A composition with synergistic antioxidant and anti-glycation properties, its preparation method and application

Abstract

This invention provides a composition with synergistic antioxidant and anti-glycation properties, its preparation method, and its application, relating to the field of cosmetic technology. The synergistic antioxidant and anti-glycation composition comprises black tiger berries and dried tangerine peel, wherein the mass ratio of black tiger berries to dried tangerine peel is 1:3. The synergistic antioxidant and anti-glycation composition of this invention exhibits excellent anti-glycation, antioxidant, and free radical scavenging abilities, and can be widely applied in the cosmetic field.

Description

Technical Field

[0001] This invention provides a composition with synergistic antioxidant and anti-glycation properties, its preparation method, and its application, relating to the field of cosmetic technology. Background Technology

[0002] Free radicals, also known as hydroxyl radicals, can cause skin to lose elasticity and develop wrinkles. Glycation is one of the factors contributing to aging and dull skin. Anti-glycation and anti-oxidation are two important concepts in skincare, helping to delay skin aging, maintain skin health, and promote a brighter complexion.

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

[0004] Liu Lina et al. published an analysis of flavonoid components and evaluation of antioxidant activity in tangerine peel from different years in the *Southern Agricultural Journal*. They reported that tangerine peel possesses DPPH free radical scavenging ability and antioxidant capacity. However, they did not conduct research on skin antioxidant and anti-glycation effects; further research is needed to determine whether tangerine peel can be used for skin antioxidant and anti-glycation applications.

[0005] Li Yajun et al. published their research on the extraction process and antioxidant activity of total flavonoids from *Gynostemma pentaphyllum* leaves in *Guangxi Plants*. They reported that *Gynostemma pentaphyllum* possesses DPPH free radical scavenging ability. However, they did not conduct research on its effects on skin anti-oxidation and anti-glycation; whether tangerine peel can be used for skin anti-oxidation and anti-glycation requires further investigation. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by providing a composition with synergistic antioxidant and anti-glycation properties, its preparation method, and its application. This composition has excellent free radical scavenging ability and can also prevent skin from glycation and oxidation.

[0007] To achieve the above objectives, 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 tangerine peel and dried tangerine peel; wherein the mass ratio of black tiger tangerine peel to dried tangerine peel is 1:3.

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

[0010] Secondly, the present invention provides a method for preparing a composition with synergistic antioxidant and anti-glycation properties, comprising the following steps:

[0011] (1) Extract the black tiger to obtain a crude extract of black tiger, and purify the crude extract of black tiger to obtain a black tiger extract.

[0012] (2) Extract the tangerine peel to obtain a crude tangerine peel extract, and purify the crude tangerine peel extract to obtain a tangerine peel extract.

[0013] (3) Mix the black tiger extract and tangerine peel extract thoroughly.

[0014] Further, the extraction in step (1) is as follows: petroleum ether is refluxed with black tiger extract, filtered, the residue is added to ethanol, and ultrasonically extracted to obtain crude black tiger extract.

[0015] Further, the extraction in step (2) is as follows: petroleum ether is refluxed with tangerine peel for extraction, filtered, and the residue is added to ethanol and ultrasonically extracted to obtain a crude extract of tangerine peel.

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

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

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

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

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

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

[0022] Furthermore, the black tiger uses 70% ethanol by mass.

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

[0024] Furthermore, the conditions for the black tiger ultrasound are: 40℃, 100W, ultrasound for 5 minutes followed by reflux at 60℃ for 1 hour.

[0025] Furthermore, the ultrasonic conditions for the dried tangerine peel are: 50℃, 300W, and ultrasonication for 40 minutes.

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

[0027] Fourthly, the present invention provides an antioxidant and anti-glycation cosmetic, comprising the above-described composition.

[0028] The technical effects achieved by this invention are:

[0029] The specific dosage relationship of black tiger tangerine peel and dried tangerine peel in this invention can avoid the antagonistic effect between the two raw materials, so that their effective components can work synergistically, enhance the free radical scavenging ability of the composition, enhance the skin's antioxidant and anti-glycation ability, and have excellent repair ability for oxidatively damaged cells. It can be widely used in the cosmetic field. Attached Figure Description

[0030] Figure 1 The DPPH radical scavenging ability is shown in the examples and comparative examples.

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

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

[0033] Figure 4 The lipid peroxidation inhibition capacity is shown in the examples and comparative examples.

[0034] Figure 5 For the pre-protective ability against H2O2 oxidative damage in the examples and comparative examples, #### indicates p < 0.0001 compared to the control group; ** indicates p < 0.01 compared to the model group; *** indicates p < 0.001 compared to the model group; **** indicates p < 0.0001 compared to the model group; & indicates p < 0.05 compared to Comparative Example 1; &&&& indicates p < 0.0001 compared to Comparative Example 1. The ns values ​​for the 0.0125 mg / mL group were not significantly different from those of the model group; the ns values ​​for the 0.1 mg / mL group were not significantly different from those of Comparative Example 1; the ns values ​​for the 0.2 mg / mL group were not significantly different from those of Comparative Example 1.

[0035] Figure 6 For the cellular oxidative damage repair capacity of the examples and comparative examples, #### indicates p < 0.0001 compared to the control group; * indicates p < 0.05 compared to the model group; ** indicates p < 0.01 compared to the model group; *** indicates p < 0.001 compared to the model group; **** indicates p < 0.0001 compared to the model group; & indicates p < 0.05 compared to Comparative Example 1; &&&& indicates p < 0.0001 compared to Comparative Example 1; ns indicates no significant difference between the examples and Comparative Example 1.

[0036] Figure 7The GSH content of the examples and comparative examples is shown below. #### indicates p < 0.0001 compared with the control group; ** indicates p < 0.01 compared with the model group; **** indicates p < 0.0001 compared with the model group; &&&& indicates p < 0.0001 compared with Comparative Example 1; ns indicates no significant difference compared with the model group. Detailed Implementation

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

[0038] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0039] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

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

[0041] I. Examples and Comparative Examples

[0042] 1. Example 1

[0043] Preparation method:

[0044] (1) Add black tiger berries to petroleum ether at a material-to-liquid ratio of 1:8 (mass ratio), reflux at 50℃ for 2.5h, filter, add 70% ethanol to the filter residue at a material-to-liquid ratio of 1g:50mL, sonicate at 40℃ and 100w for 5min, reflux for 1.0h, filter after reflux, collect the filtrate, and repeat the above steps once more with the extracted residue. Mix and concentrate the extracts from the two sonic refluxes to obtain the crude extract of black tiger berries;

[0045] Add tangerine peel to petroleum ether at a mass ratio of 1:8, reflux at 50℃ for 2.5 h, filter, and add 60% ethanol to the residue at a mass ratio of 1 g:25 mL. Sonicate at 50℃ and 300 W for 40 min, then reflux for 1.0 h. After reflux, filter again, collect the filtrate, and repeat the above steps with the extracted residue. Mix and concentrate the two ultrasonic reflux extracts to obtain a crude tangerine peel extract.

[0046] (2) Take the pretreated macroporous resin AB-8 wet packing column and purify the crude extract of black tiger and the crude extract of tangerine peel respectively. Specifically, the loading time is 4-7h, the sample is eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless. The ethanol eluent is collected, evaporated by rotary evaporation and freeze-dried to obtain black tiger extract and tangerine peel extract respectively.

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

[0048] 2. Comparative Example

[0049] Comparative Example 1

[0050] Preparation method:

[0051] (1) Add black tiger berries to petroleum ether at a material-to-liquid ratio of 1:8 (mass ratio), reflux at 50℃ for 2.5h, filter, add 70% ethanol to the filter residue at a material-to-liquid ratio of 1g:50mL, sonicate at 40℃ and 100w for 5min, reflux for 1.0h, filter after reflux, collect the filtrate, and repeat the above steps once more with the extracted residue. Mix and concentrate the extracts from the two sonic refluxes to obtain the crude extract of black tiger berries.

[0052] (2) Take the pretreated macroporous resin AB-8 wet packing column and purify the crude extract of black tiger. Specifically, the loading time is 4-7h, wash with water until colorless, and finally wash with 80% ethanol until colorless and odorless. Collect the ethanol eluent, evaporate by rotary evaporation and freeze dry to obtain black tiger extract.

[0053] Comparative Example 2

[0054] Preparation method:

[0055] (1) Add tangerine peel to petroleum ether at a material-to-liquid ratio of 1:8 (mass ratio), reflux at 50℃ for 2.5h, filter, add 60% ethanol to the filter residue at a material-to-liquid ratio of 1g:25mL, sonicate at 50℃ and 300w for 40min, reflux for 1.0h, filter after reflux, collect the filtrate, and repeat the above steps once more with the extracted residue. Mix and concentrate the extracts from the two sonic refluxes to obtain crude tangerine peel extract.

[0056] (2) Take the pretreated macroporous resin AB-8 wet packing column and purify the crude extract of tangerine peel. Specifically, the loading time is 4-7h, wash with water until colorless, and finally wash with 80% ethanol until colorless and odorless. Collect the ethanol eluent, evaporate by rotary evaporation and freeze dry to obtain tangerine peel extract.

[0057] Comparative Example 3

[0058] Preparation method:

[0059] (1) Add black tiger berries to petroleum ether at a material-to-liquid ratio of 1:8 (mass ratio), reflux at 50℃ for 2.5h, filter, add 70% ethanol to the filter residue at a material-to-liquid ratio of 1g:50mL, sonicate at 40℃ and 100w for 5min, reflux for 1.0h, filter after reflux, collect the filtrate, and repeat the above steps once more with the extracted residue. Mix and concentrate the extracts from the two sonic refluxes to obtain the crude extract of black tiger berries;

[0060] Add tangerine peel to petroleum ether at a mass ratio of 1:8, reflux at 50℃ for 2.5 h, filter, and add 60% ethanol to the residue at a mass ratio of 1 g:25 mL. Sonicate at 50℃ and 300 W for 40 min, then reflux for 1.0 h. After reflux, filter again, collect the filtrate, and repeat the above steps with the extracted residue. Mix and concentrate the two ultrasonic reflux extracts to obtain a crude tangerine peel extract.

[0061] (2) Take the pretreated macroporous resin AB-8 wet packing column and purify the crude extract of black tiger and the crude extract of tangerine peel respectively. Specifically, the loading time is 4-7h, the sample is eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless. The ethanol eluent is collected, evaporated by rotary evaporation and freeze-dried to obtain black tiger extract and tangerine peel extract respectively.

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

[0063] Comparative Example 4

[0064] Preparation method:

[0065] (1) Add black tiger berries to petroleum ether at a material-to-liquid ratio of 1:8 (mass ratio), reflux at 50℃ for 2.5h, filter, add 70% ethanol to the filter residue at a material-to-liquid ratio of 1g:50mL, sonicate at 40℃ and 100w for 5min, reflux for 1.0h, filter after reflux, collect the filtrate, and repeat the above steps once more with the extracted residue. Mix and concentrate the extracts from the two sonic refluxes to obtain the crude extract of black tiger berries;

[0066] Add tangerine peel to petroleum ether at a mass ratio of 1:8, reflux at 50℃ for 2.5 h, filter, and add 60% ethanol to the residue at a mass ratio of 1 g:25 mL. Sonicate at 50℃ and 300 W for 50 min, then reflux for 1.0 h. After reflux, filter again, collect the filtrate, and repeat the above steps with the extracted residue. Mix and concentrate the two ultrasonic reflux extracts to obtain a crude tangerine peel extract.

[0067] (2) Take the pretreated macroporous resin AB-8 wet packing column and purify the crude extract of black tiger and the crude extract of tangerine peel respectively. Specifically, the loading time is 4-7h, the sample is eluted with water until colorless, and finally eluted with 80% ethanol until colorless and odorless. The ethanol eluent is collected, evaporated by rotary evaporation and freeze-dried to obtain black tiger extract and tangerine peel extract respectively.

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

[0069] 3. Antioxidant efficacy test

[0070] 3.1 DPPH free radical scavenging experiment

[0071] Take 3 mL of each of the examples and comparative examples 1-4 with concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, and add 3 mL of 95% ethanol solution of 0.1 mol / L DPPH under light-protected conditions.

[0072] The reaction was carried out in the dark at 37°C for 30 min, and the absorbance was quickly measured at 517 nm. The absorbance of the reaction between DPPH and the sample was recorded as A1. The absorbance of the 95% ethanol solution of DPPH was recorded as A2 when 95% ethanol solution was used instead of 95% ethanol solution; and the absorbance of the sample was recorded as A0 when ultrapure water was used instead of 95% ethanol solution.

[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, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0079] Figure 1The compound solution of black tiger shavings and tangerine peel exhibits stronger DPPH free radical scavenging ability, with a scavenging rate of 94.85% at a dosage concentration of 2.0 mg / ml. The DPPH free radical scavenging ability of both black tiger shavings extract and the compound solution of black tiger shavings and tangerine peel increases with increasing dosage concentration, and there is a good linear relationship between dosage concentration and scavenging rate in each group.

[0080] 3.3 PTIO free radical scavenging experiment

[0081] Step a: Dilute the examples and comparative examples 1-4 with PBS at pH 7.4 to concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively, in 3 mL each.

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

[0083] Step c: React in a 37°C water bath for 2 hours, and measure the absorbance A1 at a wavelength of 557 nm; replace the PTIO solution with PBS at pH 7.4; replace the sample with an equal volume of ultrapure water; measure the absorbance using the same procedure and record them as A2 and A1 respectively. 0， The free radical scavenging rate of PTIO is 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.*p<0.001.

[0089] like Figure 2 As shown, the PTIO free radical scavenging ability of both Black Tiger and Black Tiger and Tangerine Peel Compound Solution increased with increasing dosage concentration, and there was a good linear relationship between each concentration and the scavenging rate. Among them, the Black Tiger and Tangerine Peel Compound Solution had a stronger PTIO free radical scavenging ability, with a scavenging rate of 98.27% at a dosage concentration of 2.0 mg / ml.

[0090] 3.5 Anti-glycation experiment

[0091] Step a: Take 2 mL of each 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: After incubation at 37℃ for 7 days, the fluorescence intensity A1 at 440nm was measured after excitation with 370nm excitation light, with an excitation and emission slit width of 5nm. The fluorescence intensity was recorded as A2 when PBS was used instead of BSA; A3 when pure water was used instead of the sample solution; and A4 when pure water was used instead of the sample solution and PBS was used instead of BSA. The fluorescence intensity was calculated using 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*p<0.001.

[0099] Experimental results are as follows Figure 3 As shown, the AGEs inhibition ability of both Black Tiger and Black Tiger and Tangerine Peel Compound Solution increased with increasing dosage concentration, and there was a good linear relationship between each concentration and the inhibition rate. Among them, the Black Tiger and Tangerine Peel Compound Solution had stronger anti-glycation ability, with an inhibition rate of 97.04% at a dosage concentration of 2.0 mg / ml.

[0100] 3.7 Anti-lipid peroxidation experiment

[0101] Step a: Prepare TCA-TBA-HCl mixture: Add 15g TCA, 0.375g TBA and 2.1mL concentrated HCl to 100mL pure water in sequence.

[0102] Step b: Take 1.5 mL of the example and comparative solutions 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: Under light-protected conditions, add 1.5 mL of 10 mg / mL lecithin solution and 1.5 mL of 0.4 mmol / L ferrous sulfate solution sequentially. Step d: Incubate at 37°C in a water bath for 1 hour in the dark. Add 3 mL of TBA-TCA-HCl mixture, incubate at 95°C for 15 minutes, then immediately cool. Centrifuge and collect the supernatant. Measure the absorbance A0 at 535 nm. If pure water is used instead of the lecithin solution, the absorbance is recorded as A1; if pure water is used instead of the sample solution, the absorbance is recorded as A2. Calculate the scavenging rate using the formula: "Scavenging 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, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0109] Experimental results are as follows Figure 4 The lipid peroxidation inhibition capacity of both the black tiger broth and the black tiger broth compound with tangerine peel increased with increasing dosage concentration, and there was a good linear relationship between each concentration and the inhibition rate. Among them, the black tiger broth compound with tangerine peel showed stronger lipid peroxidation inhibition capacity, with an inhibition rate of 93.76% at a dosage concentration of 2.0 mg / ml.

[0110] 4. Cellular antioxidant efficacy test

[0111] 4.1 Cellular H2O2 Oxidative Damage Pre-protection Experiment

[0112] L929 cells were divided into 1×10 4 The samples were seeded at the specified density in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours, after which the culture medium was discarded. The control group consisted of DMEM medium without extract; the model group consisted of DMEM medium without extract; the positive control group consisted of DMEM medium diluted to 1 mM VC solution; and the experimental group consisted of DMEM medium diluted to the dosage concentration of the herbal extracts from the examples and comparative examples, with 100 μL of sample added to each well, and three replicates for each concentration. After incubation at 37°C with 5% CO2 for 24 hours, the culture medium was discarded, and the samples were washed three times with 100 μL PBS. Except for the control group, 100 μL of H2O2 solution diluted to 0.6 mM with DMEM medium was added to each well.

[0113] The positive control group concentration was the concentration with the highest efficacy and lowest toxicity.

[0114] The control group, model group, and experimental group were incubated in a cell culture incubator at 37℃ and 5% CO2 for 4 hours. The culture medium was then discarded, and MTT reagent was added. The cells were incubated in the incubator at 37℃ and 5% CO2 for another 4 hours, then the MTT reagent was discarded. 100 μL LDMSO was added to each well, and the cells were shaken for 10 minutes. The absorbance was measured at 490 nm, and the cell viability was calculated using the formula "Cell viability = A". 给药孔 / A 空白孔 The cell viability is calculated as "×100%".

[0115] 4.2 Experimental Results

[0116] The results are shown in Table 5.

[0117] Table 5

[0118]

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

[0120] 4.3 Cellular Oxidative Damage Repair Experiment

[0121] L929 cells were divided into 1×10 4 The cells were seeded at a density of 1 / 2 well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours, after which the culture medium was discarded. Except for the control group, 100 μL of H2O2 solution diluted to 0.6 mM with DMEM was added to each well, and the cells were incubated at 37°C with 5% CO2 for 4 hours, after which the culture medium was discarded.

[0122] The control group consisted of DMEM medium without extract; the model group consisted of DMEM medium without extract; the positive control group consisted of VC solution diluted to 1 mM with DMEM medium; and the experimental group consisted of DMEM medium diluted to the dosage concentration of the traditional Chinese medicine extracts from the examples and comparative examples, with 100 μL of sample added to each well, and 3 replicates for each concentration.

[0123] The positive control group concentration was the concentration with the highest efficacy and lowest toxicity.

[0124] The control group, model group, and experimental group were incubated in a cell culture incubator at 37℃ and 5% CO2 for 24 hours. The culture medium was then discarded, and MTT reagent was added. The cells were incubated in the same incubator at 37℃ and 5% CO2 for 4 hours. The MTT reagent was then discarded, and 100 μL LDMSO was added to each well. The cells were shaken for 10 minutes, and the absorbance was measured at 490 nm. The cell viability was then calculated using the formula: "Cell viability = A..." 给药孔 / A 空白孔 The cell viability is calculated as "×100%".

[0125] 4.4 Experimental Results

[0126] The results are shown in Table 6.

[0127] Table 6

[0128]

[0129]

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

[0131] 4.5 Determination of Trace Reduced Glutathione (GSH) Content

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

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

[0134] After incubating the control group, model group, and experimental group in a 37℃, 5% CO2 cell culture incubator for 24 hours, the culture medium was discarded. The cells were then washed three times with 1 mL PBS. Except for the control group, 1 mL of H2O2 solution diluted to 0.6 mM with DMEM was added to each well. The cells were incubated in a 37℃, 5% CO2 cell culture incubator for 4 hours, after which the culture medium was discarded. The cells were then washed three times with 1 mL PBS. 350 μL PBS was added to each well, and the cells were scraped off and the cell suspension was collected. After sonication and centrifugation, the supernatant was collected. The GSH content was then determined according to the instructions of the micro-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 The cell GSH content is calculated by multiplying the result by 2 and then dividing by Cpr.

[0135] 4.6 Experimental Results

[0136] The results are shown in Table 7.

[0137] Table 7

[0138]

[0139] Experimental results are as follows Figure 7 As shown, compared with the model group (i.e., the H2O2 treatment group), the GSH content of the drug treatment group was significantly increased, and the GSH content of the compound solution was significantly increased.

[0140] Example 1 of the present invention has high DPPH and PTTO scavenging rates, high AGEs inhibition rates, and high peroxidation inhibition rates, and shows significant differences compared with Comparative Examples 1-4; Example 1 of the present invention has strong oxidative damage pre-protection and repair capabilities, high GSH content, and shows significant differences compared with Comparative Example 1.

[0141] The synergistic antioxidant and anti-glycation composition of the present invention uses a specific dosage relationship to enable black tiger tangerine peel and dried tangerine peel to work synergistically, enhance the free radical scavenging ability of the composition, enhance the skin's antioxidant and anti-glycation ability, and has excellent repair ability for oxidatively damaged cells. It can be widely used in the cosmetic field.

[0142] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a composition having synergistic antioxidant anti-glycation in the preparation of an antioxidant anti-glycation cosmetic, characterized in that, The composition comprises black tiger extract and dried tangerine or orange peel extract in a mass ratio of 1:3; The preparation method of the composition is: (1) extracting black tiger to obtain black tiger crude extract, purifying the black tiger crude extract to obtain black tiger extract; (2) extracting dried tangerine or orange peel to obtain dried tangerine or orange peel crude extract, purifying the dried tangerine or orange peel crude extract to obtain dried tangerine or orange peel extract; (3) mixing and uniformly mixing the black tiger extract and the dried tangerine or orange peel extract; In step (1), the extraction is: refluxing petroleum ether with black tiger, after filtration, adding ethanol to the filter residue, and ultrasonic extraction to obtain black tiger crude extract; In step (2), the extraction is: refluxing petroleum ether with dried tangerine or orange peel, after filtration, adding ethanol to the filter residue, and ultrasonic extraction to obtain dried tangerine or orange peel crude extract; The extraction is repeated 1-2 times.

2. Use according to claim 1, characterized in that, The mass ratio of the black tiger to petroleum ether is 1:6-8; The mass ratio of the dried tangerine or orange peel to petroleum ether is 1:6-8.

3. Use according to claim 1, characterized in that, The refluxing time is 1 h.

4. Use according to claim 1, characterized in that, The ratio of the black tiger to ethanol is 1 g:50 mL; The ratio of the dried tangerine or orange peel to ethanol is 1 g:25 mL; The black tiger uses ethanol with a mass fraction of 70%, and the dried tangerine or orange peel uses ethanol with a mass fraction of 60%.

5. The use according to claim 1, characterized in that, The ultrasonic conditions are: 40 ℃, 100 w, ultrasonic extraction for 5 min, and then refluxing at 60 ℃ for 1 h.

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