Composition having anti-glycation effect and application thereof

Abstract

The present invention relates to a composition having an anti-glycation effect and an application thereof. The anti-glycation composition includes the following constituents in parts by mass: 0.1 to 5 parts of Osmanthus fragrans extract, 0.1 to 5 parts of Punica granatum extract and 0.1 to 2 part of Olea europaea extract, wherein in the Osmanthus fragrans extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of verbascoside is more than or equal to 10%; in the Punica granatum extract, a mass content of polyphenol is more than or equal to 30%, and a mass content of punicalagin is more than or equal to 8%; and in the Olea europaea extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of hydroxytyrosol is more than or equal to 3%.

Claims

1. A skin care emulsion, comprising the constituents in parts by mass in the following ratio: 0.5 to 3 parts of Osmanthus fragrans extract; 0.5 to 5 parts of Punica granatum extract; and 0.1 to 2 parts of Olea europaea extract, wherein in the Osmanthus fragrans extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of verbascoside is more than or equal to 10%; in the Punica granatum extract, a mass content of polyphenol is more than or equal to 30%, and a mass content of punicalagin is more than or equal to 10%; and in the Olea europaea extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of hydroxytyrosol is more than or equal to 3%, wherein the skin care emulsion has an anti-glycation effect.

2. The skin care emulsion according to claim 1, wherein the skin care emulsion comprises the following constituents in parts by mass: 1 to 3 parts of Osmanthus fragrans extract, 1 to 2 parts of Punica granatum extract and 0.1 to 2 parts of Olea europaea extract.

3. The skin care emulsion according to claim 2, wherein the skin care emulsion comprises the following constituents in parts by mass: 3 parts of Osmanthus fragrans extract, 1 part of Punica granatum extract and 0.1 part of Olea europaea extract.

4. The skin care emulsion according to claim 3, wherein in the skin care emulsion: in the Osmanthus fragrans extract, the mass content of polyphenol is 10%, and the mass content of verbascoside is 10%; in the Punica granatum extract, the mass content of polyphenol is 30%, and the mass content of punicalagin is 10%; and in the Olea europaea extract, the mass content of polyphenol is 10%, and the mass content of hydroxytyrosol is 3%.

5. A method of preparing anti-glycation products for skin, comprising using the skin care emulsion according to claim 1.

6. A preparation, wherein the preparation consists of the skin care emulsion according to claim 1 and other acceptable auxiliary materials.

7. A method of preparing anti-glycation products for skin, comprising using the skin care emulsion according to claim 2.

8. A method of preparing anti-glycation products for skin, comprising using the skin care emulsion according to claim 3.

9. A method of preparing anti-glycation products for skin, comprising using the skin care emulsion according to claim 4.

10. A preparation, wherein the preparation consists of the skin care emulsion according to claim 2 and other acceptable auxiliary materials.

11. A preparation, wherein the preparation consists of the skin care emulsion according to claim 3 and other acceptable auxiliary materials.

12. A skin care cream, comprising the constituents in parts by mass in the following ratio: 0.5 to 3 parts of Osmanthus fragrans extract; 0.5 to 5 parts of Punica granatum extract; and 0.1 to 2 parts of Olea europaea extract, wherein in the Osmanthus fragrans extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of verbascoside is more than or equal to 10%; in the Punica granatum extract, a mass content of polyphenol is more than or equal to 30%, and a mass content of punicalagin is more than or equal to 10%; and in the Olea europaea extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of hydroxytyrosol is more than or equal to 3%, wherein the skin care cream has an anti-glycation effect.

13. A skin care lotion, comprising the constituents in parts by mass in the following ratio: 0.5 to 3 parts of Osmanthus fragrans extract; 0.5 to 5 parts of Punica granatum extract; and 0.1 to 2 parts of Olea europaea extract, wherein in the Osmanthus fragrans extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of verbascoside is more than or equal to 10%; in the Punica granatum extract, a mass content of polyphenol is more than or equal to 30%, and a mass content of punicalagin is more than or equal to 10%; and in the Olea europaea extract, a mass content of polyphenol is more than or equal to 10%, and a mass content of hydroxytyrosol is more than or equal to 3%, wherein the skin care lotion has an anti-glycation effect.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows expression of CMLs in glycation-induced fibroblasts.

(2) FIG. 2 shows expression of AGEs in an aging mouse model.

(3) FIG. 3 shows expression of SOD in the aging mouse model.

DESCRIPTION OF THE EMBODIMENTS

(4) The invention is further described hereinafter with reference to the specific embodiments, but the embodiments are not intended to limit the invention in any form. Unless defined otherwise, the raw material reagents used in the embodiments of the invention are conventionally purchased raw material reagents.

Embodiment 1

(5) A composition having an anti-glycation effect includes the following constituents in parts by mass: 3 parts of Osmanthus fragrans extract, 1 part of Punica granatum extract and 0.1 part of Olea europaea extract.

(6) In the Osmanthus fragrans extract, a mass content of polyphenol is 10%, and a mass content of verbascoside is 10%;

(7) in the Punica granatum extract, a mass content of polyphenol is 30%, and a mass content of punicalagin is 10%; and

(8) in the Olea europaea extract, a mass content of polyphenol is 10%, and a mass content of hydroxytyrosol is 3%.

(9) A method for extracting the Osmanthus fragrans extract was as follows:

(10) Dry and pulverized Osmanthus fragrans powder was mixed with one or more of water, alcohol and other food processing aids, and subjected to extraction, concentration and drying to obtain the Osmanthus fragrans extract. The Osmanthus fragrans of the present invention may be selected from semperflorens (Osmanthus fragrans var. semperflorens), aurantiacus (Osmanthus fragrans (Thunb.) Lour.), thunbergii (Osmanthus fragrans (Thunb.) Lour) and odoratus (Osmanthus fragrans cv. Latifoliu).

(11) A method for extracting the Punica granatum extract was as follows:

(12) Powder obtained by pulverizing Punica granatum fruits or drying Punica granatum fruit juice was mixed with one or more of water, alcohol and other food processing aids, and subjected to extraction, concentration and drying to obtain the Punica granatum extract.

(13) A method for extracting the Olea europaea extract was as follows:

(14) Powder obtained by pulverizing Olea europaeas or drying Olea europaea juice was mixed with one or more of water, alcohol and other food processing aids, and subjected to extraction, concentration and drying to obtain the Olea europaea extract.

(15) Determination of polyphenol and verbascoside in the Osmanthus fragrans extract referred to a determination method of Q/HSHLY0001S-2018.

(16) The hydroxytyrosol in the Olea europaea extract was determined by a HPLC method, wherein the determination method was as follows:

(17) 1. Reagents: methanol (AR), acetonitrile (chromatographically pure), ultrapure water and standard;

(18) 2. Standard: hydroxytyrosol (purchased from Tokyo Chemical Industry Co. Ltd., 98%);

(19) 3. Instruments and appliances: electronic balance ( 1/100000), glassware: volumetric flask, 0.45 μm millipore filter and syringe filter, ultrasonic cleaner, high performance liquid chromatograph, Agilent 1100 infusion pump, Agilent 1100 variable-wavelength ultraviolet detector, and Agilent 1100 ChemStation;

(20) 4. Chromatographic conditions

(21) Chromatographic column: Lnua Cns. 250 m×4.6 mm, 5 μm

(22) Mobile phase: acetonitrile (B): water (D)=5:95 (V/V)

(23) TABLE-US-00003 Time (min) B % D % 0.00 5 95 10.00 8 92 25.00 15 85 26.00 5 95
Flow rate: 1.0 mL/min
Detection wavelength: 276 nm
Sample size: 5μ
Column temperature: 25° C.

(24) 5. Operating steps

(25) Preparation of test solution: about 60 mg (20%) of the product was weighed accurately, placed in a 25 ml volumetric flask, and added with 30% methanol, then the mixture was ultrasonically extracted for 15 minutes, cooled to room temperature, then diluted to a certain volume with 30% methanol, shaken evenly, and filtered through a 0.45 μm filter membrane for detection.

(26) Preparation of standard solution: a proper amount of hydroxytyrosol was weighed accurately and added with 30% methanol to prepare a solution with a concentration of about 0.5 mg/mL.

(27) 5 μL of the standard solution and 5 μL of the test solution were respectively injected into a HPLC instrument respectively.

(28) 6. Result calculation:

(29) $\mathrm{Content}\mathrm{of}\mathrm{hydroxytyrosol}\left(\%\right)=\frac{A_{\mathrm{sample}}\times C_{\mathrm{standard}}\times V}{A_{\mathrm{sample}}\times M_{\mathrm{standard}}}\times P$
wherein:
A.sub.sample: peak area of sample;
A.sub.standard: peak area of standard;
C.sub.standard: concentration of standard (mg/mL); Mn: mass of sample (mg);
V.sub.sample: certain volume of sample (mL); P: purity of standard (%).

(30) Content of the polyphenol in the Punica granatum extract and content of the polyphenol in the Olea europaea extract were determined according to Q/WXJ0031 S-2015.

(31) The punicalagin in the Punica granatum extract was determined by HPLC, wherein the specific determination method was as follows:

(32) 1. Chemical reagents: methanol (chromatographically pure), glacial acetic acid (guaranteed reagent) and distilled water; and standard: punicalagin (98.00%, Sigma-Aldrich)

(33) 2. Chromatographic conditions

(34) TABLE-US-00004 Vacuumizing system Shimadzu LC-20AT Syringe and Shimadzu sil-htc autosampler autosampler Probe Shimadzu SP D-M20A Column Phenomenex Luna, C18, 5 μm, 4.6 × 150 mm Filtration system Advance Micro-devices Pvt. Ltd, polytetrafluoroethylene, 0.45 μm Wavelength (λ.sub.max) 378 nm Injection volume 20 μL Flow rate 10 mL/min Mobile phase Mobile phase A (MPA) 2% (v/v) aqueous solution of glacial acetic acid Mobile phase B (MPB) 2% (v/v) methanol solution of glacial acetic acid Diluent 2% aqueous solution of glacial acetic acid Separation mode Gradient separation Gradient design Time (min) MP A MP B 0.01 99 01 5.00 99 01 20.00 40 60 26.00 22 78 26.01 99 01 32.00 99 01

(35) 3. Preparation of standard solution:

(36) Preparation of standard solution of punicalagin: about 5 mg of pure punicalagin was accurately weighed and placed in a 10 ml volumetric flask, added with 5 ml of 2% glacial acetic acid, then the standard was ultrasonically dissolved, diluted to a scale with 2% glacial acetic acid; the solution was thoroughly mixed and filtered with a 0.2 μm membrane filter, and placed in a clean high performance liquid chromatography bottle; 20 μL of the solution was injected and a chromatogram was recorded.

(37) Preparation of sample solution: 150 mg of pure sample was accurately weighed and placed in a 50 ml volumetric flask, added with 2% glacial acetic acid to a half volume approximately, then the mixture was subjected to ultrasonic treatment for 20 minutes, cooled and diluted to a scale with 2% glacial acetic acid; the solution was thoroughly mixed and filtered with a 0.2 μm membrane filter, and placed in a clean high performance liquid chromatography bottle; 20 μL of the solution was injected and a chromatogram was recorded.

(38) Preparation of mobile phases: mobile phase A (2% (V/V) aqueous solution of glacial acetic acid): a 100 mL volumetric flask was taken, added with 50 mL of water, and 2 mL of glacial acetic acid, and then the mixture was diluted to a certain volume, mixed evenly, and filtered with a 0.45 μm membrane filter.

(39) Mobile phase B (2% (V/V) methanol solution of glacial acetic acid): a 100 mL volumetric flask was taken, added with 50 mL of methanol, and 2 mL of glacial acetic acid; then the mixture was diluted to a certain volume with methanol, mixed evenly, filtered with a 0.45 μm membrane filter, and ultrasonically degassed.

(40) 4. Analysis

(41) Given chromatographic conditions were used to record high performance liquid chromatograms of the standard solution and the sample solution prepared above.

(42) The running time of the analysis was 32 minutes, the retention time (RT) of punicalagin-1 was 10.9 minutes, and the retention time of punicalagin-2 was 13.8 minutes. Due to changes in the mobile phase compositions and chromatographic columns, the retention time of analytes may change.

(43) 5. Content calculation of punicalagin
Content of punicalagin (%)=sample area×concentration of standard×purity of standard/(area of standard×concentration of standard).

Embodiment 2

(44) A composition having an anti-glycation effect includes the following constituents in parts by mass: 2 parts of Osmanthus fragrans extract, 5 parts of Punica granatum extract and 1 part of Olea europaea extract;

(45) wherein, the Osmanthus fragrans extract, the Punica granatum extract and the Olea europaea extract are the same as that in the embodiment 1.

Embodiment 3

(46) A composition having an anti-glycation effect includes the following constituents in parts by mass: 1 part of Osmanthus fragrans extract, 2 parts of Punica granatum extract and 2 parts of Olea europaea extract,

(47) wherein, the Osmanthus fragrans extract, the Punica granatum extract and the Olea europaea extract are the same as that in the embodiment 1.

Embodiment 4

(48) A composition having an anti-glycation effect includes the following constituents in parts by mass: 0.5 part of Osmanthus fragrans extract, 0.5 part of Punica granatum extract and 1 part of Olea europaea extract,

(49) wherein, the Osmanthus fragrans extract, the Punica granatum extract and the Olea europaea extract are the same as that in the embodiment 1.

Embodiment 5

(50) A composition having an anti-glycation effect includes the following constituents in parts by mass: 2 parts of Osmanthus fragrans extract, 2 parts of Punica granatum extract and 0.1 part of Olea europaea extract,

(51) wherein, the Osmanthus fragrans extract, the Punica granatum extract and the Olea europaea extract are the same as that in the embodiment 1.

Embodiment 6

(52) A composition having an anti-glycation effect includes the following constituents in parts by mass: 3 parts of Osmanthus fragrans extract, 1 part of Punica granatum extract and 0.1 part of Olea europaea extract.

(53) wherein, in the Osmanthus fragrans extract, a mass content of polyphenol is 10%, and a mass content of verbascoside is 10%;

(54) in the Punica granatum extract, a mass content of polyphenol is 40%, and a mass content of punicalagin is 8%; and

(55) in the Olea europaea extract, a mass content of polyphenol is 10%, and a mass content of hydroxytyrosol is 3%.

Comparative Example 1

(56) An extract having an anti-glycation effect, wherein the extract is an Osmanthus fragrans extract.

Comparative Example 2

(57) An extract having an anti-glycation effect, wherein the extract is a Punica granatum extract.

Comparative Example 3

(58) An extract having an anti-glycation effect, wherein the extract is an Olea europaea extract.

Comparative Example 4

(59) A composition having an anti-glycation effect includes the following constituents in parts by mass: 3 parts of Osmanthus fragrans extract, 1 part of Punica granatum extract and 0.1 part of Olea europaea extract.

(60) wherein, in the Osmanthus fragrans extract, a mass content of polyphenol is 8%, and a mass content of verbascoside is 8%;

(61) in the Punica granatum extract, a mass content of polyphenol is 40%, and a mass content of punicalagin is 10%; and

(62) in the Olea europaea extract, a mass content of polyphenol is 10%, and a mass content of hydroxytyrosol is 3%.

Comparative Example 5

(63) A composition having an anti-glycation effect includes the following constituents in parts by mass: 3 parts of Osmanthus fragrans extract, 1 part of Punica granatum extract and 0.1 part of Olea europaea extract,

(64) wherein, in the Osmanthus fragrans extract, a mass content of polyphenol is 8%, and a mass content of verbascoside is 8%;

(65) in the Punica granatum extract, a mass content of polyphenol is 20%, and a mass content of punicalagin is 8%; and

(66) in the Olea europaea extract, a mass content of polyphenol is 8%, and a mass content of hydroxytyrosol is 2%.

(67) Result Detection

(68) (1) Verification and Test of Anti-Glycation Ability of Sample by Using Skin Cells

(69) Analysis on Expression of Glycation Marker Gene in Skin Fibroblasts:

(70) N-ε-carboxy methyl lysine (CML) was an advanced glycation end product, and the glycation degree of the skin cells was reflected by detecting a CML level.

(71) Detection Method:

(72) Fibroblasts were seeded in a 24-well plate and treated with 400 μM methylglyoxal and glyoxal to induce glycation. After the cells were treated with a sample group for 7 days, an immunofluorescence experiment was carried out. 800 μM aminoguanidine was a positive control, and experimental groups were compositions of the embodiments and the comparative examples with a mass concentration of 0.01%.

(73) The expression of CMLs after treatment with active ingredients was observed.

(74) Results are shown in FIG. 1, wherein histograms show the analysis results on red pixels after fluorescence microscope shooting. The Embodiment 1 has the most significant effect on reducing a glycation reaction of the fibroblasts.

(75) (2) Verification and Test of the Anti-Glycation Ability of Test Sample by Using a Mouse Aging Model

(76) D-galactose (1 g/kg) was used to make the model. D-galactose was freshly prepared and used every day. A normal group received subcutaneous injection of physiological saline with the same administration volume of 0.1 mL/10 g, and continuously received daily subcutaneous injection on the back for 42 days. The test samples (compositions of the embodiments and the comparison examples) were administered by gavage every day; and an induction group was given normal saline every day. After 42 days, the animals were killed to take blood for detecting biochemical indexes and pathology (SOD activity, AGEs).

(77) Results are shown in FIG. 2 and FIG. 3, wherein the Embodiment 1 has the most significant effect on reducing the generation of AGEs and increasing the expression of SODs in the aging mice.

(78) (3) Data Processing

(79) All the groups were compared with a glycation-induced control group for significant difference analysis, and T test was used for statistical difference analysis. The results are shown in Table 1 and Table 2. Results are indicated by asterisks *P<0.05; **P<0.01; and ***P<0.001.

(80) TABLE-US-00005 TABLE 1 CML % AGE/ng/mL SOD/U/mL Blank control 0 ± 0 27.83 ± 7.76 1.415 ± 0.058 P = 0.002 P = 0.001 P = 0.004 Glycation-induced 0.072 ± 0.03  42.42 ± 9.53  1.279 ± 0.070 control group Embodiment 1 0.050 ± 0.012 33.00 ± 9.46 1.377 ± 0.033 P = 0.014 P = 0.022 P = 0.033 Embodiment 2 0.058 ± 0.025 34.28 ± 5.63  1.295 ± 0.818 P = 0.061 P = 0.053 P = 0.818 Embodiment 3 0.070 ± 0.013 34.10 ± 4.96  1.333 ± 0.401 P = 0.245 P = 0.052 P = 0.401 Embodiment 4 0.064 ± 0.013 33.20 ± 2.95  1.256 ± 0.647 P = 0.168 P = 0.049 P = 0.647 Embodiment 5 0.061 ± 0.014 37.35 ± 9.08  1.346 ± 0.043 P = 0.165 P = 0.238 P = 0.043 Embodiment 6 0.069 ± 0.02  36.98 ± 2.46  1.32 ± 0.12 P = 0.162 0.32  0.103  Comparative 0.078 ± 0.013 39.57 ± 7.76  1.298 ± 0.070 example 1 P = 0.326 P = 0.312 P = 0.762 Comparative 0.066 ± 0.014 38.92 ± 9.53  1.288 ± 0.075 example 2 P = 0.232 P = 0.247 P = 0.659 Comparative 0.084 ± 0.015 36.74 ± 9.46  1.312 ± 0.16 example 3 P = 0.363 P = 0.179 P = 0.085 Comparative 0.063 ± 0.01  37.19 ± 6.29  1.29 ± 0.03 example 4 P = 0.126 0.301 0.1821 Comparative 0.059 ± 0.02  38.23 ± 6.75  1.34 ± 0.03 example 5 P = 0.102 P = 0.242 P = 0.1213

(81) From the data in Table 1 above, it can be seen that the anti-glycation composition provided by the present invention can well inhibit the generation of AGEs which are glycation end products, through the synergistic effect of the Osmanthus fragrans extract, the Punica granatum extract and the Olea europaea extract. The content of the AGEs is close to 27.83±7.76 in a blank group, which is significantly lower than 42.42±9.53 in the glycation-induced control group. The corresponding advanced glycation end products CMLs of the fibroblasts are also significantly reduced, and the expression effect of SODs is significantly increased. The anti-glycation ability of skin is improved from three dimensions of CMLs, AGEs and SODs.

(82) Obviously, the above-mentioned embodiments of the invention are merely examples for clearly illustrating the invention, but are not intended to limit the implementations of the invention. For those of ordinary skills in the art, other different forms of changes or variations can be made on the basis of the above description. It is not necessary or possible to exhaust all the embodiments here. Any change, equivalent substitution, and improvement made within the spirit and principle of the invention shall fall within the protection scope of the claims of the invention.