CN1451369A - Use of cyclodextrin derivs. in cosmetics - Google Patents

Abstract

An use of cyclodextrin derivative in the cosmetics for eliminating wrinkles, delaying skin senility and preventing and treating pigmentation, chloasma, etc is disclosed. Said cyclodextrin derivative is an artificial analogue enzyme with the activity of glutathion peroxidase.

Description

Application of cyclodextrin derivative in cosmetics

The invention relates to the field of cosmetics, in particular to application of cyclodextrin derivatives with Glutathione Peroxidase (GPX) activity in the field of cosmetics.

The micro-environment of the active site of the mimic enzyme on the molecular level, the factors which play a leading role in the catalytic reaction in the natural enzyme are introduced, and the design and synthesis of the organic molecule with simple structure and stable activity as the artificial mimic enzyme are one of the leading subjects in the field of natural science at present. As artificial enzyme molecules may have the same specificity and catalytic function as natural enzymes, and have the advantages of stable physicochemical properties, wide and easily-obtained raw materials, simple and convenient preparation process and the like, the artificial enzyme molecules indicate that the artificial enzyme molecules have wide application prospects in the fields of chemical engineering, enzymology, medicine and pharmacology and the like. For this reason, artificial simulation of enzymes is included in the U.S. and many European countries for the development of future studies. In recent years, many small molecule enzyme-like systems have been established, such as cyclodextrins, crown ethers, cyclophanes, cycloarenes and porphyrins.

Aging is an inevitable process in the biological life process, and human bodies are influenced by internal and external factors, and active oxygen free radicals are formed in skin cells to damage nucleic acids, proteins, polysaccharides and biological membranes in the skin cells; resulting in the change of the supporting tissue of the skin, namely the dermis, so that the water retention capacity of the epidermis of the skin is reduced, and the arrangement sequence of the collagen elastic fibers is abnormal, thereby having reduced mechanical force and elasticity. Ultraviolet rays are the main culprit for promoting skin cells to generate active oxygen free radicals.

Glutathione peroxidase is an important selenium-containing enzyme in organisms, and GPX utilizes Glutathione (GSH) to catalyze and reduce hydroperoxide, so that the glutathione peroxidase has excellent oxidation resistance. However, the enzyme has the defects of instability, limited source, high molecular weight, human body immunoreaction and the like, and greatly limits the development and application of the enzyme in the fields of medicines, chemical engineering, foods and the like. Cyclodextrin was chosen to mimic GPX, successfully yielding a series of cyclodextrin derivatives with GPX activity. They have small molecular weight and good membrane permeability, and can easily enter skin cells and utilize GSH in the cells to remove oxygen free radicals. We find that the cyclodextrin derivative with GPX activity can effectively protect cells, resist lipid peroxidation, clear free radicals in skin cells of human bodies, and play roles in reducing and eliminating skin wrinkles and fine lines, delaying senility, preventing and treating pigmentation, chloasma, butterfly spots, skin keratinization, rough skin and the like when being applied to the field of cosmetics.

The object of the present invention is therefore to provide the use of the cyclodextrin derivatives to which the invention relates as additives in cosmetics;

another object of the present invention is to provide a cosmetic containing the cyclodextrin derivative of the present invention.

The structural general formula of the cyclodextrin derivative is as follows:

wherein:

the structural formula of the parent cyclodextrin is shown as follows:

the parent cyclodextrin in the present invention may be α -cyclodextrin (n ═ 5), β -cyclodextrin (n ═ 6), or γ -cyclodextrin (n ═ 7), and the structure thereof may be used

Or

To represent;

substituent R₁May be in the 2-or 6-position, substituent R₂Or 2 or 6, provided that R is₁Can be in cyclodextrinAt the same substitution position as the glucose ring, R₂Can be at the same substitution position of different glucose rings of cyclodextrin;

R₁can be-Se, -Te, -SeCys and-TeCys, wherein Cys represents cysteine, SeCys represents selenocysteine, and TeCys represents tellurocysteine;

R₂can be-OH, -SeO₂H、-TeO₂H, wherein SeO₂H represents selenious acid, TeO₂H represents tellurite.

The structural formula of the preferred cyclodextrin derivative related to the invention is:

2-SeCD, i.e. 2-selenium bridged cyclodextrins

6-SeCD, i.e. 6-selenium bridged cyclodextrins

2-SeCysCD, i.e. 2, 2 '-N, N' -selenocysteine-selenocyclodextrin6-SeCysCD, i.e. 6, 6 '-N, N' -selenocysteine-selenocyclodextrin2-diseCD, i.e. 2A, 2B-selenious acid-2A ', 2B' -selenium-bridged cyclodextrins

6-diseCD, i.e. 6A, 6B-selenious acid-6A ', 6B' -selenium-bridged cyclodextrins

2-TeCD, i.e. 2-tellurium-bridged cyclodextrins6-TeCD, i.e. 6-tellurium-bridged cyclodextrins2-TeCysCD, i.e. 2, 2 '-N, N' -tellurocysteine-tellurium bridged cyclodextrins

6-TeCysCD, i.e. 6, 6 '-N, N' -tellurocysteine-tellurium bridged cyclodextrins2-diteCD, 2A, 2B-ditellurite-2A ', 2B' -tellurium bridged cyclodextrins6-ditec, namely 6A, 6B-ditellurite-6A ', 6B' -tellurium bridged cyclodextrin (in the structural formula, n is 5, 6, 7), wherein CD represents cyclodextrin.

β cyclodextrin with N ═ 6 is preferably used in the present invention, more preferably 2-Se- β -CD, i.e. 2-selenium bridged- β 0-cyclodextrin, 6-Se- β 1-CD, i.e. 6-selenium bridged- β 2-cyclodextrin, 2-Te- β 3-CD, i.e. 2-tellurium bridged- β 4-cyclodextrin, 2-diSe- β -CD, i.e. 2A, 2B-selenious acid-2A ', 2B' -selenium bridged- β -cyclodextrin, 2-secs- β -CD, i.e. 2, 2 '-N, N' -selenocysteine-selenium bridged- β -cyclodextrin, 6-secs- β -CD, i.e. 6, 6 '-N, N' -selenocysteine-bridged-selenium- β -cyclodextrin, 2-diTe- β -CD, i.e. 2A, 2B-ditellurite-2A ', 2B' -tellurite- β -cyclodextrin.

We have found that the cyclodextrin derivatives with GPX activity of the invention can be applied to cosmetics as additives, can effectively protect cells, resist lipid peroxidation, remove free radicals in human skin cells, and play roles in reducing and eliminating skin wrinkles and fine lines, delaying senility, preventing and treating pigmentation, chloasma, butterfly spot, skin keratinization, rough skin and the like.

The invention is further illustrated by the following examples. It should be understood that the examples are for illustrative purposes only and are not intended to limit the invention, and that the invention is not limited thereto, as modifications may be made without departing from the spirit of the invention and within the scope of the appended claims. Percentages in the present invention are by weight unless otherwise indicated.

Preparation examples of Cyclodextrin derivatives

The compound of the present invention can be prepared by the following method. Example 1: preparation method of NaHSe

100mg of NaBH₄Dissolved in a vial containing 5ml of water, and 100mg of selenium powder was slowly added to NaBH₄The solution of (2) is covered with a rubber cover inserted with a needle, so that a large amount of gas generated in water can be seen, and heat is released along with the gas. Since the reaction is too vigorous, it is necessary to control the reaction rate by performing the reaction in an ice bath, which is a reaction equationThe following were used: due to NaBH₄And H⁺Reaction, with a small amount of H₂Se gas is generated and must be reacted in a fume hood to prevent poisoning. After the reaction is completed, a large amount of salt is precipitated. NaHSe is very easy to oxidize, so N should be introduced₂Example 2 Synthesis of 2-Se- β -CD (i.e., 2-seleno-bridged β cyclodextrin) the synthetic route is as follows:(1) β -CD 2-position sulfonylation (product β -CD-2-position hydroxyl benzene sulfonic acid ester, 2-OTs- β -CD for short):

dissolving 2.0g of β -CD in 100ml of NaOH (0.3mol/L) solution, dropwise adding 5ml of acetonitrile solution containing 2.0g of P-TsCl at room temperature, continuously adding 1mol/L of NaOH to ensure that the pH value of the solution is more than 12.5 in the dropwise adding process, after the dropwise adding is finished for 3 hours, stirring for 1 hour, adding 1mol/L of HCl for neutralization to neutrality, adding 100ml of methanol, filtering out insoluble substances, evaporating the methanol and most of water to 20ml under reduced pressure, putting the solution in a refrigerator at 5 ℃ for one week, salting out, filtering, performing Bio-gel P-2 column chromatography by using water as an eluent, collecting a first peak, and freeze-drying to obtain a product, (2) 2-OTs- β -CD selenizing:

dissolving 100mg of 2-OTs- β -CD in 50mM phosphate buffer, introducing high-purity nitrogen to remove oxygen, adding 100 mu l of 1M NaHSe, reacting at 60 ℃ for 36 hours, fully exposing the reaction system to air for oxidation, centrifuging to remove selenium, separating and purifying the supernatant by a Bio-gel P-2 column (5.0 multiplied by 50cm), collecting a first peak by ultraviolet monitoring at 254nm, freeze-drying, washing with acetone for three times, and drying in vacuum to obtain the title compound 2-Se- β -CD, example 3, 6-Se- β -CD (namely 6-Se-bridged β cyclodextrin) synthesis method has the following synthesis route:

(1) synthesis of 6-p-toluenesulfonyl- β -cyclodextrin (6-OTs- β -CD)

β -CD is dried in vacuum at 120 ℃ for one day after recrystallization, pyridine is refluxed for 17 hours by KOH, then dried by BaO overnight, the used pyridine is distilled under normal pressure, p-TsCl 3.6g is dissolved in pyridine 10mL, the pyridine solution of p-TsCl is slowly dripped into β -CD pyridine solution 27g and 160mL under the ice bath condition, the mixture reacts for 24 hours at room temperature after the dripping is finished, acetone is used for precipitation after the reaction is finished, the filtration is carried out, solid powder is recrystallized by water, finally, the solid powder is soaked by ether, and the product is brown white, (2), 6-OTs- β -CD selenization and separation and purification

Dissolving 100mg of 6-OTs- β -CD in a mixed solution of 1mL of 50mM PBS and 1mL of DMF, deoxidizing by introducing high-purity nitrogen, adding 100 μ L of 1M NaHSe, reacting at 60 ℃ for 36 hours, then fully exposing the reaction system to air for oxidation, centrifuging to remove selenium, separating and purifying the supernatant by a Biogel P-2 column, monitoring by ultraviolet rays at 254nm, collecting a first peak, lyophilizing, washing three times with acetone to obtain a pale yellow powder, separating and appearing three absorption peaks by the P-2 column, wherein the first peak is a selenium-bridged β -CD absorption peak (i.e., a target product peak), the second peak is mono-selenohydrogenated β -CD, and the third peak is unreacted 6-OTs- β -CD and a small-molecular impurity peak, example 4: 2-Te- β -CD (i.e., 2-tellurium-bridged 2-CD) and 6-Te- β -CD (i.e., 6-tellurium- β) synthetic method (1), 6-tosyl-6-5-CD (i.e-5-CD) synthetic method (1), 6-tosyl-5-CD), synthesis example 3-5-CD) (see the synthetic method for sodium-6-tosyl-5-6-CD-5-CD) (see (OTH-5-CD-5

3.5g of tellurium powder and 2g of NaBH₄Added to 70mL of absolute ethanol, gas was generated with heat evolution. Introducing nitrogen for several minutes to keep the system oxygen-free. Heating and refluxing for about 1 hour on small fire, and mixing tellurium powder and NaBH₄Dissolving completely, making the solution purple red, cooling to room temperature, adding 4.5mL oxygen-free glacial acetic acid, heating to boil, generating black precipitate, making the solution colorless and transparent, cooling, and synthesizing (6-Te- β -CD) using (4). 6-position-tellurium bridge β -CD

Dissolving 100mg of 6-OTs- β -CD in a mixed solution of 0.5mL of 50mM PBS (pH7) and 0.5mL of DMF, deoxidizing for 25 minutes by introducing high-purity nitrogen, adding 2mL of the NaHTe solution, reacting for 36 hours at 60 ℃, fully exposing the reaction system to the air for oxidation, generating black precipitate, centrifuging the solution at 6000rpm for 10 minutes, separating and purifying the yellow solution by a Sephadex G-25 (phi 5 xA 50cm) column, using deionized water as eluent, monitoring at 254nm ultraviolet, collecting the first peak solution, washing the first peak solution with acetone for three times after freeze-drying, and drying to obtain a light yellow powder target compound, (5) synthesis of 2-position-tellurium-bridged β -CD (2-Te- β -CD)

Dissolving 100mg of 2-OTs- β -CD in 1mL of 50mM PBS (pH7), introducing high-purity nitrogen to remove oxygen for 25 minutes, adding 2mL of the above NaHTe solution, reacting at 60 ℃ for 36 hours, then fully exposing the reaction system to air for oxidation, generating black precipitate, centrifuging the solution at 6000rpm for 10 minutes, separating and purifying the yellow solution through a Sephadex G-25 (phi 5 xA 50cm) column, using deionized water as an eluent, monitoring the ultraviolet at 254nm, collecting a first peak solution, washing the solution with acetone for three times after freeze-drying, and drying to obtain a light yellow powder target compound in example 5. Synthesis method of 2-SeCys- β -CD (namely 2, 2 '-N, N' -selenocysteine-selenium- β -cyclodextrin) (1), β -CD 2-position sulfonylation (product is β -CD-2-position hydroxybenzenesulfonate, abbreviated as 2-OTs- β -CD)

Dissolving 2.0g of β -CD in 100ml of NaOH (0.3mol/L) solution, dropwise adding 5ml of acetonitrile solution containing 2.0gp-TsCl at room temperature, continuously adding 1mol/L of NaOH during dropwise adding to ensure that the pH of the solution is more than 12.5, stirring for 1h after dropwise adding is finished for 3h, adding 1mol/L of HCl for neutralizing to be neutral, adding 100ml of methanol, filtering out insoluble substances, evaporating the methanol and most of water to be 20ml under reduced pressure, putting the solution in a refrigerator at 5 ℃ for one week to precipitate out salts, filtering, performing Bio-gel P-2 column chromatography by using water as eluent, collecting a first peak, and freeze-drying to obtain a product, (2) 2-iodine- β -cyclodextrin (2-I- β -CD)

DMF was dried over phosphorus pentoxide for several days, and was distilled under reduced pressure 2-OTs- β -CD 8.5g was dissolved in DMF (50mL), KI (6.8g) was added, and N was passed through the reaction solution₂To exclude O₂Reacting under sealed condition at 80-90 deg.C under vigorous stirring for 8 hr, adding chloroform to precipitate iodo β -CD after reaction, suction filtering, dissolving the solid with water, adding tetrachloroethylene to precipitate, and collecting the solid.(3) Synthesis of 2, 2 '-N, N' -selenocysteine-selenium bridged- β -Cyclodextrin (2-SeCys- β -CD)

SeCyss (selenocysteine) 14mg dissolved in 6mL water followed by 17mg Na₂CO₃Adding the solution into DMF solution 510mg of 2-I- β -CD, reacting at 65 deg.C for 20 hr under oxygen-free condition, treating the reactant by oxidizing in air, centrifuging, subjecting the supernatant to Sephadex G-25 (phi 5 × A50cm) column chromatography, collecting the first peak, lyophilizing, and lyophilizing to obtain lyophilized powderEXAMPLE 6-diTe- β -CD (i.e., 6A, 6B-ditellurite-6A ', 6B' -tellurium bridged β cyclodextrin) Synthesis method (1), Synthesis of 6A, 6B-Cap- β -CD (i.e., 6A, 6B-Capped- β -CD)

β -CD 7.0g pyridine solution dissolved in 50mL pyridine 1, 3-m-benzene sulfonyl chloride 1.28g, slowly added dropwise to β -CD solution at room temperature, reacted at room temperature for 20 hours under vigorous stirring, after the reaction is finished, distilled under reduced pressure, soaked with acetone overnight, filtered, dried under vacuum, and 8g crude product is added to 50mLH₂O-ethanol (3: 2), filtering off insoluble matter, and dropwise adding 700mL of CH under stirring₃CN-H₂O (6: 1), filtering off white precipitate, distilling the filtrate under reduced pressure, dissolving the final product in 60mL of 20% ethanol aqueous solution, adding 50mL of DEAE-52, stirring at room temperature for 0.5 hour, filtering, distilling the filtrate under reduced pressure to obtain a white brown product (2), synthesizing 6A, 6B-ditellurite-6A ', 6B' -tellurium bridge- β -cyclodextrin (6-diTe- β -CD)

6A, 6B-Capped- β -CD (100mg) is dissolved in 1mL of 50mM phosphate buffer solution with pH of 7.0, 1mL of DMF is added for dissolution promotion, nitrogen is introduced into the reaction mixture for 20 minutes to remove oxygen, the reaction mixture issealed, then 100 muL of NaHTe solution is added by a syringe, the reaction is carried out for 36 hours at 60 ℃ under the protection of nitrogen, the oxidation and the centrifugation are carried out in the air, the supernatant is purified by a Sephadex G-25 column, three peaks appear, the first peak is collected and freeze-dried, the freeze-dried powder is a light yellow target compound, the example 7 is a synthetic method of 6-SeCys- β -CD (6, 6 '-N, N' -selenocysteine-cyclodextrin) (1), 6-p-toluenesulfonyl- β -cyclodextrin (6-OTs- β -CD)

β -CD is dried in vacuum for one day at 120 ℃, pyridine is refluxed for 17 hours by KOH, then dried by BaO overnight, the used pyridine is distilled under normal pressure, p-TsCl 3.6g is dissolved in pyridine 10mL, pyridine solution of p-TsCl is slowly dripped into pyridine solution of β -CD 27g and 160mL under the ice bath condition, the reaction is carried out for 24 hours at room temperature after the dripping is finished, the reaction is precipitated by acetone after the reaction is finished, the filtration is carried out, solid powder is recrystallized by water, finally, the solid powder is soaked by ether, and the product is brown white, (2) 6-iodine- β -cyclodextrin (6-I- β -CD) synthesis

DMF was dried over phosphorus pentoxide for several daysWhen the reaction solution was used, 6-OTs- β -CD 8.5g was dissolved in DMF (50mL) and KI (6.8g) was added thereto, and N was passed through the reaction solution₂To exclude O₂Under the sealing condition, reacting for 4 hours under the condition of vigorous stirring at 80-90 ℃, adding trichloromethane to precipitate iodo β -CD after the reaction is finished, filtering, dissolving the solid with water, adding tetrachloroethylene to precipitate, and collecting the solid (3).6, 6 '-N, N' -selenocysteine-selenium bridging- β -cyclodextrin synthesis (6-SeCys- β -CD)

SeCyss (10mg, 0.03mmol) was dissolved in 4mL of water, followed by addition of 13mg (0.12mmol) Na₂CO₃Adding the solution into DMF solution (400mg, 32mmol) of 6-I- β -CD, reacting at 65 ℃ for 20 hours under oxygen-free conditions, treating the reactant, namely oxidizing in air, centrifuging, carrying out Sephadex G-25 (phi 5 xA 50cm) column chromatography on the supernatant, collecting the first peak, freeze-drying, and synthesizing the freeze-dried powder into a light yellow target compound (example 8: 2-diSe- β -CD (2A, 2B-selenite-2A ', 2B' -selenium bridge β cyclodextrin) (1) synthesizing method (2A, 2B-selenite-2A ', 2B' -selenium bridge β -cyclodextrin (2-diSe- β -CD)

Dissolving 6A, 6B-Capped- β -CD (100mg) in 1mL of 50mM phosphate buffer solution with pH of 7.0, adding 1mL of dissolution promoting solution, introducing nitrogen into the reaction mixture for 20 minutes to remove oxygen, sealing, adding 100 mu L of NaHSe solution by using a syringe, reacting at 60 ℃ for 36 hours under the protection of nitrogen, oxidizing in the air, centrifuging, purifying the supernatant by a Sephadex G-25 column to obtain three peaks, collecting the first peak, freeze-drying to obtain a light yellow target compound, and adding a cyclodextrin derivative-containing cosmetic preparation example and a stability test thereof

When the cyclodextrin derivative is applied to cosmetics, the cyclodextrin derivative is prepared into general composition content ranges and specific preparation examples of cosmetics such as emulsion, cream, aqua and the like:

1. general compositional content ranges: 0.5 to 10.0 percent

2. Specific formulation examples: the cyclodextrin derivative has stable physicochemical properties, no toxicity, small molecular weight, good mold permeability, and strong water solubility, and can be made into cosmetic dosage forms such as emulsion, cream,and aqua.

① emulsion:

the component w percent

Stearic acid 1.5-3.5

Cetyl alcohol 1.0-2.0

3.0-6.0 parts of vaseline

Liquid paraffin 8.0-12.0

Polyoxyethylene (n ═ 10) oleate 1.0-3.0

Polyethylene glycol (molecular weight 1500) 1.5-4.5

0.5 to 1.5 parts of triethanolamine

0.5 to 1.0% of a perfume

2-Se-β-CD 0.5～1.5

Balance of deionized water

Adding polyethylene glycol, triethanolamine, and 2-Se- β -CD into distilled water, heating for dissolving, maintaining 70 deg.C (water phase), mixing other components, heating for dissolving, maintaining 70 deg.C (oil phase), adding oil phase into water phase, stirring, and cooling to 30 deg.C.

② vanishing cream formulation:

the component w percent

Stearic acid 2.0-4.0

Cetyl alcohol 1.0-3.0

3.0-5.0 parts of vaseline

7.0-9.0 parts of liquid paraffin

3.0-5.0% of glycerin

6-SeCys-β-CD 0.5～1.5

0.5-1.0 part of urea

0.5 to 1.5 parts of triethanolamine

0.5 to 1.0% of a perfume

Balance of deionized water

Adding glycerol, triethanolamine, urea, and 6-SeCys- β -CD into distilled water, heating to dissolve, maintaining 80 deg.C (water phase),mixing other components, heating to dissolve, maintaining 80 deg.C (oil phase), adding oil phase into water phase, stirring, and cooling to 30 deg.C.

③ aqueous dosage form:

the component w percent

Alcohol 10.0-25.0

Ethoxylated lanolin 1.0-2.0

2.0-5.0 parts of propylene glycol

0.2-0.5% of essence

2-diTe-β-CD 0.5～1.5

Balance of deionized water

3. Stability test of cosmetic:

3.1 method:

1) heat resistance test: and (3) bottling 100 g of the cyclodextrin derivative emulsion and the vanishing cream, putting each of the three bottles in a water bath at 40 +/-0.5 ℃ for 24 hours, taking out, and observing the phenomenon.

2) Cold resistance test: and (3) bottling 100 g of the cyclodextrin derivative emulsion and the vanishing cream, putting each of the three bottles in a refrigerator with the temperature of-10 +/-0.5 ℃ for 24 hours, taking out the bottles, and observing the phenomenon.

3) And (3) centrifugal test: and (3) respectively taking 20 g of the cyclodextrin derivative emulsion and 20 g of the vanishing cream, filling the cyclodextrin derivative emulsion and the vanishing cream into 50ml centrifuge tubes, centrifuging each three tubes at 4000prm for 30 minutes, taking out the tubes, and observing the phenomenon.

4) GPX residual Activity (C)_r%) was examined: dissolving 10g of the cosmetic before and after stability test in 500ml redistilled water, centrifuging, collecting supernatant as determination solution, and respectively measuring GPX activity (C) before test₀) And GPX activity after assay (C). Then, the residual activities (C) were determined_r%) is calculated as C_r％＝C/C₀×100％

3.2 results

Phenomenon residual Activity (C)_r% of oil-water separation phenomenon in heat resistance test (24 hours in water bath at 40 ℃) 92.4 cold resistance test (24 hours at 10 ℃) 94.7 centrifugal test (30 minutes at 4000 prm) with 90.5 oil-water separation phenomenon

3.3 conclusion: the product has stable physical and chemical properties, and is suitable for adding into cosmetic matrix without compatibility change.

4. Skin irritation test method:

4.1 method

1) Rabbit irritation test: the method is carried out by multiple skin irritation test, namely applying 0.5g of the preparation to the range of about 2.5cm × 2.5cm on either side of the skin of shaved rabbit, applying the same dose of ointment matrix without GPX additive to the same part on the other side, observing the reaction of the skin-applied part once every morning and afternoon for seven consecutive days, and carrying out pathological and histological examination.

2) Human body irritation test, four stimulation points of about 2.5cm × 2.5cm were selected on the medial skin surface of the left arm, and high, medium and low doses of 2-Se- β -CD ointment and placebo were applied by spotting for 24 hours, and skin irritation response at the application site was observed.

4.2 results: see tables 1 and 2 below

TABLE 1 rabbit irritation test results

Examining content

Test animal

Control side

Experimental side

Visual inspection	4 are	No obvious erythema and edema	No obvious erythema and edema
				Histological examination	4 are	The layers of the skin are clearly visible and not found Keratinization transition and thick spinous layer	The layers of the skin are clearly visible and not found Keratinization transition and thick spinous layer
Pathological examination	4 are	No pathological change of skin	No pathological change of skin

TABLE 2 results of human skin irritation test

Grouping	Number of examples (n)	Observation results
				Control points	Test point
		High dose group	5			No erythema and edema formation	No erythema and edema formation
Middle dose group	5			No erythema and edema formation	No erythema and edema formation
		Low dose group	5			No erythema and edema formation	No erythema and edema formation

4.3 conclusion: the product can be used as cosmetic additive without irritation to skin. Test of using cyclodextrin derivative as active component of cosmetics to eliminate free radical in human skin and to prevent ultraviolet ray and senility

1. Test name-cell protective action (taking 2-Se- β -CD as an example)

1.1 Experimental methods

Succinate Dehydrogenase (SDH) in living cells can reduce MTT (methyl tetrazolium blue) to a bluish purple crystal, which is the substance at OD₄₉₀Has characteristic absorption peaks. SDH is viable in living cells, so that a larger value indicates the number of cells that surviveThe more the mesh is.

(1) UV-B irradiation of epidermal cell culture plate with 300. mu.l (5X 10) of cells⁵～1×10⁶One/well), negative and positive control and administration groups (6 wells/group) were set, and CO at 37 ℃ after administration₂Culturing in incubator for 4 hr, and applying ultraviolet-B (UV-B) (dosage rate 6J/m)²Min) cells were irradiated for 17 minutes, 700. mu.l of RPMI 1640 cell culture medium was added, and the mixture was placed in CO at 37 ℃₂Incubate for 18 hours. (UV-B dose for irradiation 100J/m²The mimic is Ebselen, 2-Se- β -CD at 8. mu. mol/L)

(2) MTT method determination of cell viability culture plate Add 40. mu.l of MTT reaction solution per well (2 ml of 10mmol/L PBS (pH7.4) to MTT10mg, 0.22 μm membrane filtration after dissolution), incubate 4 hours at 37 ℃, centrifuge 10 minutes at 1500rpm, discard supernatant, add 600. mu.l of dimethyl sulfoxide (DMSO), shake for 10 minutes, determine OD₄₉₀。

1.2 the results are given in Table 3 below

Table 3: 100J/m²Effect of UV-B irradiation plus 8 μ M mock on cell viability

Group of

OD490

Negative control	O.410±0.031
		Positive control	0.293±0.007
PZ51	0.359±0.016
		2-Se-β-CD	0.372±0.010

1.3 conclusion shows that the 2-Se- β -CD has the functions of protecting cells, increasing the survival rate of epidermal cells, prolonging the service life of cells, reducing and eliminating skin wrinkles and fine lines and delaying senility.

2. Test name anti-lipid peroxidation (exemplified by 6-SeCys- β -CD)

2.1 Experimental methods

Determination of lipid peroxide in free radical induction System 1ml of cell sap was added with 625. mu.l MDA reaction solution (thiobarbituric acid 0.8g was added with trichloroacetic acid 16g, then added with 8.33ml concentrated HCl, constant volume was adjusted to 1000ml for future use), cells were incubated in 95 ℃ water bath for 30 minutes, cooled, centrifuged at 2500rpm for 10 minutes, and supernatant was measured for OD₅₃₂。

2.2 the results are given in Table 4 below

Table 4: pairs of different simulants 100J/m²Effect of UV-B irradiation on changes in cellular lipid peroxidation

Group of	MDA％
		Negative blank	100±1.2
Positive blank	220±4.7
		PZ51	140±2.2
6-SeCys-β-CD	113±2.0

2.3 conclusion, the above data show that the percentage of MDA content is obviously reduced after 6-SeCys- β -CD is added, and a good effect of resisting lipid peroxidation is achieved, which indicates that 6-SeCys- β -CD has the functions of preventing and treating pigmentation, chloasma, butterfly spot and the like caused by lipid peroxidation in skin cells, 3. the test name is the effect of removing free radicals (taking 2-diTe- β -CD as an example)

3.1 Experimental methods

Flow cytometry to determine cell cycle, apoptosis and Reactive Oxygen Species (ROS):

(1) measurement of cell cycle and apoptotic cells were washed 2 times with 10mmol/L PBS pH7.4 (centrifugation at 1500rpm for 5 minutes), the supernatant was discarded, 80. mu.l of RNase was added and 150. mu.l of PI fluorochrome was added for staining, and the reaction was carried out in the dark for 30 minutes, and the fluorescence intensity was measured on a flow cytometer.

(2) Assay ROS plates were incubated at 37 ℃ for 20 minutes with 10. mu.l of DCFH/DA fluorochrome per well, at 37 ℃ for 15 minutes with 60. mu.l of pancreatin, at 1500rpm for 5 minutes, the cells were harvested, washed 2 times with PBS (1500rpm for 5 minutes), the supernatant discarded, and 200. mu.l of PBS was added and the fluorescence intensity was measured on a flow cytometer.

3.2 the results are given in Table 5 below

TABLE 5.100J/m²Effect of UV-B irradiation plus 0.8 μ M mimic on ROS

Group of	ROS
		Negative control	29.72±3.72
Positive control	39.29±9.341
		2-diTe-β-CD	29.29±4.72

3.3 conclusion, the data show that the 2-diTe- β -CD has better effect on eliminating free radicals, and the 2-diTe- β -CD has the effects of preventing ultraviolet rays from damaging the skin, preventing and treating skin keratinization, rough skin and the like.

In conclusion, the cyclodextrin derivative provided by the invention has the advantages of simple preparation process, high yield, mass production, higher GPX activity, good stability and small molecular weight, can overcome the defects of difficult and unstable transmembrane of natural enzyme, and the like, can effectively protect cells, resist lipid peroxidation, eliminate free radicals in human skin cells, and play a role in reducing and eliminating skin wrinkles and fine lines, delaying aging, preventing and treatingpigmentation, chloasma, butterfly spot, skin keratinization, rough skin and the like when being applied to the field of cosmetics.

Claims (12)

1. Use of a cyclodextrin derivative as an additive in cosmetics, said cyclodextrin derivative having the structure:wherein: the parent cyclodextrin has the following structural formula:

the parent cyclodextrin may be α -cyclodextrin (n ═ 5), β -cyclodextrin (n ═ 6), or γ -cyclodextrin (n ═ 7), and its structure can be used

OrTo represent; substituent R₁May be in the 2-or 6-position, substituent R₂Or in the 2-or 6-position, with the proviso that R is₁Is at the same substitution position of different glucose rings of cyclodextrin, R₂Is at the same substitution position of different glucose rings of cyclodextrin; r₁Can be-Se, -Te, -SeCys and-TeCys, wherein Cys represents cysteine, SeCys represents selenocysteine, and TeCys represents tellurocysteine; r₂Can be-OH, -SeO₂H、-TeO₂H, wherein SeO₂H represents selenious acid, TeO₂H represents tellurite.

2. Use according to claim 1, wherein the cyclodextrin derivative is selected from:2-selenium bridged cyclodextrin

6-selenium bridged cyclodextrin2, 2 '-N, N' -selenocysteine-selenium bridged cyclodextrin

6, 6 '-N, N' -selenocysteine-selenium bridged cyclodextrin

2A, 2B-selenious acid-2A ', 2B' -selenium bridged cyclodextrin6A, 6B-selenious acid-6A ', 6B' -selenium bridged cyclodextrin

2-tellurium-bridged cyclodextrins6-tellurium-bridged cyclodextrins2, 2 '-N, N' -tellurocysteine-tellurium bridged cyclodextrin6, 6 '-N, N' -tellurocysteine-tellurium bridged cyclodextrin2A, 2B-ditellurite-2A ', 2B' -tellurite bridged cyclodextrin, or

6A, 6B-ditellurite-6A ', 6B' -tellurium-bridged cyclodextrins, wherein: n is 5, 6, 7.

3. Use according to claim 2, wherein a cyclodextrin derivative of n-6 is selected.

4. Use according to claim 3, wherein the cyclodextrin derivative is selected from 2-seleno- β -cyclodextrin, 6-seleno- β -cyclodextrin, 2-telluro- β -cyclodextrin, 6A, 6B-selenious acid-6A ', 6B' -seleno- β -cyclodextrin, 2, 2 '-N, N' -selenocysteine-seleno-bridge- β -cyclodextrin, 6, 6 '-N, N' -selenocysteine-seleno-bridge- β -cyclodextrin, or 6A, 6B-ditelluro-6A ', 6B' -telluro-bridge- β -cyclodextrin.

5. Use according to one of claims 1 to 4, for reducing and eliminating skin wrinkles, fine lines, delaying ageing, preventing and treating pigmentation, chloasma, butterfly rash, skin keratinization or skin roughness.

6. A cosmetic composition comprises a cyclodextrin derivative having the structure

Wherein: the parent cyclodextrin has the following structural formula:

the parent cyclodextrin may be α -cyclodextrin (n ═ 5), β -cyclodextrin (n ═ 6), or γ -cyclodextrin (n ═ 7), and its structure can be usedOr

To represent; substituent R₁May be in the 2-or 6-position, substituent R₂Or in the 2-or 6-position, with the proviso that R is₁Is at the same substitution position of different glucose rings of cyclodextrin, R₂Is at the same substitution position of different glucose rings of cyclodextrin; r₁Can be-Se, -Te, -SeCys and-TeCys, wherein Cys represents cysteine, SeCys represents selenocysteine, and TeCys represents tellurocysteine; r₂Can be-OH, -SeO₂H、-TeO₂H, wherein SeO₂H represents selenious acid, TeO₂H represents tellurite.

7. The composition according to claim 6, wherein the cyclodextrin derivative is selected from the group consisting of:

2-selenium bridged cyclodextrin6-selenium bridged cyclodextrin

2, 2 '-N, N' -selenocysteine-selenium bridged cyclodextrin6, 6 '-N, N' -selenocysteine-selenium bridged cyclodextrin

2A, 2B-selenious acid-2A ', 2B' -selenium bridged cyclodextrin

6A, 6B-selenious acid-6A ', 6B' -selenium bridged cyclodextrin

2-tellurium-bridged cyclodextrins6-tellurium-bridged cyclodextrins2, 2 '-N, N' -tellurocysteine-tellurium bridged cyclodextrin6, 6 '-N, N' -tellurocysteine-tellurium bridged cyclodextrin

2A, 2B-ditellurite-2A ', 2B' -tellurite bridged cyclodextrin, or6A, 6B-ditellurite-6A ', 6B' -tellurium bridged cyclodextrin wherein: n is 5, 6, 7.

8. A composition according to claim 7, wherein a cyclodextrin derivative of n-6 is selected.

9. The composition according to claim 8, wherein the cyclodextrin derivative is selected from 2-seleno- β -cyclodextrin, 6-seleno- β -cyclodextrin, 2-telluril- β -cyclodextrin, 6A, 6B-selenious acid-6A ', 6B' -seleno- β -cyclodextrin, 2, 2 '-N, N' -selenocysteine-selenium bridge- β -cyclodextrin, 6, 6 '-N, N' -selenocysteine-selenium bridge- β -cyclodextrin, or 6A, 6B-ditelluride-6A ', 6B' -telluril- β -cyclodextrin.

10. The cosmetic composition according to claims 6 to 9, wherein the cyclodextrin derivative is contained in the cosmetic in an amount ranging from 0.5 to 10.0% by weight.

11. Cosmetic composition according to one of claims 6 to 10, which is in the form of a cream, an emulsion or an aqueous preparation.

12. Use of a cosmetic composition according to one of claims 6 to 11 for protecting cells, combating lipid peroxidation, scavenging free radicals in human skin cells, reducing and eliminating skin wrinkles, fine lines, delaying ageing, preventing and treating pigmentation, chloasma, butterfly rash, skin keratinization or skin roughness.