is a disaccharide formed by the combination of D-galactose and D-glucose 1,6. Its chemical system name is O-α-galactopyranosyl-(1→6)-D-glucopyranose. It exists in floating wheat, honey, and legumes, but the content is relatively small. In industry, yeast is often used to ferment raffinose to produce melibiose. Melibiose has anti-inflammatory properties and can slow down the allergic reaction of the skin. It has good permeability on the skin, can improve skin condition, increase skin moisture, slow down the degradation of elastin, maintain skin elasticity, and improve eyelid swelling and eye bags. Conventional cell experiments show that melibiose exerts anti-inflammatory and anti-aging effects by promoting the production of type 3 collagen, inhibiting the activity of elastase, the activity of B-16 melanocytes, and the production of interleukin IL-8. Exploring the immune anti-aging mechanism of melibiose in postpartum skin has important scientific and application value.

[0004] Abalone peptide is a small molecule peptide (<1000Da) obtained by proteolysis technology using abalone as raw material. The Compendium of Materia Medica records that abalone is flat in nature, sweet and salty in taste, can improve eyesight, nourish deficiency, clear heat and nourish yin, nourish blood and benefit the stomach, and nourish the liver and kidneys, and is known as the "eye-improving fish". The Annotations to the Classic of Materia Medica records that abalone can improve eyesight, regulate menstruation and moisten the intestines, and is mainly used to treat diseases such as irregular menstruation and leukorrhea, which is related to the blood stasis disease recorded in the Classic of Internal Medicine. Abalone peptide contains rich peptides, amino acids, natural polyphenols, xanthines, ceramides and other ingredients, and has the effects of anti-oxidation, delaying aging, improving immunity, improving sleep quality, and relieving fatigue. With the improvement of people's living standards, abalone peptides, which are constantly developed under the medicinal value of abalone, have entered the research field of scientific researchers, confirming that the anti-aging, whitening and wrinkle-removing effects of abalone peptides on the skin are produced by removing free radicals in the body and preventing tyrosine in the skin from converting into melanin. In recent years, relevant literature reports at home and abroad have mainly focused on new technologies and industrialization of abalone peptide production and abalone peptide active substances. The actual mechanism and mode of action of the active substances need to be studied urgently.

[0005] Changes in female hormones have an important impact on women's lives. Female aging is caused by changes in hormone levels in women. Women gradually enter the aging period from the age of 35, which is mainly manifested in: menopausal syndrome, uterine atrophy, vulvar and vaginal atrophy, pelvic organ ptosis, breast atrophy, skin changes, etc. Among female hormones, estrogen and progesterone play a dominant role in the regulation of female aging, and estrogen and progesterone have become the research hotspots of female hormone anti-aging. Estrogen, also known as female hormones and female hormones, is a major type of female hormone. Estrogen is divided into two major categories, both of which are steroid hormones, namely estrogen (also known as estrogen) and progesterone. Changes in estrogen and progesterone levels are closely related to skin aging. Pregnant women have higher levels of estrogen and progesterone in their bodies during pregnancy. After delivery, the secretion of estrogen and progesterone gradually decreases, and the hormone level begins to decline, so the skin becomes loose, dry, and wrinkles appear. Therefore, it can be seen that the reduction of estrogen and progesterone does accelerate the aging process of the human body. Therefore, it is crucial to deeply study the mechanism of action of estrogen and progesterone on female aging.

[0006] The skin is composed of three layers, epidermis, dermis and subcutaneous layer. The dermis is mainly composed of extracellular matrix (ECM), such as collagen. In addition, the dermis also contains structures such as blood vessels, lymphatic vessels, nerves, sweat glands and sebaceous glands. Within the dermis, there are diverse populations of mononuclear phagocytes, including dermal dendritic cells and dermal macrophages. Dendritic cells are sentinels of the immune system that sample the microenvironment and either present antigens to resident T cells or migrate to lymph nodes through lymphatic vessels to initiate an immune response.

[0007] Calreticulin (CRT) is an endoplasmic reticulum (ER) Ca2+ Binding protein, molecular chaperone, widely distributed, also exists in the cell nucleus, nuclear membrane, cell membrane surface and extracellular matrix. CRT has different functions in different locations. CRT in ER has the function of regulating intracellular Ca 2+ Homeostasis, assisting protein correct folding, regulating cell apoptosis, etc. Non-endoplasmic reticulum CRT regulates cell adhesion, migration, proliferation and fibrosis, mediates the recognition and phagocytosis of apoptotic cells, and also participates in antigen presentation in the adaptive immune response and tumor cell recognition process. It has good clinical application prospects in wound repair, tumor and autoimmune disease treatment. Studies have found that CRT can appear on the surface of almost all immune cells, such as monocytes, macrophages, neutrophils, T cells, DCs, fibroblasts and endothelial cells, and the expression of CRT changes with age, suggesting that CRT plays an important immunological function in the extracellular environment and aging immunity. Although great progress has been made in the study of the immunobiological function of CRT in recent years, there are still many unresolved issues, such as how does CRT on the surface of immune cells play an immune response function? How does CRT affect the activation process of immune cells? What is the mechanism by which CRT affects immunity? I believe that with the continuous deepening of research, our understanding of the immunoregulatory effect of CRT and the body's immune regulatory network will be clearer.

[0008] NF-κB is a family of transcription factors. After cells are stimulated by inflammatory mediators, viral infection, oxidative stress, etc., NF-κB can be activated, thereby initiating the transcription of related genes and participating in various biological processes, including immune response, inflammation, cell growth, survival and development. According to the different activation pathways, NF-κB signals can be divided into classical activation pathways and atypical activation pathways. The latter helps to express pleiotropic biological functions by controlling the fine-tuning of its own and / or common NF-κB target genes, which are mainly related to the development and function of immune cells, namely T cells, B cells, thymocytes, macrophages, fibroblasts, and neurons.

[0009] The skin is the interface between the body's internal environment and the outside world, and the immune system plays an important role in this interface. Lymphocyte homing (LH), that is, lymphocytes selectively cross the capillary high endothelial venules, migrate in a directional manner and enter peripheral organs or specific tissue areas, is an important process in the body's immune system, especially the homing of T lymphocytes, which is crucial for immune surveillance. Peripheral mature T lymphocytes are divided into CD4 + and CD8 +T lymphocytes, but the initial T cells (TN) after antigen activation can be divided into central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF). The three types of T lymphocytes each express different constitutive receptors and ligands, allowing them to interact with microvessels or epithelial cells in different regional tissues, and then migrate to specific sites to play a role. TN can preferentially migrate to secondary lymphoid tissues. TCM can preferentially migrate to secondary lymphoid tissues and bone marrow, while TEM and TEFF migrate to peripheral non-lymphoid tissues such as skin, intestinal mucosal lamina propria, liver, lungs and inflammatory sites to play an effector role in killing target cells. Different T lymphocyte subsets migrate to different lymphoid and non-lymphoid tissues, allowing the body to perfectly exert its immune defense, immune surveillance and immune self-stabilization capabilities.

[0010] The average length of telomeres shortens with the increase of cell division times and age. The proliferation capacity of normal human fibroblasts in vitro culture is not unlimited. Cell division and proliferation will appear aging after 40 to 60 generations. This is because DNA polymerase cannot completely replicate the 3' end of linear chromosomes, resulting in the continuous loss of telomeric DNA repeat sequences with each round of DNA replication. When it shortens to a certain length, the cell enters the aging stage. A large number of studies have shown that telomeres play an important role in individual aging. Telomere length is one of the key factors of aging. The shortened telomere length in aging T cells may be one of the reasons why aging leads to abnormal immune function of the body.

[0011] Because the skin is exposed, it is easily affected by external environmental factors (ultraviolet radiation, air pollution, smoking, etc.), and with the increase of age, the human immune system undergoes characteristic changes, which often lead to weakened immune ability and cause immune aging. When aging causes the immune system to decline in function, it can lead to a decrease in the body's ability to clear harmful substances such as toxins and pathogens, presenting a chronic inflammatory state. In addition, changes in various characteristic cells of the skin's immune system also affect the skin, the largest immune organ, causing the epidermis and dermis to become thinner, water loss to increase, and collagen and elastin to break, which seriously damages the skin's immune defense ability. Under the combined effect of many factors, skin aging occurs.

[0012] Aging is the final outcome that is difficult for individuals of any species to avoid. With the development of society, people's pursuit of anti-aging has intensified, but our research on anti-aging mechanisms is still quite limited. Given its importance, the development of anti-aging compositions with novel mechanisms, outstanding activity, significant efficacy, and high safety is one of the hot topics in biological research. Summary of the invention

[0013] The purpose of the present invention is to solve the above problems and provide a pharmaceutical composition with anti-aging activity and its application.

[0014] In order to achieve the above-mentioned object, the technical scheme of the present invention is as follows: a composition for treating skin deterioration, characterized in that the composition consists of cactus flower extract, melibiose and abalone peptide.

[0015] Furthermore, the composition is composed of 0.01% to 16% of cactus flower extract, 0.05% to 12% of melibiose, and 0.01% to 15% of abalone peptide in terms of mass fractions.

[0016] Furthermore, the composition exhibits anti-aging activity by activating calreticulin in fibroblasts in postpartum skin, enhancing the antigen presentation ability of dendritic cells, activating the "non-classical" NF-κB signaling pathway, promoting lymphocyte homing, and extending T cell telomeres.

[0017] Furthermore, the composition is combined with auxiliary materials or solvents to prepare skin care and anti-aging products such as facial masks, eye creams, essence water, essence milk, facial creams, body lotions, oral liquids, capsules, etc.

[0018] The present invention also provides an anti-aging skin care product containing cactus flower extract, melibiose and abalone peptide. The product contains 0.01% to 16% of cactus flower extract, 0.05% to 12% of melibiose, and 0.01% to 15% of abalone peptide in terms of mass fraction, and can treat skin deterioration problems caused by postpartum hormonal changes.

[0019] Furthermore, the cactus flower extract is a water extract of cactus flower, which is obtained by freezing and crushing fresh cactus flowers in a low-temperature refrigerator, stirring them evenly under 60-65°C water, adding activated carbon and letting them stand for 24 hours, and removing the activated carbon by centrifugation at 4000r / min for 5 minutes.

[0020] Furthermore, the product activates fibroblast calreticulin in the skin, enhances the antigen presentation ability of dendritic cells, activates the "non-classical" NF-κB signaling pathway, promotes lymphocyte homing, and prolongs T cell telomeres, thereby exerting an anti-aging effect.

[0021] Furthermore, the product includes the following forms: facial mask, eye cream, essence water, essence lotion, facial cream, body lotion, oral liquid or capsule.

[0022] The present invention also provides a method for preparing an anti-aging skin care product containing cactus flower extract, melibiose and abalone peptide, comprising the following steps:

[0023] Step 1, after freezing and crushing fresh cactus flowers in a low-temperature refrigerator, stir them evenly in water at 60-65° C., add activated carbon and let them stand for 24 hours, then centrifuge at a speed of 4000 r / min for 5 minutes to remove the activated carbon, and obtain a light yellow cactus flower water extract;

[0024] Step 2: Mix the above-mentioned cactus flower water extract, melibiose and abalone peptide according to a predetermined mass ratio, stir evenly, add appropriate amount of auxiliary materials or solvents to prepare an anti-aging skin care product.

[0025] Furthermore, in step one, through the method of low-temperature freezing and appropriate temperature heating extraction, the structural changes of the natural active ingredients of cactus flowers are minimized, their natural aroma is retained, and the extraction efficiency is effectively improved.

[0026] Compared with the prior art, this solution has the following beneficial effects:

[0027] The composition of the invention has anti-aging function and is suitable for anti-aging of postpartum skin.

[0028] The melibiose and abalone peptide in the composition of the present invention have certain immune-enhancing activity, but it is weak, and the activity of the combination of the two is significantly higher than that of any single component, which is innovative. Mechanism research found a new mechanism of the composition, that is, melibiose and abalone peptide activate calreticulin in fibroblasts in postpartum skin, enhance the antigen presentation ability of dendritic cells, and activate the "non-classical" NF-κB signaling pathway, promote lymphocyte homing, and extend T cell telomeres, showing anti-aging activity, which is innovative. The anti-aging mechanism of melibiose and abalone peptide is different from the new mechanism produced by the composition of the present invention. Previous studies have found that melibiose has anti-aging activity by promoting the production of type 3 collagen and inhibiting the activity of elastase. Abalone peptides exert anti-aging effects by scavenging free radicals in the body through their own natural polyphenols and xanthines, and no new mechanism of the composition of the present invention has been found. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The effect of each group on postpartum skin repair in mice (a is the HE staining picture of the mouse back tissue, and b is the epidermal thickness)

[0030] Figure 2 is the effect of the present invention on the expression of estrogen and progesterone levels in serum;

[0031] Figure 3 Effects of the present invention on the expression of calreticulin in fibroblasts;

[0032] Figure 4 is the effect on the antigen presenting ability of dendritic cells (DCs) in the embodiments of the present invention;

[0033] Figure 5 is the effect on the NF-κB signaling pathway in the embodiments of the present invention;

[0034] Figure 6 is the effect on lymphocyte homing in the embodiments of the present invention;

[0035] Figure 7 This is the effect on T cell telomeres in the embodiments of the present invention. DETAILED DESCRIPTION

[0036] The present invention and its implementation methods are described below, which are not restrictive and the actual implementation methods are not limited thereto. Based on the embodiments of the present invention, if ordinary technicians in the field are inspired by them and design structural methods and implementation methods similar to the technical solution without creative design without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

[0037] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0038] Example 1 A composition with anti-aging effect

[0039] S1: After cleaning the container suitable for the reaction, perform high temperature sterilization;

[0040] S2: Add appropriate amount of water, glycerin and emulsifier into the sterilized container and heat to 30-45°C while stirring to fully dissolve and mix them to obtain a compound of group A;

[0041] S3: 40 g of cactus flower extract, 35 g of melibiose and 10 g of abalone peptide were added into a sterilized container containing the compound of group A in sequence, and heated to 30-45° C. while stirring to obtain a compound of group B;

[0042] S4: After the two-phase composite material AB is fully dissolved and mixed evenly, it is cooled to room temperature to obtain the anti-aging composition.

[0043] Example 2

[0044] S1: After cleaning the container suitable for the reaction, perform high temperature sterilization;

[0045] S2: Add appropriate amount of water, glycerin and emulsifier into the sterilized container and heat to 30-45°C while stirring to fully dissolve and mix them to obtain a compound of group A;

[0046] S3: 50 g of cactus flower extract, 25 g of melibiose and 5 g of abalone peptide were added into a sterilized container containing the compound of group A in sequence, and heated to 30-45° C. while stirring to obtain a compound of group B;

[0047] S4: After the two-phase composite material AB is fully dissolved and mixed evenly, it is cooled to room temperature to obtain the anti-aging composition.

[0048] Example 3

[0049] S1: After cleaning the container suitable for the reaction, perform high temperature sterilization;

[0050] S2: Add appropriate amount of water, glycerin and emulsifier into the sterilized container and heat to 30-45°C while stirring to fully dissolve and mix them to obtain a compound of group A;

[0051] S3: 48 g of cactus flower extract, 12 g of melibiose, 20 g of milk exosomes and 20 g of abalone peptide were added into a sterilized container containing the compound of group A in sequence, and heated to 30-45° C. while stirring to obtain a compound of group B;

[0052] S4: After the two-phase composite material AB is fully dissolved and mixed evenly, it is cooled to room temperature to obtain the anti-aging composition.

[0053] Pharmacodynamics studies

[0054] 1. Experimental Materials

[0055] Experimental animals: SPF grade BALB / c postpartum female mice, 12 weeks old, 23.7±7.5g, were adaptively raised in the animal room environment for 7 days before the experiment, with an ambient temperature of 20-25°C, a relative humidity of 40-70°C, and light and darkness alternating for 24 hours per day. The sample was the composition obtained in Example 3, and the cactus flower extract, melibiose extract and abalone peptide extract were prepared respectively by the method of Example 3.

[0056] Experimental model: SPF BALB / c postpartum female mice were divided into 6 groups, 10 mice in each group, including model group, β-nicotinamide mononucleotide NMN positive control group, cactus flower extract group, melibiose extract group, abalone peptide extract group, and combination administration group. The prepared combination or saline was directly applied to the back and ear skin of mice, 10 mice in each group, 3 times a day, for 30 consecutive days.

[0057] 2. Experimental methods

[0058] 2.1 Detection methods of serum estrogen and progesterone

[0059] 0.5-1 mL of blood was collected from the eyeballs of fasting mice and centrifuged at 1500 r / min for 5 minutes. The upper serum was collected and stored at ultra-low temperature for sample detection of estrogen (E2) / progesterone (PRGE).

[0060] 2.2 HE staining method to detect the pathological condition of mouse back skin tissue

[0061] The skin of the back of the mouse (2×2cm) was surgically removed, and the subcutaneous fat, blood vessels and other redundant tissues were removed and placed in 4% tissue fixative. The paraffin sections were placed in xylene I for 10min-xylene II for 10min-xylene III for 10min-anhydrous ethanol I for 5min-anhydrous ethanol II for 5min-90% alcohol for 5min-80% alcohol for 5min-70% alcohol for 5min-50% alcohol for 5min gradient dewaxing. The sections that had been put into distilled water were placed in a hematoxylin aqueous solution for staining for several minutes, and then placed in acid water and ammonia water for color separation for several seconds each. After rinsing with running water for 1h, they were immersed in distilled water for a while, and then placed in 70% and 90% alcohol for dehydration for 10min each, and stained with alcohol eosin staining solution for 2-3min. The stained sections were dehydrated with pure alcohol, and then made transparent with xylene. The transparent sections were dripped with gum, covered with coverslips for sealing, and after the gum was slightly dry, labels were attached and observed under a microscope.

[0062] 2.3 Immunochemical staining and enzyme-linked immunosorbent assay (ELISA) detection of CRT expression

[0063] The back skin of the mouse was surgically removed and placed in a PBS buffer containing double antibodies. The subcutaneous fat, blood vessels and other redundant tissues were removed, washed with PBS, and digested with trypsin for 1 hour. The true epidermis was separated, removed, and washed with PBS three times. The dermis was cut into pieces and inoculated in a culture dish, and an appropriate amount of trypsin was added. It was transferred to a 15mL centrifuge tube and digested in a constant temperature water bath at 37°C for 1 hour. After digestion, DMEM culture medium containing 10% bovine serum was added to terminate the reaction, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. Wash once with PBS, centrifuge and discard the supernatant. The washed tissue blocks were evenly spread in a culture dish, and DMEM culture medium containing 10% bovine serum was added.

[0064] Base, 37°C 5% CO 2 After static culture, change the medium when fibroblasts grow out of the tissue block, remove the culture medium, wash gently with PBS, fix with 2% paraformaldehyde for 10 minutes, react with 0.3% hydrogen peroxide for 5 minutes, and wash again with PBS; incubate with secondary antibody serum at 37℃ for 20 minutes, act with anti-CRT antibody at 37℃ for 45 minutes, react with HRP-anti-sheep IgG at 37℃ for 1 hour, and develop color with DAB; observe the color signals of each group under a microscope.

[0065] The following detection steps were performed using an enzyme-linked immunosorbent assay (ELISA) kit according to the instructions: each group was digested with 0.25% trypsin, and the cells were collected and the cell concentration was adjusted to 10 5 / mL. Add 100μL of the sample to be tested to each well, mix the reaction plate thoroughly and place it at 37℃ for 120 minutes; wash the reaction plate thoroughly 4-6 times with washing solution, blot dry on filter paper, and add 100μL of the first antibody working solution to each well. Mix the reaction plate thoroughly and place it at 37℃ for 60 minutes; after washing the plate as before, add 100μL of enzyme-labeled antibody working solution to each well, mix the reaction plate thoroughly and place it at 37℃ for 30 minutes; after washing the plate as before, add 100μL of substrate working solution to each well, place it at 37℃ in the dark for 15 minutes: add 100μL of stop solution to each well and mix well, and measure the absorbance at 450nm with an enzyme reader within 30 minutes;

[0066] 2.4 DCs antigen presentation ability detection

[0067] 2.4.1 Isolation of dendritic cells

[0068] The skin on the back of the mouse was scraped and collected in a culture dish. PMI-1640 medium containing double antibodies was added and incubated at 37°C with 5% CO 2 Incubate for 45 minutes, discard the supernatant, add new PMI-1640 medium containing double antibodies, culture for 45 minutes, discard the supernatant, transfer the skin tissue to a cell bottle, add cRPMI-1640, culture for 12 hours, transfer to a 250mL Erlenmeyer flask, stir magnetically for 3 minutes, filter with 150 mesh and 300 mesh steel sieves, collect the filtrate in a centrifuge tube, centrifuge at 2000r / min for 5 minutes, place the suspended cells on 2 times the volume of lymphocyte separation solution, centrifuge at 2000r / min for 20 minutes, and collect the middle cell layer. Resuspend the separated skin-derived dendritic cells with RPMI140 complete culture medium solution and adjust the cell concentration to 5x10 5 / mL, in 4 culture flasks.

[0069] 2.4.2 Preparation of Responder Cells (Allogeneic Splenic T Cells)

[0070] (1) OVA solution and complete Freund's adjuvant were mixed in equal volumes to form an emulsion. Each mouse was immunized with 100 μg of OVA each time. OVA protein was subcutaneously immunized in BALB / c (H-2 d ) mice.

[0071] (2) On days 7 and 14, equal volumes of OVA and incomplete Freund's adjuvant were mixed into an emulsion and the immunization was repeated once at the above doses.

[0072] (3) Three days after the last immunization, the spleen was removed aseptically, the outer surface was washed with PBS, the surrounding adipose tissue was removed, and the spleen was placed in a culture dish with a 200-mesh stainless steel cell sieve (previously sterilized by high pressure). 10 mL of PBS was added to the culture medium, and the spleen was gently ground with the needle of a 5-mL glass syringe to allow lymphocytes to flow into the dish through the omentum. The remaining white spleen capsule and connective tissue were removed.

[0073] (4) Add the cell suspension to a centrifuge tube. After centrifugation, add 10 times the volume of red blood cell lysis buffer and treat for 3 to 5 minutes to remove red blood cells. Wash twice by centrifugation (200 x g, 5 min). Discard the supernatant, resuspend the cells in RPMI1640 complete medium, add 3 mL of lymphocyte separation buffer, centrifuge (300 x g, 30 min, 20°C), carefully aspirate the white mist in the middle, and wash three times with RPMI1640 basal medium.

[0074] (5) Add OVA solution to the lymphocytes to a final concentration of 100 g / mL and adjust the cell concentration to 1x10 with RPMI1640 complete medium. 6 100 μL / well was added to a round-bottom 96-well plate.

[0075] 1x10 4 , 5x10 3 The collected DCs were added to each well, and the total volume was adjusted to 200 μL / well. Finally, 10 OVA-sensitized lymphocytes were added. 5 Each group was set up with 4 replicate wells. The cells were placed at 37°C and 5% CO 2 Incubate for 72 hours in an incubator, add 5 mg / mL MTT, 20 μL / well, in the last 4 hours, centrifuge at 300 x g for 5 minutes, remove the supernatant, add DMSO 150 μL / well, shake for 10 minutes, and measure the OD value at 570 nm on an ELISA reader. The results are expressed as the average of 4 wells.

[0076] 2.5 Detection of p52 expression in the NF-κB pathway using Western blot technology

[0077] According to the grouping method of model group, positive control group, cactus flower extract group, melibiose extract group, abalone peptide extract group, and combination administration group, fibroblasts of model mice with good growth status and culture dish coverage rate of 70%-80% were selected and cultured at a rate of 1×10 6 The cells were seeded at a density of 100 cells / well in a six-well plate. After culturing for 24-48 hours, the original culture medium was discarded, and 2 mL of serum-free culture medium was added to each well after rinsing with PBS. 5 μM B0222 mL of NF-κB-inducing kinase (NIK) inhibitor diluted with normal saline was added to the fibroblasts of the other five groups except the model group as the control group. The control groups were divided into C1, C2, C3, C4, C5, and C6 and cultured in a normal incubator for six hours.

[0078] 2.5.1 Nuclear protein extraction method

[0079] Take out the six-well plate, discard the supernatant, rinse twice with PBS, add 1mL PBS to each well, scrape the cells with a cell scraper to separate the cells from the bottom of the culture dish, add the PBS containing cells to the marked EP tubes, centrifuge at 4℃, 1000rpm for 3min, carefully aspirate the supernatant, wash the cells twice with cold PBS, and aspirate the supernatant as much as possible after each wash. Add 200μL cold BufferA and 15μL 10% NP-40 to each centrifuge tube, vortex at high speed for 15 seconds, put on ice for 10 minutes, vortex at high speed for 15 seconds every 3min, shake for 30min, centrifuge at 4℃, 14800rpm for 30min, and discard the supernatant. Add 200 μL cold Buffer B and 15 μL 10% NP-40 to the precipitate, vortex at high speed for 15 seconds, place on ice for 10 minutes, vortex at high speed for 15 seconds every 10 minutes, shake for 40 minutes, centrifuge at 4°C, 14800 rpm for 10 minutes, quickly aspirate the supernatant into another pre-cooled clean centrifuge tube to obtain the nuclear protein.

[0080] 2.5.2 BCA method to measure protein content:

[0081] Weigh 20 mg BSA standard, dissolve it in protein standard solution and prepare a 15 mg / mL protein standard solution. Take 2 μL of 15 mg / mL protein standard solution and dilute it with saline to a final concentration of 0.15 mg / mL. Add 20 μL of saline to each well of the 96-well plate and 2 μL of protein sample. Add 200 μL of BCA working solution to each well and place it at 37°C for 30 minutes. After 30 minutes, take out the 96-well plate and place it in an ELISA reader to detect its absorbance. Detect the absorbance at 570 nm and calculate the final sample protein concentration based on the standard curve calculated by the computer and the sample volume used.

[0082] 2.5.3 Western blot detection steps

[0083] (1) SDS-PAGE gel preparation: Prepare the required SDS-PAGE gel according to the instructions.

[0084] (2) Add 5× bµffer to the protein sample and heat at 100°C for 15 minutes.

[0085] (3) Loading: Add the cooled protein sample into the gel wells as required.

[0086] (4) Electrophoresis: Connect the equipment as needed and set the voltage to 80 V and the time to 80 minutes.

[0087] (5) Transfer: Soak the PVDF membrane in methanol, take out the gel, place it neatly in a "sandwich" pattern and insert it into the transfer tank, fill it with transfer buffer, place the transfer tank on ice, and transfer at 80V for 1.5 hours. The entire transfer process requires an ice bath.

[0088] (6) Blocking: Block the PVDF membrane with 5% skimmed milk powder for 1 h.

[0089] (7) Primary antibody incubation: After blocking is complete, discard the blocking solution and add TBST buffer to wash four times to remove loosely bound proteins. Prepare the primary antibody according to the antibody instructions, add the transfer membrane and 2 mL of antibody to the sealing film, place the sealing film on a shaker, and incubate overnight at 4°C.

[0090] (8) Secondary antibody incubation: After the primary antibody incubation is complete, the primary antibody is recovered and washed four times with TBST to remove the loosely bound primary antibody. The secondary antibody is prepared according to the antibody instructions, the transfer membrane and 2 mL of antibody are added to the sealing film, and the sealing film is placed on the bed, incubated at room temperature for 1 hour, and then washed three times with TBST, each time for 15 minutes.

[0091] (9) Development: Incubate with the prepared color developing solution for 2 min in a dark environment, then expose to light for development. The grayscale value of the experimental results was analyzed using ImageJ and then graphed using GraphPad.

[0092] 2.6 Lymphocyte homing

[0093] 2.6.1 Adoptive T lymphocyte experiment

[0094] Take 15 normal mice of the same age, kill them by cervical dislocation, and disinfect them with 75% alcohol for 10 minutes. Remove the submandibular lymph nodes of the mice on a clean bench and place them on a moistened 200-mesh stainless steel filter. Cut the lymph nodes into pieces and gently grind them with a sterilized glass needle. Add pre-cooled PBS containing 1% fetal bovine serum to rinse the filter while grinding. Collect as much suspension as possible in a centrifuge tube and centrifuge at 2000r for 5 minutes at room temperature. Discard the supernatant and collect the cell precipitate. Take about 10 6 Resuspend the cells in 90 μL PBS, add 10 μL mouse anti-CD8 magnetic beads to make a 100 μL system, and incubate in a refrigerator at 4°C for 30 min. Take out the incubated centrifuge tube, add 10 times the volume of 1% fetal bovine serum in PBS, centrifuge at 2000r for 10 min at room temperature, collect the cell pellet, and resuspend in 5 mL PBS. Use a miniMACS sorter equipped with LS sorting columns to collect CD8 + Place T cells in the same centrifuge tube, pipette and mix, pipette 10 μL of the suspension into a cell counting plate, count the number of cells, centrifuge at 2000r for 10 min at room temperature, and collect the cell precipitate. + T cells were added to PBS and adjusted to 1x104 / mL, repeatedly blow the cells. Pass through a 200-mesh filter, add 5mM CFSE mother solution to the filtered single-cell suspension to make it a 5μM working solution, and incubate at 37°C for 30min. Add 10 times the volume of PBS to wash, and at the same time, draw 10μL on the cell counting plate and count, and at the same time, draw 10μL and place the counting plate under a laser microscope to check the CESE staining. Centrifuge the remaining cell solution at 2000r for 10min, collect the cell pellet, and return 5x10 6 Count and resuspend the cells at 100 cells / 200 μL. Dispense into sterilized EP tubes, 40 μL per tube, for other experiments. Inject 20 μL of CFSE-labeled cell suspension into the auricular vein of mice in the control and experimental groups according to the dose.

[0095] 2.6.2 Flow cytometry detection of CFSE-labeled CD8 + T cell homing

[0096] 24 hours after the mice were adoptively transferred with CFSE-labeled T cells, the mice were retrieved, killed by cervical dislocation, and soaked in 75% alcohol for 10 minutes. Using a clean bench, the submandibular lymph nodes of the mice were placed in collagenase working solution and cut into pieces. Place in a 37°C water bath and incubate for 3 minutes. Use a pipette to blow 10 times every 15 minutes. Collect the lymph node digestion fluid, pass it through a 200-mesh sieve, collect the lymph single cell suspension, add 10 times the volume of FACS solution to wash, and centrifuge it in a normal temperature centrifuge at 2000r for 10 minutes. If there are incompletely digested cell clumps, continue to add collagenase and digest for 30 minutes as above. Collect all cell precipitates. Resuspend the lymph single cells and dilute them with FACS solution. After counting on a counting plate, dilute to 10 6 / 100μL, according to experimental needs, directly detect adoptive CFSE + CD8 + T cell homing behind the mouse ear.

[0097] 2.7 CFSE detection by flow cytometry fluorescence in situ hybridization (F-FISH) + CD8 + T cell telomere length

[0098] Follow the instructions of the telomere length detection kit and wash the CFSE in the control and experimental groups with PBS. + CD8 + T cells were counted using a counting plate, and 1×10 6 CFSE + CD8 +T cells were added to 6L of PBS and mixed evenly. The cell suspension was evenly added into labeled flow cytometry centrifuge tubes with caps, centrifuged at 1000r / min for 5 minutes, and the supernatant was discarded.

[0099] Add 300 μL of hybridization solution 1 (Via1) containing telomere probe to the control group and experimental group flow tubes, cover them and mix them with a vortex oscillator, put the flow tubes in a water bath preheated to 82°C for 10 minutes, then mix the cells in the test tubes again and keep them in a dark room at room temperature overnight. Preheat the water bath to 40°C in advance, take out the test tubes, add 1mL of washing solution (ViaL2, 1:10 dilution) to each of them, cover them, and mix them with an oscillator. Put them in a preheated water bath for 10 minutes, then centrifuge at 500×g for 5 minutes, and discard the supernatant as much as possible. Then repeat the above steps and wash again. Add DNA dye solution 3 (ViaL3, 1:00 dilution) to the flow tube, cover it, mix it with an oscillator, and let it stand at 4°C and avoid light for 3 hours. Detect and analyze with a flow cytometer, take Linear for FL3-H and Log for FLI-H, and count 20,000 cells. Calculate relative telomere length.

[0100] 2.8 Skin efficacy test methods

[0101] 2.8.1 Skin patch allergy test

[0102] Test method According to the relevant requirements of "Human Skin Patch Test" in the 2015 edition of "Technical Specification for Safety of Cosmetics", 40 female volunteers two weeks postpartum were selected to test the anti-aging compositions prepared in Examples 1 to 3 of the present invention, and the volunteers were observed at 1h, 12h, and 24h for adverse skin reactions such as redness, swelling, and itching.

[0103] 2.8.2 Determination of moisture content of stratum corneum (CPA)

[0104] The moisture content of the stratum corneum represents the degree of skin dryness. Comparing the moisture content of the stratum corneum before and after use can reflect the moisturizing effect of the product. If the skin is very dry, the CPA value will be lower. If the skin returns to normal dryness, the higher the moisture content, the higher the CPA value.

[0105] Test method: 40 postpartum female volunteers with healthy skin and stratum corneum moisture content <30 were selected, one person was selected from each age group, and 10 people were selected in a group, for a total of 4 groups. During the experiment, the subjects applied the same amount of anti-aging composition to the skin behind the ears every morning and evening, and no other cosmetics were applied. The changes in skin hydration rate (MMV) before using cosmetics and on the 14th and 28th days were tested using Corneometer, and repeated 5 times to obtain the average value.

[0106] 2.8.3 Skin melanin determination

[0107] The determination of melanin content is the most powerful method for testing whitening and anti-freckle products. The difference in human skin color mainly depends on the content and distribution of melanin. Melanin is mainly formed in melanocytes in the basal layer of the epidermis of human skin. Under the action of enzymes such as tyrosinase, tyrosine in melanocyte tissue is gradually converted into true melanin through intermediates such as dopa, dopaquinone, dopachrome, and dihydroxyindole. The lower the melanin content, the better the whitening and anti-freckle effect.

[0108] Test method: 40 postpartum female volunteers with healthy skin and stratum corneum moisture content <30 were selected, with 10 people in each group, for a total of 4 groups. During the experiment, the subjects applied the same amount of anti-aging composition to the skin behind the ears every morning and evening, and no other cosmetics were applied. The MEXAMETERMX18 skin pigmentation meter was used to test the skin melanin content before using cosmetics and on the 14th and 28th days, and the average value was obtained by repeating 5 times.

[0109] 2.8.4 Skin elasticity measurement

[0110] The commonly used method for elasticity measurement experiments is the suction method designed based on the principles of suction and stretching. The elastic parameters of the skin are obtained through the relationship curve between skin stretching length and time. This method is simple and rapid, and the measurement is not affected by skin thickness. It is a good indicator for studying skin aging.

[0111] Test method: 40 postpartum female volunteers with healthy skin and stratum corneum moisture content <30 were selected, with 10 people in each group, for a total of 4 groups. During the experiment, the subjects applied the same amount of anti-aging composition to the skin on the inside of their arms every morning and evening, and no other cosmetics were applied. According to the group standard T / ZHCA 005-2019 "Test method for the effect of cosmetics on skin elasticity", the skin elasticity characterization values ​​were measured before trial and 14 days and 28 days after trial. The results are expressed as the elasticity parameter R2, without units.

[0112] 2.8.5 Determination of skin wrinkle area ratio

[0113] The wrinkle area ratio indicates the size of the wrinkle area. The smaller the wrinkle area ratio, the fewer wrinkles there are. The larger the difference in this indicator, the better the improvement effect of wrinkle reduction.

[0114] Test method: 40 postpartum female volunteers with healthy skin and stratum corneum moisture content <30 were selected, with 10 people in each group, for a total of 4 groups. During the experiment, the subjects applied the same amount of anti-aging composition to their facial skin every morning and evening, and were not allowed to apply any other cosmetics. The skin wrinkle area was measured before the trial and 14 days and 28 days after the trial using a skin wrinkle meter.

[0115] 2.9 Statistical analysis methods

[0116] The data of each group were expressed as (X±SD). One-way analysis of variance (ANOVA) was used to compare the differences between the experimental groups and the model group. The LSD-t test was used for pairwise comparison. P<0.05 indicated that the difference was statistically significant.

[0117] 3 Experimental results

[0118] 3.1 Skin anti-aging test results

[0119] 3.1.1 Pathological results of mouse back skin tissue detected by HE staining

[0120] Figure 1 HE staining results of the back skin tissue of postpartum mice Note: P < 0.0001 vs model group, *P < 0.05 vs positive control group, P < 0.0001 vs cactus flower group, *P < 0.001 vs melibiose group, P < 0.01 vs abalone peptide group Figure 1 As shown, it can be seen that the epidermis of the model group mice after childbirth becomes thinner, the number of cell layers decreases, and the basal cell bodies become smaller; the thickness of the collagen fibers in the dermis becomes thinner, the arrangement is loose, the collagen bundles are slender and broken; the elastic fibers become shorter and reduced. Compared with the model group, the epidermal thickness and the number of cell layers of the mice in each group of drug administration after anti-aging drugs are increased, and the arrangement is more compact; the collagen fibers increase and thicken, and the arrangement is denser and regular, and they are mostly interwoven into a network, running parallel to the epidermis. It can be seen that the composition provided by the present invention can significantly and effectively repair the skin damage caused by postpartum, thereby achieving the purpose of anti-aging.

[0121] 3.1.2 Results of serum estrogen and progesterone test

[0122] Figure 2 Effects of cactus flower extract, melibiose and abalone peptide combination on serum estrogen and progesterone Note: *P<0.001vs model group

[0123] Depend on Figure 2 It can be seen that the comparison of hormone levels of the model group mice before and after the treatment with the composition showed that E2 was higher than that of the model group mice, and PRGE was also higher, indicating that the composition of the present invention can adjust the levels of estrogen and progesterone to achieve anti-aging effects.

[0124] 3.1.3 Effects of cactus flower extract, melibiose and abalone peptide combination on CRT expression in fibroblasts

[0125] Figure 3Effects of cactus flower extract, melibiose and abalone peptide combination on CRT expression in fibroblasts Note: P < 0.0001 vs model group, *P < 0.05 vs positive control group, P < 0.0001 vs cactus flower group, *P < 0.001 vs melibiose group, P < 0.01 vs abalone peptide group

[0126] The results of immunocytochemical staining of CRT expression in fibroblasts showed that the CRT expression signal strength in fibroblasts of different experimental groups and model groups was stronger than that in fibroblasts of the model group. Figure 3 The results of ELISA detection of CRT expression showed that the CRT expression value of the control group was 2.21±0.17 (A), the model group was 1.14±0.07 (A), and the CRT expression values ​​of different experimental groups were 1.85±0.10 (A), 1.55±0.09 (A), 1.67±0.11 (A), 1.82±0.15 (A), and 2.14±0.13 (A), respectively, all showing different abilities to promote CRT expression, and the differences in CRT expression values ​​between the combination group and each group had different significance. This suggests that the composition of the present invention can activate and enhance the expression of CRT signals in fibroblasts.

[0127] 3.1.4 Effects of cactus flower extract, melibiose and abalone peptide combination on DCs antigen presentation ability

[0128] Figure 4 Effect of cactus flower extract, melibiose and abalone peptide combination on DCs antigen presentation ability Note: P < 0.0001 vs model group, P < 0.0001 vs cactus flower group, P < 0.01 vs melibiose group, *P < 0.001 vs abalone peptide group, *P < 0.05 vs positive control group

[0129] Depend on Figure 4 It can be seen that in the experiment, each group of DCs can stimulate the proliferation of T cells to varying degrees in the presence of antigen (OVA), and each experimental group has a good ability to promote the proliferation of sensitized lymphocytes. The combination experimental group can significantly enhance the antigen presentation ability of DCs (P < 0.0001), indicating that DCs stimulated by the combination group have a good antigen presentation function. It suggests that the composition of the present invention can enhance the antigen presentation ability of dendritic cells.

[0130] Figure 5 Effects of cactus flower extract, melibiose and abalone peptide combination on the expression of p52, a specific protein in the NF-κB signaling pathway Note: P < 0.0001 vs model group, P < 0.0001 vs cactus flower group, P < 0.0001 vs melibiose group

[0131] Figure 5 The results showed that the expression of p52, a specific protein of the NF-κB signaling pathway, was enhanced to varying degrees in the experimental groups of fibroblasts under different treatments, and the p52 protein content in the nucleus of the experimental group of the combination was significantly higher than that of the model group, indicating that the experimental group significantly promoted the nuclear translocation of NF-κB (P < 0.0001). In subsequent experiments, we added the NF-κB inducing kinase (NIK) inhibitor B022 for reverse verification, and the results showed that after the addition of B022, the NF-κB activation process was significantly inhibited. In summary, the composition of the present invention can promote the nuclear translocation of p52 in cells by activating NF-κB.

[0132] Figure 6 Effect of cactus flower extract, melibiose and abalone peptide on CFSE + CD8 + Effect of T quantity Note: P < 0.0001 vs model group, P < 0.01 vs cactus flower group

[0133] Calculate homing CFSE based on cell ratio + CD8 + The total number of T cells was counted in 5 mice per group. The total number of cells homing in the model group and the experimental group was (14±1.99, 27±2.27, 23±2.02, 26.7±4.4, 27±3.02, 33±2.80)^104, respectively. Flow cytometry was used to detect the CFSE expression in the back of the ear of mice homing to the submandibular lymph nodes. + CD8 + T cell results Figure 6 As shown, CFSE was given to different experimental groups + CD8 + The proportion of T cells was higher than that of the model group, and the CFSE + CD8 + There are different significant differences in the number of T cells (P < 0.01), which shows that the composition of the present invention can increase the number of T cells and promote lymphocyte homing.

[0134] Figure 7 Effect of cactus flower extract, melibiose and abalone peptide on CFSE + CD8 + Effect of T on telomere length Note: P < 0.0001 vs model group, P < 0.0001 vs cactus flower group, P < 0.0001 vs melibiose group, P < 0.01 vs abalone peptide group

[0135] CFSE was measured by FLow-FISH method + CD8 +The relative telomere length of T cells was calculated by the formula. The relative telomere lengths of T cells in the control group and the experimental group were (12.10±2.80, 57.21±5.04, 30.75±2.31, 38.37±4.62, 54.55±9.78, 76.57±6.80)%, respectively. Figure 7 The results showed that CFSE + CD8 + The relative telomere length of T cells was significantly longer than that of other groups, and the CFSE + CD8 + The relative telomere lengths of T cells were compared, and there were different degrees of statistical differences, indicating that the composition of the present invention can promote the extension of T cell telomeres and has anti-aging activity.

[0136] 3.2 Skin efficacy test results

[0137] 3.2.1 Results of skin patch allergy test

[0138] Table 1 Effect of the present invention on skin allergic reactions

[0139] Allergic reactions Number of subjects 0h 12h 24h Abalone peptide group 10 0 0 0 Cactus flower set 10 0 0 0 Melibiose group 10 0 0 0 Combination group 10 0 0 0

[0140] The results of the patch allergy test are shown in Table 1 below. The results of the human skin patch test show that none of the 10 people in each group had adverse skin reactions, indicating that the monomer components and compositions of the present invention are highly safe and mild without irritation.

[0141] 3.2.2 Results of measurement of stratum corneum moisture content (CPA)

[0142] Table 2 Effect of the present invention on moisture content of skin stratum corneum

[0143]

[0144]

[0145] Note: Compared with the model group, *P<0.05, P<0.01, *P<0.001

[0146] As can be seen from Table 2, after using the product with anti-aging efficacy for 14 days, the moisture content of the stratum corneum of the skin showed a significant increase. After using the product for 28 days, the moisture content of the stratum corneum further increased, indicating that the composition with anti-aging efficacy provided by the present invention has good moisturizing and anti-aging effects.

[0147] 3.2.3 Skin melanin measurement results

[0148] Table 3 Effect of the present invention on skin melanin

[0149] Melanin content difference 0d 14d 28d Control group 91.3±0.82 90.8±0.84 90.9±1.14 Model Group 167.8±1.49 166.6±1.19 166.4±1.82 Cactus flower set 164.2±1.03 163.8±1.92 162.0±1.87 Melibiose group 166.6±1.14 163.2±2.17 161.8±0.84 Abalone peptide group 164.2±2.39 163.0±1.22 161.0±1.22* Combination group 157.6±2.3 150.6±1.34 148.6±0.89 Positive control group 138.6±1.82 135.8±0.84 131.6±1.14

[0150] Note: Compared with the model group, *P<0.05, P<0.01, *P<0.001

[0151] As shown in Table 3, the melanin in the skin of the subjects was effectively reduced at 14 days and further reduced at 28 days, and the combination group was significantly different from the model group, indicating that the anti-aging composition provided by the present invention has good moisturizing and anti-aging effects.

[0152] 3.2.4 Skin elasticity test results

[0153] Table 4 Effect of the present invention on skin elasticity

[0154] Skin elasticity characterization value 0d 14d 28d Control group 3.2±0.10 3.5±0.24 3.3±0.15 Model Group 0.7±0.18 0.7±0.14 0.7±0.15 Cactus flower set 0.7±0.18 1.1±0.15 1.2±0.27* Melibiose group 0.6±0.10 1.2±0.19 1.4±0.15* Abalone peptide group 0.6±0.15 1.6±0.18 1.9±0.25 Combination group 0.6±0.13 1.6±0.21 1.9±0.37 Positive control group 0.7±0.15 1.9±0.33 2.1±0.38

[0155] Note: Compared with the model group, *P<0.05, P<0.01, *P<0.001

[0156] As shown in Table 4, the skin elasticity of the subjects was significantly improved at 14 days, and the skin elasticity of the test samples within 28 days was significantly different from that of the model group, indicating that long-term continuous use of the anti-aging composition provided by the present invention has a good moisturizing and anti-aging effect.

[0157] 3.2.5 Skin anti-wrinkle test results

[0158] Table 5 Effect of the present invention on skin wrinkle area

[0159] Skin wrinkle area ratio 0d 14d 28d Control group 22.3±1.07 23.0±0.56 22.6±0.47 Model Group 37.8±1.28 37.3±1.09 36.8±0.80 Cactus flower set 36.2±0.75 35.3±0.85. 34.2±0.27* Melibiose group 36.9±1.11 34.8±1.55 34.4±0.51* Abalone peptide group 35.6±0.27 33.7±2.00 34.9±0.19 Combination group 36.1±1.25 31.4±2.09 31.1±0.97 Positive control group 35.9±0.77 32.3±0.01 30.6±1.99

[0160] As shown in Table 5, the skin wrinkle area ratio of the subjects was significantly reduced at 14 days, and the skin wrinkle area ratio of the test samples within 28 days was significantly different from that of the model group. This shows that long-term continuous use of the anti-aging composition provided by the present invention has a good anti-wrinkle effect.

[0161] In summary, the experiment proves that the composition of cactus flower extract, melibiose, milk exosomes, and abalone peptides significantly improves the efficacy of the drug, and significantly improves the antigen presentation ability of dendritic cells and macrophages in postpartum skin. The effect of the cactus flower extract, melibiose, milk exosomes, and abalone peptides is significantly better than that of single administration. The experiment found that the composition activated the calreticulin of fibroblasts in the skin, enhanced the antigen presentation ability of dendritic cells, activated the "non-classical" NF-κB signaling pathway, promoted lymphocyte homing, and extended T cell telomeres, showing postpartum anti-aging activity, which belongs to the new mechanism of the composition of the present invention.

[0162] The following are four specific embodiments, describing the preparation methods and use effects of different proportions and applications:

[0163] Example 1: Preparation of anti-aging cream

[0164] Raw material ratio:

[0165] Cactus flower extract: 5%

[0166] Melibiose: 8%

[0167] Abalone peptide: 10%

[0168] Preparation steps:

[0169] 1. Freeze fresh cactus flowers in a -20°C low-temperature refrigerator for 12 hours and then crush them. Then stir them in 60°C distilled water for 1 hour, add activated carbon and let it stand for 24 hours. Then, centrifuge to remove the activated carbon to obtain a cactus flower water extract.

[0170] 2. According to the above ratio, mix the cactus flower extract, melibiose and abalone peptide, add appropriate amount of emulsifier and moisturizer, and stir evenly to form a cream base.

[0171] 3. The mixture is homogenized under low speed stirring to finally obtain the anti-aging cream.

[0172] Example 2: Preparation of anti-aging serum

[0173] Raw material ratio:

[0174] Cactus flower extract: 3%

[0175] Melibiose: 6%

[0176] Abalone peptide: 5%

[0177] Preparation steps:

[0178] 1. The preparation of cactus flower extract was the same as in Example 1, but the extract was extracted at 50°C to retain more active ingredients.

[0179] 2. Mix cactus flower extract, melibiose and abalone peptide in the above proportions, add appropriate amount of sodium hyaluronate solution, stir well to make essence.

[0180] 3. Fill the essence into a small bottle and store it in a sealed condition away from light.

[0181] Example 3: Preparation of anti-aging oral liquid

[0182] Raw material ratio:

[0183] Cactus flower extract: 2%

[0184] Melibiose: 4%

[0185] Abalone peptide: 3%

[0186] Preparation steps:

[0187] 1. The preparation of cactus flower extract was the same as in Example 1, using 65° C. water for extraction.

[0188] 2. Mix the cactus flower extract, melibiose and abalone peptide according to the above ratio, add appropriate amount of fruit juice concentrate and sweetener, stir evenly, and prepare an oral solution.

[0189] 3. Aseptically fill the mixed solution to prepare the anti-aging oral solution.

[0190] Example 4: Preparation of anti-aging facial mask

[0191] Raw material ratio:

[0192] Cactus flower extract: 10%

[0193] Melibiose: 7%

[0194] Abalone peptide: 8%

[0195] Preparation steps:

[0196] 1. The preparation of cactus flower extract is the same as that in Example 1.

[0197] 2. Mix the cactus flower extract, melibiose and abalone peptide, add appropriate amount of collagen and moisturizing factor, and stir into a mask base.

[0198] 3. Spread the mask matrix evenly on the mask paper and vacuum pack it.

[0199] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition for treating skin deterioration, characterized in that: The composition consists of cactus flower extract, melibiose and abalone peptide.

2. The composition for treating skin deterioration caused by postpartum hormone changes according to claim 1, characterized in that: The composition consists of 0.01% to 16% of cactus flower extract, 0.05% to 12% of melibiose and 0.01% to 15% of abalone peptide in terms of mass fractions.

3. The composition for treating skin deterioration according to claim 1, characterized in that: The composition activates calreticulin in fibroblasts in postpartum skin, enhances the antigen presenting ability of dendritic cells, activates the "non-classical" NF-κB signaling pathway, promotes lymphocyte homing, and prolongs T cell telomeres, thereby showing anti-aging activity.

4. The composition for treating skin deterioration according to claim 1, characterized in that: The composition is combined with auxiliary materials or solvents to prepare skin care and anti-aging products such as facial masks, eye creams, essence water, essence milk, facial creams, body lotions, oral liquids, capsules, etc.

5. An anti-aging skin care product containing cactus flower extract, melibiose and abalone peptide, characterized in that: The product contains 0.01% to 16% of cactus flower extract, 0.05% to 12% of melibiose, and 0.01% to 15% of abalone peptide in terms of mass fractions, and can treat skin deterioration problems caused by postpartum hormone changes.

6. The anti-aging skin care product according to claim 5, characterized in that: The cactus flower extract is a water extract of cactus flowers, which is obtained by freezing and crushing fresh cactus flowers in a low-temperature refrigerator, stirring them evenly under 60-65° C. water, adding activated carbon and letting them stand for 24 hours, and removing the activated carbon by centrifugation at 4000 r / min for 5 minutes.

7. The anti-aging skin care product according to claim 5, characterized in that: The product exerts its anti-aging effect by activating fibroblast calreticulin in the skin, enhancing the antigen presentation ability of dendritic cells, activating the "non-classical" NF-κB signaling pathway, promoting lymphocyte homing, and extending T cell telomeres.

8. The anti-aging skin care product according to claim 5, characterized in that: The products include the following forms: facial mask, eye cream, essence water, essence lotion, facial cream, body lotion, oral liquid or capsule.

9. A method for preparing an anti-aging skin care product containing cactus flower extract, melibiose and abalone peptide, characterized in that: The following steps are involved: Step 1, after freezing and crushing fresh cactus flowers in a low-temperature refrigerator, stir them evenly in water at 60-65° C., add activated carbon and let them stand for 24 hours, then centrifuge at a speed of 4000 r / min for 5 minutes to remove the activated carbon, and obtain a light yellow cactus flower water extract; Step 2: Mix the above-mentioned cactus flower water extract, melibiose and abalone peptide according to a predetermined mass ratio, stir evenly, add appropriate amount of auxiliary materials or solvents to prepare an anti-aging skin care product.

10. The method according to claim 1, characterized in that In step one, through the method of low-temperature freezing and appropriate temperature heating extraction, the structural changes of the natural active ingredients of cactus flowers are minimized, their natural aroma is retained, and the extraction efficiency is effectively improved.

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