TWI795561B - Use of mango extracts and bioactive ingredients obtained therefrom for skincare and healthcare - Google Patents

Abstract

The present invention provides use of a mango extract for manufacture of a composition for UV protection and whitening of skin and anti-glycation. The present invention also provides bioactive ingredients that include hydrolyzed tannins obtained from the early-harvested mango extract, and use of said bioactive ingredients for manufacture of a composition for anti-glycation.

Description

Use of mango fruit extract and bioactive substances derived therefrom for skin care and health care

The present invention relates to a kind of mango fruit extract and its use to obtain biologically active substances containing hydrolyzed tannin compounds, especially to the use of a kind of mango fruit extract for preparing skin anti-ultraviolet, whitening and anti-glycation compositions, and The bioactive substance is used for preparing an anti-glycation composition.

The skin provides the body's first line of protection against ultraviolet radiation in the sun. The skin consists of the epidermis (including the outermost stratum corneum), the dermis, and the subcutaneous tissue. The epidermis is the outermost layer of the skin and is constantly renewed. There are continuously dividing cells (such as fibroblasts, keratinocytes, melanocytes) between the epidermis and dermis, and the activities of these cells are very sensitive to ultraviolet light. The dermis contains collagen and elastin, which give the skin its elasticity and support. When exposed to high levels of UV rays (mainly UVA), collagen fibers and elastin are damaged, resulting in decreased skin elasticity, wrinkle formation, and the appearance of aging skin. In order to delay skin aging, it is extremely important to reduce the damage to the skin from UV exposure.

A fair and translucent complexion is also a symbol of youth and vitality. In order to show their personal vitality, modern people maintain their skin bright and white through means such as makeup and skin care. Skin color is mainly determined by the amount of melanin produced by melanocytes. Congenital inheritance, endocrine disorders, lifestyle, sun exposure, and drug use may all cause melanocytes to produce melanin, which in turn leads to dull skin or localized spots. Therefore, developing ways to actively inhibit melanin production is key to maintaining skin lightness.

Another cause of skin aging is glycation, which is a non-enzymatic reaction between aldehyde (ketone) groups of reducing sugars and amine groups of amine-containing substances (such as proteins, nucleic acids, etc.). When the human body is in a state of high blood sugar for a long time, the protein in the body is likely to undergo glycation reaction with glucose, and finally form advanced glycation end products (AGEs). In addition to the body's own synthesis, glycation end products Drugs can also enter the body through dietary intake. Foods that are overcooked or processed often contain higher levels of end-glycation products, such as grilled foods, fried foods, bread, etc. Proteins that form glycation end products will lose their normal molecular structure and physiological functions. At the same time, glycation end products will also increase cellular oxidative stress and induce the secretion of pro-inflammatory cytokines, which may cause various metabolic diseases that cause body aging, such as porridge Arteriosclerosis, neurodegenerative diseases, cataracts, kidney failure, etc. As far as skin aging is concerned, the glycation reaction will affect the aggregation of collagen to form fibers, and the glycated collagen fibers will become stiff and brittle, making the skin prone to wrinkles. Therefore, inhibiting the occurrence of glycation reaction is also a countermeasure to slow down skin aging.

Most of the sunscreen, whitening, or anti-glycation ingredients in commercially available skin care products are chemically synthesized. If these ingredients are used improperly, they may be harmful to skin health. Therefore, it is necessary to develop new types of skin care products by developing natural ingredients with skin whitening, anti-ultraviolet and anti-glycation properties.

One object of the present invention is to provide a mango fruit extract for the preparation of skin anti-ultraviolet and whitening and anti-glycation compositions, wherein the mango fruit extract is obtained by extracting a mango fruit with a solvent, wherein the mango fruit is Refers to the mango fruit that is in the young fruit stage or the rapid growth stage, and the peel has not turned yellow.

In one embodiment of the present invention, the weight ratio of the solvent to the mango fruit ranges from 20:1 to 1:1, and the extraction is performed at 55°C to 100°C.

In one embodiment of the present invention, the solvent is water, and the concentration of the mango fruit extract is at least 0.25 mg/mL.

In one embodiment of the present invention, the mango fruit extract inhibits the formation of melanin, prevents and repairs damage to skin fibroblasts caused by ultraviolet A, and inhibits glycation of collagen.

Another object of the present invention is to provide a biologically active substance comprising a compound represented by formula (I):

Another object of the present invention is to provide a use of a biologically active substance comprising a compound represented by formula (I) or formula (II) for preparing an anti-glycation composition.

In one embodiment of the present invention, the compounds of formula (I) and formula (II) are isolated from the aforementioned mango fruit extract.

In one embodiment of the present invention, the compound of formula (I) or formula (II) inhibits glycation of collagen.

The invention discloses that the mango fruit extract not only has ultraviolet protection and whitening effects on skin cells, but also can inhibit protein glycosylation and prevent protein from losing its normal structure and function due to glycosylation. In addition, the present invention also discloses a biologically active substance comprising compounds of formula (I) and formula (II) with significant anti-glycation activity. In view of the fact that ultraviolet radiation, excessive melanin precipitation, and saccharification reaction can all lead to the appearance of skin aging, especially saccharification reaction is one of the reasons for body aging, and the mango fruit extract disclosed by the present invention and the biologically active substance can be used to prepare Skin anti-ultraviolet, whitening and anti-glycation composition, therefore, the present invention provides a new strategy to delay the aging of skin and even individuals. The above skin anti-ultraviolet, whitening and anti-glycation composition can be powder, granule, solution, colloid or paste, And it can be made into medicine, food, drink, or nutritional supplement, and given to an individual by oral administration, skin application or other methods.

The implementation of the present invention will be further described in conjunction with the drawings below. The following examples are used to illustrate the characteristics and applications of the present invention, but not to limit the scope of the present invention. Anyone who is familiar with this technology will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made, so the protection scope of the present invention should be defined by the scope of the appended patent application.

FIG. 1 shows the relative content of melanin in melanoma cells B16F10 treated with kojic acid or mango fruit extract according to an embodiment of the present invention.

Fig. 2 shows the cell survival rate of dermal fibroblasts pretreated with mango fruit extract according to an embodiment of the present invention and then irradiated with ultraviolet A.

FIG. 3 shows the survival rate of dermal fibroblasts after being irradiated with ultraviolet A and then treated with mango fruit extract according to an embodiment of the present invention.

Fig. 4 shows the effect of the mango fruit extract of an embodiment of the present invention on the production of end-products of collagen glycation.

Figure 5 shows the mass spectrum of compound 1.

FIG. 6A shows the hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of compound 1. FIG.

FIG. 6B shows the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of compound 1. FIG.

FIG. 6C shows the hydrogen-hydrogen correlation (COZY) spectrum of compound 1. FIG.

FIG. 6D shows the carbon-hydrogen heteronuclear single quantum correlation (heteronuclear single quantum correlation, HSQC) spectrum of Compound 1. FIG.

FIG. 6E shows the carbon-hydrogen heteronuclear multiple bond correlation (heteronuclear multiple bond correlation, HMBC) spectrum of Compound 1. FIG.

Fig. 7 shows the effects of the compounds of formula (I) and formula (II) on the production of collagen glycation end products.

definition

As used herein, "a", "the" and similar terms should be understood to include both singular and plural forms unless otherwise stated.

The numerical values used herein are approximate values, and all experimental data are expressed within the range of 20%, preferably within the range of 10%, and most preferably within the range of 5%.

The pharmaceutical compositions described herein can be formulated into a dosage form suitable for parenterally or orally administered using techniques well known to those skilled in the art, including but not limited to : injection (for example, sterile aqueous solution (sterile aqueous solution) or dispersion (dispersion)], powder (sterile powder), lozenge (tablet), tablet (troche), buccal lozenge (lozenge) , pill, capsule, dispersible powder, granule, solution, suspension, emulsion, syrup, elixir, concentrated pulp (slurry) and the like.

The pharmaceutical compositions described herein can be administered by parenteral routes, including but not limited to: intraperitoneal injection, subcutaneous injection, intramuscular injection, and Intravenous injection.

The pharmaceutical compositions described herein may comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier), which is widely used in pharmaceutical manufacturing techniques. The pharmaceutically acceptable carrier may contain one or more agents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer , binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant lubricants, absorption delaying agents, liposomes, and the like. The selection and amounts of such reagents are within the professionalism and routine skill of those skilled in the art.

The aforementioned pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), sugar solution, Aqueous solution containing alcohol, and combinations thereof.

Materials and Methods Material

Purchase DMEM medium (Gibco Dulbecco's modified Eagle's medium), Eagle's minimal essential medium with Earle's balanced salt solution from Thermo Fisher Scientific (Gibco Eagle's minimum essential medium, referred to as MEM medium), fetal bovine serum (Gibco fetal bovine serum, FBS), penicillin/streptomycin (Gibco penicillin/streptomycin), phosphate buffered saline (Gibco PBS), sodium pyruvate, carbonic acid sodium hydrogen, and non-essential amino acids. 3-(4,5-Dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide, MTT). Dimethyl sulfoxide (DMSO) was purchased from Echo chemical.

Solvents were purchased from Merck, Taiwan, including n -hexane, ethyl acetate, acetone, methanol, ethanol, acetonitrile, and chloroform- d ₁ (deuteration degree 99.5%), methanol- d ₆ (deuteration degree 99.5%), heavy water (deuterium oxide, deuteration degree>99.8%), and dimethyl sulfoxide- d ₆ (dimethyl sulfoxide- d ₆ , deuterium The degree of transformation >99.9%).

Chemical Analysis Instruments

The compounds were separated using column chromatography (column chromatography) and thin layer chromatography (thin layer chromatography, TLC). The medium pressure liquid chromatography (MPLC) system is Combi Flash ^® Rf+ (Teledyne ISCO); the column system is selected from Sephadex LH-20 (Amersham Biosciences), Diaion HP-20 (Mitsubishi Chemical), Merck Kieselgel 60 (40-63μm, Art.9385), and Merck LiChroprep® RP-18 (40-63μm, Art.0250). High performance liquid chromatography (HPLC) system equipped with Hitachi L-2310 series pump, Hitachi L-2420 UV-VIS detector (detection wavelength is 200nm to 380nm), and D-2000 Elite data processing Software; the column system is selected from analytical grade Discovery® HS C ₁₈ (250×4.6mm, 5μm; SUPELCO) and Mightysil RP-18 GP 250 (250×4.6mm, 5μm; Kanto Chemical), and semi-preparative grade Discovery® HS C ₁₈ (250×10.0 mm, 5 μm; SUPELCO) and preparative grade Discovery® HS C ₁₈ (250×21.0 mm, 5 μm; SUPELCO). The chromatography system is equipped with UV lamp UVP UVGL-25 (wavelength 254nm and 365nm). Thin-layer chromatography film is an aluminum film coated with silica gel 60 F ₂₅₄ (0.25mm; Merck) or RP-18 F _254S (0.25mm; Merck).

The chemical structure of the compound was determined by mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR). Specifically, two-dimensional ion trap tandem Fourier transform mass spectrometry (Bruker amaZon SL system) and electrospray ionization tandem mass spectrometry (ESI-MS/MS; Thermo Scientific Orbitrap Elite system) were used; and a 400MHz Varian 400 FT-NMR was used to obtain a Two-dimensional and two-dimensional NMR spectra, tetramethylsilane (tetramethylsilane, TMS) as an internal standard (δ = 0).

cell culture

The cells used in the following examples include mouse melanoma cell line B16F10 (ATCC CRL-6475) and human dermal fibroblasts CCD-966SK (ATCC CRL-1881) purchased from the American Type Culture Collection (ATCC). ). B16F10 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% carbon dioxide, hereinafter referred to as DMEM cell culture medium. CCD-966SK cells were cultured in 10% FBS, 1mM sodium pyruvate, 1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids, and 1% penicillin/streptomycin at 37°C and 5% carbon dioxide. The MEM medium, hereinafter referred to as MEM cell culture medium.

melanin production test

The melanin content determination method of the melanoma cell line B16F10 is briefly described as follows. Cells were harvested from indicated treated cell cultures. The cells were washed with PBS solution and treated with trypsin solution for 3 minutes. The resulting suspended cells were collected by centrifugation (400 x g, 5 minutes), washed twice with PBS solution, and then resuspended in 200 μL PBS solution. The cell suspension was frozen with liquid nitrogen for 10 minutes, and then placed at room temperature for about 30 minutes until completely thawed, and then the supernatant was removed by centrifugation (12,000 g, 3 minutes). The remaining cell pellet was mixed evenly with 120 μL of sodium hydroxide, and then heated at 60° C. for 1 hour to obtain a cell lysate. 100 μL of the cell lysate was transferred into a 96-well plate, and the absorbance value of the cell lysate at 450 nm (OD 450 ) was measured using an ELISA disk reader (enzyme-linked immunosorbent assay reader; BioTek). The relative content of melanin was calculated according to the following formula: relative content of melanin (%)=(OD450 value of each group/OD450 value of blank control group)×100%.

MTT analysis

(formazan)結晶。最終，使用ELISA讀盤機(BioTek)測量該細胞混合物在570nm的吸光值(OD 570)。細胞存活率係依下列公式計算：細胞存活率=(各組的OD 570值/空白對照組的OD 570值)×100%。 Cell viability was determined by MTT assay. Briefly, 15 μL/well of MTT solution (4 mg/ml MTT dissolved in PBS solution) was added to cells in a 96-well plate, and reacted at room temperature for 4 hours. After removing the reaction solution, add DMSO to the cells at 50 μL/well and shake the reaction for 10 minutes to dissolve the formed formazan

(formazan) crystallization. Finally, the absorbance of the cell mixture at 570 nm (OD 570 ) was measured using an ELISA disk reader (BioTek). The cell survival rate was calculated according to the following formula: cell survival rate=(OD 570 value of each group/OD 570 value of blank control group)×100%.

Statistical Analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using Excel software, and statistically significant differences among the data were judged by student's t-test.

Example 1 Preparation of mango fruit extract

The mango ( Mangifera indica ) mentioned in this article refers to the mango variety produced in Taiwan, but not limited thereto. Generally speaking, the growth and development of mango fruit is divided into four stages, which are (1) young fruit stage: after the mango flower fades, the fruit begins to grow slowly and turns green; Starch gradually accumulates; (3) Maturity period: the fruit enters the maturity period after the endocarp hardens. At this time, the appearance of the fruit does not change much, but the weight of the fruit continues to increase, and some physical and chemical changes are still in progress. The hardness decreases, the sugar content increases, and the peel turns yellow, making the fruit close to the ripe and edible stage; (4) aging period: the fruit begins to age after it is fully ripe. The mango fruit described herein refers to the mango fruit in the young fruit stage or the rapid growth stage and the pericarp has not turned yellow.

To obtain a mango fruit extract, unripe mango fruit with a length of about 3 to 7 cm is ground with a homogenizer. Thereafter, the mango fruit homogeneous substance is extracted with water, alcohols, or an alcohol-water mixture as a solvent, and the solvent can be added with a mixed acid of 0.1% to 5% organic acid (such as acetic acid and citric acid) and hydrochloric acid, Wherein, the weight ratio of the solvent to the homogeneous mango fruit is 20:1 to 1:1. The extraction temperature is between 55°C and 100°C, preferably between 55°C and 85°C. The mango fruit extracts described in Examples 2-5 below are all extracted with an aqueous solution containing 0.1 to 0.5% acetic acid, citric acid and hydrochloric acid, and the extraction time is 0.5 to 3 hours.

After the mango fruit extract obtained through the above extraction steps is cooled to room temperature, it can be further centrifuged at room temperature at a speed of 3000 to 5000 rpm for 5 to 10 minutes to obtain a supernatant, and the supernatant can be used in 400 mesh (mesh ) through a strainer to remove residual solids. The filtered mango fruit extract can be further concentrated under reduced pressure at 45° C. to 70° C. to obtain a concentrated product. In order to obtain a solid mango fruit extract, the above-mentioned concentrated mango fruit extract can be dried by freeze-drying, spray drying, etc. to remove the solvent, thereby obtaining a powdered mango fruit extract.

Example 2 Mango Fruit Extract Inhibits Melanin Formation

In order to examine the effect of the mango fruit extract on melanin production, the melanin content of the melanoma cell line B16F10 treated with the mango fruit extract described in Example 1 was measured by a melanin production test. Briefly, B16F10 cells were seeded in 6-well culture plates at 1.5×10 ⁵ cells/well, and each well contained 3 mL of DMEM cell culture medium. After culturing the cells at 37°C for 24 hours, the cell culture medium was removed, and 3 mL of DMEM cell culture medium containing 0.25 mg/mL mango fruit extract (experimental group) or 0.25 mg/mL kojic acid (positive control group) was added to each Pore cells. Another blank control group was set up, in which the cells were only treated with 3 mL of DMEM cell culture medium. After culturing at 37°C for 48 hours, the aforementioned three groups of cells were collected to measure the melanin content (triple experiment).

Figure 1 shows the relative content of melanin in the aforementioned three groups of melanoma cells. According to the figure, compared with the blank control group, the application of mango fruit extract significantly reduced the relative content of black by about 18%, and the inhibitory effect of this melanin formation was comparable to the inhibitory effect of the same concentration of kojic acid (a known whitening bioactive substance) . This result shows that the mango fruit extract has melanin formation inhibitory effect no less than that of conventional whitening bioactive substances.

Example 3 Mango fruit extract prevents and reduces the death of dermal fibroblasts induced by ultraviolet A irradiation

In order to test the effect of mango fruit extract on protecting the skin against ultraviolet radiation, human dermal fibroblasts CCD-966SK were pre-treated with the mango fruit extract described in Example 1 and then irradiated with ultraviolet A (UVA protection) by cell survival assay (MTT analysis). test), or the cell survival rate of the mango fruit extract treatment (UVA damage repair test) after being irradiated with ultraviolet A first.

3.1 UVA protection test

CCD-966SK cells were seeded in 96-well culture plates at 5×10 ³ cells/well, and each well contained 200 μL of MEM cell culture medium. After culturing the cells at 37°C for 24 hours, remove the cell culture medium, add 200 μL of MEM cell culture medium without or containing 0.5 mg/mL mango fruit extract to the cells in each well, and then culture the cells at 37°C for 24 hours, as follows The aforementioned cells were treated in a row: (a) cells without pretreatment of mango fruit extract were cultured for 1 hour without ultraviolet irradiation (blank control group); (b) cells without pretreatment of mango fruit extract were placed under ultraviolet irradiation 1 hour UVA irradiation at 15J/ ^cm2 in a box (Vilber) (UVA group), which causes half of the cells to die; or (c) 15J/ ^cm2 UVA irradiation for cells pretreated with mango fruit extract 1 hour (mango fruit extract + UVA group). Thereafter, MTT analysis was performed on the cells of each group to calculate the cell viability.

Figure 2 shows the cell viability of dermal fibroblasts after different treatments. According to Figure 2, compared with the blank control group, the cell survival rate of the UVA group was about 61.8%, indicating that ultraviolet A irradiation would cause a large number of skin fibroblasts to die. Compared with the UVA group, the pretreatment of mango fruit extract increased the survival rate of cells under UV irradiation to about 84.1%. This result shows that the application of mango fruit extract can prevent damage or death of skin cells caused by ultraviolet rays.

3.2 UVA damage repair test

CCD-966SK cells were seeded in 96-well culture plates at 5×10 ³ cells/well, and each well contained 200 μL of MEM cell culture medium. After culturing the cells at 37°C for 24 hours, the cells in each well were treated and the cell culture medium was replaced in the following manner: (a) 200 μL of MEM cell culture medium without mango fruit extract was applied after culturing for 1 hour without mango fruit extract (blank control group) ); (b) irradiated with 15J/cm ² ultraviolet A for 1 hour and then applied 200 μL MEM cell culture medium without mango fruit extract (UVA group); or (c) irradiated with 15J/cm ² ultraviolet A for 1 hour and then applied With 200 μL of MEM cell culture medium containing 0.5 mg/mL mango fruit extract (UVA+mango fruit extract group). Cells in each group were cultured at 37°C for 24 hours, and MTT analysis was performed to calculate the cell viability.

Figure 3 shows cell viability of dermal fibroblasts after different treatments. According to Figure 3, compared with the blank control group, the cell survival rate of the UVA group was about 42.9%, but after the cells were irradiated with mango fruit extract, the survival rate rose to about 78.4%. This result shows that the application of mango fruit extract can reduce or repair the damage of skin cells caused by ultraviolet rays.

Example 4 Mango Fruit Extract Inhibits Collagen Glycation

In order to test the anti-glycation activity of the mango fruit extract, the anti-glycation assay was used to measure the inhibitory effect of different concentrations of the mango fruit extract described in Example 1 on the glycation reaction of porcine collagen. Briefly, 60mg/mL collagen solution (containing 0.06% sodium azide) and 1.5M fructose solution were first prepared using 200mM phosphate buffer solution (pH 7.4). For collagen glycosylation, mix 0.2 mL of collagen solution and 0.2 mL of fructose solution with 0.2 mL of 140, 70, 14, 1.4, or 0.14 mg/mL mango fruit extract samples (diluted with deionized water) Or deionized water (blank control group) was uniformly mixed, reacted at 50° C. for 24 hours, and then added aminoguanidine (AG, purchased from Sigma) to stop the saccharification reaction. Use a spectrofluorometer (FLx 800, BioTek) to measure the fluorescence intensity (excitation wavelength 360nm, detection wavelength 460nm) of the aforementioned reaction solution (0.1mL) at 0 hour and 24 hours, and calculate the end product of collagen glycation according to the following formula (AGEs) generation rate: AGEs generation rate=[(sample fluorescence intensity _{24 hours} -sample fluorescence intensity _{0 hours} )/(blank control group fluorescence intensity _{24 hours} -blank control group fluorescence intensity _{0 hours} )]×100% .

Fig. 4 shows the effect of mango fruit extract on the production of collagen end-glycation products. According to the figure, compared with the blank control group, the treatments of 140, 70, 14, 1.4, and 0.14mg/mL mango fruit extract significantly reduced the production of end-saccharification products to about 3.6%, 9.6%, 39.7%, and 59.2% respectively , And 62.9%, showing that mango fruit extract can inhibit the glycation reaction of protein in the body, thereby delaying the aging of individuals.

Example 5 Preparation and identification of compounds of formula (I) and formula (II)

In order to obtain the anti-glycation active ingredients in the mango fruit extract, first, 1 L of mango fruit extract was prepared according to the method described in Example 1, and the extract was extracted three times by liquid phase partitioning of ethyl acetate and water in equal proportions. The resulting ethyl acetate layer extracts were combined, then concentrated and dried under reduced pressure to obtain about 4.7 g of the ethyl acetate layer extract.

According to the bioassay guided fractionation method, the ethyl acetate layer extract (about 4.7g) was subjected to column chromatography using a Diaion HP-20 column and an eluent mixed with water and methanol with a gradient of decreasing polarity , separated into three divided layers (marked as F1 to F3 respectively). Thereafter, use Sephadex LH-20 column and methanol eluent to carry out column chromatography on F2 or F3 divided layers, and the resulting effluent is separated by thin-layer chromatography to obtain 3 divided layers (marked respectively are F2-1 to F2-3 and F3-1 to F3-3). F3-2 was divided into layers and further performed high-performance liquid chromatography using a mixture of water and methanol at a volume ratio of 1:1 as the mobile phase, and about 13.0 mg of compound 1 could be isolated. In addition, F2-1 was divided into layers and further subjected to high-performance liquid chromatography using a mixture of water and methanol at a volume ratio of 2:1 as the mobile phase, and about 23.0 mg of compound 2 could be separated.

The chemical structures of compound 1 and compound 2 were determined by mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). Compound 1 is a light brown oil. A pseudo-molecular ion peak m/z 907 [MH] ^- was observed in the mass spectrum shown in Figure 5, so the molecular weight of Compound 1 is speculated to be 908Da. According to the proton NMR spectrum shown in Figure 6A, compound 1 has a main structure consisting of one sugar group and five aromatic rings. According to the carbon NMR spectrum shown in Figure 6B, Compound 1 has a total of 41 carbon absorption signals, including 5 carbonyl absorption signals, 5 benzene ring absorption signals, and 1 sugar group absorption signal. In addition, according to the two-dimensional nuclear magnetic resonance spectra (COSY, HSQC, HMBC) shown in Figure 2C to 2E, the connection mode and substituent position of the single sugar group and five aromatic rings in compound 1 can be deduced, so it is confirmed that compound 1 is a Hydrolyzed tannin has a structure as shown in formula (I). Compound 2 was confirmed to be a hydrolyzed tannin according to its mass spectrum and nuclear magnetic resonance spectrum, which has the structure shown in formula (II).

Example 6 Anti-glycation activity of compounds of formula (I) and formula (II)

In order to test the anti-glycation activity of the compounds of formula (I) and formula (II), the anti-glycation assay as described in Example 4 was used to determine the inhibitory effect of these compounds on porcine collagen glycation reaction. In short, the mixture of 0.2mL collagen solution and 0.2mL fructose solution was uniformly mixed with 0.2mL 100μg/mL aqueous solution sample of the compound of formula (I) or formula (II) or deionized water (blank control group), and mixed at 50 °C for 24 hours, and measure the fluorescence intensity of the aforementioned reaction solution (0.1 mL) at 0 hour and 24 hours to calculate the production rate of collagen glycation end products.

Fig. 7 shows the effects of the compounds of formula (I) and formula (II) on the production of collagen glycation end products. According to the figure, compared with the blank control group, the treatment of the compounds of formula (I) and formula (II) significantly reduced the production of glycation end products to about 60.2% and 73.7%, respectively, showing that the two compounds have significant anti-glycation activity.

To sum up, the present invention discloses that the mango fruit extract not only has ultraviolet protection and whitening effects on skin cells, but also can inhibit protein glycosylation and prevent protein from losing its normal structure and function due to glycosylation. In addition, the present invention also discloses a compound of formula (I) and formula (II) with significant anti-glycation activity. In view of the fact that ultraviolet radiation, excessive melanin precipitation, and saccharification reaction can all lead to the appearance of skin aging, especially saccharification reaction is one of the reasons for body aging, and the mango fruit extract and bioactive substances disclosed in the present invention can be used to prepare skin based on their characteristics. Anti-ultraviolet, whitening and anti-glycation composition, therefore, the present invention provides a new countermeasure to delay the aging of the skin and even the individual. The aforementioned anti-ultraviolet, whitening and anti-glycation compositions can be powders, granules, solutions, colloids or pastes, and can be made into medicines, foods, drinks, or nutritional supplements, and administered by oral administration, skin application, or other methods a body.

Claims (3)

A mango fruit extract is used to prepare a skin anti-ultraviolet, whitening and collagen saccharification inhibiting composition, wherein the mango fruit extract is obtained by extracting a mango ( Mangifera indica ) fruit with a solvent, wherein the mango ( The fruit of Mangifera indica ) refers to mango ( Mangifera indica ) fruit in young fruit stage or rapid growth stage, wherein the skin anti-ultraviolet includes preventing and repairing the damage of skin fibroblasts caused by ultraviolet A, wherein the solvent and the mango The weight ratio of the fruit is 20:1 to 1:1, the solvent is an aqueous solution containing 0.1 to 0.5% acetic acid, citric acid and hydrochloric acid, the extraction temperature of the mango fruit extract is between 55°C and 85°C, the mango The extraction time of the fruit extract is 0.5 to 3 hours. A mango fruit extract is used to prepare a skin anti-ultraviolet, whitening and collagen saccharification inhibiting composition, wherein the mango fruit extract is obtained by extracting a mango ( Mangifera indica ) fruit with a solvent, wherein the mango ( The fruit of Mangifera indica ) refers to the mango ( Mangifera indica ) fruit in the young fruit stage or the rapid growth stage, wherein the weight ratio of the solvent to the mango fruit is 20:1 to 1:1, and the solvent contains 0.1 to 0.5 % acetic acid, citric acid and hydrochloric acid aqueous solution, the extraction temperature of the mango fruit extract is between 55°C and 85°C, the extraction time of the mango fruit extract is 0.5 to 3 hours, and the mango fruit extract contains the formula The compound shown in (I),

or

The use as described in claim 1 or 2, wherein the concentration of the mango ( Mangifera indica ) fruit extract is at least 0.25 mg/mL.

Images