Composition with synergistic anti-oxidation and anti-saccharification functions as well as preparation method and application of composition

Abstract

The invention provides a composition with synergistic anti-oxidation and anti-saccharification functions as well as a preparation method and application of the composition, and relates to the technical field of cosmetics. The synergistic anti-oxidation and anti-saccharification composition comprises kadsura coccinea and pericarpium citri reticulatae, and the mass ratio of the kadsura coccinea to the pericarpium citri reticulatae is 1: 3. The synergistic anti-oxidation and anti-saccharification composition has good anti-saccharification, anti-oxidation and free radical scavenging capabilities, and can be widely applied to the field of cosmetics.

Description

Technical Field

[0001] The invention provides a composition with synergistic antioxidant and anti-glycation properties, a preparation method and application thereof, and relates to the technical field of cosmetics. Background Art

[0002] Free radicals, also called free radicals, can cause the skin to lose elasticity and produce wrinkles. Glycation reaction is one of the factors that cause human aging and dull skin. Skin anti-glycation and anti-oxidation are two important concepts in skin care, which help delay skin aging, maintain skin health and whiten the skin.

[0003] Tangerine peel is pungent, bitter, and warm in nature. It enters the spleen and lung meridians and can regulate qi and strengthen the spleen, dry dampness and resolve phlegm. Black tiger peel is pungent, slightly bitter, and warm in nature. It can be used for stomachache, rheumatic pain, bruises, fractures, dysmenorrhea, abdominal pain due to postpartum blood stasis, and hernia pain.

[0004] Liu Lina and others published in the Southern Journal of Agriculture: Analysis of flavonoid components and evaluation of antioxidant activity of dried tangerine peel from different years. They reported that dried tangerine peel has DPPH free radical scavenging ability and antioxidant capacity. However, they did not conduct research on skin antioxidant and anti-glycation effects. Whether dried tangerine peel can be used for skin antioxidant and anti-glycation effects requires further research.

[0005] Li Yajun et al. published the extraction process of total flavonoids from black tiger leaves and their antioxidant activity in Guangxi Plants. They reported that black tiger has the ability to scavenge DPPH free radicals. However, they did not conduct research on skin antioxidant and anti-glycation effects. Whether tangerine peel can be used for skin antioxidant and anti-glycation effects requires further research. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a composition with synergistic antioxidant and anti-glycation properties, a preparation method and application thereof, which has excellent free radical scavenging ability and can make the skin anti-glycation and anti-oxidation.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a composition with synergistic antioxidant and anti-glycation properties, comprising black tiger and tangerine peel; the mass ratio of the black tiger to tangerine peel is 1:3.

[0009] Furthermore, the black tiger is a black tiger extract; and the tangerine peel is a tangerine peel extract.

[0010] In a second aspect, the present invention provides a method for preparing a composition having synergistic antioxidant and anti-glycation effects, comprising the steps of:

[0011] (1) extracting black tiger to obtain a black tiger crude extract, and purifying the black tiger crude extract to obtain a black tiger extract;

[0012] (2) extracting tangerine peel to obtain a tangerine peel crude extract, and purifying the tangerine peel crude extract to obtain a tangerine peel extract;

[0013] (3) The black tiger extract and the tangerine peel extract are mixed and mixed.

[0014] Furthermore, the extraction in step (1) is as follows: petroleum ether and black tiger are refluxed for extraction, after filtering, ethanol is added to the filter residue, and ultrasonic extraction is performed to obtain a black tiger crude extract.

[0015] Furthermore, the extraction in step (2) is as follows: petroleum ether and tangerine peel are refluxed for extraction, and after filtering, ethanol is added to the filter residue, and ultrasonic extraction is performed to obtain a tangerine peel crude extract.

[0016] Furthermore, the extraction is repeated 1-2 times.

[0017] Furthermore, the mass ratio of black tiger to petroleum ether is 1:6-8.

[0018] Furthermore, the mass ratio of tangerine peel to petroleum ether is 1:6-8.

[0019] Furthermore, the reflux time of the black tiger is 1 hour, and the reflux temperature is 60°C.

[0020] Furthermore, the ratio of black tiger to ethanol is 1g:50mL.

[0021] Furthermore, the ratio of tangerine peel to ethanol is 1g:25mL.

[0022] Furthermore, the black tiger uses ethanol with a mass percentage of 70%.

[0023] Furthermore, the ethanol used in the dried tangerine peel is 60% by mass.

[0024] Furthermore, the black tiger ultrasonic conditions are: 40° C., 100W, ultrasonic for 5 minutes and then reflux at 60° C. for 1 hour.

[0025] Furthermore, the ultrasonic conditions of the tangerine peel are: 50°C, 300w, and ultrasonication for 40min.

[0026] In a third aspect, the present invention provides use of a composition in the preparation of antioxidant and anti-glycation cosmetics.

[0027] In a fourth aspect, the present invention provides an antioxidant and anti-glycation cosmetic comprising the above-mentioned composition.

[0028] The technical effects achieved by the present invention are:

[0029] The black tiger and tangerine peel in the specific dosage relationship of the present invention can avoid the antagonistic effect between the two raw materials, so that the effective ingredients thereof can play a synergistic role, enhance the free radical scavenging ability of the composition, enhance the anti-oxidation and anti-glycation ability of the skin, and have excellent repairing ability for oxidatively damaged cells, and can be widely used in the field of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the DPPH free radical scavenging ability of the embodiment and the comparative example.

[0031] Figure 2 The PTIO free radical scavenging ability of the examples and comparative examples.

[0032] Figure 3 The AGEs inhibition ability of the examples and comparative examples is shown in FIG.

[0033] Figure 4 It is the lipid peroxidation inhibition ability of the examples and comparative examples.

[0034] Figure 5 are the cell H2O2 oxidative damage pre-protection abilities of the embodiments and comparative examples, #### is compared with the control group, p<0.0001; ** is compared with the model group, p<0.01, *** is compared with the model group, p<0.001, **** is compared with the model group, p<0.0001; & is the embodiment compared with comparative example 1, p<0.05, &&&& is the embodiment compared with comparative example 1, p<0.0001; the ns of the 0.0125 mg / mL group embodiment and comparative example 1 is: there is no significant difference compared with the model group; the ns of the 0.1 mg / mL group is: there is no significant difference compared with the comparative example 1; the ns of the 0.2 mg / mL group is: there is no significant difference compared with the comparative example 1.

[0035] Figure 6 is the cell oxidative damage repair ability of the embodiment and the comparative example, #### is compared with the control group, p<0.0001; * is compared with the model group, p<0.05, ** is compared with the model group, p<0.01, *** is compared with the model group, p<0.001, **** is compared with the model group, p<0.0001; & is compared with the embodiment and comparative example 1, p<0.05, &&&& is compared with the embodiment and comparative example 1, p<0.0001; ns means: there is no significant difference between the embodiment and comparative example 1.

[0036] Figure 7is the GSH content of the embodiment and the comparative example, #### is compared with the control group, p<0.0001; ** is compared with the model group, p<0.01, **** is compared with the model group, p<0.0001; &&&& is compared with the embodiment and comparative example 1, p<0.0001; ns means: there is no significant difference compared with the model group. DETAILED DESCRIPTION

[0037] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0039] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0040] It is worth noting that the raw materials used in the present invention are all common commercially available products, so their sources are not specifically limited.

[0041] 1. Examples and Comparative Examples

[0042] 1. Example 1

[0043] Preparation method:

[0044] (1) Add petroleum ether to the black tiger according to a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 70% ethanol to the residue according to a solid-liquid ratio of 1g:50mL, ultrasonicate for 5 minutes at 40°C and 100w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude black tiger extract;

[0045] Add petroleum ether to tangerine peel at a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 60% ethanol to the residue at a solid-liquid ratio of 1g:25mL, ultrasonicate for 40 minutes at 50°C and 300w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude tangerine peel extract.

[0046] (2) The pretreated macroporous resin AB-8 was loaded into a wet column, and the crude extract of black tiger and tangerine peel was purified respectively, specifically: the loading time was 4-7 hours, eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless, the ethanol eluate was collected, rotary evaporated and freeze-dried to obtain black tiger extract and tangerine peel extract respectively.

[0047] (3) Mix the black tiger extract and the tangerine peel extract in a mass ratio of 1:3.

[0048] 2. Comparative Example

[0049] Comparative Example 1

[0050] Preparation method:

[0051] (1) Add petroleum ether to the black tiger according to a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 70% ethanol to the residue according to a solid-liquid ratio of 1g:50mL, ultrasonicate for 5 minutes at 40°C and 100w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude black tiger extract.

[0052] (2) The pretreated macroporous resin AB-8 was loaded into a wet column and the crude black tiger extract was purified by the following steps: the sample was loaded for 4-7 hours, eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless, the ethanol eluate was collected, rotary evaporated and freeze-dried to obtain the black tiger extract.

[0053] Comparative Example 2

[0054] Preparation method:

[0055] (1) Add petroleum ether to dried tangerine peel at a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 60% ethanol to the residue at a solid-liquid ratio of 1g:25mL, ultrasonicate for 40 minutes at 50°C and 300w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude dried tangerine peel extract.

[0056] (2) The pretreated macroporous resin AB-8 was loaded into a wet column to purify the tangerine peel crude extract, specifically: the sample was loaded for 4-7 hours, eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless, the ethanol eluate was collected, rotary evaporated and freeze-dried to obtain the tangerine peel extract.

[0057] Comparative Example 3

[0058] Preparation method:

[0059] (1) Add petroleum ether to the black tiger according to a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 70% ethanol to the residue according to a solid-liquid ratio of 1g:50mL, ultrasonicate for 5 minutes at 40°C and 100w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude black tiger extract;

[0060] Add petroleum ether to tangerine peel at a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 60% ethanol to the residue at a solid-liquid ratio of 1g:25mL, ultrasonicate for 40 minutes at 50°C and 300w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude tangerine peel extract.

[0061] (2) The pretreated macroporous resin AB-8 was loaded into a wet column, and the crude extract of black tiger and tangerine peel was purified respectively, specifically: the loading time was 4-7 hours, eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless, the ethanol eluate was collected, rotary evaporated and freeze-dried to obtain black tiger extract and tangerine peel extract respectively.

[0062] (3) Mix the black tiger extract and the tangerine peel extract in a mass ratio of 1:1.

[0063] Comparative Example 4

[0064] Preparation method:

[0065] (1) Add petroleum ether to the black tiger according to a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 70% ethanol to the residue according to a solid-liquid ratio of 1g:50mL, ultrasonicate for 5 minutes at 40°C and 100w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude black tiger extract;

[0066] Add petroleum ether to tangerine peel at a solid-liquid ratio of 1:8 (mass ratio), reflux for 2.5 hours at 50°C, filter, add 60% ethanol to the residue at a solid-liquid ratio of 1g:25mL, ultrasonicate for 50 minutes at 50°C and 300w, reflux extract for 1.0 hour, filter after reflux, collect the filtrate and repeat the above steps for the extracted medicinal residue. Mix and concentrate the extracts from the two ultrasonic refluxes to obtain a crude tangerine peel extract.

[0067] (2) The pretreated macroporous resin AB-8 was loaded into a wet column, and the crude extract of black tiger and tangerine peel was purified respectively, specifically: the loading time was 4-7 hours, eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless, the ethanol eluate was collected, rotary evaporated and freeze-dried to obtain black tiger extract and tangerine peel extract respectively.

[0068] (3) Mix the black tiger extract and the tangerine peel extract in a mass ratio of 1:4.

[0069] 3. Antioxidant efficacy test

[0070] 3.1DPPH free radical scavenging experiment

[0071] Take 3 mL of each of Examples and Comparative Examples 1-4 with a concentration of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, and add 3 mL of 0.1 mol / LDPPH 95% ethanol solution respectively under light-proof conditions.

[0072] React for 30 minutes at 37°C in the dark, and quickly measure the absorbance at 517nm. The absorbance of the reaction between DPPH and the sample is recorded as A1. The absorbance of the 95% ethanol solution of DPPH is replaced by 95% ethanol solution, and recorded as A2; the absorbance of the sample is replaced by ultrapure water, and recorded as A0.

[0073] Then calculate according to "DPPH clearance rate = [1-(A1-A2) / A0]×100%".

[0074] 3.2 Experimental Results

[0075] The results are shown in Table 1.

[0076] Table 1

[0077]

[0078] Compared with Example 1, * means p<0.05, ** means p<0.01, *** means p<0.001.

[0079] Figure 1The DPPH free radical scavenging ability of the black tiger and tangerine peel compound liquid was stronger, and its scavenging rate was 94.85% when the dosage concentration was 2.0mg / ml. The DPPH free radical scavenging ability of the black tiger extract and the black tiger and tangerine peel compound liquid increased with the increase of dosage concentration, and there was a good linear relationship between the dosage concentration and the scavenging rate in each group.

[0080] 3.3 PTIO free radical scavenging experiment

[0081] Step a: Dilute Examples and Comparative Examples 1-4 with PBS having a pH of 7.4 to a concentration of 3 mL each of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL.

[0082] Step b: Add 2 mL of 0.64 mmol / L PTIO aqueous solution in sequence under light-protected conditions.

[0083] Step c: Place in a 37°C water bath for 2 hours, and detect the absorbance A1 at a wavelength of 557 nm; replace the PTIO solution with PBS with a pH of 7.4; replace the sample with an equal amount of ultrapure water; perform the same operation to detect the absorbance and record it as A2 and A 0， ; Calculated using the formula “PTIO free radical scavenging rate = [1-(A1-A2) / A0]×100%”.

[0084] 3.4 Experimental Results

[0085] The experimental results are shown in Table 2.

[0086] Table 2

[0087]

[0088] Compared with Example 1, * means p<0.05, ** means p<0.01, and *0.* means p<0.001.

[0089] like Figure 2 As shown in the figure, the PTIO free radical scavenging ability of the black tiger and the black tiger and tangerine peel compound solution increased with the increase of the dosage concentration, and there was a good linear relationship between each concentration and the clearance rate. Among them, the PTIO free radical scavenging ability of the black tiger and tangerine peel compound solution was stronger, and the clearance rate was 98.27% when the dosage concentration was 2.0 mg / ml.

[0090] 3.5 Anti-glycation experiment

[0091] Step a: Take 2 mL of the examples and comparative examples with concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively.

[0092] Step b: Add 2 mL of 20 mg / mL BSA and 2 mL of 0.5 mol / mL glucose solution in sequence and mix well.

[0093] Step c: Incubate at 37°C for 7 days, and measure the fluorescence intensity A1 at 440nm after excitation with 370nm excitation light, with the excitation and emission slit width of 5nm. Replace BSA with PBS, and record the fluorescence intensity as A2; replace the sample solution with pure water, and record the fluorescence intensity as A3; replace the sample solution with pure water, and replace BSA with PBS, and record the fluorescence intensity as A4; calculate with the formula "AGEs inhibition rate (%) = [1-(A1-A2) / (A3-A4)]×100%".

[0094] 3.6 Experimental Results

[0095] The results are shown in Table 3.

[0096] Table 3

[0097]

[0098] Compared with Example 1, * means p<0.05, ** means p<0.01, and *0* means p<0.001.

[0099] The experimental results are as follows Figure 3 As shown in the figure, the AGEs inhibition ability of Black Tiger and Black Tiger and Tangerine Peel Compound Liquid increases with the increase of the dosage concentration, and there is a good linear relationship between each concentration and the inhibition rate. Among them, the anti-glycation ability of Black Tiger and Tangerine Peel Compound Liquid is stronger, and the inhibition rate is 97.04% when the dosage concentration is 2.0mg / ml.

[0100] 3.7 Anti-lipid peroxidation experiment

[0101] Step a: Prepare TCA-TBA-HCl mixed solution: add 15 g TCA, 0.375 g TBA, and 2.1 mL concentrated HCl into 100 mL pure water in sequence.

[0102] Step b: Take 1.5 mL of solution of the examples and comparative examples with concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1.0 mg / mL, and 2.0 mg / mL.

[0103] Step c: Add 1.5mL 10mg / mL lecithin solution and 1.5mL 0.4mmol / L ferrous sulfate solution in sequence under light-proof conditions. Step d: Place in a 37°C water bath to react in the dark for 1h, add 3mL TBA-TCA-HCl mixed solution, cool immediately after 15min in a 95°C water bath, centrifuge to obtain the supernatant, and detect the absorbance A0 at a wavelength of 535nm. Replace the lecithin solution with pure water, and the absorbance is recorded as A1; replace the sample solution with pure water, and the absorbance is recorded as A2. Calculate with the formula "clearance rate (%) = (A0-A1+A2) / A0×100%".

[0104] 3.8 Experimental Results

[0105] The results are shown in Table 4.

[0106] Table 4

[0107]

[0108] Compared with Example 1, * means p<0.05, ** means p<0.01, *** means p<0.001.

[0109] The experimental results are as follows Figure 4 The results show that the ability of the black tiger and the black tiger and tangerine peel compound solution to inhibit lipid peroxidation increases with the increase of the dosage concentration, and there is a good linear relationship between each concentration and the inhibition rate. The ability of the black tiger and tangerine peel compound solution to inhibit lipid peroxidation is stronger, and the inhibition rate is 93.76% when the dosage concentration is 2.0 mg / ml.

[0110] 4. Cellular antioxidant efficacy test

[0111] 4.1 Cellular H2O2 oxidative damage pre-protection experiment

[0112] L929 cells were cultured at 1×10 4 The cells were seeded at a density of 100 μL / well in a 96-well plate, incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours, and then the culture medium was discarded. The control group was DMEM culture medium without extract; the model group was DMEM culture medium without extract; the positive control group was VC solution diluted to 1 mM with DMEM culture medium; the experimental group was: the Chinese medicine extracts of the embodiment and the comparative example were diluted to the dosing concentration with DMEM culture medium, 100 μL of sample was added to each well, 3 replicates were set for each concentration, incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours, and then the culture medium was discarded, and then washed three times with 100 μL PBS, and 100 μL of H2O2 solution diluted to 0.6 mM with DMEM culture medium was added to each well except the control group.

[0113] Among them, the concentration of the positive control group is the concentration with the highest efficacy and the lowest toxicity.

[0114] The control group, model group and experimental group were incubated in a cell culture incubator at 37°C and 5% CO2 for 4 h, the culture medium was discarded, MTT reagent was added, and the cell culture incubator at 37°C and 5% CO2 was incubated for 4 h. The MTT reagent was discarded, and 100 μL DMSO was added to each well, and the cells were shaken on a shaker for 10 min. The absorbance was measured at 490 nm, and the formula "cell survival rate = A 给药孔 / A 空白孔 × 100%” to calculate the cell survival rate.

[0115] 4.2 Experimental Results

[0116] The results are shown in Table 5.

[0117] Table 5

[0118]

[0119] The experimental results are as follows Figure 5 As shown, compared with the model group (i.e., H2O2-treated group), the cell survival rates of the drug pre-protection groups were significantly improved, and the cell survival rate of the compound solution was improved more significantly.

[0120] 4.3 Cellular oxidative damage repair experiment

[0121] L929 cells were cultured at 1×10 4 The cells were seeded at a density of 100 μL / well in a 96-well plate, incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h, and then the culture medium was discarded. Except for the control group, 100 μL of H2O2 solution diluted to 0.6 mM with DMEM medium was added to each well, and the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 4 h, and then the culture medium was discarded.

[0122] The control group was DMEM medium without extract; the model group was DMEM medium without extract; the positive control group was VC solution diluted to 1 mM with DMEM medium; the experimental group was the Chinese medicine extracts of the examples and comparative examples were diluted to the dosing concentration with DMEM medium, 100 μL of sample was added to each well, and 3 replicate wells were set for each concentration.

[0123] Among them, the concentration of the positive control group is the concentration with the highest efficacy and the lowest toxicity.

[0124] The control group, model group and experimental group were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours, the culture medium was discarded, MTT reagent was added and incubated in a cell culture incubator at 37°C and 5% CO2 for 4 hours, the MTT reagent was discarded, 100uLDMSO was added to each well, and the shaker was shaken for 10 minutes. The absorbance value was measured at 490nm, and the formula "cell survival rate = A 给药孔 / A 空白孔 × 100%” to calculate the cell survival rate.

[0125] 4.4 Experimental Results

[0126] The results are shown in Table 6

[0127] Table 6

[0128]

[0129]

[0130] The experimental results are as follows Figure 6 As shown, compared with the model group (i.e., H2O2-treated group), the cell survival rates of the drug-repair groups were significantly improved, and the cell survival rate of the compound solution was improved more significantly.

[0131] 4.5 Experiment on determination of trace reduced glutathione (GSH) content

[0132] HaCaT cells were cultured at a rate of 1×10 6 The cells were seeded at a density of 1.5 × 10 / well in a 6-well plate and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h, after which the culture medium was discarded.

[0133] The control group was: DMEM medium without extract; the model group was: DMEM medium without extract; the positive control group was: VC solution diluted to 1 mM with DMEM medium; the experimental group: the Chinese medicine extracts of the examples and comparative examples were diluted to the dosing concentration with DMEM medium, and 1 mL of sample was added to each well.

[0134] The control group, model group and experimental group were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours, the culture medium was discarded, and the cells were washed three times with 1 mL PBS. Except for the control group, 1 mL of H2O2 solution diluted to 0.6 mM with DMEM culture medium was added to each well, and the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 4 hours, the culture medium was discarded, and the cells were washed three times with 1 mL PBS. 350 μL PBS was added to each well, the cells were scraped and the cell suspension was collected, the cells were ultrasonically disrupted and centrifuged to obtain the supernatant, and then the GSH content was determined according to the instructions of the trace reduced glutathione (GSH) assay kit, and the formula "GSH content = (A 测定 -A 空白 ) / (A 标准 -A 空白 )×C 标准 ×2÷Cpr” to calculate the cellular GSH content.

[0135] 4.6 Experimental Results

[0136] The results are shown in Table 7.

[0137] Table 7

[0138]

[0139] The experimental results are as follows Figure 7 As shown, compared with the model group (i.e., H2O2-treated group), the GSH content in the drug-treated groups was significantly increased, and the GSH content in the compound solution was increased more significantly.

[0140] Example 1 of the present invention has high DPPH and PTTO clearance rates, high AGEs inhibition rates, high peroxidation inhibition rates and is significantly different from Comparative Examples 1-4; Example 1 of the present invention has strong oxidative damage pre-protection and repair capabilities, high GSH content and is significantly different from Comparative Example 1.

[0141] The synergistic antioxidant and anti-glycation composition of the present invention enables black tiger and tangerine peel to work synergistically in a specific dosage relationship, thereby enhancing the free radical scavenging ability of the composition, enhancing the antioxidant and anti-glycation abilities of the skin, and having excellent repairing ability for oxidatively damaged cells, and can be widely used in the field of cosmetics.

[0142] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A composition with synergistic antioxidant and anti-glycation effects, characterized in that: It comprises black tiger and tangerine peel; the mass ratio of the black tiger and tangerine peel is 1:

3.

2. The composition according to claim 1, characterized in that The black tiger is a black tiger extract; and the tangerine peel is a tangerine peel extract.

3. A method for preparing the composition according to any one of claims 1 to 2, characterized in that: The steps are: (1) extracting black tiger to obtain a black tiger crude extract, and purifying the black tiger crude extract to obtain a black tiger extract; (2) extracting tangerine peel to obtain a tangerine peel crude extract, and purifying the tangerine peel crude extract to obtain a tangerine peel extract; (3) The black tiger extract and the tangerine peel extract are mixed and mixed.

4. The preparation method according to claim 3, characterized in that: The extraction in step (1) comprises: refluxing and extracting petroleum ether and black tiger, filtering, adding ethanol to the filter residue, and ultrasonically extracting to obtain a black tiger crude extract; The extraction in step (2) comprises: refluxing petroleum ether and tangerine peel for extraction, filtering, adding ethanol to the filter residue, and ultrasonically extracting to obtain a tangerine peel crude extract; The extraction was repeated 1-2 times.

5. The preparation method according to claim 4, characterized in that: The mass ratio of black tiger to petroleum ether is 1:6-8; The mass ratio of tangerine peel to petroleum ether is 1:6-8.

6. The preparation method according to claim 4, characterized in that: The reflux time is 1 h.

7. The preparation method according to claim 4, characterized in that: The ratio of black tiger to ethanol is 1g:50mL; The ratio of tangerine peel to ethanol is 1g:25mL; The black tiger uses ethanol with a mass percentage of 70%, and the tangerine peel uses ethanol with a mass percentage of 60%.

8. The preparation method according to claim 4, characterized in that: The ultrasonic conditions are: 40° C., 100W, ultrasonication for 5 minutes followed by reflux at 60° C. for 1 hour.

9. Use of the composition according to any one of claims 1 to 2 or the composition prepared by the preparation method according to any one of claims 3 to 8 in the preparation of antioxidant and anti-glycation cosmetics.

10. An anti-oxidation and anti-glycation cosmetic, characterized in that: The invention comprises the composition according to any one of claims 1 to 2 or the composition prepared by the preparation method according to any one of claims 3 to 8.

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