Application of epiphyllum callus extract in preparation of cosmetics

Abstract

The invention provides application of epiphyllum callus extract in preparation of cosmetics, and belongs to the technical field of new biological materials. Compared with a common epiphyllum extract, the epiphyllum callus extract provided by the invention is rich in high-content active ingredients, such as kaempferol, general flavone, total polyphenol, total protein and total sugar; the antioxidant skincare effect is achieved, and the removal efficiency of cells on DPPH free radicals can be improved. The skin care effect of promoting ATP synthesis is achieved, ATP synthesis can be remarkably promoted, and energy metabolism of damaged mitochondria is improved. Effective components of epiphyllum calluses are extracted, the effect of the epiphyllum calluses on human immortal keratinocytes is evaluated through cell experiments, it is proved that the epiphyllum calluses have excellent effects of resisting oxidation, resisting inflammation and repairing the keratinocytes, and the epiphyllum calluses are suitable for being used as raw materials to be applied to the cosmetic industry.

Description

Technical Field

[0001] The present invention relates to the technical field of new biological materials, and in particular to application of an epiphyllum callus extract in preparing cosmetics. Background Art

[0002] Botanical skincare ingredients are extracted from plants, processed using modern technology, and then incorporated into skincare products. Compared to traditional chemical skincare products, skincare formulated with plant extracts as core ingredients offers many unique advantages, such as aloe vera, which fights acne, reduces swelling, and inhibits inflammation; calendula, which is an antioxidant and soothes sensitive skin; rose, which effectively scavenges free radicals and promotes blood circulation; and dendrobium, which effectively hydrates and locks in moisture, regulating water and oil balance.

[0003] Chinese patent CN117427008A discloses the use of Epiphyllum cyrtonema extract for oil control, hair loss prevention, and / or acne treatment. The extract is prepared by decoction using water as the extraction solvent. The invention also provides the use of Epiphyllum cyrtonema extract in the preparation of a topical skin preparation with oil control, hair loss prevention, and / or acne treatment effects.

[0004] However, the efficacy of the above products needs to be further improved. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of epiphyllum callus extract in the preparation of cosmetics. The callus extract produced by the present invention through callus culture technology has significantly improved production efficiency and skin care efficacy, can comprehensively obtain and enrich the effective active ingredients in epiphyllum, and prepare epiphyllum callus extract in a green and sustainable manner, thereby expanding the application of epiphyllum callus in cosmetic raw materials.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides the use of an epiphyllum callus extract in preparing cosmetics with anti-inflammatory effects.

[0007] The present invention also provides the use of the epiphyllum callus extract in preparing cosmetics with repairing effects.

[0008] The present invention also provides the use of the epiphyllum callus extract in preparing cosmetics with antioxidant effects.

[0009] The present invention also provides a method for preparing an epiphyllum callus extract, comprising the following steps: The Epiphyllum callus tissue was ground into powder in liquid nitrogen and subjected to alcohol extraction to obtain an alcohol extract; The ethanol extract is subjected to ultrasonic treatment, and the solvent is removed to obtain the Epiphyllum cyrtonema callus extract.

[0010] Preferably, the material-liquid ratio of the alcohol extraction treatment is 1:3~8; The alcohol extraction treatment uses a methanol solution, and the concentration of the methanol solution is 50-100%; The power of the ultrasonic treatment is 1800~2200W; The temperature of the ultrasonic treatment is 20-45°C; The ultrasonic treatment time is 100-400 min; The number of ultrasonic treatments is 2 to 5 times.

[0011] Preferably, the Epiphyllum cyrtonema callus is pretreated as follows: Freeze-dry after freezing at -40~-20℃ for 12~48h; The freeze-drying parameters include: cold trap temperature of -100 to -60°C, vacuum degree of 0 to 10 Pa, and time of 12 to 48 hours.

[0012] Preferably, the Epiphyllum callus is obtained by induction culture using the following induction medium: The induction medium includes the following components: MS basal medium, 6-BA 0.3~0.8 mg / L, NAA 0.3~0.8 mg / L, TDZ 0.3~0.8 mg / L.

[0013] The present invention also provides an epiphyllum callus extract prepared by the preparation method.

[0014] Preferably, the Epiphyllum cereus callus extract is the Epiphyllum cereus callus extract obtained by the preparation method of the present invention.

[0015] The present invention also provides a cosmetic comprising the epiphyllum callus extract and cosmetically acceptable excipients.

[0016] Beneficial effects of the present invention: Compared with common epiphyllum extracts, the epiphyllum callus extract provided by the present invention is rich in active ingredients such as kaempferol, total flavonoids, total polyphenols, total protein, and total sugars; and has the following skin care benefits: antioxidant skin care benefits. The raw material prepared by the above process can improve the efficiency of cells in scavenging DPPH free radicals. It has the skin care benefit of promoting ATP synthesis. The raw material prepared by the above process can significantly promote ATP synthesis and improve the energy metabolism of damaged mitochondria. The present invention extracts the active ingredients from epiphyllum callus and conducts cell experiments on the active ingredients to evaluate the efficacy of the extract on human immortalized keratinocytes. Subsequently, comparative experiments were conducted to test the antioxidant, anti-inflammatory, and repair effects of the epiphyllum callus extract and traditional epiphyllum extracts. The results show that the epiphyllum callus extract is suitable for use as a raw material in the cosmetics industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 HPLC spectrum of the Epiphyllum callus extract of Example 1 of the present invention; Figure 2 HPLC spectrum of the Epiphyllum scabra extract prepared in Comparative Example 1 of the present invention; Figure 3 This is a comparison chart of the total flavonoids, total polyphenols, total protein, and total sugar content of the Epiphyllum cyrtonema callus extract and the Epiphyllum cyrtonema extract of Example 1 of the present invention and Comparative Example 1; Figure 4 This is a graph showing the effect of the Epiphyllum cyrtonema callus extract on TNF-α gene expression in Example 1 of the present invention; Figure 5 This is a diagram showing the effect of the Epiphyllum cyrtonema callus extract on IL-1β gene expression in Example 1 of the present invention; Figure 6 This is a comparison chart of the DPPH radical scavenging rates of the Epiphyllum cyrtonema callus extract and the Epiphyllum cyrtonema extract according to Example 1 of the present invention and Comparative Example 1; Figure 7 This is a comparison chart of the effects of the epiphyllum callus extract and the epiphyllum extract of Example 1 of the present invention and Comparative Example 1 on promoting ATP synthesis. DETAILED DESCRIPTION

[0018] The present invention provides the use of an extract from Epiphyllum cyrtonema callus in the preparation of cosmetics with anti-inflammatory efficacy. In the present invention, the Epiphyllum cyrtonema callus extract refers to an extract obtained by water extraction or alcohol extraction using Epiphyllum cyrtonema callus as the raw material. The present invention also provides the use of an extract from Epiphyllum cyrtonema callus in the preparation of cosmetics with repair efficacy. The present invention also provides the use of an extract from Epiphyllum cyrtonema callus in the preparation of cosmetics with antioxidant efficacy. Compared to conventional extracts from Epiphyllum cyrtonema, the present invention demonstrates that the callus extract has superior activity and can achieve better application prospects.

[0019] The present invention also provides a method for preparing an extract of Epiphyllum cereus callus, comprising the following steps: grinding Epiphyllum cereus callus into a powder in liquid nitrogen, subjecting the extract to an alcohol extraction process to obtain an alcohol extract; subjecting the alcohol extract to ultrasonic treatment, and removing the solvent to obtain the extract of Epiphyllum cereus callus. Preferably, the alcohol extraction process comprises a solid-liquid ratio of 1:3 to 8; a methanol solution having a concentration of 50 to 100% is used for the alcohol extraction process; the ultrasonic treatment process comprises a power of 1800 to 2200 W; a temperature of 20 to 45°C; a duration of 100 to 400 minutes; and two to five ultrasonic treatments. Preferably, the Epiphyllum cereus callus undergoes the following pretreatment: freezing at -40 to -20°C for 12 to 48 hours, followed by freeze-drying; freeze-drying parameters include a cold trap temperature of -100 to -60°C, a vacuum of 0 to 10 Pa, and a drying time of 12 to 48 hours. Preferably, the Epiphyllum callus is obtained by induction culture in the following induction medium: the induction medium comprises the following components: MS basal medium, 6-BA 0.3~0.8 mg / L, NAA 0.3~0.8 mg / L, TDZ 0.3~0.8 mg / L.

[0020] The present invention also provides an Epiphyllum occidentalis callus extract obtained by the above preparation method. Preferably, the Epiphyllum occidentalis callus extract is an Epiphyllum occidentalis callus extract obtained by the preparation method of the present invention.

[0021] The present invention also provides a cosmetic comprising the above-mentioned Epiphyllum callus extract and cosmetically acceptable excipients. The dosage form of the cosmetic is preferably a cream, a facial mask, an emulsion, a toner, a shower gel and a facial cleanser, etc. The amount of the Epiphyllum callus extract added to the cosmetic is preferably 5 to 100%, and more preferably 40 to 90%. In the present invention, the excipients include moisturizers, emulsifiers, thickeners, skin conditioners, pH regulators, preservatives and water, etc.; the emulsifier is preferably sodium lauroyl glutamate, lauroamidopropyl betaine, isopropyl myristate or cocoyl glucoside; the moisturizer is preferably sodium hyaluronate, glycerin, propylene glycol, butylene glycol, dipropylene glycol, sorbitol, inositol, β-glucan or trehalose; the thickener is preferably ... The thickener is preferably disodium EDTA, carbomer, xanthan gum, ammonium acryloyldimethyltaurate / VP copolymer or sodium polyacrylate grafted starch; the skin conditioner is preferably caprylhydroxamic acid, allantoin, ceramide, niacinamide, vitamin C derivatives, arbutin, tranexamic acid, yeast / hydrolyzed yeast; the pH adjuster is preferably arginine, sodium citrate, citric acid, phosphoric acid, tartaric acid, sodium dihydrogen phosphate, triethanolamine, the preservative is preferably methylparaben, butylparaben, ethylparaben, isobutylparaben, propylparaben, potassium sorbate, sodium benzoate, and the excipients preferably do not affect the efficacy of the Epiphyllum callus extract of the present invention.

[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1 This example provides a method for preparing an extract of Epiphyllum callus: Induction of Epiphyllum Callus Fresh stem segments and leaves of Epiphyllum spp. were cut into approximately 10 mm pieces using a scalpel and inoculated onto solid culture medium. The callus induction basal medium contains 2 g / L MS medium powder (Coolaber, PM1011), 25-30 g / L sucrose (Coolaber, CS10581), and 6-8 g / L agar (Coolaber, CA1331). The solution is adjusted to pH 5.8 and sterilized at high temperature before use. The final concentrations of plant hormones are: 0.5 mg / L 6-BA (6-Benzylaminopurine); 0.5 mg / L NAA (Naphthylacetic acid); and 0.5 mg / L TDZ (Thidiazuron). The plant hormone stock solution is sterilized by filtration and added when the basal culture medium temperature drops to approximately 65°C. Callus induction is carried out by dark culture at about 25°C, and subculture is carried out after about 20-30 days. The callus is picked up with tweezers and placed in a new proliferation culture medium tissue culture bottle. Then it is flattened little by little with tweezers and cultured in a dark place.

[0024] Preparation of freeze-dried Epiphyllum callus Fresh Epiphyllum callus was pre-frozen in a -30°C constant temperature refrigerator for 24 h, and then placed in a vacuum freeze dryer for drying. The cold trap temperature was -80°C, the freezing chamber temperature was -45°C to -35°C, the vacuum degree was 0-10 Pa, and the vacuum freezing was carried out for 48 h. During the 48 h drying process, the freezing chamber temperature and vacuum degree gradually changed with the freezing time, showing the following trends: Program 1 (1 h, -45°C, vacuum degree 0), Program 2 (0.5 h, -45°C, vacuum degree 0), Program 3 (0.5 h, -45°C, vacuum degree 0-10 Pa), Program 4 (0.5 h, -30°C, vacuum degree 0-10 Pa), Program 5 (1 h, -10°C, vacuum degree 0-10 Pa), Program 6 (5 h, 0°C, vacuum degree 0-10 Pa), Program 7 (4 h, 5°C, vacuum degree 0-10 Pa), Program 8 (3 h, 10°C, vacuum 0-10 Pa), program 9 (5 h, 15°C, vacuum 0-10 Pa), program 10 (5 h, 20°C, vacuum 0-10 Pa), program 11 (5 h, 25°C, vacuum 0-10 Pa), program 12 (5 h, 30°C, vacuum 0-10 Pa), program 13 (5 h, 30°C, vacuum 0-10 Pa), program 14 (7.5 h, 35°C, vacuum 0-10 Pa). The material obtained at this time is freeze-dried Epiphyllum cyrtonema callus tissue, and the finished product is stored in a refrigerator at -20°C or -80°C in the dark.

[0025] Preparation of Epiphyllum callus extract Precool the mortar and pestle (add liquid nitrogen to the mortar to precool it, the amount of liquid nitrogen added should be at least two-thirds of the mortar volume, and stir gently with the pestle. Repeat the operation twice after the liquid nitrogen evaporates). Take the freeze-dried Epiphyllum occidentalis callus tissue, grind the Epiphyllum occidentalis callus tissue in liquid nitrogen until it is powdery, and weigh 1 kg of the ground callus tissue powder.

[0026] Add 5 kg of 70% methanol at a (m / m) material-liquid ratio of 1:5. Repeat extraction three times at an ultrasonic power of 2000 W, a temperature of 25-45°C, and a duration of 120 min. Filter the combined extracts using a Buchner funnel and pass through a 0.22 μm filter membrane.

[0027] The filtered solution is passed through a rotary evaporator to remove the solvent. The rotary evaporation process requires three steps. The first rotary evaporation conditions are: 35-42℃, 80-120 rpm, and 35-40 minutes to ensure that the liquid in the rotary evaporation bottle does not overflow; the second rotary evaporation conditions are: 48℃, 80-120 rpm, and 30 minutes; the third rotary evaporation conditions are: 48℃, 80-120 rpm, and 30-45 minutes. Select an appropriate rotary evaporation time until the solvent is completely removed. The substance obtained at this time is the epiphyllum callus extract, and the finished product is stored in a refrigerator at 4℃ or -20℃ in the dark.

[0028] Comparative Example 1: A method for preparing an epiphyllum extract, comprising the following steps: The difference from Example 1 is that the extraction raw materials are different. Comparative Example 1 uses Epiphyllum as the extraction raw material.

[0029] Preparation of freeze-dried Epiphyllum Fresh Epiphyllum was pre-frozen in a -30°C constant temperature refrigerator for 24 h, and then placed in a vacuum freeze dryer for drying. The cold trap temperature was -80°C, the freezing chamber temperature was -45°C to -35°C, the vacuum degree was 0-10 Pa, and the vacuum freezing was carried out for 48 h. During the 48 h drying process, the freezing chamber temperature and vacuum degree gradually changed with the freezing time, showing the following trends: Program 1 (1 h, -45°C, vacuum degree 0), Program 2 (0.5 h, -45°C, vacuum degree 0), Program 3 (0.5 h, -45°C, vacuum degree 0-10 Pa), Program 4 (0.5 h, -30°C, vacuum degree 0-10 Pa), Program 5 (1 h, -10°C, vacuum degree 0-10 Pa), Program 6 (5 h, 0°C, vacuum degree 0-10 Pa), Program 7 (4 h, 5°C, vacuum degree 0-10 Pa), Program 8 (3 h, 10°C, vacuum degree 0-10 Pa), and Program 9 (10 h, 15°C, vacuum degree 0-10 Pa). pa), program 9 (5 h, 15°C, vacuum 0-10 Pa), program 10 (5 h, 20°C, vacuum 0-10 Pa), program 11 (5 h, 25°C, vacuum 0-10 Pa), program 12 (5 h, 30°C, vacuum 0-10 Pa), program 13 (5 h, 30°C, vacuum 0-10 Pa), and program 14 (7.5 h, 35°C, vacuum 0-10 Pa). The resulting material is a freeze-dried Epiphyllum cyrtonema sample, which should be stored in a -20°C or -80°C refrigerator protected from light.

[0030] Preparation of Epiphyllum plant extract Pre-cool the mortar and pestle (add liquid nitrogen to the mortar to pre-cool it, the amount of liquid nitrogen added should be at least two-thirds of the mortar volume, and stir it gently with the pestle. Repeat the operation twice after the liquid nitrogen evaporates). Take the freeze-dried Epiphyllum plants, grind the Epiphyllum tissue in liquid nitrogen until it is powdery, and weigh 1 kg of the ground powder.

[0031] Add 5 kg of 70% methanol at a (m / m) material-liquid ratio of 1:5. Repeat extraction three times at an ultrasonic power of 2000 W, a temperature of 25-45°C, and a duration of 120 min. Filter the combined extracts using a Buchner funnel and pass through a 0.22 μm filter membrane.

[0032] The filtered solution is then passed through a rotary evaporator to remove the solvent. This rotary evaporation process involves three steps. The first step involves evaporating at 35-42°C and 80-120 rpm, ensuring that the liquid in the evaporator does not overflow. The second step involves evaporating at 48°C for 30 minutes. The third step involves evaporating at 48°C for 30-45 minutes, ensuring that the solvent is completely removed. The resulting extract is the Epiphyllum cyrtonema plant extract. Store the product in a refrigerator at 4°C or -20°C, away from light.

[0033] Experimental Example 1 Detection of active ingredients in Epiphyllum callus extract The cryopreserved Epiphyllum callus extract mentioned in Example 1 was used as the test material, and the specific steps were as follows: Test sample preparation Epiphyllum callus extract was dissolved in methanol and sonicated for 20 minutes to completely dissolve. The extract was then centrifuged and the sonication and centrifugation steps were repeated three times. The supernatant obtained from these three cycles was collected and used as the test sample. A control group consisted of pure methanol solution without any sample added.

[0034] HPLC determination of active ingredients The substances in the extract of Epiphyllum callus were qualitatively analyzed by HPLC, and the experimental operations were carried out under light-proof conditions.

[0035] The resulting supernatant was filtered through a 0.22 μm filter. A binary gradient elution was used with mobile phases A: acetonitrile and B: double-distilled water (0.2% phosphoric acid). The column temperature was 25°C, the injection volume was 20 μL, the flow rate was 1 mL / min, and the detection wavelength was 360 nm. The elution procedure was as follows: 0-4 min, 80%-76% B; 4-20 min, 76%-69% B; 20-30 min, 69%-60% B. The sample was repeated three times to obtain the HPLC chromatogram of the Epiphyllum callus extract ( Figure 1 ).

[0036] Detection of active ingredients in Epiphyllum extract The epiphyllum extract mentioned in Comparative Example 1 was used as the test raw material, and the specific steps were as follows: Test sample preparation Epiphyllum flower extract was dissolved in methanol and ultrasonically shaken for 20 minutes to promote dissolution. The solution was then centrifuged and the ultrasonic and centrifugal steps were repeated three times. The supernatant was collected and evaporated to dryness. The dried sample was used as the test sample. A control group consisted of pure methanol solution without any sample added.

[0037] HPLC determination of active ingredients in extracts The substances in the Epiphyllum extract were qualitatively analyzed by HPLC, and the experimental operations were carried out under light-proof conditions.

[0038] The resulting supernatant was filtered through a 0.22 μm filter. Binary gradient elution was used with mobile phases A and B, acetonitrile and double-distilled water (0.2% phosphoric acid). The column temperature was 25°C, the injection volume was 20 μL, the flow rate was 1 mL / min, and the detection wavelength was 360 nm. The elution procedure was as follows: 0-4 min, 80%-76% B; 4-20 min, 76%-69% B; 20-30 min, 69%-60% B. The sample was repeated three times to obtain the HPLC chromatogram of the Epiphyllum cyrtonema extract ( Figure 2 ).

[0039] The chromatogram shows that kaempferol, the main active ingredient, was detected in the epiphyllum callus extract, but not in the epiphyllum extract. This result demonstrates that callus culture can effectively enrich the active ingredients in the raw material.

[0040] Experimental Example 2 Comparison of the main components of Epiphyllum callus extract and Epiphyllum extract Through plant callus culture technology, the main active substances such as flavonoids, total polyphenols, total protein, and total sugars were enriched in the Epiphyllum callus extract. The content of these substances was measured to compare the component differences between Epiphyllum callus extract and Epiphyllum extract.

[0041] The Epiphyllum callus extract in Example 1 and the Epiphyllum plant extract in Comparative Example 1 were used as test samples, and the method was as follows: Total flavonoids determination The benzopyran ring of the flavonoid compound reacts with aluminum salt to form a yellow complex under alkaline conditions. The absorbance of the solution is measured at the maximum absorption wavelength, and rutin is used as a calibration standard to quantitatively determine the total flavonoid content in the Epiphyllum callus extract and Epiphyllum extract.

[0042] 0.5 mL each of the diluted Epiphyllum callus extract and Epiphyllum cyrtonema extract were placed in a 25 mL volumetric flask (with 0.5 mL of 50% ethanol as a blank control). 1 mL of 5% NaNO₂ solution and 1 mL of 10% Al(NO₃)₃ solution were added, followed by shaking and allowing to stand for 6 minutes. 10 mL of 4% NaOH solution was then added and mixed thoroughly. Finally, 50% ethanol was added to the volume to 25 mL, shaken and allowed to stand for 20 minutes. The absorbance was measured at the maximum absorption wavelength of 510 nm. The total flavonoid content in the sample was calculated using a regression equation.

[0043] Total polyphenols determination Folin phenol reagent oxidizes the -OH groups in polyphenols and develops a blue color. The absorbance of the solution is measured at the maximum absorption wavelength. Gallic acid is used as a calibration standard to quantitatively determine the total polyphenol content in Epiphyllum callus extracts and Epiphyllum occidentalis extracts.

[0044] Take 1 mL each of the diluted Epiphyllum callus extract and Epiphyllum cyrtonema extract, add 5 mL of distilled water, then add 1 mL of Folin phenol reagent and shake thoroughly. Add 3 mL of 7.5% Na₂CO₃ over 8 minutes. Incubate in the dark for 2 hours and measure the absorbance at the wavelength of maximum absorption. Calculate the total polyphenol content using the regression equation.

[0045] Total protein determination Peptide bond structure in protein molecules under alkaline environment and Cu 2+ Complex and Cu 2+ Reduction to Cu + , BCA specifically binds to Cu 2+ The combination forms a stable purple-blue complex with a maximum light absorption value at 562 nm, which can be used to calculate the protein enzyme content in Epiphyllum callus extract and Epiphyllum extract.

[0046] Weigh 0.1 g of tissue sample and dissolve it in methanol. Ultrasonicate for 20 minutes, and the supernatant is the test sample. A control group consists of pure methanol solution without any sample. Twenty wells on an ELISA plate were selected and divided into standard and sample groups. In 16 of these wells, one replicate of each standard was set up, and 5 μL of the standard protein solution of the corresponding concentration was added to each well. The remaining four wells were divided into two replicate groups, with the replicates numbered identically. The test sample was added to two wells with different numbers for total polysaccharide determination.

[0047] Total sugar determination Total sugars are hydrolyzed into reducing sugars. Reducing sugars are reduced to amino compounds after being heated with DNS reagent under alkaline conditions. They appear orange-red in alkaline solution. The amount of reducing sugars is proportional to the depth of the orange-red substance. The reducing sugar content in Epiphyllum callus extracts and Epiphyllum cyrtonema extracts can be calculated.

[0048] Weigh 0.1 g of tissue sample, add 1 mL of extract A and 1.5 mL of distilled water, homogenize thoroughly, treat in a boiling water bath for 30 min (seal to prevent water loss), cool to room temperature, add 1 mL of extract B and mix thoroughly, dilute to 10 mL with distilled water, mix thoroughly, and pipette 1 mL into a centrifuge tube. Centrifuge at 8000 g for 10 min at room temperature. Take the supernatant and determine the total polysaccharide content in the sample.

[0049] Figure 3 This is a comparison of the total flavonoids, total polyphenols, total protein and total sugar content of Epiphyllum callus extract and Epiphyllum extract: the total flavonoids content of Epiphyllum callus extract is 8.61 ± 0.82 mg / g, the total polyphenols content is 16.31 ± 0.35 mg / g, the total protein content is 54.36 ± 1.07 mg / g; the total sugar content is 122.11 ± 1.27 mg / g; the total flavonoids content of Epiphyllum extract is 6.27 ± 0.47 mg / g, the total polyphenols content is 10.18 ± 0.62 mg / g, the total protein content is 44.95 ± 0.75 mg / g, and the total sugar content is 106.52 ± 0.95 mg / g.

[0050] Among them, the total flavonoids content in Epiphyllum callus was significantly higher than that in Epiphyllum (P<0.05), the total polyphenols content in Epiphyllum callus was significantly higher than that in Epiphyllum (P<0.01), the total protein content in Epiphyllum callus was significantly higher than that in Epiphyllum (P<0.05), and the total sugar content in Epiphyllum callus was significantly higher than that in Epiphyllum (P<0.05).

[0051] In summary, the content of main components of Epiphyllum oxypetalum callus extract is significantly higher than that of Epiphyllum oxypetalum extract, and can be used as a raw material in skin care products.

[0052] Experimental Example 3 Epiphyllum callus extract inhibits the expression of inflammatory factors TNF-α and IL-1β are two key pro-inflammatory factors that regulate gene expression and mediate inflammatory responses by activating downstream NF-κB and MAPK signaling pathways. In Experimental Example 1, the epiphyllum callus extract in Example 1 was used as the test sample, and the method was as follows: Preparation of test samples Epiphyllum callus extracts and Epiphyllum cyrtonema extracts were prepared and diluted to different sample concentrations using DMEM medium and labeled into groups. To prepare the modeling stimulant solution: 1 μg / mL LPS solution: Dissolve 1 mg of LPS powder in 1 mL of sterile water, filter through a membrane, and dispense into EP tubes. Store at -20°C. During the experiment, dilute the 1 mg / mL LPS solution to 1 μg / mL using DMEM medium.

[0053] Inflammatory factor expression test Collect samples from each group and extract; all prepared reagents should be equilibrated at room temperature for 30 minutes, dilute samples and standards; add 50 μL of prepared samples and standards, 50 μL of biotin antigen working solution, and react at 37°C for 30 minutes; wash the plate five times, add 50 μL of avidin-HRP working solution, and react at 37°C for 30 minutes; wash the plate five times, add 50 μL each of color development solution A and B, and react at 37°C for 10 minutes; add 50 μL of stop solution; read the OD value at 450 nm on a microplate reader within 10 minutes; calculate the content of TNF-α and IL-1β inflammatory factors in each group according to the standard curve.

[0054] Figure 4 This is the result of the effect of Epiphyllum callus extract on TNF-α gene expression. Figure 5 The results show that the epiphyllum callus extract can effectively regulate the expression of TNF-α and IL-1β genes and mediate inflammatory responses. Compared with the model group, 1%, 2%, and 4% epiphyllum callus extracts all inhibited the expression of inflammatory factors TNF-α and IL-1β, with inhibition rates of 23.75%, 42.72%, and 47.73% for TNF-α, respectively. 1%, 2%, and 4% epiphyllum callus extracts inhibited TNF-α by 41.09%, 24.36%, and 59.27%, respectively. These experimental results demonstrate that the epiphyllum callus extract of the present invention can effectively regulate the expression of TNF-α and IL-1β genes and mediate inflammatory responses, and can be used as a raw material in cosmetics.

[0055] Comparison of the antioxidant effects of DPPH free radicals of Epiphyllum callus extract and Epiphyllum extract: 1. The Epiphyllum callus extract in Example 1 and the Epiphyllum plant extract in Comparative Example 1 were used as test samples, and the method was as follows: Preparation of test samples: Epiphyllum callus extract and Epiphyllum cyrtonema extract were prepared and diluted into different sample concentrations using DMEM culture medium, and the samples were marked and grouped.

[0056] 2. Determination of free radical scavenging rate by DPPH method Weigh a certain amount of DPPH and prepare a 0.04 mg / mL DPPH solution with anhydrous ethanol. Take 2 mL of the solution of different test samples, add 2 mL of DPPH solution, mix well, leave at room temperature for 30 minutes, and centrifuge at 5000 r / min for 10 minutes. Take the supernatant and measure the absorbance at 517 nm. Use 1% concentration of VC solution as a positive control. The scavenging rate of the sample on DPPH free radicals is calculated using the following formula: Inhibition rate = (1- (A sample - A blank ) / A control ) × 100%. Paired differences between samples at the same concentration were tested using the T-test. P < 0.05 (*) was considered to indicate a significant difference, P < 0.01 (**) was considered to indicate a very significant difference, and P < 0.001 (***) was considered to indicate a very strong significant difference.

[0057] The results are as follows Figure 6 The experimental results showed that the DPPH scavenging rates of 0.1%, 1%, and 5% Epiphyllum callus extracts were 20.82%, 59.44%, and 85.90%, respectively; and the DPPH scavenging rates of 0.1%, 1%, and 5% Epiphyllum extracts were 14.71%, 23.57%, and 83.92%, respectively. The Epiphyllum callus extract of the present invention is more effective in scavenging free radicals in the body than Epiphyllum extract.

[0058] Comparative experiment on the efficacy of Epiphyllum callus extract and Epiphyllum extract in promoting ATP synthesis: ATP helps promote skin metabolism and remove yellowness and dullness. The Epiphyllum callus extract in Example 1 and the Epiphyllum plant extract in Comparative Example 1 were used as test samples, and the method was as follows: Epiphyllum callus extract and Epiphyllum cereus extract were prepared and diluted into different sample concentrations using DMEM culture medium, and the samples were marked and grouped.

[0059] ATP synthesis test method: Human skin fibroblasts (HSF) cells in logarithmic growth phase were cultured at a rate of 1*10 6 Cells were seeded at 100 μM Braco-19 per well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 h. The culture medium was discarded, and the cells were treated with 10 μM Braco-19 (the blank control group did not require Braco-19 treatment). Samples at concentrations of 0.1% and 0.1% were added to the experimental group, while serum-free culture medium was added to the blank control group and model group, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 h. The supernatant was discarded, and the cells were washed twice with PBS and collected. Finally, RNA was extracted, reverse transcribed into cDNA, and then tested by RT-qPCR.

[0060] The results are as follows Figure 7 As shown, after H2O2 treatment, cellular ATP levels were significantly reduced. Compared with the model group, 0.1% and 1% Epiphyllum occidentalis callus extract significantly increased cellular ATP levels by 179.67% and 242.28%; 0.1% and 1% Epiphyllum occidentalis extract significantly increased cellular ATP levels by 101.62% and 168.29%. The Epiphyllum occidentalis callus extract of the present invention is more effective in promoting ATP synthesis than Epiphyllum occidentalis extract, indicating that Epiphyllum occidentalis callus extract can significantly promote ATP synthesis and improve energy metabolism in damaged mitochondria.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of Epiphyllum callus extract in the preparation of cosmetics with anti-inflammatory effects.

2. Application of Epiphyllum callus extract in the preparation of cosmetics with repairing effects.

3. Application of Epiphyllum callus extract in the preparation of cosmetics with antioxidant effects.

4. A method for preparing an extract of Epiphyllum callus, characterized in that: The following steps are involved: The Epiphyllum callus tissue was ground into powder in liquid nitrogen and subjected to alcohol extraction to obtain an alcohol extract; The ethanol extract is subjected to ultrasonic treatment, and the solvent is removed to obtain the Epiphyllum cyrtonema callus extract.

5. The preparation method according to claim 4, characterized in that The material-liquid ratio of the alcohol extraction process is 1:3~8; The alcohol extraction treatment uses a methanol solution, and the concentration of the methanol solution is 50-100%; The power of the ultrasonic treatment is 1800~2200W; The temperature of the ultrasonic treatment is 20-45°C; The ultrasonic treatment time is 100-400 min; The number of ultrasonic treatments is 2 to 5 times.

6. The preparation method according to claim 4, characterized in that The epiphyllum callus is pretreated as follows: Freeze-dry after freezing at -40~-20℃ for 12~48h; The freeze-drying parameters include: cold trap temperature of -100 to -60°C, vacuum degree of 0 to 10 Pa, and time of 12 to 48 hours.

7. The preparation method according to claim 4, characterized in that The epiphyllum callus was obtained by induction culture on the following induction medium: The induction medium includes the following components: MS basal medium, 6-BA 0.3~0.8 mg / L, NAA 0.3~0.8 mg / L, TDZ 0.3~0.8 mg / L.

8. Epiphyllum callus extract prepared by the preparation method according to any one of claims 4 to 7.

9. The use according to any one of claims 1 to 3, characterized in that The epiphyllum callus extract is the epiphyllum callus extract according to claim 8.

10. A cosmetic, characterized in that: The invention comprises the epiphyllum callus extract according to claim 8, and cosmetically acceptable excipients.

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