CN116650579A

CN116650579A - Application of oil tea flower extract in aspects of freckle removal, whitening and anti-saccharification

Info

Publication number: CN116650579A
Application number: CN202310634963.6A
Authority: CN
Inventors: 沈建福; 金蒙; 陈士国; 蒋志林; 徐旭平; 王欣如
Original assignee: Changshan Fuerkang Camellia Oil Co ltd
Current assignee: Changshan Fuerkang Camellia Oil Co ltd
Priority date: 2023-05-31
Filing date: 2023-05-31
Publication date: 2023-08-29

Abstract

The invention belongs to the field of daily chemicals, and particularly relates to application of an oil tea flower extract in removing freckles, whitening skin and resisting saccharification. The camellia oleifera flower extract is used for preparing medicines or daily chemical products for removing freckles and whitening and preparing medicines or daily chemical products for resisting saccharification. The invention determines the activity of the oil tea flower extract for inhibiting tyrosinase and inhibiting melanin synthesis, has anti-saccharification effect, determines the effect of the oil tea flower extract on preventing and treating diseases related to melanin and AGEs, such as freckle, macula lutea, dark complexion and the like, and expands the application of the oil tea flower extract in medicines and daily chemicals.

Description

Application of oil tea flower extract in aspects of freckle removal, whitening and anti-saccharification

Technical Field

The invention belongs to the field of daily chemicals, and particularly relates to application of an oil tea flower extract in removing freckles, whitening skin and resisting saccharification.

Background

Many women worldwide have problems with skin darkness, and current research into human skin tone is mostly focused on melanin. The skin is protected from harmful stimulus such as ultraviolet radiation, however, if melanin is accumulated excessively in human body, it may be deposited on skin to cause freckle, macula, etc. to affect beauty, and it may cause lesions such as black nevus or melanoma. In addition, advanced glycation end products (AGEs) which are products of the glycation reaction are yellow or brown in color and have fluorescence, and accumulate in facial epidermis to cause dark yellow skin, and AGEs can promote melanin generation by activating RAGE in melanocytes to influence skin color change. Saccharification refers to the covalent bonding of proteins, lipids or nucleic acids to a reducing sugar molecule (typically glucose or fructose) that occurs without enzymatic catalysis. The function of the protein, lipid or nucleic acid molecule that undergoes saccharification is inhibited. Anti-glycation, that is, resistance to glycation reactions.

The whitening and freckle-removing products on the market at present mostly realize the whitening effect by inhibiting the activity of tyrosinase, a key melanin synthesis enzyme, have a single inhibition path, and have the problems of poor stability of whitening accepted components such as kojic acid and the like. The natural plant extracts such as the camellia oleifera have the advantages of wide sources, mildness, no irritation and the like, and have wide market prospect.

The Camellia oleifera (Camellia oleifera Abel.) is a Camellia (Camellia) evergreen small arbor or shrub in Theaceae (Theaceae), is a Chinese specific oil crop, and has various pharmacological activities such as bacteriostasis, antioxidation, anti-inflammatory, anti-tumor, tyrosinase activity inhibition and the like. The camellia oleifera is a reproductive organ of camellia oleifera (Camellia oleifera Abel) tree, can be collected from 10 months to 3 months of the next year, is rich in nutrients and bioactive components such as proteins, amino acids, reducing sugar, polysaccharides, polyphenols, flavonoids, anthocyanin and vitamins, and has the effect of being beneficial to human health. Since the 80 s of the last century, the planting area of oil tea in China is continuously increased, and the trend of further expansion is that oil tea flowers are supplied sufficiently, but research on active ingredients, development process, application and the like of the oil tea flowers is not reported at present, and the development and the utilization of the oil tea flowers are also in a starting stage.

Disclosure of Invention

The invention aims to provide a new application of an oil tea flower extract.

In order to solve the technical problems, the invention provides application of an oil tea flower extract in aspects of freckle removal, whitening and saccharification resistance.

Improvement of use of the oil tea flower extract of the present invention: the method is used for preparing the freckle-removing and whitening drugs or daily chemical products and preparing the anti-glycation drugs or daily chemical products.

Further improvement of the use of the oil tea flower extract of the present invention: the oil tea flower extract is oil tea flower alcohol extract.

The inventor of the invention discovers that the oil tea flower extract has the capability of inhibiting tyrosinase and inhibiting the growth of melanoma cells in the research process, can reduce the synthesis of melanin and the synthesis of AGEs, can achieve the effects of removing spots, whitening and resisting saccharification, and the content has not been reported before.

The camellia oleifera flower extract can be applied to the fields of medicines and daily chemicals for preventing and treating diseases related to melanin and AGEs.

The invention provides a plant extract, namely an oil tea flower extract, which has wide sources, safety, effectiveness, economy and applicability, and systematic research on the effect of preventing and treating diseases related to melanin and AGEs is carried out, so that the oil tea flower extract has obvious effects of inhibiting tyrosinase activity, inhibiting melanin synthesis and resisting saccharification. Previous researches show that the oil tea flower extract has remarkable physiological and pharmacological activities such as free radical resistance, oxidation resistance and the like, so that the oil tea flower extract has the advantages of safety, no toxicity, stable performance and the like. The above researches are integrated, and the Chinese medicinal composition can be singly or compounded with other auxiliary materials to prepare medicines or daily chemical products for removing spots, whitening and resisting saccharification.

The invention has the main advantages that:

1. the activity of the camellia oleifera flower extract for inhibiting tyrosinase and the synthesis of melanin are determined, the anti-glycation effect is achieved, the effect of the camellia oleifera flower extract on preventing and treating diseases related to melanin and AGEs, such as freckle, macula lutea, dark complexion and the like, is determined, and the application of the camellia oleifera flower extract in medicines and daily chemicals is expanded.

2. The camellia oleifera flower is developed and utilized, the added value of the camellia oleifera flower is improved, the comprehensive utilization value of the camellia oleifera flower is improved, and the economic benefit is increased.

When the camellia oleifera flower extract is taken orally as a freckle-removing whitening/anti-saccharification medicament, the dosage is about 0.5-0.7 g/person day, and when the camellia oleifera flower extract is used for daily chemical products, the weight content of the camellia oleifera flower extract in daily chemical products is about one to five parts per million.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a graph showing the inhibition of tyrosinase activity by COFE.

FIG. 2 is a graph showing the effect of COFE on the formation of melanin on the body surface of zebra fish;

note that: a-Control; b-10 μg/mL COFE; c-50 μg/mL COFE; d-100 μg/mL COFE; e-100 μg/mL KA; each group was repeated with 40 zebra fish.

FIG. 3 is a graph showing the effect of COFE on the relative pigment values of zebra fish body surfaces;

note that: the complete difference in letters between the groups indicates that there is a significant difference (p < 0.05), denoted by a, b, c, d and e.

FIG. 4 shows the inhibition of tyrosinase activity in zebra fish by COFE;

note that: the complete difference in letters between groups indicates that there is a significant difference (p < 0.05), denoted by a, b, c and d.

FIG. 5 is a graph showing the effect of COFE on relative melanin content in zebra fish;

FIG. 6 is the effect of COFE on proliferation rate of A375 cells;

note that: a, B, C in fig. 6 is survival of a375 cells at 24h, 48h, 72h, respectively;

FIG. 7 shows the inhibition of tyrosinase activity in A375 cells by COFE;

note that: p <0.01 compared to the same concentration KA set.

FIG. 8 is the effect of COFE on relative melanin content in A375 cells;

note that: p <0.01 compared to the same concentration KA set.

FIG. 9 shows the effect of COPE on the relative protein expression level of MITF;

note that: the complete difference in letters between groups indicates that there is a very significant difference (p < 0.01), denoted by a, b and c.

FIG. 10 shows the effect of COFE on the relative protein expression levels of TYR, TRP-1 and TRP-2;

note that: a, B, C in FIG. 10 is the relative protein expression levels of TYR, TRP-1, TRP-2, respectively; the complete difference in letters between groups indicates that there is a very significant difference (p < 0.01), denoted by a, b, c and d.

FIG. 11 shows the effect of COFE on Amadori product formation.

FIG. 12 shows the effect of COFE on the amount of dicarbonyl compound produced.

FIG. 13 is a graph showing the effect of COFE on the CML content of non-fluorescent AGEs;

note that: p <0.01 compared to AG group.

FIG. 14 is a graph showing the effect of COFE on the amount of fluorescent AGEs;

note that: p <0.01 compared to AG group.

FIG. 15 is a graph showing the effect of COPE on GLA-induced AGEs production;

note that: p compared to Control group<0.01; in contrast to the GLA-treated group, ^## p<0.01

FIG. 16 is a graph showing the effect of COFE on RAGE expression.

Note that: comparison with Control group,/p<0.01; in comparison with the GLA-treated group, ^## p<0.01。

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1, preparation of 50% ethanol oil tea flower extract (conventional technique):

drying healthy tea-oil camellia flower (without pathological change) at 37deg.C under low temperature to water content of about 10%, pulverizing, and sieving with 60 mesh sieve. 10g of camellia oleifera powder is taken according to the following formula 1:10 (W: v, g/mL) adding ethanol with volume fraction of 50%, magnetically stirring at room temperature (rotation speed of 400 rpm) for 3h, ultrasonically extracting at 50deg.C (power of 130-150W) for 2h, centrifuging at 4000r/min for 10min, collecting supernatant, spin-evaporating to remove ethanol in the supernatant, and vacuum freeze-drying at-20deg.C for 72h to dry powder (water content of less than 6.0%), to obtain lyophilized powder of tea-oil camellia flower extract, named COFE.

Experiment 1: effect of COFE on tyrosinase activity experiments

The experimental method comprises the following steps: COPE was reconstituted with water to a series of concentration gradients of 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0 mg/mL. 200. Mu.L of sample solution, 200. Mu.L of PBS buffer (pH=6.80) and 100. Mu.L of 0.2mg/mL (i.e. 100U/mL) tyrosinase solution are sequentially added into a 1.5mL centrifuge tube, and after uniform mixing, the mixture is incubated in a constant temperature water bath at 37 ℃ for 10min. After 200. Mu.L of 1.0mg/mL L-dopa solution was added, each tube of the reaction solution was rapidly transferred into a 96-well plate, and absorbance at 475nm was detected every 30 seconds, and the change in absorbance per unit time was used as a criterion for the level of enzyme activity. The sample solution in the reaction system was changed to PBS as a blank control. The formula for the inhibition rate of tyrosinase activity is as follows:

k in ₀ Slope, k of the curve of absorbance at 475nm for a blank _i Absorbance of the sample solution at 475nmSlope of the change curve.

As is clear from FIG. 1, when the concentration of the COPE solution is 0.2 to 1.0mg/mL, the inhibitory effect of COPE on tyrosinase activity increases with increasing concentration. The IC50 value of COFE for inhibiting tyrosinase activity was calculated to be 0.49mg/mL according to the inhibition curve. Thus, the following description: COFE has a good tyrosinase inhibition effect.

Experiment 2: efficacy experiment of COFE in inhibiting melanin synthesis based on zebra fish embryo model

1. Preparing a solution:

1) Kojic Acid (KA) zebra fish broth solution: weighing 10.0mg KA, dissolving with conventional zebra fish embryo culture solution, and fixing volume to 10mL to obtain 1.0mg/mLKA solution; precisely diluting the zebra fish embryo culture solution by 10 times to obtain 100 mug/mLKA solution;

zebra fish embryo culture solution: embryo rearing medium, ERM;1L deionized water containing 8g NaCl,0.4g KCl,0.035gNa ₂ HPO ₄ ,0.6g KH ₂ PO ₄ ,0.14g CaCl ₂ ,0.12g MgSO ₄ ,0.35g NaHCO ₃ ,pH 7.2。

2) COFE zebra fish broth solution: weighing 10.0mg of COFE, dissolving with zebra fish embryo culture solution, and fixing the volume to 10mL to obtain 1.0mg/mL of COFE solution; according to the pre-experiment result, the applicable concentration of the zebra fish embryo culture solution is determined, and the COFE solution is precisely diluted into low, medium and high concentration COFE solutions by the zebra fish embryo culture solution: 10 μg/mL, 50 μg/mL, 100 μg/mL;

2. grouping and administration modes: the developmental stage of zebra fish embryos and larvae is expressed in hours after fertilization (hpf). 6-well plates were used as experimental carriers, and 40 zebra fish embryos with 6hpf-8hpf developing normally were added to each well. 3.0mL of sample solutions to be tested (10. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL COPE) with different exposure concentrations are added to each well, and the culture solution is replaced every 24 hours at constant temperature of 28+/-0.5 ℃. The sample liquid is changed into common zebra fish culture liquid (-Control) and 0.1mg/mL KA to be used as different Control groups. Each group was provided with 3 wells simultaneously as parallel.

1) Effect of COFE on relative pigment values on zebra fish body surface

At the time of zebra fish embryo development to 72hpf, the melanin granule distribution of the embryo was observed and recorded under a stereoscopic microscope. The zebra fish embryo is placed in a concave glass slide, 1-2 drops of anesthetic (0.05 mg/mL MS-222) are dripped, the zebra fish is adjusted to the condition that eyes and body segments at two sides coincide, the tail is at the same level with the body, and the side view is shot under the same magnification and illumination intensity. Analysis was performed using Image J software to calculate the relative pigment values of the body surface. The calculation formula is as follows:

wherein: s is S _{Blank space} The melanin area ratio of the Control group; s is S _{Sample of} Melanin area ratio for the sample group.

As can be seen from fig. 2 and 3, the higher the COFE exposure concentration, the fewer melanin particles on the surface of the zebra fish, the lighter the color, the significantly reduced the relative pigment value (p < 0.05) on the surface of the zebra fish, and the more significant the effect with increasing concentration. In addition, the relative pigment value of the COFE group with 100 mug/mL to the body surface of the zebra fish is 66.02 +/-0.75 percent, which is similar to that of the KA group with the same concentration (64.26 +/-1.27 percent), and no significant difference exists (p is more than 0.05). Description: the COFE has good inhibiting effect on the synthesis of the zebra fish melanin.

2) Effect of COFE on tyrosinase activity in zebra fish

After the zebra fish embryos developed to 72hpf, 40 zebra fish embryos were collected per group, placed at-80℃for 24 hours, then washed 3 times with PBS, 100. Mu.L of PBS buffer containing 1% Triton X-100 by volume was added, and sonicated for 5min. Finally, centrifuging for 15min at the temperature of 10000r/m and the temperature of 4 ℃ to obtain supernatant, namely the crude extract of the zebra fish tyrosinase. The tyrosinase activity in the zebra fish body is measured by adopting a dopa oxidation method. 50 mu L of zebra fish tyrosinase crude extract is taken and placed in a 96-well plate, 150 mu L of 1mg/mL L-DOPA is added, L-DOPA is used as background for correction, zebra fish embryos of a Control group are used as blank Control, absorbance values are detected every 30s at 37 ℃, enzymatic reaction curves at 475nm in 30min are recorded, change of absorbance values in unit time is used as a standard of enzyme activity, and 3 parallel concentrations are set. The calculation formula is as follows:

k in ₀ Slope, k of the curve of absorbance at 475nm for a blank _i The slope of the curve for the absorbance of the sample solution at 475 nm.

As can be seen from fig. 4, COFE has a significant inhibitory effect on tyrosinase activity in zebra fish (p < 0.05) and is concentration dependent. When the concentration of COFE is 100 mug/mL, the inhibition rate of tyrosinase activity is maximum and reaches 32.42% + -0.35%, which is equivalent to the action of KA (33.47% + -0.40%) with the same concentration (p is more than 0.05). Description: the COFE has good inhibition effect on tyrosinase activity in zebra fish bodies.

3) Effect of COFE on melanin content in zebra fish

And (5) determining the melanin content of the zebra fish by adopting a NaOH cracking method. 600. Mu.L of 1mol/L NaOH solution containing 10% DMSO by volume was added to the centrifuged precipitate obtained in step (2) and the mixture was resuspended in 80℃water bath for 2 hours. Transferring into 96-well culture plate after cooling, and measuring absorbance A at 405nm with Control group zebra fish embryo as blank Control ₄₀₅ Each concentration was set in 3 replicates.

Wherein: a is that _{Blank space} The light absorption value is the Control group; a is that _{Sample of} The absorbance after the addition of the sample.

As can be seen from fig. 5, COFE has a significant inhibitory effect on melanin formation in zebra fish (p < 0.05). At a concentration of 100 μg/mL, the relative melanin content of the COFE group zebra fish was reduced to 64.39% ± 0.85%, comparable to the inhibition of the same concentration KA group (62.58% ± 0.94%), without significant differences (p > 0.05). Description: the COFE can effectively inhibit the synthesis of melanin in the zebra fish body.

Experiment 3: efficacy research experiment of inhibiting melanin synthesis of COFE based on A375 cell model

1. Preparing a solution:

1) Complete medium: 50mL of fetal bovine serum and 5mL of penicillin-streptomycin mixed solution (double antibody) are added into 500mL of DMEM culture medium, and the mixture is fully and uniformly shaken;

2) KA cell culture solution: weighing 10.0mg KA, dissolving with complete culture medium, and fixing volume to 10mL to obtain 1.0mg/mLKA solution; it was diluted precisely with complete medium to a series of concentration gradient kojic acid solutions: 10 μg/mL, 40 μg/mL, 160 μg/mL;

3) COFE cell culture solution: weighing 10.0mg of COPE, dissolving with a complete culture medium, and fixing the volume to 10mL to obtain 1.0mg/mL of COPE solution; it was diluted precisely with complete medium to a series of concentration gradient COFE solutions: 10. Mu.g/mL, 40. Mu.g/mL, 160. Mu.g/mL.

2. Grouping and experimental contents:

1) Effect of COFE on proliferation Rate of A375 cells

A375 cells in the logarithmic growth phase were taken for 5 passages at 2.5X10 ⁴ Inoculating 96-well plate (200 μl per well), adhering supernatant, adding oil tea flower extract and kojic acid (200 μl per well) at concentration of 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, 160 μg/mL and 320 μg/mL, respectively, and adding 5% CO at 37deg.C ₂ Culturing for 24h, 48h and 72h respectively. After the culture is finished, removing the supernatant, adding 180 mu L/hole of DEME culture medium, adding 20 mu L/hole of MTT in dark place, incubating for 4 hours, removing the supernatant, adding 150 mu L/hole of DMSO, oscillating for 5 minutes by a shaker to promote the dissolution of crystals, changing the tested sample medicine into a complete culture medium as a control, and measuring the absorbance at the wavelength of 570 nm. The cell proliferation rate was calculated as follows:

in which A ₀ For the absorbance of the Control group at 570nm, A _i Absorbance at 570nm was determined for the sample group.

As shown in FIG. 6, in the concentration range of 10 to 160. Mu.g/mL, COFE and KA did not significantly inhibit the growth of A375 cells during the culture for 24 hours, and the cell proliferation rate was 90% or more. When the concentration reached 320. Mu.g/mL, the cell proliferation rates of the COFE group and the KA group were reduced to 87.35.+ -. 2.63% and 89.30.+ -. 8.44%, respectively. Culturing for 48h, wherein the cell proliferation is affected in the concentration range of 40-320 mug/mL, and the proliferation rate is lower than 90%. When the concentration reaches 320 mug/mL, the COFE and KA have obvious inhibition effect on the proliferation of A375 cells, the proliferation rate is respectively reduced to 51.26+/-7.21% and 72.37 +/-8.79%, and the inhibition effect of the COFE on the proliferation of the cells is obviously stronger than that of the same concentration KA. Description: the COFE can reduce melanin synthesis by inhibiting proliferation of melanoma cells, so as to achieve the whitening effect.

2) Effect of COFE on intracellular tyrosinase activity of A375 cells

Taking 5 th generation of A375 cells in the logarithmic growth phase at 5×10 ⁵ The concentration of each well was inoculated into a 6-well plate (2 mL per well), the supernatant was removed after adherence, COFE and kojic acid (2 mL per well) were added at concentrations of 10. Mu.g/mL, 40. Mu.g/mL and 160. Mu.g/mL, respectively, and incubated at 37℃under 5% CO2 for 24 hours. At the end of the culture, 0.25% pancreatin digests were performed to collect cell pellets.

Washing the precipitate with PBS for 2 times, adding 200 μL of PBS buffer solution containing 1% Triton X-100, freezing at-80deg.C for 30min, thawing at room temperature, centrifuging at 10000rpm at 4deg.C for 15min, and collecting supernatant to obtain tyrosinase crude extract. 50. Mu.L of crude enzyme solution was placed in a 96-well plate, 150. Mu.L of 1.0mg/mL L-DOPA was added thereto, the reaction was carried out at 37℃for 2 hours, the sample was changed to a complete medium as a blank, and the absorbance at a wavelength of 475nm was measured. The inhibition rate of COPE on the intracellular tyrosinase activity of A375 cells is calculated as follows:

As can be seen from fig. 7, the higher the concentration, the greater the inhibition of tyrosinase by COFE and KA, and the relative inhibition of tyrosinase activity by COFE was significantly stronger than that of KA at the same concentration (p < 0.01). At 160 mug/mL, the inhibition rate of COFE to the intracellular tyrosinase activity of A375 cells reaches 34.30 +/-0.24%. Description: COFE has excellent effect of inhibiting intracellular tyrosinase activity.

3) Effect of COFE on intracellular melanin content of A375 cells

Cell pellet was collected as in 2), washed 2 times with PBS, and 1mL ethanol was added: diethyl ether solution [ V (ethanol)/V (diethyl ether) =1:1 ] to solubilize non-melanin opaque particles, water bath at 37℃for 15min, centrifugation at 3000r/min for 5min and discarding supernatant, and precipitation adding 600. Mu.L of 1mol/L NaOH solution (containing 10% DMSO by volume) at 95℃for 30min to completely solubilize the cell pellet. After cooling, the sample was transferred to a 96-well plate, and the absorbance at 405nm was measured by changing the sample drug to a complete medium as a blank. The relative melanin content was calculated as follows:

in which A ₀ For the absorbance of the Control group at 405nm, A _i The absorbance at 405nm was used for the sample group.

As can be seen from fig. 8, COFE can reduce the formation of melanin in cells. The higher the action concentration, the greater the inhibition rate of COFE and KA on melanin formation, with the Control group melanin content being 100%, and the COFE effect being significantly stronger than KA at the same concentration (p < 0.01). At a COFE concentration of 160. Mu.g/mL, the relative melanin content in the cells was reduced by 26.41.+ -. 0.35%. Description: COFE can inhibit melanin synthesis in a375 cells.

4) Effect of COFE on melanin synthesis-related protein expression

Collecting cell sediment by the method in the step 2), and detecting the expression level of melanin synthesis-related proteins MITF, TYR, TRP-1 and TRP-2 in A375 cells by using a Western blot technology.

As can be seen from FIGS. 9 and 10, the intervention of COFE and KA at different concentrations can significantly reduce the relative expression levels of the four proteins (p < 0.01) compared with the Control group. And the reduction degree of the protein expression of the high concentration COPE (160 mug/mL) is more obvious than the intervention of the low concentration COPE (p < 0.01). Description: the COFE can effectively reduce the expression of MITF, TYR, TRP-1 and TRP-2, thereby reducing the tyrosinase activity, reducing the generation amount of the dopa pigment, inhibiting the conversion of the dopa pigment into the eumelanin, reducing the synthesis amount of the melanin and achieving the whitening effect.

Experiment 4: anti-glycation experiment of COFE based on bovine serum albumin-glucose model

1. Preparing a solution:

1) PBS phosphate buffered saline: adding 0.2% proclin 300 as preservative into 0.2mol/L PBS phosphate buffer solution, and fully mixing to obtain 0.2mol/L PBS phosphate buffer antibacterial solution (pH=7.4);

2) COFE and positive drug Aminoguanidine (AG) were prepared into 0.8mg/mL solution with 0.2mol/L PBS phosphate buffer antibacterial solution, and tested.

2. Grouping and model establishment:

under aseptic conditions, 20mg/mL of an aqueous Bovine Serum Albumin (BSA), 0.5mol/L of an aqueous glucose (Glu) solution and a sample solution to be tested were passed through a 0.22. Mu. Mol/L filter membrane. Solutions of each group were prepared according to table 1, and a bovine serum albumin-glucose model reaction system was established. AG was used as positive control, PBS was used as blank control, and Glu solution was replaced with PBS as the corresponding negative control for each group. The prepared solutions of each group were placed in a sterile incubator at 37℃for 10d.

TABLE 1 formulation of solutions for each group of bovine serum albumin-glucose model reaction system

1) Effect of COFE on Amadori product content

The first stage product of non-enzymatic saccharification of proteins is called Amadori product, and accumulation of this product promotes the production of AGEs. The amount of Amadori product in each group was measured at 1d, 3d, 5d, 7d, 9d, and 10d of culture. 2mL of carbonate buffer was added to each of the groups of 0.2mL of the culture solution and 0.8mL of 0.3mmol/L NBT reagent, and the mixture was mixed uniformly, reacted in a water bath at 40℃for 1 hour, and then absorbance was measured at 530 nm.

As can be seen from FIG. 11, the Amadori product content in the system was increased continuously during the culture, the Amadori product content in the COFE group was lower than that in the blank control group, and the positive control group was slightly higher than that in the blank control group. Description: COFE can inhibit synthesis of Amadori products.

2) Effect of COFE on dicarbonyl Compound content

The second stage of the non-enzymatic saccharification reaction of the protein is the synthesis of carbonyl intermediates, which have high physiological damage hazard, can directly act on free amino groups of the protein and induce protein crosslinking, and is a main way for generating AGEs in vivo. The content of each group of dicarbonyl compounds was determined by ultraviolet spectrophotometry at 1d, 3d, 5d, 7d, 9d, 10d.

As can be seen from fig. 12, the dicarbonyl compound content in the positive control group and the blank control group system is significantly increased, and the dicarbonyl compound content in the COFE group is increased more slowly. Description: COFE can inhibit the synthesis of dicarbonyl compounds.

3) Effect of COFE on non-fluorescent AGEs (CML) content

The formation of AGEs is the final stage of the non-enzymatic saccharification reaction of proteins. AGEs can be classified into fluorescent AGEs and non-fluorescent AGEs according to whether they are fluorescent or not. At 10d, the non-fluorescent AGEs (CML) content of each group was determined using ELISA kit.

As shown in fig. 13, the inhibition effect of COFE on CML (inhibition rate 22.81 ±2.46%) was significantly higher than AG. Description: COFE can inhibit CML synthesis.

4) Effect of COFE on fluorescent AGEs content

At 10d, measuring fluorescence values of each group of culture solutions at excitation/emission wavelength (Ex/Em) of 340/420nm by using a fluorescence spectrophotometer, namely total fluorescence AGEs content; simultaneously determining the fluorescence value of the characteristic fluorescence AGEs: pentososides (Ex 335nm/Em 385 nm) and glycosylated collagens (Ex 370nm/Em 440 nm).

As shown in FIG. 14, the inhibition ratios of COFE to total fluorescence AGEs, pentososides and glycosylated collagens were 98.04.+ -. 1.91%, 92.67.+ -. 1.01% and 94.70.+ -. 1.39%, respectively, which were slightly lower than those of the AG group. Description: COFE can inhibit the synthesis of fluorescent AGEs. The anti-glycation means that AGEs are inhibited from being generated.

Experiment 5COFE anti-glycation experiment based on HaCaT cell glycation model

1. Preparing a solution:

2) COFE solution: weighing 10.0mg of COPE, dissolving with a complete culture medium, and fixing the volume to 10mL to obtain 1.0mg/mL of COPE solution; it was diluted precisely with complete medium to a series of concentration gradient COFE solutions: 20. Mu.g/mL, 80. Mu.g/mL, 320. Mu.g/mL.

2. Grouping and model establishment: taking 5 th generation of cells at 5 x 10 ⁵ The concentration of each cell/well was seeded in 6-well plates, and after 24 hours the cells were divided into Control, GLA and cofe+gla treatment groups, each group being specifically treated as follows:

(1) Control group: after the DMEM culture medium is pretreated for 3 hours, a new DMEM culture medium is added for culturing for 24 hours;

(2) GLA group: after pretreatment for 3 hours in DMEM medium, 400 mu mol/L GLA is added for culture for 24 hours;

(3) 20 μg/mL COFE+GLA treatment group: after pretreatment with 20. Mu.g/mL COFE for 3 hours, 400. Mu. Mol/LGLA was added for 24 hours;

(4) 80 μg/mL COFE+GLA treatment group: after pretreatment with 80. Mu.g/mL COFE for 3 hours, 400. Mu. Mol/LGLA was added for 24 hours;

(5) 320 μg/mL cofe+gla treated group: after pretreatment with 320. Mu.g/mL COFE for 3h, 400. Mu. Mol/LGLA was added for 24h.

The pretreatment is as follows: after cell adhesion, the supernatant is discarded, and corresponding treatment culture solutions are respectively added, and the culture (pretreatment) is carried out for 3 hours under the conditions of 37 ℃ and 5% CO 2; culturing: culturing at 37deg.C under 5% CO2 for 24 hr.

1) Effect of COFE on AGEs content of cell saccharification model

After the culture is finished, collecting cell supernatant, and detecting the content of AGEs in the cell supernatant according to the step of the AGEs ELISA detection kit.

As can be seen from fig. 15, the GLA-treated group had significantly increased AGEs compared to the control group. The AGEs content of each of cofe+gla treatment groups was reduced as compared with GLA treatment groups, and there was an inhibitory effect on the generation of AGEs, and there was a dose-dependency of the inhibitory effect. Wherein 80 μg/mL of COFE+GLA treated group and 320 μg/mL of COFE+GLA treated group have significantly lower AGEs than GLA treated group. Description: on the cellular level, COPE can inhibit GLA-induced AGEs production, and has good anti-saccharification effect.

2) Effect of COFE on HaCaT cell RAGE protein expression

And detecting the expression level of RAGE of the HaCaT cells by adopting a Western blot technology. Description: RAGE is a receptor for AGEs, and the reduction of the expression level of RAGE can further reflect that the synthesis of AGEs is inhibited.

As can be seen from FIG. 16, 400. Mu. Mol/L GLA treatment significantly increased the expression of the RAGE protein factor in the cells compared to the Control group. And COPE can effectively inhibit the increase of the RAGE expression level caused by GLA, and the higher the concentration of COPE is, the stronger the inhibition effect on the RAGE protein expression is. And the high dose COFE (320. Mu.g/mL) treatment works best, with a 50.35.+ -. 0.26% decrease in the expression level of the RAGE protein.

Description: COFE may be effective in reducing RAGE expression.

Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.

Claims

1. Application of oil tea flower extract in resolving macula, whitening skin and resisting saccharification is provided.

2. Use according to claim 1, characterized by at least any one of the following: the method is used for preparing the freckle-removing and whitening drugs or daily chemical products and preparing the anti-glycation drugs or daily chemical products.

3. The use according to claim 2, characterized in that: the oil tea flower extract is oil tea flower alcohol extract.

4. A use according to claim 3, characterized in that: the oil tea flower alcohol extract is obtained by extracting oil tea flowers with 50% ethanol.