Preparation method of peony stamen composite microcrystalline capsule composition and its use in resisting blue light, protecting cells, resisting allergy and resisting aging

Abstract

The present invention relates to a peony stamen composite microcrystal capsule composition, which comprises: a carrier forming a shell structure with protective and shielding effects; and a peony stamen extract embedded in or coated by the shell structure. The composition of the present invention coats the peony stamen extract with the carrier of the shell structure to prevent the peony stamen extract from deteriorating or being contaminated, thereby providing a stable effect. In addition, the effects of the composition of the present invention include: anti-blue light, cell protection, cell repair, anti-allergy, promotion of cell energy generation, anti-aging, and inhibition of pigmentation.

Description

Technical Field

[0001] The present invention relates to a microcrystalline capsule composition, and in particular to a peony stamen composite microcrystalline capsule composition. In addition, the present invention also relates to a preparation method of the above composition and its use in resisting blue light, protecting cells, repairing cells, resisting allergies, promoting cell energy generation, resisting aging, and inhibiting the expression of pigmentation mechanism genes in melanoma cells. Background Art

[0002] Peony flowers were once known as "plant gold". Peony stamens are part of peony flowers and the most concentrated part of peony growth. Therefore, they are the essence of peony essence and are extremely nutritious. They are rich in amino acids, active polysaccharides, flavonoid compounds and other ingredients that can nourish yin and yang. In addition, peony stamens contain a large amount of vitamin B3, which is the highest content among all plant foods. Its effects include: replenishing qi and blood, ruddy complexion, full spirit, relieving physical pain, lowering blood pressure, improving anemia, and beautifying the skin, etc. Summary of the invention

[0003] One object of the present invention is to provide a peony stamen composite microcrystal capsule composition, comprising: a carrier, which is formed into a shell-like structure; and a peony stamen extract, which is embedded in the shell-like structure or coated by the shell-like structure.

[0004] More preferably, the carrier comprises phosphatidylcholine.

[0005] More preferably, the proportion of the phosphatidylcholine in the components of the carrier is greater than 50% by weight.

[0006] More preferably, the carrier further comprises phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, lecithin, hydrogenated lecithin and D-α-tocopheryl polyethylene glycol 1000 succinate.

[0007] Another object of the present invention is to provide a method for preparing a peony stamen composite microcrystal capsule composition, the method comprising: mixing the peony stamen extract with the carrier, and dispersing the carrier to form a colloidal complex.

[0008] More preferably, the method further comprises: a shaking step of shaking the colloidal complex using a high-power ultrasonic oscillator.

[0009] More preferably, the temperature condition of the shaking step is 35°C ~55°C.

[0010] More preferably, the shaking step lasts for at least 30 minutes.

[0011] Another object of the present invention is to provide a use of a peony stamen composite microcrystal capsule composition, which is used to prepare a composition that resists blue light, protects cells, repairs cells, is anti-allergic, promotes cell energy generation, resists aging, and inhibits the expression of pigmentation mechanism genes in melanoma cells.

[0012] More preferably, the composition is used to administer an effective dose of the peony stamen extract to the skin cells of an individual to achieve the effects of resisting blue light, protecting cells, repairing cells, anti-allergy, promoting cell energy production, anti-aging, and inhibiting the expression of pigmentation mechanism genes in melanoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Hereinafter, “peony stamen extract” is referred to as “PSB” and “encapsulated peony stamen microcapsules” is referred to as “PSBL”;

[0014] Figure 1 is a photograph showing the “average particle size of coated peony stamen microcapsules”;

[0015] Figure 2 is a graph showing the activity stability of PSBL and PSB in water;

[0016] Figure 3 is a photographic diagram, which represents the “photohealth function of encapsulated peony stamen microcapsules (PSBL)”;

[0017] Figure 4 A photo showing "the expression of anti-aging protein Lamin A regulated by encapsulated peony stamen microcapsules (PSBL)";

[0018] Figure 5 It is a bar graph, which represents the "gene expression of Tyrosinase, Mtor, and TOP1 after the action of PSBL". DETAILED DESCRIPTION

[0019] In order to make the above and / or other purposes, effects, and features of the present invention more clearly understood, preferred embodiments are specifically described below in detail:

[0020] The present invention provides a peony stamen composite microcrystalline capsule composition, comprising: a carrier, which is formed into a shell-like structure; and a peony stamen extract, which is embedded in the shell-like structure or coated by the shell-like structure. In a preferred embodiment, the peony stamen extract type includes: peony stamen water extract or peony stamen ethanol extract, but is not limited thereto. In another preferred embodiment, the peony stamen extract can be used to prepare a composition with anti-inflammatory, mitochondrial regeneration promotion, collagen production promotion, cell protection or cell repair function, but is not limited thereto. In another preferred embodiment, the mitochondria include animal cell mitochondria, plant cell mitochondria, fungal cell mitochondria or protist cell mitochondria, but is not limited thereto. In another preferred embodiment, the collagen type includes type I collagen, type II collagen, type III collagen, type IV collagen or type V collagen, but not limited thereto. In another preferred embodiment, the cell type includes skin cells, skin fibroblasts, epithelial cells, nerve cells, red blood cells, white blood cells, platelets, phagocytes (neutrophils, basophils, eosinophils), B lymphocytes, effector B cells, memory B cells, T lymphocytes, memory T cells, effector T cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, cardiomyocytes, osteoblasts, glial cells, hepatocytes, kidney cells, glandular cells or endocrine cells (thyroid cells, thymocytes, pancreatic B cells, pancreatic islet cells), but not limited thereto.

[0021] More preferably, the raw material of the carrier includes: phosphatidylcholine. In another preferred embodiment, the weight percentage of the phosphatidylcholine in the total carrier raw material is greater than 50 weight%. In another preferred embodiment, the weight percentage of the phosphatidylcholine in the total carrier raw material is 60 weight% to 80 weight%. In another preferred embodiment, the weight percentage of the phosphatidylcholine in the total carrier raw material is 70 weight%. In another preferred embodiment, the carrier raw material further includes phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidic acid (PA), egg phosphatidylcholine (EPC), hydrogenated lecithin (hydoegenatedlecithin) or D-alpha tocopherylpolyethylene glycol 1000 succinate (TPGS), but is not limited thereto.

[0022] In addition, another embodiment of the present invention provides a method for preparing a peony stamen composite microcrystal capsule composition, as described in detail below:

[0023] First, the peony stamen extract is mixed with a carrier raw material to disperse the carrier raw material to form a colloidal complex. Specifically, the carrier raw material is dissolved in a solvent, and the solvent is removed by a rotary vacuum concentrator to form a lipid film, and then the peony stamen extract is added to the lipid film to disperse the lipid into a colloidal complex.

[0024] In a preferred embodiment, the preparation method of the peony stamen extract has two embodiments. In the first embodiment, the preparation method of the peony stamen extract includes: a soaking step, covering and soaking the peony stamens with water or ethanol solution; and an extraction step, continuously and rapidly oscillating and extracting the water or ethanol solution soaked with the peony stamens with ultrasonic energy of a total energy of 500-800 w at low temperature. In a preferred embodiment of the first embodiment, the temperature condition of the low temperature is -4°C~25°C. In another preferred embodiment, the continuous rapid oscillation time is accumulated for 60-120 minutes. In another preferred embodiment, the process of the continuous rapid oscillation is: rest for 2 minutes every 5 minutes of oscillation until the cumulative oscillation time is 60-120 minutes. In another preferred embodiment, in the soaking step, when the peony stamens are covered and soaked with water, the weight ratio of the peony stamens to water is 1:7~1:11, the temperature condition is 4°C~50°C, and the soaking time is 72~120 hours; when the peony stamens are covered and soaked with an ethanol solution, the weight ratio of the peony stamens to the ethanol solution is 1:7~1:11, the temperature condition is 4°C~50°C, and the soaking time is 72~120 hours. In another preferred embodiment, the weight ratio of the peony stamens to water is 1:9. In another preferred embodiment, the water can be pure water, crystal water, ultrapure water, groundwater, natural water, biological water, mineral water or deionized water, but is not limited thereto. In another preferred embodiment, the weight ratio of the peony stamens to the ethanol solution is 1:9. In another preferred embodiment, the ethanol solution contains 50% by weight to 75% by weight of ethanol. In the second embodiment, the preparation method of the peony stamen extract comprises: a freezing step, freezing and solidifying the peony stamens rapidly (-195.79°C); a grinding step, grinding the peony stamens with a freezing grinder to grind the peony stamens into powder; a soaking step, covering and soaking the peony stamen powder with water or ethanol solution; and an extraction step, extracting the water or ethanol solution soaked with the peony stamen powder by continuous rapid oscillation with a total energy of 500-800 w of ultrasonic energy at low temperature. In a preferred embodiment of the second embodiment, the temperature condition of the low temperature is -4°C~25°C. In another preferred embodiment, the continuous rapid oscillation time is accumulated for 60-120 minutes. In another preferred embodiment, the process of the continuous rapid oscillation is: rest for 2 minutes every 5 minutes of oscillation until the cumulative oscillation time is 60-120 minutes. In another preferred embodiment, in the soaking step, when the peony stamen powder is covered and soaked in water, the weight ratio of the peony stamen powder to water is 1:7~1:11, the temperature condition is 4°C~50°C, and the soaking time is 72~120 hours; when the peony stamen powder is covered and soaked in an ethanol solution, the weight ratio of the peony stamen to the ethanol solution is 1:7~1:11, the temperature condition is 4°C~50°C, and the soaking time is 72~120 hours.In another preferred embodiment, the weight ratio of the peony stamen powder to water is 1:9. In another preferred embodiment, the water can be pure water, crystal water, ultrapure water, groundwater, natural water, biological water, mineral water or deionized water, but is not limited thereto. In another preferred embodiment, the weight ratio of the peony stamen powder to the ethanol solution is 1:9. In another preferred embodiment, the ethanol solution contains 50% to 75% by weight of ethanol. In addition, the preparation method of the peony stamen extract described in the above two embodiments further includes: a centrifugation step, at a low temperature (-20°C), the water or ethanol solution after rapid oscillation extraction is centrifuged at high speed at 15,000g for 10 minutes to remove the bottom precipitate and collect the supernatant. In another preferred embodiment, the method for preparing the peony stamen extract further comprises: a filtering step, filtering the supernatant with a microporous structure filter membrane with a pore size of 0.22-0.45 μm and using a vacuum filter to obtain a clear filtrate. In another preferred embodiment, the method for preparing the peony stamen extract further comprises: a drying step, freeze-drying the clear filtrate using a vacuum concentrator and a vacuum freeze dryer.

[0025] In another preferred embodiment, the solvent is Ethanol. In another preferred embodiment, the carrier material comprises phosphatidylcholine. In another preferred embodiment, the weight percentage of the phosphatidylcholine in the total carrier material is greater than 50 weight %. In another preferred embodiment, the weight percentage of the phosphatidylcholine in the total carrier material is 60 weight % to 80 weight %. In another preferred embodiment, the weight percentage of the phosphatidylcholine in the total carrier material is 70 weight %. In another preferred embodiment, the carrier material further comprises phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidic acid (PA), eggphosphatidylcholine (EPC), hydrogenated lecithin (hydoegenatedlecithin) or D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS). In another preferred embodiment, phosphatidylcholine is an important component for building nerve cell membranes, which is very important in the growth and development stage of the body; after phosphatidylcholine is decomposed by enzymes, choline is produced, and it combines with acetyl groups in the human body to produce acetylcholine, which is an important neurotransmitter and beneficial to nerve cell activity; phosphatidylcholine can repair damaged cell membranes and also help acetylcholine synthesis, making the brain more flexible and improving memory. In another preferred embodiment, the effects of phosphatidylethanolamine include: improving concentration, slowing down hyperactivity, reducing stress, preventing Alzheimer's disease and cognitive decline, or increasing memory and learning ability, but not limited to this. In another preferred embodiment, the main components of lecithin include: choline, fatty acids, glycerol, glycolipids, phosphoric acid, triglycerides and phospholipids; its effects include: activating metabolism, preventing premature cell aging, helping brain development, regulating nerve tissue, improving memory, soothing emotions, promoting digestion and absorption of oils and fat-soluble vitamins, lowering blood lipids or helping liver metabolism, but not limited to this. In another preferred embodiment, phospholipids are key structural and functional components of all plant and animal cell membranes, including: phosphatidylserine, phosphatidylcholine, and phosphatidylinositol.

[0026] Next, the colloidal complex is shaken by a high-power ultrasonic oscillator to obtain a peony stamen composite microcrystal capsule composition. In another preferred embodiment, the high power is an ultrasonic energy with a total energy of 20 to 100 w. In another preferred embodiment, the shaking time is 30 to 60 minutes, and the shaking temperature is 30°C to 60°C. In another preferred embodiment, the shaking time is 30 to 40 minutes, and the shaking temperature is 30°C to 60°C. In another preferred embodiment, the shaking time is 40 to 50 minutes, and the shaking temperature is 30°C to 60°C. In another preferred embodiment, the shaking time is 40 to 60 minutes, and the shaking temperature is 30°C to 60°C. In another preferred embodiment, the shaking time is 50 to 60 minutes, and the shaking temperature is 30°C to 60°C. In another preferred embodiment, the shaking time is 30 minutes, and the shaking temperature is 45°C.

[0027] More preferably, the carrier of the peony stamen microcrystal capsule composition prepared according to the above method has a protective effect, which can prevent the peony stamen extract contained therein from deteriorating or being contaminated. In a preferred embodiment, since the carrier is fat-soluble, it is more easily absorbed by the skin, thus greatly improving the absorption effect of the peony stamen extract contained in the carrier by human skin.

[0028] In addition, another embodiment of the present invention provides a use of a peony stamen composite microcrystal capsule composition, as described in detail below:

[0029] The peony stamen composite microcrystal capsule composition proposed in the present invention can be used to prepare a composition with the effects of anti-blue light, cell protection, cell repair, anti-allergy, promotion of cell energy generation, anti-aging or inhibition of the expression of pigmentation mechanism genes, but not limited thereto. In another preferred embodiment, the composition can be a raw material for pharmaceuticals, skin care products, cosmetics, health foods or skin care products, but not limited thereto. In another preferred embodiment, the composition is used to administer an effective dose of the peony stamen extract to the skin cells of an individual to achieve anti-blue light, cell protection, cell repair, anti-allergy, promotion of cell energy generation, anti-aging, and inhibition of the expression of pigmentation mechanism genes.

[0030] In order to understand the technical features, technical means and expected effects of the present invention, the relevant embodiments of the extract of the present invention are described as follows:

[0031] Example 1: Extraction method of peony stamen growth point tissue extract

[0032] Extraction method 1: Soak peony stamen tissue in pure water

[0033] After the fresh stamens were collected, the peony stamen tissue was rapidly frozen and solidified by liquid nitrogen at −195.79°C (77 K), and the peony stamen tissue was ground into powder by a freezing grinder; pure water and peony stamen tissue powder were extracted in a weight ratio of 9:1, and the peony stamen tissue powder was soaked in pure water at a temperature of 4°C~50°C for 72~120 hours; the mixture of the soaked peony stamen tissue powder and pure water was continuously and rapidly oscillated with a total energy of 500~800 w at a temperature of -4°C~25°C, wherein the oscillation was continuously carried out in a manner of 5 minutes of shaking and 2 minutes of rest until the total oscillation time reached 60~120 minutes; the bottom sediment was removed by high-speed centrifugation at 15,000g for 10 minutes at a temperature of -20°C and the supernatant was collected; the supernatant was centrifuged at a pore size of 0.22-0.45 The mixture was filtered through a microporous membrane with a volume of 100 μM and filtered using a vacuum filter to obtain a clear filtrate. The clear filtrate was freeze-dried using a vacuum concentrator and a vacuum freeze dryer to obtain a peony stamen growth point tissue extract (PSB).

[0034] Example 2: Preparation of encapsulated peony stamen microcapsules (PSBL)

[0035] In a spherical glass flask, the carrier materials: phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidic acid (PA), egg phosphatidylcholine (EPC), hydrogenated lecithin and D-alpha-tocopherylpolyethylene glycol 1000 succinate (D-alphatocopherylpolyethylene glycol 1000 succinate, The invention discloses a method for preparing a peony stamen extract (PSB) and a peony stamen extract (TPGS) in an ethanol mixture, wherein phosphatidylcholine is the main component of the carrier, and accounts for 70% by weight of the total weight of the carrier components; the ethanol is removed by a rotary vacuum concentrator to form a lipid film on the wall of a spherical glass flask; a peony stamen extract (PSB) solution is added to mix with the lipid film for interaction, so that the lipid is dispersed in the solution and a colloidal complex is formed; the colloidal complex is oscillated by a high-power ultrasonic oscillator with a total ultrasonic energy of 20-100 W, wherein the temperature is set to 45° C. and the duration is 30 minutes to form a coated peony stamen microcapsule (PSBL); the concentration of the PSBL is 0.1-5 mM to enhance the stability and transdermal penetration ability of the active ingredient. In another preferred embodiment, phosphatidylcholine is an important component for constructing nerve cell membranes and is very important during the growth and development stage of the body. After being decomposed by enzymes, phosphatidylcholine produces choline, which combines with acetyl groups in the human body to produce acetylcholine, which is an important neurotransmitter and is beneficial to nerve cell activity. Phosphatidylcholine can repair damaged cell membranes and also helps in the synthesis of acetylcholine, making the brain more flexible and improving memory.

[0036] Example 3: The shape of the encapsulated peony stamen microcapsule (PSBL)

[0037] The structure of particles in the colloidal dispersion was observed by transmission electron microscope (TEM). The sample was pre-treated by dropping lipid dispersion droplets on a carbon-coated copper mesh on a support film (formvar film) and letting it stand for two minutes to allow the sample particles to embed on the copper mesh. The excess sample was then wiped off with a clean paper. Uranium acetate (0.5 wt%) was then dropped on the copper mesh to stain the sample. The sample was left standing for two minutes and then wiped off with a clean paper. The sample was then stored in a drying oven for 12 hours. The morphological structure was then analyzed using a transmission electron microscope. The results of the transmission electron microscope analysis are as follows: Figure 1As shown, the encapsulated peony stamen microcapsules (PSBL) formed nano-scale colloidal complexes with an average particle size of 50~250nm.

[0038] Example 4: Particle size and interface potential analysis of coated peony stamen microcapsules (PSBL)

[0039] The particle size and interface potential of PSBL were measured by dynamic light scattering and interface potential analyzer (DLS), wherein the interface potential analyzer model is Brookhaven 90Plus Particle Size Analyzer, purchased from Brookhaven Inc., USA. The measurement results are shown in Table 1: the average particle size (AP.S.) of the coated peony stamen microcapsules (PSBL) is 170.7 nm; the polydispersity index (Pd.I) is 0.24; the average zeta potential (AZP) is -5.46 mV; the ratio of the PSB concentration after the peony stamen extract (PSB) and the microcapsules (L) are combined to the PSB concentration before the combination is 94.39%, and the combined percentage (CP) is 94.39%.

[0040] Table 1

[0041]

[0042] Example 5: Analysis of active ingredient content in coated peony stamen microcapsules (PSBL)

[0043] High performance liquid chromatography (HPLC) was used to quantitatively and qualitatively analyze the active components of PSB. The pump model was L-7100, the automatic injector model was L-7200, and the UV-vis model was L-4200, all purchased from Hitachi; the column model was Microsorb-MV 100-5C18 (250 mm ×4.6 mm id, 5 μm particle size), purchased from Getech. The relationship between the active concentration of PSB aqueous solution and PSBL complex over time is shown in Figure 2. Figure 2 The results show that the nano-scale colloidal complex PSBL can effectively improve the activity stability of PSB in water; PSBL can improve the storage stability of PSB activity.

[0044] Example 6: Safety evaluation test of coated peony stamen microcapsules (PSBL) Safety evaluation of coated peony stamen microcapsules (PSBL) and cell viability test were performed. Human skin keratinocytes (HaCaT cells) were quickly moved to a 37°C water bath to be thawed rapidly within 40 to 60 seconds, and then cell freezing solution (cell culture medium containing 10% DMSO) was added to the cell culture medium, and then the cells were broken up and moved to 25 cm 2 The cell culture flasks were grown in a 37°C, 5% CO2 cell culture incubator, and the culture medium was changed every 2 days. Human skin keratinocytes and human skin fibroblasts (HaCaT and Hs68 cells) (1.5×10 5 Cells were cultured in 96-well plates and incubated at 37°C and 5% CO2 for at least 24 hours. To evaluate whether PSBLs affect cell viability in skin cells: PSBs were added and exposed to the cells for a specified time. After the reaction time, the old culture medium was removed, washed once with PBS, and replaced with new culture medium. 10 μL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution was added for reaction. After 4 hours of reaction at 37°C and 5% CO2, the culture medium was removed, 100 μL of DMSO was added to dissolve the formazan precipitate, and the absorbance was measured at a wavelength of 570 nm (BioTek, Synergy TM 2, USA). Experimental results: After the human skin keratinocytes and human skin fibroblasts (HaCaT and Hs68 cells) were treated with the encapsulated peony stamen microcapsules (PSBL) for 24 hours, the cell viability was 102.5% and 101.6%, respectively.

[0045] Example 7: Skin membrane protection test (anti-blue light test) of encapsulated peony stamen microcapsules (PSBL)

[0046] Human keratinocytes (HaCaT cells) (1.5×10 5Cells were cultured in 96-well plates and incubated at 37°C and 5% CO2 for at least 24 hours. To evaluate whether PSBL has a protective function on the skin membrane layer of skin cells. The cells were irradiated with blue light for 3 hours, and then PSBL was added and reacted for a specified time. After the reaction time, the old culture medium was removed, washed once with PBS, and replaced with new culture medium. 10 μL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide) solution was added for reaction. After 4 hours of reaction at 37°C and 5% CO2, the culture medium was removed, 100 μL of DMSO was added to dissolve the formazan precipitate, and the absorbance was measured at a wavelength of 570 nm (BioTek, Synergy TM 2, USA). Test results: The cell viability of human skin keratinocytes in the action group (control group) was 100.0%; the cell viability after blue light irradiation was 62.5%; the cell viability after PSBL treatment increased to 95.6%, indicating that PSBL has the function of protecting the skin membrane against blue light.

[0047] Table 2

[0048]

[0049] Example 8: Photohealth function of encapsulated peony stamen microcapsules (PSBL)

[0050] To evaluate whether PSBL can reduce the expression of UVB photoproduct cyclobutane pyrimidine dimer (CPD), protect and repair damaged skin cells, and thus achieve skin photohealth function. Human keratinocytes (HaCaT cells) (1.5×10 5 After culturing in a 24-well plate for 24 hours, the samples were added and cultured for 4 hours. The supernatant was drained and UVB (50 mJ / cm 2) irradiation, then add serum-free culture medium containing samples and react in the incubator for 4 hours. After the reaction time, remove the supernatant, wash with PBS, add 500 μL 4% formalin, and place at room temperature for 10 minutes for fixation. Then remove formalin, wash twice with PBS, add 500 μL 0.5% triton-X100 and react on ice for 5 minutes, then remove and wash twice with PBS. Add 1 mL 2 M HCL and act at room temperature for 30 minutes to denature the DNA in the cells, and then wash five times with PBS. Then add 1 mL 20% FBS and block at 37˚C for 30 minutes to prevent non-specific protein binding. After the time, wash five times with PBS. Add 70μL CPD (1:100) and shake evenly at 37˚C for 30 minutes, and then wash five times with PBS. Finally, add an equal amount of PBS and measure the absorbance value with an enzyme immunoassay at Excitation: 530nm, Emission: 630nm. Then, add 70 μL of Hoechst 33342 staining solution (10 μg / mL) at a ratio of 1:250 and continue shaking for 5 minutes. Immediately wash with PBS 5 times and observe the fluorescence performance under a fluorescence microscope. Figure 3 As shown: CPD is a photochemical product, and the test is carried out using the characteristic of being detectable by fluorescence. After being irradiated by UVB, the cells are damaged and broken, and photochemical products accumulate, with a CPD induction of 138.7%. After the skin cells are exposed to the coated peony stamen microcapsules (PSBL) and then irradiated by UVB, only a few photochemical products are produced, and the CPD induction is 102.2%. The results show that the coated peony stamen microcapsules (PSBL) have the function of protecting and repairing damaged skin cells, thereby achieving the function of skin photohealth.

[0051] Table 3

[0052]

[0053] Example 9: Anti-allergic evaluation test of encapsulated peony stamen microcapsules (PSBL)

[0054] Allergy is an abnormal reaction of the body, which is an abnormal reaction to allergens. Allergies will occur only when allergens come into contact with people with allergic constitutions. Allergens include hundreds of chemicals, ultraviolet rays, pollen, dust, foreign proteins, etc. In the process of allergic reactions, allergic mediators play a direct role. Allergens are the external cause of allergic symptoms, while low immunity and the oxidative damage of mast cells and basophils by a large number of free radicals are the internal causes of allergic symptoms. There are two types of cells in the human body, namely mast cells and basophils, which are widely distributed in the nasal mucosa, bronchial mucosa, gastrointestinal mucosa, and around the microvessels in the connective tissue under the skin and in the capsule of the internal organs. These two types of cells contain allergic mediators such as histamine, leukotrienes, 5-hydroxytryptamine, and kinase. Mast cell lines (RBL-2H3 cells) were used to evaluate the anti-allergic efficacy of coated peony stamen microcapsules (PSBL). When mast cells are stimulated by IgE antibodies or calcium ion carriers, they can release a large amount of β-glucuronidase (β-hexosaminidase) through degranulation. The amount released can be used to determine whether the peony extract (PSE) to be tested has the potential to inhibit allergic reactions. 4 cells / well) were cultured in a 96-well plate and treated with coated peony stamen microcapsules (PSBL) for 24 hours, followed by the addition of anti-DNP IgE Ab (1 μg / mL) and incubation for 6-12 hours to sensitize mast cells. After washing the supernatant, DNP-BSA (100 ng / mL) was added and incubated for 1 hour to activate mast cells. The supernatant was collected and frozen for storage. An equal volume of supernatant and p-NAG substrate (1 μM) were added to the 96-well plate and the absorbance at 405 nm was measured after incubation at 37°C for 1 hour (BioTek, Synergy TM 2, USA). The experimental results showed that the release rate of β-hexosaminidase was 62.3% when calcimycin A23187 (1 µM) was used to induce allergic reactions. After PSBL was applied to mast cells, the activity of β-hexosaminidase was 49.6%, indicating that PSBL could inhibit the activity of β-hexosaminidase and had anti-allergic effect.

[0055] Table 4

[0056]

[0057] Example 10: Experiment on promoting cell energy production by encapsulated peony stamen microcapsules (PSBL)

[0058] Determination of intracellular adenosine triphosphate (ATP) content. ATP is the most abundant energy carrier molecule in the body. It can use the chemical energy in food molecules and release it to provide fuel for work in cells. To evaluate whether the encapsulated peony stamen microcapsules (PSBL) can induce the generation of cellular energy ATP, the ApoSENSORTM Cellviability assay kit was used to quantify the amount of ATP induced. Human skin keratinocytes (1.5×10 5 cell / mL) in a 96-well plate and grown in a 37˚C, 5% CO2 incubator for more than 24 hours. Then, PSBL was added and reacted for 24 hours. The supernatant was removed and washed with PBS. 100 μL Nuclear releasing reagent was added and reacted for 5 minutes. Then, 10 μL ATP monitoring enzyme was added and the ATP content was measured by cold light instrument (BioTek, Synergy TM 2, USA). Test results: The ATP content of untreated human skin keratinocytes was 100% in the control group. After being treated with PSBL, the ATP content in the cells increased to 108.7%. The test results showed that the cells were stimulated by PSBL and the ATP content in the cells increased by 8.7%, indicating that PSBL has the effect of promoting the production of cellular energy ATP.

[0059] Table 5

[0060]

[0061] Example 11: PSBL-encapsulated peony stamen microcapsules regulate the expression of anti-aging protein Lamin A

[0062] Mutations in the lamin A gene can damage DNA repair function, making the genome unstable and leading to premature aging. The anti-aging mechanism found that Lamin A protein can stimulate the longevity gene SIRT1, allowing cells to self-protect, self-renew and delay aging. To evaluate the effect of coated peony stamen microcapsules (PSBL) on regulating the expression of anti-aging protein Lamin A, thereby delaying cell aging. Human skin keratinocytes (HaCaT cells) (1.5×10 5cell / mL) was cultured in a 10 cm diameter dish and cultured in a 37°C and 5% CO2 incubator for at least 24 hours. The coated peony stamen microcapsules (PSBL) were added and placed in the incubator for 24 hours. When the reaction time was reached, protein extraction was performed using RIPA buffer, and then SDS-PAGE electrophoresis was performed until the target protein was separated. After adding 0.5% skim milk for 2 minutes, the first-level antibody was added for reaction for 10 minutes, and then rinsed 3 times with 0.1% phosphate-buffered saline (PBST), and then the second-level antibody was added for reaction for 10 minutes, and then rinsed 3 times with 0.1% PBST. Finally, the ECL cold light sensing reagent was added, the cold light camera system was placed to take pictures, and image J was used to quantitatively analyze the protein expression. The experimental results are shown in the following table. Figure 4 As shown: After human skin keratinocytes (HaCaT cells) were treated with PSBL, the expression of Lamin A protein in the cells was analyzed. The cytoskeletal protein β-actin was used as a quantitative control group. The results showed that compared with the control group (Lamin A expression was 1.0), the expression of Lamin A increased to 3.8 after PSBL treatment, indicating that PSBL can activate Lamin A protein, which is speculated to have anti-aging effects.

[0063] Example 12: Encapsulated Peony Stamen Microcapsules (PSBL) regulate the expression of Tyrosinase, a gene that inhibits pigmentation; regulates the expression of mTOR, a gene that inhibits aging; and regulates the expression of TOP1, a gene that promotes telomerase

[0064] The experiment was conducted by real-time quantitative qPCR. The expression of the tyrosinase gene in the mechanism of melanin production regulated by PSBL was analyzed; the expression of the gene that inhibits aging (mTOR) was regulated; and the expression of the gene that promotes telomerase (TOP1) was regulated. Tyrosinase is an oxidase and the rate-limiting enzyme that regulates melanin production. This enzyme participates in melanin synthesis. By regulating and inhibiting the activation of Tyrosinase, melanin production and pigmentation can be inhibited. mTOR is a serine-threonine protein kinase, which is an important factor causing aging and canceration. It promotes cells to absorb heat, shuts down the autophagy mechanism, and prevents cell renewal. Therefore, in recent years, studies on the inhibition of mTOR expression have pointed out that by inhibiting the expression of mTOR in aging cells, cells can have the potential for regeneration. The function of telomeres is to maintain the integrity of eukaryotic chromosomes and control the cell division cycle. After cell division, telomeres will stably replicate the post-division DNA to ensure the integrity of the genetic sequence. However, each time a cell divides, the telomeres will shorten. When the telomeres are exhausted, the cell will start apoptosis. Therefore, telomeres are considered the best marker of aging. Telomerase can replenish telomeres, extend the number of cell divisions, and slow down the aging process. TOP1 is a telomere protection protein. Promoting its expression can enhance the protection of telomeres. Melanoma cell lines (B16 cells) and human skin keratinocytes (HaCaT cells) (1.5×10 5 cell / mL) was 1×10 5The cells were inoculated at a density of 100 cells / mL in a 6-well plate and cultured for 24 hours. RNA of melanoma cell lines was extracted after PSBL was mixed with serum-free culture medium for 72 hours; RNA of human skin keratinocytes was extracted after PSBL was mixed with serum-free culture medium for 24 hours. The supernatant was collected into a 15 mL centrifuge tube, cells were removed with Trypsin, and cells were collected with PBS and centrifuged to remove the supernatant. 1 mL REzolTMC&T was added and mixed evenly for 5 minutes at room temperature to dissolve the cells. 200 μL chloroform was added and mixed evenly for 15 seconds. Total RNA was purified and isolated by reacting at 4°C for 5 minutes. Centrifugation was performed at 12,000 rpm at 4°C for 10 minutes. The top transparent layer in the centrifuge tube was taken into a 1.5 mL microcentrifuge tube soaked in DEPC-H2O and sterilized. Add an equal volume of isopropanol and mix well. Let stand at 4°C for 5 minutes to precipitate RNA. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Remove the supernatant. Add 200 μL of 75% ice alcohol to wash RNA. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Completely remove the supernatant and invert in the air to dry in a sterile operating table to completely evaporate the moisture in the centrifuge tube. Add 100 μL of 0.1% DEPC-H2O to disperse RNA and store in a -80°C refrigerator. Quantify RNA: Take 2 μL of RNA in 198 μL of 0.1% DEPC-H2O, mix well, take 100 μL in a micro quartz tube, and measure the absorbance at OD260 nm and OD280 nm using a spectrophotometer (BioTek, Synergy TM2, USA). Reverse transcription (RT): 0.1% DEPC-H2O was added to adjust the concentration to 2 mg RNA, and the volume was made up to 12.5 μL. After adding 1 μL OligodT primer and mixing evenly, 70 μL was added for 2 minutes, and then quickly placed on ice. Then 4 μL 5× MMLV-RT buffer solution, 1 μL 10mM Each dNTP, 0.5 μL RNase inhibitor and 1 μL MMLV RTase were added, and the reaction was incubated at 42°C for 1 hour, and then at 94°C for 5 minutes. Then, ice-cold ddH2O was added to make the volume up to 100 μL, and the cDNA preparation was completed and stored in a -80°C refrigerator. The first step of qRT-PCR was completed using a fluorescence detection system (prism 7700 sequencing detection system) and Qiagen's Quantitect SYBR Green RT-PCR kit according to the manufacturer's procedures. The primer sequences were the same as those used to generate arrayed cDNAs. The amplification conditions were as follows: reverse transcription at 50°C for 30 minutes, followed by PCR activation at 95°C for 15 minutes. Melting temperature was 95°C for 15 seconds, and annealing / extension was performed at 60°C for 1 minute, for a total of 40 cycles. The size of the amplified product was confirmed by the cycle threshold (CT) (ΔΔCT) method, and the CT values ​​of the control group and the laboratory were compared. The β-actin gene was used for quantitative analysis. The fold change of gene expression was calculated. Take 10 μL cDNA into a microcentrifuge tube, add 5 μL 10× reaction buffer, 2 μL 25 mM MgCl2, 0.8 μL 10 mM dNTP and 1 μL each of 50 μM upstream and downstream primer, Taq DNA polymerase (5 unit / μL), and finally add deionized water to make the total volume reach 50 μL, and place it in an automatic temperature cycler (MyGo Pro real-time PCR) for PCR reaction. The test results are as follows Figure 5 As shown: Genetic analysis experiments have shown that the coated peony stamen microcapsules (PSBL) regulate and inhibit the expression of the pigmentation mechanism gene Tyrosinase in melanoma cells; regulate and inhibit the expression of the aging gene mTOR in human skin keratinocytes, and regulate the expression of the telomerase-promoting gene TOP1.

[0065] However, what is described above is only a preferred embodiment of the present invention, but it cannot be used to limit the scope of protection of the present invention; therefore, all simple equivalent changes and modifications made according to the claims and description of the present invention are still within the scope of protection of the present invention.

Claims

1. A peony stamen composite microcrystal capsule composition, characterized in that: include: A carrier formed into a shell structure, wherein the components of the carrier include: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, lecithin, hydrogenated lecithin, and D-α-tocopheryl polyethylene glycol 1000 succinate, and wherein the proportion of phosphatidylcholine in the components contained in the carrier is greater than 50% by weight; and The peony stamen extract is coated by the shell-like structure, wherein the preparation method of the peony stamen extract comprises: A freezing step, freezing and solidifying the peony stamens with liquid nitrogen; a grinding step of grinding the solidified peony stamens into peony stamen powder; A soaking step, mixing the peony stamen powder and water in a weight ratio of 1:7 to 11, and soaking at a temperature of 4 to 50° C. for 72 to 120 hours to obtain a mixture; as well as The extraction step is to extract the mixture by continuous rapid oscillation at a total ultrasonic energy of 500 to 800 W at a temperature of -4 to 25°C for 60 to 120 minutes.

2. A method for preparing the composition according to claim 1, characterized in that: The method comprises: mixing the peony stamen extract with the carrier, and dispersing the carrier to form a colloidal complex.

3. The method according to claim 2, characterized in that The method further comprises a shaking step of shaking the colloidal complex using a high-power ultrasonic oscillator.

4. The method according to claim 3, characterized in that The temperature condition of this shaking step is 35°C ~ 55°C.

5. The method according to claim 4, characterized in that The duration of this shaking step lasts at least 30 minutes.

6. A use of the composition according to claim 1, characterized in that: Used to prepare anti-blue light, anti-allergic and anti-aging cosmetics.

7. The use according to claim 6, characterized in that The composition is used to administer the peony stamen extract at an effective dose to individual skin cells to achieve the effects of resisting blue light, protecting cells, repairing cells, resisting allergies, promoting cell energy generation, resisting aging, and inhibiting the expression of pigmentation mechanism genes.

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