TWI568451B - Skin care products - Google Patents

Description

Skin care products

The present invention relates to a method for improving, conditioning and/or repairing skin Active ingredients and their use, especially regarding the use of the active ingredients in inhibiting the activity of matrix metalloproteinases (MMPs).

Collagen is the main ingredient that maintains skin and muscle elasticity. Currently The discovered animal collagen can be roughly divided into twenty-one types, which have different collagens depending on different tissues. Among them, type I collagen is the most abundant and is the most widely used collagen. In skin tissue, type I collagen accounts for about 80%, while type III collagen accounts for about 20%. The fibroblasts in the dermis mainly produce type I collagen and type III collagen for skin use.

The structure of the skin is from top to bottom, followed by the epidermis, Dermis and hypodermis. As people age, the aging of skin aging will gradually appear, such as skin relaxation, wrinkle formation and skin tone. The causes of skin aging can be divided into internal factors and external factors. Endogenous aging refers to the natural aging process of the body, such as aging due to increased age, decreased hormones, and reduced immunity; external aging refers to external factors such as Sun exposure, pollution, free radical damage and aging caused by smoking.

The aging factors of aging and the aging factors of exogenous factors promote the phosphorylation of the mitogen-activated protein kinase pathway (MAPK pathway) and increase the content of matrix metalloproteinases (MMPs) in the dermis. Matrix metalloproteinases break down collagen and elastin, reducing the amount of collagen and elastin in the skin. If the skin lacks the support of collagen and elastin, its appearance will become slack and cause excessive hyperplasia of the stratum corneum, which will make the skin sink and wrinkle. Among them, the mechanism of action on skin aging is generally considered to be the most relevant to MMP-1, so the recent suppression of skin aging, the first inhibition of MMP-1, can be found, for example, in the literature of Moon et al. (Fucoidan inhibits UVB- Induced MMP-1 expression in human skin fibroblasts. Biol . Pharm.Bull. 2008;31(2):284-289),Dong et al. (UV-induced DNA damage initiates release of MMP-1 in human skin. Exp . Dermatol. 2008; 17(12): 1037-1044), US2010197759A1, US2012065159A1, and US2014045777A1.

Past studies have found that components that inhibit matrix metalloproteinases can be extracted from plants. For example, ziyuglycoside-I obtained from the roots of Sanguisorba officinalis inhibits the expression of matrix metalloproteinase-1 (MMP-1); by Selaginella tamariscina The obtained sumaflavone and amentoflavone can also inhibit the expression of matrix metalloproteinase-1 (MMP-1). However, there is still a great demand for ingredients which can inhibit the activity of matrix metalloproteinases (MMPs) and have a better anti-aging effect.

The inventors of the present invention found that the formula (I) and formula (II) of the present invention The compound has excellent efficacy in inhibiting the activity of matrix metalloproteinases (MMPs), and particularly can effectively inhibit the activity of MMP-1/-2/-9, effectively slowing/eliminating the decomposition and/or denaturation of collagen and elastin, and thus can be used for Improves, conditioned, and/or repairs the skin, for example, to combat skin aging, particularly to prevent skin wrinkles, reduce skin wrinkles, firm skin, and/or increase skin elasticity.

It is an object of the present invention to provide an use of an active ingredient for the manufacture of a skin care product, wherein the active ingredient is selected from the group consisting of a compound of formula (I), a compound of formula (II), a compound of formula (I) or A cosmetically acceptable salt or ester, a pharmaceutically or cosmetically acceptable salt or ester of a compound of formula (II), and combinations of the foregoing, Wherein R1, R2, R3, and R4 are each independently H, -OH, or C1-6 alkoxy; and n is an integer from 1 to 20. Preferably, in formula (I) and formula (II), R1, R2, R3, and R4 are each independently H, -OH, or C1-3 alkoxy, and n is an integer from 4 to 16; More preferably, R1, R2, R3, and R4 are each independently H, -OH, or methoxy, and n is an integer from 8 to 14.

The detailed technical and some specific embodiments of the present invention will be described in the following, and the technical contents of the present invention will be apparent to those skilled in the art.

1 is an HPLC analysis chart of the compound (1) of the present invention; FIG. 2 is an HPLC analysis chart of the compound (2) of the present invention; and FIG. 3 is an HPLC analysis chart of the compound (3) of the present invention; HPLC analysis pattern of the inventive compound (4); Figure 5 is an HPLC analysis pattern of the compound (5) of the present invention; and Figure 6 is an HPLC analysis pattern of the compound (6) of the present invention.

The invention will be described in detail below with reference to the specific embodiments of the present invention. The present invention may be practiced in various different forms without departing from the spirit and scope of the invention. The presenter. In addition, the terms "a", "an" and "the" and "the" It refers to the amount of a compound that is effective at least partially to improve the condition of a suspected individual when administered to an individual; the so-called "individual" is a human.

As mentioned above, matrix metalloproteinases (MMPs) in the skin cause The function of collagen and elastin is destroyed. Therefore, if the activity of matrix metalloproteinase in the skin can be inhibited, the decomposition and/or denaturation of collagen and elastin can be effectively alleviated/exempted.

The inventors have found that the compounds of the following formula (I) and formula (II) have the effect of inhibiting the activity of matrix metalloproteinases (MMPs) in the skin:

Wherein R1, R2, R3, and R4 are each independently H, -OH, or C1-6 alkoxy, and n is an integer of 1 to 20. Preferably, the above compounds inhibit the activity of at least one of the matrix metalloproteinases: MMP-1, MMP-2 and MMP-9.

Accordingly, the present invention relates to the use of a compound of the following formula (I) and formula (II), a pharmaceutically or cosmetically acceptable salt or ester thereof, and a combination of the foregoing for the manufacture of a skin care product: Wherein R1, R2, R3, and R4 are each independently H, -OH, or C1-6 alkoxy, and n is an integer of 1 to 20.

In the compounds of formula (I) and formula (II) used in the present invention, preferably, R1, R2, R3, and R4 are each independently H, -OH, or C1-3 alkoxy, and n is An integer from 4 to 16; more preferably, R1, R2, R3, and R4 are each independently H, -OH, or methoxy, and n is an integer from 8 to 14.

Specific aspects of the compounds of formula (I) and formula (II) used in the present invention include, but are not limited to: (1) 1,6-dihydroxy-3,5,7,8-tetramethoxy Ketone (1,6-dihydroxy-3,5,7,8-tetramethoxyxanthone); (2) 1,5-dihydroxy-3-methoxy Ketone (1,5-dihydroxy-3-methoxyxanthone); (3) 1,7-dihydroxy-3,8-dimethoxy Ketone (1,7-dihydroxy-3,8-dimethoxyxanthone); (4) 1,7-dihydroxy-3-methoxy Ketone (1,7-dihydroxy-3-methoxyxanthone); (5) 1-hydroxy-3,5,6,7,8-pentamethoxy Ketone (1-hydroxy-3,5,6,7,8-pentamethoxyxanthone); and (6) 15,18,21-tetracosatrienoic acid (15Z, 18Z, 21Z-tetracosatrienoic acid).

The compounds of the formula (I) and the formula (II) used in the present invention can be obtained by a chemical synthesis method or can be isolated and purified from plants. Among them, examples of plants which can be used to obtain the compounds of the formula (I) and formula (II) of the present invention include Centaurium linarifolium , Swertia punicea , Swertia macrosperma , and Swertia macrosperma . , Tripterospermum chinense (double butterfly), Gentianopsis paludosa (wet flat bud) , Swertia nervosa (G. Don ) Wall. ( Jumbo sinensis ) , Juglans mandshurica Maxim (walnut) , Swertia punicea Hemsl (purple red sauerkraut ) (variant)) , Swertia bifolia ( Gentiana sylvestris ) , Gentiana kochiana , Halenia elliptica , Swertia przewalskii , Swertia davida Franch Tooth) , Swertia thomsonii , Gentianopsis barbata var.stennocalyx HWLi ex TNHo , Gentiana ciliata L. , Anthocleista vogelii ) , Gentiana bauarica , Gentiana acaulis , Calophyllum inophyllum L. , Calophyllum dryobalanoides , Poly Gala tenuifolia , Swertia petiolata Royle , Calophyllum brasiliense , Mammea siamensis , Polygala telephioides , Garcinia linii ), Eustoma grandiflorum (Raf.) Shinners in Texas , small centaury ( Centa genus erythraea ), and Eustoma grandiflorum (Eustoma), but not limited to this. In some embodiments of the invention, the compounds of formula (I) and formula (II) are obtained from an enzyme extract and/or an aqueous extract of Eustoma grandiflorum ( Eustoma ).

The skin care products of the present invention are useful for inhibiting the activity of matrix metalloproteinases (MMPs). Matrix metalloproteinases can be mainly classified into collagenase, stromelysin, gelatinase, matrilysin, and transmembrane type-MMP. Common matrix metalloproteinases include MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12 MMP-12), matrix metalloproteinase-13 (MMP-13), and matrix metalloproteinase-14 (MMP-14). In some embodiments of the invention, the skin care product is for inhibiting the activity of at least one of: matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase -9 (MMP-9).

It is known that by inhibiting the activity of matrix metalloproteinases (MMPs) in the skin, decomposition and/or denaturation of collagen and elastin can be effectively alleviated/exempted. Accordingly, the skin care products of the present invention can be used to improve, condition and/or repair the skin, for example, for combating skin aging, particularly for preventing skin wrinkles, reducing skin wrinkles, firming the skin, and/or increasing skin elasticity.

The skin care product of the present invention may be in any convenient form and is not particularly limited. For example, but not limited thereto, the skin care product may be in the form of a lotion for external use, a cream, a gel (such as a hydrogel), a paste (such as a dispersion cream, an ointment), a spray, or a solution. (eg lotion, suspension), etc. Alternatively, the skin care product of the present invention can be prepared in the form of a food product that can be swallowed or consumed, such as a healthy food. Products, beauty drinks, etc. Further, the skin care product of the present invention can also be provided in the form of a medicament for oral or subcutaneous injection.

For example, in the form of a medicament suitable for oral administration, the medicament may contain Any pharmaceutically acceptable carrier which does not adversely affect the desired skin care efficacy of the medicament, such as a solvent (water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof), Oily solvents, diluents, stabilizers, absorption delaying agents, disintegrating agents, emulsifiers, antioxidants, binders, binders, dispersants, suspending agents, lubricants, hygroscopic agents, solid carriers (eg starch, soap) Soil (bentonite) and so on. The agent may be provided in a form suitable for oral administration by any convenient method, for example, a tablet (for example, a sugar-coated tablet), a pill, a capsule, a granule, a powder, a flow extract, a solution, a syrup, a suspension. , emulsions, and tinctures.

As for the injection type or the drip type suitable for subcutaneous administration, the medicine can be used The agent contains one or more components such as an isotonic solution, a salt buffer (such as a phosphate buffer or a citrate buffer), a solubilizer, an emulsifier, a 5% sugar solution, and other carriers, and a dry powder. The agent is provided in a dosage form such as an injection, a suspension injection, or a dry powder suspension injection. Alternatively, the medicament is prepared as a pre-injection solid, the pre-injection solid is provided in a dosage form, or emulsifiable, which is soluble in the other solution or suspension, and is administered prior to administration to the individual in need thereof. The solid is dissolved in other solutions or suspensions or emulsified to provide the desired injection.

The formula (I) and/or formula (II) of the present invention may be used as needed. The skin care product prepared by the compound additionally contains a suitable amount of an additive, for example, a flavoring agent, a toner, a coloring agent, etc., which can improve the mouthfeel and visual sense of the skin care product, and can improve the stability of the skin care product. Sexual and storage buffer Agents, tackifiers, stabilizers, preservatives, preservatives, antibacterial agents, antifungals, etc. In addition, the skin care product may optionally contain one or more other active ingredients, or may be combined with a drug containing the one or more other active ingredients to further enhance the efficacy of the skin care product or increase the flexibility and formulation of the formulation. In principle, as long as the type and amount of other ingredients or additives added do not adversely affect the desired efficacy of the skin care product of the present invention. For example, when preparing a skin care product that reduces skin wrinkles, any suitable and appropriate amount of emulsifier, flavor, and/or other active ingredients that reduce wrinkles, such as collagen, elastin, and the like, may be added.

The invention is further illustrated by the following examples. The embodiments are provided by way of illustration only and are not intended to limit the scope of the invention. The scope of the invention is shown in the appended claims.

[Examples] [Experimental Materials]

The materials, reagents and instruments used in the examples are as follows:

(1) Arena strain Eustoma plant material: provided by Taiwan Sugar Company's Jingnong Business Department; Abbe strain, Robella strain and Voyage strain eustoma plant: purchased from Xiaoxian farmers in Chiayi County, Taiwan.

(2) Methanol for HPLC: produced by Merck, HPLC grade or equivalent.

(3) Ethanol: 95%, provided by Taiwan Sugar Company Biotechnology Division.

(4) Trifluoroacetic acid (TFA): 99%, Alfa Aesar.

(5) Deionized water: Mill-Q (MQ water).

(6) dimethyl sulfoxide (DMSO): ACS grade, 99.9%, JTBaker.

(7) Ethyl acetate (EtOAc): produced by Merck, reagent grade or equivalent.

(8) n-Hexane: produced by Merck, at the drug level or equivalent.

(9) Acetic acid: produced by Merck, the reagent grade or the same grade.

(10) SPE Diaion Hp20: Taiyang Chemical Company.

(11) Quantitative bottles: 1 liter, 500 ml and 5 ml.

(12) 3-L concentration equipment: Tokyo Physical and Chemical Equipment Co., Ltd.

(13) Centrifuge: Hitachi Industrial Co., Ltd.

(14) Blender: Tokyo Physical and Chemical Equipment Co., Ltd.

(15) Ultrasonic oscillator: 150W, Delta D150H.

(16) Bruker AV-500 high-resolution nuclear magnetic resonance spectrometer: Brooke Life Science Co., Ltd., Taiwan.

(17) Thermo Nicolet IS-10 Infrared Spectrometer: Nichols Instruments, USA.

(18) Shimadzu QP high-resolution mass spectrometer: Taiwan Shimadzu Industrial Equipment Co., Ltd.

(19) MEM-α: Gibco (cat. 12571-063), for culturing WS1 cells.

(20) DMEM: Gibco (cat. 11965-084), for culturing NIH-3T3 cells.

(21) MTS (4-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt ;MTS]: Promega Corporation (cat.G111A).

(22) 96-well plate: BD Falcon (cat. 353072).

(23) 24-well plate: BD Falcon (cat. 353047).

(24) Gelatin: USB company (cat.16045).

(25) MMP-1/-2//-9 standard: R&D Company (cat. WBC024/cat.WBC025/cat.WBC031).

Operating conditions for high performance liquid chromatography (HPLC) in the examples:

(1) Analytical instrument: American Waters High Performance Liquid Chromatography System, (Waters 2690)-PDA (Waters 996).

(2) chromatographic column: Inertsil ODS3V‧C ₁₈ chromatography column (250 × 4.6 mm, 5 [mu] m) available from GL Sciences.

(3) Chromatographic column temperature: room temperature.

(4) UV detector detection wavelength: 205 nm.

(5) Injection volume: 15 microliters; all samples were filtered through a 0.45 micron nylon membrane before injection.

(6) Analysis time: 35 minutes.

(7) Mobile phase A: 0.1% aqueous solution of trifluoroacetic acid (TFA).

(8) Mobile phase B: 100% methanol (MeOH).

(9) Gradient analysis conditions are shown in Table 1:

(10) Preparation of standard solution and standard curve: Take 5.1 mg of oleanolic acid in a 5-ml quantitative bottle with a precision balance, and then take methanol to a 5-ml quantitative bottle with a drop tube. Prepare a standard solution of 1.02 mg/ml. Subsequent 2-fold serial dilutions with methanol were prepared at seven concentrations of 0.51, 0.255, 0.128, 0.064, 0.032, 0.016, and 0.008 mg/ml. The above-mentioned different concentration standard solution was analyzed by HPLC, and the concentration of the standard product was taken as the abscissa value, and the peak area value was the ordinate value for linear regression analysis, and the XY line passing through the origin was obtained, and the linear regression determination coefficient (R ² ) was obtained. It is used from 0.999 to 1.000.

(11) Blank solution: 30% mobile phase A and 70% mobile phase B.

(12) Injection procedure: Five injections are performed first, including the secondary standard solution, the secondary sample solution and at least one blank solution to confirm the presence of non-interfering substances, and then the sample is analyzed. Once the sample has been injected six times, the standard must be analyzed again to confirm the stability of the HPLC instrument. Once all samples have been analyzed, the standard is analyzed again.

[sample preparation]

(1) different strains of eustoma extract raw materials

Take different types of eustoma plants (according to the appearance of the roots), remove the sediment and wash, then dry in an oven at 50 ° C to constant weight, grind it through a grinder to pass through 30 mesh, Used as a raw material for extracting eustoma.

(2) Extracting raw materials from different parts of eustoma

Take the orange stems of the Arena strain, which are divided into three parts: flower, stem and root according to the appearance, remove the sediment and wash separately, then dry in an oven at 50 °C to constant weight, and grind to 30 mesh through a grinder. As a raw material for extraction of eustoma.

[Preparation Example]

A. Preparation of ethanol extracts from different strains of Eustoma

The roots of different stems of the platycodon roots were extracted by the following procedure to obtain ethanol extracts of different strains of Eustoma. Approximately 50.0 g of raw material was weighed and placed in a 1 liter four-neck flat-bottomed flask. 500 ml of 95% ethanol was added to the flask, and the mixture was stirred at 200 rpm for 3 hours at room temperature, followed by centrifugation (2000 x g). The filtrate was obtained by filtration, and the filtrate was concentrated and dried to constant weight to obtain ethanol extracts of different strains of Eustoma.

The yields of the ethanol extracts of different strains of Eustoma were shown in Table 2 below, and the yield (26.9%) of the ethanol extract of the Arena strain was the best.

B. Preparation of ethanol extract samples from different parts of Eustoma

The Araceae eustoma flowers, roots and stems were extracted by the following procedure to obtain ethanol extracts from different parts. Weigh about 5.0 grams of raw material and place it in a 50 ml centrifuge tube, add 50 ml of 95% ethanol to the centrifuge tube, and extract it by ultrasonic vibration for 3 hours at room temperature, then centrifuge (2000 xg) and The filtrate was filtered, and the filtrate was concentrated and dried to constant weight to obtain a sample of ethanol extract from different parts of the eustoma.

The yields of ethanol extracts from different parts of the Arena strains of Eustoma are shown in Table 3 below, and the yield (14.9%) of the ethanol extract of the stem is the best.

C. Preparation of different strains of Eustoma water extract samples

The roots of different strains of Eustoma were extracted by the following procedure to obtain water extracts of different strains. Weigh about 25 grams of raw material and place it in a 500 ml serum bottle. Add 250 ml of pre-heated boiled deionized water to the serum bottle. Place the serum bottle in a preheated boiled water bath and stir with a glass rod. The extraction was carried out for 40 minutes, followed by centrifugation (2000 x g) and filtration to obtain a filtrate. Deionized water was added to the filtrate to a total volume of 250 ml, and concentrated to a constant weight to obtain a difference. A strain of Eustoma water extract.

The preparation yields of different strains of Eustoma water extracts are shown in Table 4 below, wherein the Abbe line water extract preparation yield (21.1%) is the best.

Example 1: MMP-1 activity inhibition test of Eustoma extract

1-1. Cell culture

The frozen cells of the human dermal layer collagen parent cell line (WS1) were placed in a water bath at 37 ° C for 1 to 2 minutes, and then inoculated into T-containing MEM-α medium containing 37 ° C back temperature. In a 75 flask, the mixture was subcultured for about 2 to 3 passages at 37 ° C in a 5% CO ₂ incubator, and the cells were allowed to grow stably (about 4 to 10 days in total) for subsequent experiments. Next, WS1 cells were seeded on a 96-well culture plate at a starting number of 1 × 10 ⁴ cells per well, and cultured in an incubator at 37 ° C, 5% CO ₂ for 24 hours. Thereafter, the ethanol extracts of different strains of Eustoma, the ethanol extracts of different parts of Eustoma, or the different extracts of Eustoma water extracts prepared by the above preparation examples are respectively taken at different concentrations (ie, 1.0, 10 ^-1 , 10 ^The aforementioned WS1 cells were treated with ^-2 , 10 ^-3 and 10 ^-4 mg/ml for 24 hours. The WS1 cells were then transferred to serum-free medium for further culture. After 24 hours, the supernatant was collected and stored at -80 °C as a sample for subsequent Bio-Rad suspension array multi-label analysis.

1-2. Cytotoxicity analysis

Take the above-mentioned cells still in the 96-well plate, and add 100 μl of a mixture containing MTS solution and complete medium (1:5 volume ratio of MTS solution to complete medium) at 37 ° C, 5% CO ₂ The culture is carried out in an incubator for 3 to 4 hours. Next, the OD _{490/655 nm} (i.e., the absorbance at a wavelength of 490 nm and 655 nm) was detected by an ELISA Reader, and the absorbance of the complete medium was used as a blank group to be corrected to analyze the cell survival rate.

1-3. Multi-target analysis of Bio-Rad suspension array

The samples provided in the above Experiment 1-1 were thawed and shaken uniformly, and the upper layer was clarified and the precipitate-free sample was taken, and the human matrix metalloproteinase analysis was carried out using a 3-plex assay kit (purchased from Bio-Rad Laboratories, Hercules, CA) (Bio- Plex Pro Human MMP Assays) to test the MMP-1 activity inhibition ability of Eustoma extract. The test procedures provided in the original operating manual are as follows: (i) take 50 μl of antibody-coupled magnetic beads and wash twice; (ii) take 50 μl of sample and step (i) The magnetic beads provided are reacted at room temperature for 60 minutes; (iii) the washing step is performed three times to remove the sample that has not bound to the antibody on the magnetic beads; (iv) the biotin-labeled antibody is added, and is placed in the chamber Warming for 30 minutes; (v) performing a washing step three times to remove unbound biotin-labeled antibody; (vi) adding the colorant streptavidin-PE and reacting at room temperature for 10 minutes; (vii) Performing a cleaning step three times to remove excess color former; and (viii) suspending the completed magnetic beads in 125 microliters of assay buffer using a multi-labeled suspension array system-bead wafer analyzer ( Bio-Plex Suspension Array System; Bio-Plex 200). Data reading and analysis were performed with Bio-Plex Manager software, and statistical analysis was performed using Mann-Whitney U Test.

Inhibition rate (%) = 100% × [(the amount of TNF-α added to the medium) - the amount of the sample test group] / (the amount of TNF-α added to the medium)

1-4. Experimental results

(A) MMP-1 inhibitory ability of ethanol extracts from different strains of Eustoma

The cytotoxicity of ethanol extracts of different strains of Eustoma chinensis and the inhibition ability against MMP-1 activity were analyzed, and the results are shown in Table 5 below, respectively. As shown in Table 5, ethanol extracts of different strains of Eustoma have the ability to inhibit MMP-1 activity.

(B) Ethanol extracts from different parts of Eustoma

The cytotoxicity of the ethanol extracts from different parts of the Arena platycodon and the inhibition ability against MMP-1 activity were analyzed, and the results are shown in Table 6 below. As shown in Table 6, the ethanol extracts from different parts of the extract of Eustoma can be inhibited. The ability of MMP-1 activity.

(C) Different strains of Eustoma water extract

The cytotoxicity of different extracts of water extracts from different strains of Eustoma was extracted by water and the inhibition ability against MMP-1 activity was analyzed. The results are shown in Table 7 below. As shown in Table 7, the different extracts of Eustoma water extract have the ability to inhibit MMP-1 activity.

Table 7. Inhibition of MMP-1 activity in different extracts of Eustoma water extract

Example 2: MMPs activity inhibitory component of ethanol extract of Eustoma

2-1. Diaion Hp20 (macroporous adsorption resin) column distinguishes sample preparation

The ethanol extract of the root stem of the Rubella strain of the above-prepared preparation example was taken, and the column was distinguished by a Diaion Hp20 column (6 cm id × 30 cm). Gradient elution with 0%, 20%, 40%, 60%, 80%, 100% ethanol (flow rate 2BV/h (1BV is 1 resin bed volume); 3BV for each gradient) Eluted samples of different ethanol concentrations were collected separately. After removing the solvent in each sample, it was dried at 50 ° C to obtain Hp20 column distinguishing samples of different ethanol concentrations.

2-2. Dialine Hp20 column to distinguish samples from MMPs activity inhibition test

(A) Cell culture

The frozen cells of the human dermal layer collagen parent cell line (WS1) were placed in a water bath at 37 ° C for 1 to 2 minutes, and then inoculated into T-containing MEM-α medium containing 37 ° C back temperature. In the 75 culture plates, they were placed in a 37 ° C, 5% CO ₂ incubator for about 2 to 3 generations of subculture, and the cells were stably grown (about 4 to 10 days in total) and then subjected to subsequent experiments. Next, WS1 cells were seeded on a 24-well culture plate at a starting cell number of 1 × 10 ⁵ cells per well, and cultured in an incubator at 37 ° C, 5% CO ₂ for about 24 hours. Thereafter, the Hp20 column samples of different ethanol concentrations prepared in the above experiment 2-1 were used to distinguish the samples at different concentrations (ie, 1.0, 10 ^-1 , 10 ^-2 , 10 ^{-3 ,} and 10 ^-4 mg / ml ) The aforementioned WS1 cells were treated for 24 hours. The WS1 cells were replaced with serum-free medium and culture was continued for 24 hours. The supernatant was collected and stored at -80 ° C as a sample for subsequent electrophoresis analysis.

(B) Cytotoxicity analysis

Take the above-mentioned cells still in the 24-well plate, and add 200 μl of a mixture containing MTS solution and complete medium (the volume ratio of MTS solution to complete medium is 1:5) in each well at 37 ° C, 5 The cultivation was carried out in an incubator of % CO ₂ for 3 to 4 hours. Next, the OD _{490/655 nm} (i.e., the absorbance at a wavelength of 490 nm and 655 nm) was detected by an ELISA Reader, and the absorbance of the complete medium was used as a blank group to be corrected to analyze the cell survival rate.

(C) Gelatin zymography

The samples provided in Experiment 2-2 (A) were thawed and shaken evenly, taking 20 μl of the upper clarified sample without precipitation and 4 μl of 6-fold SDS gel loading buffer without reducing agent. The mixture was mixed (6X SDS gel loading buffer), placed at room temperature for 10 minutes, and electrophoresed by 10% polypropylene guanamine gel (containing gelatin). The film after completion of electrophoresis was immersed in washing buffer I (washing buffer I) and shaken at room temperature for 40 minutes, then immersed in washing buffer II and shaken at room temperature. After a minute, the film was placed in Washing Buffer III and reacted at 37 ° C for 18 hours. Thereafter, the film was dyed in Coomassie Blue R-250 to make the film uniform in color, and then placed in the dyeing solution for decolorization. Finally, the film is placed under a digital image analysis system, photographed by a digital camera, and the area and intensity of the band of the MMP-2/-9 on the film are measured (after the area and strength of the strip are formed) The obtained value is the strong and weak value of the strip), and statistical analysis was performed by Mann-Whitney U Test.

(D) Experimental results

The cytotoxicity of the Hp20 column distinguishing samples of different ethanol concentrations and the inhibition ability against MMP-2/-9 activity were analyzed, and the results are shown in Table 8 below, respectively. As shown in Table 8, the 80% ethanol Hp20 column discrimination sample has the best ability to inhibit MMP-2/-9 activity.

2-3. Purification and isolation of MMPs activity inhibitory components from Hp20 column differential samples at 80% ethanol concentration

The Hp20 column distinguishing samples of 80% ethanol prepared in the above experiment 2-1 were purified by preparative thin layer chromatography at different solvent ratios, and analyzed by HPLC to obtain purified components with different Rt (residence time) values. Compound 1 (Rt: 14 minutes), Compound 2 (Rt: 19.5 minutes), Compound 3 (Rt: 20.5 minutes), Compound 4 (Rt: 18.5 minutes), Compound 5 (Rt: 23.5 minutes), and Compound 6 (Rt: 28 minutes) and other six ingredients. Finally, the above six compounds were subjected to mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy (IR spectra), and nuclear magnetic resonance spectroscopy (NMR spectrum), and the results were as follows.

Compound (1): 1,6-dihydroxy-3,5,7,8-tetramethoxy Ketone (1,6-dihydroxy-3,5,7,8-tetramethoxyxanthone)

The HPLC analysis pattern of the compound (1) is shown in Fig. 1 (detection wavelength is 254 nm), and the spectral data thereof is as follows: UV: 252, 320 nm; IR: νmax (neat): 3331 (OH), 1686 (C=O) ) cm ^-1 ; ¹ H NMR (500MHz, MeOH-d ₄ ): δ 6.20 (1H, d, 2.1Hz, H2), 6.43 (1H, d, 2.1Hz, H4), 3.873 (3H, s, OCH) ₃ ), 3.867 (3H, s, OCH ₃ ), 3.864 (3H, s, OCH ₃ ), 3.849 (3H, s, OCH ₃ ); ¹³ C-NMR (125MHz, MeOH-d ₄ ): δ 164.51 ( C1), 97.7 (C2), 166.9 (C3), 92.76 (C4), 158.33 (C4a), 150.14 (4b), 135.92 (C5), 149.85 (C6), 144.22 (C7), 150.63 (C8), 104.16 ( C8a), 103.96 (C8b), 180.43 (C9), 56.37 (OCH ₃ -C3), 62.26 (OCH ₃ -C8), 61.24 (2C, OCH ₃ -C7, C6); Mass (EI-MS): m/ z: 348 [M+].

Combine the above analysis results of HPLC, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, etc. with the literature of Jiang Yong et al. (see Polygala) The structural characteristics and spectroscopic regularity of ketone compounds, Peking University Journal (Medical Edition), 2004; 36: 94-98, the entire disclosure of which is hereby incorporated by reference, it is determined that the compound (1) is 1, 6-dihydroxy-3,5,7,8-tetramethoxy Ketone (1,6-dihydroxy-3,5,7,8-tetramethoxyxanthone).

Compound (2): 1,5-dihydroxy-3-methoxy Ketone (1,5-dihydroxy-3-methoxyxanthone)

The HPLC analysis pattern of the compound (2) is shown in Fig. 2 (detection wavelength is 254 nm), and the spectral data thereof is as follows: UV: 249, 311, 355 nm; IR: νmax (neat): 3424 (OH), 1655 (C=O) ) cm ^-1 ; ¹ H NMR (400 MHz, D6-DMSO): δ 6.38 (1H, d, 2.4 Hz, H2), δ 6.61 (1H, d, 2.4 Hz, H4), δ 7.27 (1H, dd , 7.6, 1.6 Hz, H6), δ 7.22 (1H, t, 7.6, Hz, H6), δ 7.46 (1H, d, 7.6, 1.6 Hz, H8), 3.885 (3H, s, OCH ₃ ); ¹³ C-NMR (176MHz, D6-DMSO): δ 162.09 (C1), 96.56 (C2), 165.96 (C3), 92.16 (C4), 156.77 (C4a), 144.91 (4b), 147.25 (C5), 120.42 ( C6), 123.89 (C7), 112.75 (C8), 120.42 (C8a), 102.55 (C8b), 180.23 (C9), 55.67 (OCH _{3 -} C3).

The results of the above HPLC, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy and the like were compared with the literature of Jiang Yong et al., and it was judged that the compound (2) was 1,5-dihydroxy-3-methoxy. Ketone (1,5-dihydroxy-3-methoxyxanthone).

Compound (3): 1,7-dihydroxy-3,8-dimethoxy Ketone (1,7-dihydroxy-3,8-dimethoxyxanthone)

The HPLC analysis pattern of the compound (3) is shown in Fig. 3 (detection wavelength is 254 nm), and the spectral data is as follows: UV: 238, 261, 311, 375 nm; IR: v max (neat): 3396 (OH), 1666 (C=O) ) cm ^-1 ; ¹ H NMR (500MHz, D6-DMSO): δ 6.31 (1H, d, 2.5Hz, H2), 6.51 (1H, d, 2.5Hz, H4), 7.19 (1H, d, 9Hz, H5), 7.42 (1H, d, 9Hz, H6), 3.86 (3H, s, OCH ₃ ), 3.75 (3H, s, OCH ₃ ); ¹³ C-NMR (176MHz, D6-DMSO): δ 162.18 ( C1), 96.78 (C2), 166.80 (C3), 91.53 (C4), 155.25 (C4a), 149.18 (4b), 113.13 (C5), 127.22 (C6), 145.28 (C7), 147.49 (C8), 114.91 ( C8a), 103.26 (C8b), 180.63 (C9), 56.09 (OCH 3 -C3), 60.93 (OCH 3 -C8).

The above analysis results of HPLC, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, etc. were compared with the literature of Jiang Yong et al., and it was judged that the compound (3) was 1,7-dihydroxy-3,8-dimethyl Oxyl Ketone (1,7-dihydroxy- 3,8-dimethoxyxanthone).

Compound (4): 1,7-dihydroxy-3-methoxy Ketone (1,7-dihydroxy-3-methoxyxanthone)

The HPLC analysis pattern of the compound (4) is shown in Fig. 4 (detection wavelength is 254 nm), and the spectral data thereof is as follows: UV: 237, 258, 305, 375 nm; IR: v max (neat): 3381 (OH), 1648 (C = O)cm ^-1 ; ¹ H NMR (500MHz, MeOH-d ₄ ): δ 6.31 (1H, d, 2.5Hz, H2), 6.51 (1H, d, 2.5Hz, H4), 7.39 (1H, d, 9Hz, H5), 7.26 (1H, dd, 3Hz, 9Hz, H6), 7.49 (1H, d, 3Hz, H8), 3.90 (3H, s, OCH ₃ ); ¹³ C-NMR (176MHz, MeOH-d ₄ ): δ 164.55 (C1), 98.00 (C2), 168.49 (C3), 93.42 (C4), 159.58 (C4a), 155.87 (4b), 120.17 (C5), 125.8 (C6), 151.36 (C7), 109.53 (C8), 122.59 (C8a), 104.55 (C8b), 180.49 (C9), 56.09 (OCH _{3 -} C3); Mass (E I-MS): m/z: 258 [M+].

The results of analysis by HPLC, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy and the like were compared with the literature of Jiang Yong et al., and it was judged that the compound (4) was 1,7-dihydroxy-3-methoxy. Ketone (1,7-dihydroxy-3-methoxyxanthone).

Compound (5): 1-hydroxy-3,5,6,7,8-pentamethoxy Ketone (1-hydroxy-3,5,6,7,8-pentamethoxyxanthone)

The HPLC analysis pattern of the compound (5) is shown in Fig. 5 (detection wavelength is 254 nm), and the spectral data thereof is as follows: UV: 256, 315 nm; IR: v max (neat): 3356 (OH), 1666 (C = O) cm ^-1 ; ¹ H NMR (700MHz, MeOH-d ₄ ): δ 6.32 (1H, d, 2.1 Hz, H2), δ 6.54 (1H, d, 2.1 Hz, H4), 3.094 (3H, s, OCH ₃ ), 3.909 (3H, s, OCH ₃ ), 3.927 (3H, s, OCH ₃ ), 3.977 (3H, s, OCH ₃ ), 4.109 (3H, s, OCH ₃ ); ¹³ C-NMR (125MHz, MeOH-d ₄ ): δ 164.81 (C1), 98.54 (C2), 168.33 (C3), 93.35 (C4), 158.52 (C4a), 149.08 (4b), 139.07 (C5), 150.58 (C6), 144.84 (C7), 154.60 (C8), 104.81 (C8a), 112.32 (C8b), 180.43 (C9), 56.68 (OCH ₃ -C3), 62.32 (OCH ₃ -C7), 62.36 (OCH ₃ -C6), 62.64 (OCH _{3 -} C5), 62.83 (OCH _{3 -} C8).

The results of analysis such as HPLC, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy were compared with those of Jiang Yong et al., and it was judged that the compound (4) was 1-hydroxy-3, 5, 6, 7, 8 -pentamethoxy Ketone (1-hydroxy-3,5,6,7,8-pentamethoxyxanthone).

Compound (6): 15,18,21-tetracosatrienoic acid (15Z, 18Z, 21Z-tetracosatrienoic acid)

The HPLC analysis pattern of the compound (6) is shown in Fig. 6 (detection wavelength is 205 nm), and the spectral data thereof is as follows: UV: 205 nm; IR: v max (neat): 34354 (OH), 1686 (C=O) ) cm ^-1 ; ¹ H NMR (500MHz, MeOH-d ₄ ): δ 2.16 (1H, t, 6Hz, H2), 1.60 (2H, m, H3), 1.33-1.35 (18H, m, H4-H12) , 1.37 (2H, m, H13), 2.03 (4H, m, H14, H23), 5.32-5.38 (6H, m, H15, H16, H18, H19, H21, H22), 2.80 (4H, m, H17, H20), 0.89 (3H, t, 7.7 Hz, H24); ¹³ C-NMR (125MHz, MeOH-d ₄ ): δ 188.22 (COOH), 39.1 (C2), 27.82 (C3), 30.58-30.82 (8C , C4-C12), 30.93 (C13), 28.3 (2C, C14, C23), 131.31 (C15), 128.92 (C16), 26.6 (2C, C17, C20), 29.8 (C18), 130.95 (C19), 128.4 (C21), 132.87 (C22), 14.7 (C24); Mass (E I-MS): m/z: 362 [M+].

The above HPLC, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy and other analytical results, and the literature of the 冀克俭 et al (see NMR characterization of fish oil polyene fatty acid ethyl ester, chemical analysis, 2001; 10 (3): 3 -4, the entire disclosure of which is hereby incorporated by reference in its entirety, the the the the the the the the the the the the the the the the the the the the the the the

Example 3: MMPs activity inhibition test of compounds (1) to (6)

3-1. Cell culture

Frozen cells of human dermal layer collagen mother cell line (WS1) and mouse fibroblast cell line (NIH-3T3) were placed in a water bath at 37 ° C for 1 to 2 minutes, and then inoculated separately. The T-75 flasks of MEM-α (WS1) and DMEM (NIH-3T3) medium at 37 ° C were placed in a 37 ° C, 5% CO ₂ incubator for 24 hours. Next, WS1 cells or NIH-3T3 cells were seeded on a 24-well culture plate at a number of cells starting at 1 × 10 ⁵ cells per well, and placed in an incubator at 37 ° C, 5% CO ₂ for overnight culture (about 24 hours). Thereafter, the compounds (1) to (6) provided in the above Experiment 2-3 were treated at different concentrations (i.e., 1.0, 10 ^-1 , 10 ^-2 , 10 ^{-3 ,} and 10 ^-4 mg / ml). The aforementioned cells lasted for 24 hours. The cells were then transferred to serum-free medium for further culture for 24 hours, and the supernatant was collected and stored at -80 ° C as a sample for subsequent electrophoresis enzyme assay and Bio-Rad suspension array multi-label analysis.

3-2. Cytotoxicity analysis

Take the above-mentioned cells still in the 24-well plate, and add 200 μl of MTS solution and complete medium (1:5 volume ratio of MTS solution to complete medium) to each well at 37 ° C, 5%. The cultivation was carried out in a CO ₂ incubator for 3 to 4 hours. Next, the OD _{490/655 nm} (i.e., the absorbance at a wavelength of 490 nm and 655 nm) was detected by an ELISA Reader, and the absorbance of the complete medium was used as a blank group to be corrected to analyze the cell survival rate. .

3-3. Gelatin zymography

The samples provided in the above experiment 3-1 were thawed and shaken evenly, and 20 μl of the supernatant-free, non-precipitated sample was taken, and 4 μl of a 6-fold SDS gel loading buffer containing no reducing agent ( 6X SDS gel loading buffer) was mixed and placed at room temperature for 10 minutes, and then electrophoresed by 10% polypropylene guanamine gel (containing gelatin). The film after completion of electrophoresis was immersed in washing buffer I (washing buffer I) and shaken at room temperature for 40 minutes, then immersed in washing buffer II and shaken at room temperature. After a minute, the film was placed in Washing Buffer III and reacted at 37 ° C for 18 hours. Thereafter, the film was dyed in Coomassie Blue R-250 to make the film uniform in color, and then placed in the dyeing solution for decolorization. Finally, the film is placed under a digital image analysis system, photographed by a digital camera, and the area and intensity of the band of the MMP-2/-9 on the film are measured (after the area and strength of the strip are formed) The obtained value is the strong and weak value of the strip), and statistical analysis was performed by Mann-Whitney U Test.

3-4. Multi-target analysis of Bio-Rad suspension array

The samples provided in the above experiment 3-1 were thawed and shaken uniformly, and the upper layer was clarified and the precipitate-free samples were taken, and the human matrix metalloproteinase analysis was carried out using a 3-plex assay kit (purchased from Bio-Rad Laboratories, Hercules, CA) (Bio- Plex Pro Human MMP Assays) to test the MMP-1 activity inhibition ability of Eustoma extract. The test procedures provided in the original operating manual are as follows: (i) take 50 μl of antibody-coupled magnetic beads and wash twice; (ii) take 50 μl of sample and step (i) The magnetic beads provided are reacted at room temperature for 60 minutes; (iii) the washing step is performed three times to remove the sample that has not bound to the antibody on the magnetic beads; (iv) the biotin-labeled antibody is added, and is placed in the chamber Warming for 30 minutes; (v) performing a washing step three times to remove unbound biotin-labeled antibody; (vi) adding the colorant streptavidin-PE and reacting at room temperature for 10 minutes; (vii) Performing a cleaning step three times to remove excess color former; and (viii) suspending the completed magnetic beads in 125 microliters of assay buffer using a multi-labeled suspension array system-bead wafer analyzer ( Bio-Plex Suspension Array System; Bio-Plex 200). Data reading and analysis were performed with Bio-Plex Manager software, and statistical analysis was performed using Mann-Whitney U Test.

Inhibition rate (%) = 100% × [(the amount of TNF-α added to the medium) - the amount of the sample test group] / (the amount of TNF-α added to the medium)

3-5. Experimental results

The cytotoxicity of the compounds (1) to (6) and the ability to inhibit the activity of MMP-1/-2/-9 were analyzed, and the results are shown in Tables 9, 10 and 11 below. As shown in Tables 9, 10 and 11, the compounds (1) to (6) have the ability to inhibit the activity of MMP-1, MMP-2, or MMP-9.

Table 9. Compounds (1) to (6) inhibit MMP-2 of NIH-3T3 cells

It can be seen from the above experimental results that the formula (I) and the formula (II) of the present invention The compound has excellent efficacy in inhibiting the activity of matrix metalloproteinases (MMPs), and particularly inhibits MMP-1/-2/-9 activity, thereby effectively reducing/eliminating the decomposition and/or denaturation of collagen and elastin.

The above embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be defined as defined in the appended claims.

Claims (7)

An use of an active ingredient for the manufacture of a skin care product, wherein the active ingredient is selected from the group consisting of the following compounds, their pharmaceutically or cosmetically acceptable salts, and combinations of the foregoing: 1,6-dihydroxy-3, 5,7,8-tetramethoxy Ketone (1,6-dihydroxy-3,5,7,8-tetramethoxyxanthone); 1,5-dihydroxy-3-methoxy Ketone (1,5-dihydroxy-3-methoxyxanthone); 1,7-dihydroxy-3,8-dimethoxy Ketone (1,7-dihydroxy-3,8-dimethoxyxanthone); 1,7-dihydroxy-3-methoxy And 1,10,21-tetracosatrienoic acid. The use of claim 1, wherein the skin care product is for inhibiting the activity of matrix metalloproteinases (MMPs). The use of claim 2, wherein the skin care product is for inhibiting the activity of at least one of: matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9). The use of claim 1 wherein the skin care product is for improving, conditioning and/or repairing the skin. The use of claim 1, wherein the skin care product is for use against skin aging. The use of claim 1, wherein the skin care product is for preventing skin wrinkles, reducing skin wrinkles, firming the skin, and/or increasing skin elasticity. The use of claim 1, wherein the skin care product is in a form selected from the group consisting of an emulsion, a cream, a gel, a cream, a spray, and a solution.