WO2020258775A1 - 一种透明质酸皮肤保护组合物及其制备方法和应用 - Google Patents

一种透明质酸皮肤保护组合物及其制备方法和应用 Download PDF

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WO2020258775A1
WO2020258775A1 PCT/CN2019/127012 CN2019127012W WO2020258775A1 WO 2020258775 A1 WO2020258775 A1 WO 2020258775A1 CN 2019127012 W CN2019127012 W CN 2019127012W WO 2020258775 A1 WO2020258775 A1 WO 2020258775A1
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hyaluronic acid
skin protection
protection composition
composition according
molecular weight
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PCT/CN2019/127012
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English (en)
French (fr)
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王云云
王昕宇
宋文俊
张红晨
王坤
舒晓正
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常州百瑞吉生物医药有限公司
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Priority to US17/441,915 priority Critical patent/US20220105020A1/en
Priority to EP19935112.3A priority patent/EP3991716A4/en
Publication of WO2020258775A1 publication Critical patent/WO2020258775A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations

Definitions

  • the present invention relates to the field of biomedicine, in particular to a hyaluronic acid skin protection composition, and also relates to a preparation method and application of the hyaluronic acid skin protection composition.
  • Reactive oxygen free radicals have strong oxidizing properties and can attack lipids, proteins and DNA, etc., and can damage the tissues and cells of the body, thereby triggering various chronic diseases and aging effects (Gutteridge et al., British Medical Bulletin 1999, 55:49-57).
  • the skin is the largest organ in the human body. Studies have shown that up to 80% of skin aging is related to active oxygen free radicals. Due to the destructive effect of active oxygen free radicals, the cells and extracellular matrix that maintain the skin’s balance are destroyed and reduced, and the immune repair ability is rapidly reduced. Finally, various skin aging and pathological symptoms, such as dryness, roughness, dullness, relaxation, Wrinkles and so on.
  • Moisturizing is an important part of skin care, which can effectively improve some of the symptoms of skin aging, such as dullness, fine lines, roughness, scaling and itching.
  • the moisture level of the skin is closely related to the content of hyaluronic acid. With the increase of age and the destruction of active oxygen free radicals, the content of hyaluronic acid in the skin decreases and loses, which weakens the skin's water retention function and causes aging (Agren et al., Free Radical Biol. Med. 1997, 23: 996-1001; Stern et al., Clinics in Dermatology 2008, 26: 106-122).
  • Hyaluronic acid also known as hyaluronic acid
  • Hyaluronic acid is an acidic mucopolysaccharide inherent in the human body. It is widely used in various skin care products. It is a super moisturizing agent naturally present in human skin and can maintain moisture 1000 times its own weight. Moisturizing effect is the most important role of hyaluronic acid in skin care products. Topical use of hyaluronic acid for hydrating and moisturizing is an important measure to delay skin aging symptoms and partly restore youthfulness.
  • the hyaluronic acid aqueous solution has strong viscoelasticity and lubricity. When applied to the skin, it can form a moisturizing and breathable film to keep the skin moist and shiny.
  • Hyaluronic acid has unique moisturizing properties, and the relative humidity of the surrounding environment has little effect on its moisturizing properties. Even in extreme cases where the relative humidity is very low, it can still capture water from the air and supply it to the skin; on the contrary, in extreme cases Under dry conditions, traditional moisturizers (such as sorbitol, polyethylene glycol, etc.) will absorb water from the skin, which may aggravate the symptoms of skin dehydration (Smejkalova, etc., Harry's Cosmeticology 9th Edition Vol.2.Part 4.1.3, pp.605 -622; Publisher: Chemical Publishing Company, 2015).
  • traditional moisturizers such as sorbitol, polyethylene glycol, etc.
  • the moisturizing effect of hyaluronic acid is positively correlated with molecular weight, and high molecular weight hyaluronic acid has better moisturizing, film-forming and lubricating properties.
  • the high molecular weight hyaluronic acid added in current skin care products is usually greater than 800kDa (800,000 Daltons). It not only has a good moisturizing effect on the surface of the skin, but also has a lifting and firming effect, which can effectively improve the rough skin. To make the skin more delicate and smooth (Biomatrix Inc. Hyaluronan Vol. 2: Biomedical, Medical and Clinical Aspects, pp. 285-288; Publisher: Woodhead Publishing Limited, 2002).
  • the molecular weight distribution of hyaluronic acid is very wide, and different molecular weights have different physical, chemical and biological properties. Studies have shown that low- and medium-molecular-weight hyaluronic acid with a molecular weight of less than 500KDa can penetrate the superficial stratum corneum of the skin in a small amount, hydrate and soften the stratum corneum, and establish a bridge channel with the endogenous hyaluronic acid in the deep epidermis, which is beneficial to nutrients Absorption and discharge of metabolic waste.
  • the transdermal absorption performance of hyaluronic acid is negatively correlated with molecular weight, and the decrease in molecular weight helps transdermal absorption.
  • hyaluronic acid an oligosaccharide with 6-20 disaccharide repeating units, that is, a molecular weight of 2.4KDa-8KDa
  • blood flow promote micro-angiogenesis
  • fibroblast growth promote fibroblast growth and internal The secretion and synthesis of hyaluronic acid. Therefore, skin care products containing low-molecular-weight hyaluronic acid can repair and nourish the skin base to a certain extent (Farwick et al., SOFW-Journal 2008, 134: 1-6; Smejkalova et al., Harry's Cosmeticology 9th Edition Vol.2. Part 4.1.3, pp.605-622; Publisher: Chemical Publishing Company, 2015).
  • CN 106109265B discloses a hyaluronic acid moisturizing composition, the weight percentage of hyaluronic acid or its salt with a molecular weight of 1,100kDa is 60-62%, and the weight percentage of hyaluronic acid or its salt with a molecular weight of 400kDa is 18-20 %, the weight percentage of hydrolyzed hyaluronic acid with a molecular weight of 6kDa or its salt is 18-20%.
  • CN 106137786B discloses a hyaluronic acid anti-aging composition.
  • the weight percentage of hydrolyzed hyaluronic acid or its salt with a molecular weight of 7KDa is 60%-62%, and the weight percentage of hyaluronic acid or its salt with a molecular weight of 320KDa
  • the content is 18%-20%, and the weight percentage of hyaluronic acid or its salt with a molecular weight of 1,100KDa is 18%-20%.
  • CN 101450028A discloses an efficient moisturizing hyaluronic acid stock solution, which is composed of a high molecular weight sodium hyaluronate with an average molecular weight greater than 2,600 KDa and a low molecular weight sodium hyaluronate with a molecular weight less than 10 KDa.
  • CN 101500535A discloses a composition with several hyaluronic acid fractions for cosmetic and medical purposes, comprising at least two hyaluronic acid fractions or salts thereof, one of which has an average molecular weight of 8KDa to 100KDa, and the other One fraction has an average molecular weight of 100KDa to 500KDa.
  • hyaluronic acid can also quench active oxygen free radicals, and it is destroyed and decomposed at the same time, and has a weak anti-inflammatory effect; however, the ability to quench active oxygen free radicals is very weak and is not sufficient to effectively remove active oxygen.
  • Free radicals Yamamazaki et al., Pathophysiology 2003, 9:215-220.; Stern et al., Biotechnology Advances 2007, 25:537-557; Serban et al., Biomaterials 2008, 29:1388-1399; CN 106137786B).
  • this application provides a new type of hyaluronic acid skin protection composition, which not only has hyaluronic acid moisturizing and other skin care functions, but also It also has enhanced antioxidant capacity and can effectively scavenge active oxygen free radicals, so it can more effectively improve skin aging symptoms and better solve skin aging problems.
  • a hyaluronic acid skin protection composition containing:
  • At least one molecular weight hyaluronic acid or salt thereof At least one molecular weight hyaluronic acid or salt thereof.
  • the hyaluronic acid or its salt in the present invention includes sodium salt, potassium salt, zinc salt, calcium salt, etc., preferably sodium and potassium salt, particularly preferably sodium salt.
  • the hyaluronic acid sulfhydryl derivative used contains sulfhydryl groups, which can effectively inhibit the oxidative stress directly caused by active oxygen free radicals, and can prevent lipid, protein and DNA damage; in addition, it can also be used with The divalent iron ions are complexed to indirectly inhibit the formation of hydroxyl radicals.
  • the sulfhydryl derivatives of hyaluronic acid in the present invention refer to hyaluronic acid derivatives containing sulfhydryl groups, which can be prepared by sulfhydryl modification of hyaluronic acid or its salts, and also include various hyaluronic acid derivatives further through sulfhydryl groups.
  • the sulfhydryl derivative prepared by chemical modification.
  • the side chain carboxyl groups, side chain hydroxyl groups, and reducing end groups of hyaluronic acid or its salts or derivatives are usually reactive functional groups that can be thiolated and modified.
  • WO 2009/006780A1 discloses a variety of hyaluronic acid thiol groups. Derivative preparation method.
  • the sulfhydryl modification of the carboxyl group of the side chain of hyaluronic acid can adopt the chemical method of amino (hydrazide group)/carbodiimide coupling.
  • the carbodiimide includes 1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide hydrochloride, etc.
  • Another method of sulfhydryl modification of side chain carboxyl groups is to directly combine with carbodiimides containing disulfide bonds (such as 2,2'-dithiobis (N-ethyl (N'-ethyl carbodiimide)). Amine))) prepared by reaction (Bulpitt et al., US 6,884,788).
  • carbodiimides containing disulfide bonds such as 2,2'-dithiobis (N-ethyl (N'-ethyl carbodiimide)). Amine)
  • the sulfhydryl modification of the hydroxyl group of the side chain of hyaluronic acid can be prepared by the nucleophilic ring-opening reaction of the hydroxyl group and ethylene sulfide under alkaline conditions (Serban et al., Biomaterials 2008, 29:1388-1399).
  • the sulfhydryl modification of the side chain hydroxyl of hyaluronic acid can also be carried out in an indirect manner. For example, the hydroxyl is first carboxymethylated under alkaline conditions, and then the carboxyl is modified with sulfhydryl (Prestwich et al., WO 2005/056608A1) .
  • hyaluronic acid is also an active group that can undergo sulfhydryl modification, and the terminal sulfhydryl group can be introduced chemically (Inoue et al., Carbohydrate Research 1985, 141:99-110).
  • hyaluronic acid derivatives such as acetylated sodium hyaluronate disclosed in CN 109206537A, etc., can also be modified by the above-mentioned sulfhydryl modification method to obtain the hyaluronic acid sulfhydryl derivative of the present invention.
  • the sulfhydryl content in the hyaluronic acid sulfhydryl derivative used can be determined by Ellman's reagent method or 1 H NMR absorption spectrogram (Shu et al., Biomacromolecules 2002, 3:1304-1311), expressed as The number of micromoles ( ⁇ mol/g) of sulfhydryl groups per gram of hyaluronic acid sulfhydryl derivatives.
  • the sulfhydryl grafting degree of hyaluronic acid sulfhydryl derivatives is generally defined as the percentage of the number of sulfhydryl groups introduced per 100 hyaluronic acid disaccharide repeating segments.
  • the molecular weight of the disaccharide repeating segment of the hyaluronic acid sulfhydryl derivative is approximately equal to 400 Da, it can be calculated by the sulfhydryl content and the sulfhydryl grafting degree. For example, when the content of sulfhydryl groups is 125 ⁇ mol/g, the number of sulfhydryl groups introduced per 100 disaccharide segments is 5, that is, the degree of sulfhydryl grafting is 5%.
  • various sulfhydryl grafting degrees of hyaluronic acid sulfhydryl derivatives can be used to prepare the hyaluronic acid skin protection composition of the present invention, such as the sulfhydryl grafting degree (sulfhydryl content) reported by Shu et al. ) Is 26.8% ⁇ 66.8% (670 ⁇ mol/g ⁇ 1670 ⁇ mol/g) hyaluronic acid sulfhydryl derivative (Shu et al., Biomacromolecules 2002, 3:1304-1311), the degree of sulfhydryl grafting (sulfhydryl content) reported by Serban et al.
  • the sulfhydryl content is a decisive factor for the antioxidant capacity of the hyaluronic acid sulfhydryl derivative.
  • the hyaluronic acid sulfhydryl derivative used in the present invention has significantly enhanced anti-oxidation ability, and its ability to scavenge free radicals can be increased to 10-40 times or even higher.
  • the increase in the content of sulfhydryl groups helps to enhance the scavenging ability of active oxygen radicals; but on the other hand, when the content of sulfhydryl groups is less, it helps to better maintain the original structure of hyaluronic acid and the hyaluronic acid itself Has the skin care function. Therefore, the hyaluronic acid sulfhydryl derivative used in the present invention combines the above two factors.
  • the mercapto group content is usually less than 500 ⁇ mol/g, preferably less than 100 ⁇ mol/g, and particularly preferably between 20 ⁇ mol/g and 50 ⁇ mol/g.
  • the content of sulfhydryl groups largely determines the antioxidant capacity of hyaluronic acid sulfhydryl derivatives, and the molecular weight has little effect on its antioxidant capacity.
  • the molecular weight of the hyaluronic acid thiolated derivative used is usually between 1 KDa and 10,000 KDa, preferably between 1.6 KDa and 3,000 KDa, and particularly preferably between 2.4 KDa and 1,500 KDa.
  • the molecular weight refers to the average molecular weight.
  • the concentration of the hyaluronic acid thiolated derivative contained is usually less than 5 mg/ml, preferably less than 2 mg/ml, particularly preferably less than 1 mg/ml.
  • the hyaluronic acid skin protection composition of the present invention may also contain two or more hyaluronic acid sulfhydryl derivatives, and these sulfhydryl derivatives may have different molecular weights and/or sulfhydryl content.
  • hyaluronic acid skin protection composition of the present invention also contains at least one molecular weight hyaluronic acid or its salt, and its molecular weight is usually between 1KDa and 10,000KDa, preferably between 1.6KDa and 3,000KDa , Particularly preferably between 2.4KDa and 2,800Kda.
  • the molecular weight of at least one hyaluronic acid or its salts is 1.6KDa ⁇ 500KDa and the molecular weight of at least one hyaluronic acid or salt thereof is between 800KDa and 3,000KDa, particularly preferably the molecular weight of at least one hyaluronic acid or salt thereof is between 2.4 and 8KDa and at least one of which is transparent
  • the molecular weight of acid or its salt is between 1,000KDa and 2,800KDa.
  • the concentration of hyaluronic acid or its salt contained is usually less than 10 mg/ml, preferably less than 8 mg/ml, and particularly preferably less than 4 mg/ml.
  • vitamins, amino acids, traditional moisturizers (such as sorbitol, polyethylene glycol, etc.) and antioxidants can be added to the hyaluronic acid skin protection composition.
  • the hyaluronic acid skin protection composition of the present invention can usually be terminally sterilized (such as moist heat sterilization) in a manner well known to those skilled in the art, and preservatives may not be added, which can effectively eliminate the harm caused by preservatives.
  • preservatives/bacteriostatic agents well known to those skilled in the art can also be appropriately added to the hyaluronic acid skin protection composition of the present invention.
  • the free radical scavenging ability of the composition of the present invention is more significant, and the free radical scavenging rate can be increased by 100% to 1000% or more. Therefore, the composition of the present invention is used for skin protection Time has a unique anti-aging advantage.
  • Another object of the present invention is to provide a preparation method of the hyaluronic acid skin protection composition.
  • the preparation method of the hyaluronic acid skin protection composition is characterized in that the components are uniformly mixed according to a set ratio.
  • the components can also be filled in a sealed container under the protection of an inert atmosphere after the components are uniformly mixed.
  • Another object of the present invention is to provide the application of the hyaluronic acid skin protection composition.
  • the application of the hyaluronic acid skin protection composition refers to the application in skin cleansing, care, and cosmetics, and has moisturizing and anti-oxidant effects.
  • the hyaluronic acid skin protection composition not only has the moisturizing and other skin care effects of hyaluronic acid itself, but also has strong antioxidant effects, can effectively remove active oxygen free radicals, and can effectively improve skin aging symptoms and better solve the skin Aging problem; it can be used in all forms of cosmetics and cleansing products, including softening lotions, essences, gels, lotions, creams, masks, makeup, soaps, facial cleansers and shower gels.
  • composition of the present invention contains hyaluronic acid sulfhydryl derivatives, which can effectively quench active oxygen free radicals and other active oxygen free radicals, inhibit the oxidative stress directly caused by them, prevent lipid, protein and DNA damage, and also It can indirectly inhibit the formation of active salt radicals such as hydroxyl radicals.
  • composition of the present invention also contains one molecular weight or multiple different molecular weight hyaluronic acid or its salt, which can form a combination of different molecular weights to fully exert the moisturizing, softening of the stratum corneum and repair and nourish the skin base. And other multiple skin protection effects.
  • composition of the present invention has unique advantages when used for skin protection. It not only has the multiple skin protection effects of hyaluronic acid itself, but also has strong antioxidant capacity, can effectively remove active oxygen free radicals, and can effectively improve skin aging symptoms. , Such as increasing skin elasticity and reducing wrinkles, etc., has a good skin protection effect.
  • Figure 1 The scavenging rate of DPPH free radicals of hyaluronic acid thiolated thiolated derivatives of different concentrations and sulfhydryl content.
  • Figure 3 The scavenging rate of a hyaluronic acid skin protection composition (containing a molecular weight sodium hyaluronate) on DPPH free radicals.
  • Hyaluronic acid skin protection composition (containing two molecular weight sodium hyaluronate) to DPPH free radical scavenging rate.
  • Example 1 Antioxidant effect of sulfhydryl derivatives of hyaluronic acid with different sulfhydryl content in scavenging free radicals
  • DPPH 1,1-diphenyl-2-trinitrophenylhydrazine test method was used to evaluate the free radical scavenging performance of the hyaluronic acid sulfhydryl derivative used in the present invention.
  • DPPH is widely used in the quantitative determination of the antioxidant capacity of biochemical substances; this method is based on the fact that DPPH free radicals have a single electron and have a strong absorption at a wavelength of 517nm.
  • the alcohol solution is purple; when there is a free radical scavenger At this time, the absorption gradually disappears due to its single electron pairing, and the degree of fading is quantitatively related to the number of electrons it accepts. Therefore, a spectrophotometer can be used for rapid quantitative analysis (Sharma et al., Food Chemistry 2009,113:1202-1205) .
  • Hyaluronic acid sulfhydryl groups were prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and the sulfhydryl content was 18 ⁇ mol/g, 38 ⁇ mol/g, 57 ⁇ mol/g, 108 ⁇ mol/g, 266 ⁇ mol/g, respectively , 416 ⁇ mol/g and 530 ⁇ mol/g.
  • the sodium hyaluronate raw material and the above-mentioned sodium hyaluronate sulfhydryl derivative are respectively dissolved in a phosphate buffer solvent (pH 7.4) to obtain a test sample with a final concentration of 0.1 mg/mL, 0.5 mg/mL or 1 mg/mL.
  • the free radical scavenging ability is calculated according to the following formula:
  • test results show that the scavenging rate of DPPH free radicals is positively correlated with the number of sulfhydryl groups in the test sample, and the scavenging rate of thiolated sodium hyaluronate derivatives is about 10-40 times higher than that of sodium hyaluronate raw materials.
  • Example 2 Antioxidant effect of sulfhydryl derivatives of hyaluronic acid with different molecular weights in scavenging free radicals
  • Hyaluronic acid sulfhydryl derivatives are prepared with 180KDa, 300KDa and 1,500KDa sodium hyaluronate as raw materials, and are prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and their sulfhydryl content is respectively 38 ⁇ mol/g, 33 ⁇ mol/g and 40 ⁇ mol/g.
  • the raw material of sodium hyaluronate and the sulfhydryl derivative of sodium hyaluronate were respectively dissolved in a phosphate buffer solvent (pH 7.4) to obtain the test sample solution described in the following table.
  • Test samples 1, 3, and 5 are sodium hyaluronate control solutions of different molecular weights, which have low DPPH radical scavenging rate; test samples 2, 4, and 6 are test sample solutions of hyaluronic acid sulfhydryl derivatives of different molecular weights, which have the same
  • the sulfhydryl content (38 ⁇ 10 -3 ⁇ mol/mL) and the scavenging rate of DPPH free radicals are similar, and both are about 15 times stronger than the control solution.
  • Example 3 Preparation of hyaluronic acid skin protection composition (containing sodium hyaluronate with a molecular weight)
  • the hyaluronic acid sulfhydryl derivative used is made with 180KDa sodium hyaluronate as raw material, prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and its sulfhydryl content is 18 ⁇ mol/g. , 38 ⁇ mol/g, 57 ⁇ mol/g, 108 ⁇ mol/g, 266 ⁇ mol/g, 416 ⁇ mol/g and 530 ⁇ mol/g.
  • the molecular weight of the sodium hyaluronate used is 1.6KDa, 3KDa, 8KDa, 50KDa, 180KDa, 500KDa, 800KDa, 1,500KDa, 2,700KDa.
  • any one of the above-mentioned mercapto-containing hyaluronic acid sulfhydryl derivatives is dissolved in distilled water to obtain a 6mg/mL solution; any of the above-mentioned molecular weight sodium hyaluronate is dissolved in distilled water to obtain a 12mg/mL solution; then according to 0.9: The volume ratio of 0.1-0.1:0.9 is mixed uniformly, and the hyaluronic acid skin protection composition with different proportions is obtained.
  • the content of hyaluronic acid thiolated derivatives is any concentration between 5.4 mg/mL and 0.6 mg/mL, and the content of sodium hyaluronate is between 1.2 mg/mL and 10.8 mg/mL. Any concentration between.
  • distilled water is added according to a volume ratio of 1:0.08 to 0.08:1 and mixed uniformly to obtain a diluted composition.
  • the content of hyaluronic acid thiolated derivative is any concentration between 5.0mg/mL ⁇ 0.04mg/mL
  • the content of sodium hyaluronate is between 0.09mg/mL ⁇ 10.0mg/mL Any concentration between.
  • the above composition was poured into a glass ampoule under the protection of an inert atmosphere (nitrogen) and sealed for use.
  • Example 4 Hyaluronic acid skin protection composition (containing a molecular weight of sodium hyaluronate) for scavenging free radicals and antioxidant effect
  • Example 3 Ten kinds of hyaluronic acid protective compositions prepared in Example 3 (the composition of which are shown in the following table) were selected, and their anti-oxidant effects of scavenging free radicals were measured according to the method described in Example 1.
  • compositions of various proportions have significant free radical scavenging and antioxidant capacity, which is 3.4 to 26 times stronger than the control.
  • the hyaluronic acid sulfhydryl derivative used is made with 180KDa sodium hyaluronate as raw material, prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and its sulfhydryl content is 18 ⁇ mol/g. , 38 ⁇ mol/g, 57 ⁇ mol/g, 108 ⁇ mol/g, 266 ⁇ mol/g, 416 ⁇ mol/g and 530 ⁇ mol/g.
  • the molecular weight of the sodium hyaluronate used is 1.6KDa, 3KDa, 8KDa, 50KDa, 180KDa, 500KDa, 800KDa, 1,500KDa, 2,700KDa.
  • Any one of the above-mentioned mercapto-containing hyaluronic acid sulfhydryl derivatives is dissolved in distilled water to obtain a solution of 6mg/mL; any two of the above-mentioned sodium hyaluronates (in any weight ratio) are dissolved in distilled water to obtain a total concentration of 12mg/ mL of the solution; then mix uniformly according to the volume ratio of 0.9:0.1 to 0.1:0.9 to obtain hyaluronic acid skin protection compositions with different ratios.
  • the content of hyaluronic acid thiolated derivatives is any concentration between 5.4mg/mL ⁇ 0.6mg/mL, and the total concentration of sodium hyaluronate is 1.2mg/mL ⁇ 10.8mg/mL Any concentration in between.
  • distilled water is added according to a volume ratio of 1:0.08 to 0.08:1 and mixed uniformly to obtain a diluted composition.
  • the content of the hyaluronic acid thiolated derivative is any concentration between 5.0mg/mL ⁇ 0.04mg/mL, and the total content of sodium hyaluronate is 0.09mg/mL ⁇ 10.0mg/mL Any concentration in between.
  • the above composition is poured into a glass ampoule under the protection of an inert atmosphere (helium gas), and sealed for use.
  • an inert atmosphere helium gas
  • Example 6 Antioxidant effect of hyaluronic acid skin protection composition (containing sodium hyaluronate with two molecular weights) in scavenging free radicals
  • Example 5 Ten types of hyaluronic acid protective compositions prepared in Example 5 (the composition of which are shown in the following table) were selected, and their anti-oxidant effects in scavenging free radicals were measured according to the method described in Example 1.
  • Example 7 Preparation of hyaluronic acid skin protection composition (containing sodium hyaluronate with three or more molecular weights)
  • the hyaluronic acid sulfhydryl derivative used is made with 180KDa sodium hyaluronate as raw material, prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and its sulfhydryl content is 18 ⁇ mol/g. , 38 ⁇ mol/g, 57 ⁇ mol/g, 108 ⁇ mol/g, 266 ⁇ mol/g, 416 ⁇ mol/g and 530 ⁇ mol/g.
  • the molecular weight of the sodium hyaluronate used is 1.6KDa, 3KDa, 8KDa, 50KDa, 180KDa, 500KDa, 800KDa, 1,500KDa, 2,700KDa.
  • any one of the above-mentioned mercapto-containing hyaluronic acid sulfhydryl derivatives is dissolved in distilled water to obtain a 6mg/mL solution; any of the above-mentioned sodium hyaluronate with three molecular weights and above (in any weight ratio) is dissolved in distilled water to obtain a total concentration 12mg/mL solution; then mix uniformly according to the volume ratio of 0.9:0.1 to 0.1:0.9 to obtain hyaluronic acid skin protection compositions with different ratios.
  • the content of hyaluronic acid thiolated derivatives is any concentration between 5.4mg/mL ⁇ 0.6mg/mL, and the total content of sodium hyaluronate is 1.2mg/mL ⁇ 10.8mg/mL Any concentration in between.
  • distilled water is added according to a volume ratio of 1:0.08 to 0.08:1 and mixed uniformly to obtain a diluted composition.
  • the content of the hyaluronic acid thiolated derivative is any concentration between 5.0mg/mL ⁇ 0.04mg/mL, and the total content of sodium hyaluronate is 0.09mg/mL ⁇ 10.0mg/mL Any concentration in between.
  • the above composition was poured into a glass ampoule under the protection of an inert atmosphere (nitrogen) and sealed for use.
  • Example 8 Preparation of hyaluronic acid skin protection composition (containing two or more hyaluronic acid sulfhydryl derivatives and three or more molecular weight sodium hyaluronate)
  • Hyaluronic acid sulfhydryl derivatives are prepared with 180KDa, 300KDa and 1,500KDa sodium hyaluronate as raw materials, and are prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and their sulfhydryl content is respectively 38 ⁇ mol/g, 33 ⁇ mol/g and 40 ⁇ mol/g.
  • the molecular weight of the sodium hyaluronate used is 1.6KDa, 3KDa, 8KDa, 50KDa, 180KDa, 500KDa, 800KDa, 1,500KDa, 2,700KDa.
  • any two or more of the above hyaluronic acid sulfhydryl derivatives are dissolved in distilled water to obtain a 6mg/mL solution; any of the above three or more molecular weight sodium hyaluronate (in any weight ratio) is dissolved in distilled water, A solution with a total concentration of 12 mg/mL is obtained; then, it is mixed uniformly according to a volume ratio of 0.9:0.1 to 0.1:0.9 to obtain hyaluronic acid skin protection compositions with different ratios.
  • the content of hyaluronic acid thiolated derivatives is any concentration between 5.4mg/mL ⁇ 0.6mg/mL, and the total content of sodium hyaluronate is 1.2mg/mL ⁇ 10.8mg/mL Any concentration in between.
  • distilled water is added according to a volume ratio of 1:0.08 to 0.08:1 and mixed uniformly to obtain a diluted composition.
  • the content of the hyaluronic acid thiolated derivative is any concentration between 5.0mg/mL ⁇ 0.04mg/mL, and the total content of sodium hyaluronate is 0.09mg/mL ⁇ 10.0mg/mL Any concentration in between.
  • the above composition was poured into a glass ampoule under the protection of an inert atmosphere (nitrogen) and sealed for use.
  • Example 9 Moisturizing properties of hyaluronic acid skin protection composition
  • composition numbers 5 and 6 and their respective reference substances were measured before application and 1, 2 and 4 hours after application.
  • the relative increase in skin moisture is calculated according to the following formula:
  • hyaluronic acid composition The effect of hyaluronic acid composition on skin elasticity was measured by a similar method disclosed in CN106137786B. Six subjects were recruited in each group, and the hyaluronic acid skin protection composition (composition Nos. 8 and 9 in Example 6 and their respective control substances) was applied to the cheeks and gently massaged until all was absorbed. After cleansing the face every morning and evening, for 4 weeks. The skin elasticity meter MPA580 (Courage+Khazaka, Germany) was used to measure the elastic changes of the cheeks (apple muscle) before and 4 weeks after application.
  • the elasticity index in this test includes initial elasticity, net elasticity and biological elasticity.
  • the average value of these three elasticity indexes is used to characterize skin elasticity; the relative increase rate of skin elasticity (%) is calculated according to the following formula:
  • compositions of all proportions significantly increased skin elasticity, and it was more significant than the control.
  • hyaluronic acid composition on skin wrinkles was measured by a similar method disclosed in CN106137786B. Six subjects were recruited in each group, and the hyaluronic acid skin protection composition (composition number 8 and 9 in Example 6) was applied to the corner of the eye and gently massaged until all was absorbed. After cleansing the face every morning and evening, for 4 weeks. The human skin rapid three-dimensional imaging system was used to evaluate the wrinkle depth of the subjects' right eye before use and 4 weeks after use.
  • the relative reduction value (%) of average wrinkle depth is calculated according to the following formula:

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Abstract

一种透明质酸皮肤保护组合物，含有：1)透明质酸巯基化衍生物；和2)至少一种分子量的透明质酸或其盐。该组合物用于皮肤保护时具有独特的优势，不仅具有透明质酸本身的多重皮肤保护效果，同时还具有强抗氧化能力，可有效清除活性氧自由基。

Description

一种透明质酸皮肤保护组合物及其制备方法和应用 技术领域
本发明涉及生物医药领域，特别涉及一种透明质酸皮肤保护组合物，还涉及所述透明质酸皮肤保护组合物的制备方法及应用。
背景技术
人体正常新陈代谢过程中，氧化过程和还原过程存在着动态平衡。但在多种内源性(如慢性或急性感染等)及外源性因素(如环境污染、紫外辐射等)的作用下，氧化反应可能占据优势，出现氧化应激，从而产生多种有害的活性氧自由基；活性氧自由基具有强氧化性，会进攻脂质、蛋白质及DNA等，可损害机体的组织和细胞，进而引发各种慢性疾病及衰老效应(Gutteridge等，British Medical Bulletin 1999,55:49-57)。
皮肤是人体面积最大的器官，有研究表明高达80％的皮肤老化现象与活性氧自由基有关。由于活性氧自由基的破坏作用，使维持皮肤平衡的细胞及细胞外基质被破坏和减少，免疫修复能力迅速下降，最后导致各种皮肤衰老及病变症状，如干燥、粗糙、暗沉、松弛、皱纹等等。
保湿是皮肤护理的重要环节，可有效改善皮肤衰老的部分症状，如无光泽、细纹、粗糙、脱屑及瘙痒等。皮肤的湿润水平与透明质酸含量密切相关，随着年龄的增长以及活性氧自由基的破环作用，皮肤中透明质酸含量下降流失，使皮肤保水功能减弱，产生老化(Agren等，Free Radical Biol.Med.1997,23:996-1001；Stern等，Clinics in Dermatology 2008,26:106-122)。透明质酸又名玻尿酸，是一种人体内固有的酸性黏多糖，被普遍应用于各种护肤品，是人体肌肤中天然存在的超强保湿剂，可维持自身重量1000倍的水份。保湿作用是透明质酸在护肤品中最重要的作用，局部使用透明质酸进行补水保湿是延缓皮肤老化症状及部分恢复年轻态的重要措施。透明质酸水溶液具有较强的黏弹性和润滑性，涂于皮肤表面，可形成一层保湿透气膜，保持皮肤滋润亮泽。透明质酸的保湿性能独特，周围环境的相对湿度对其保湿性能的影响较小，即使在相对湿度很低的极端情况下，仍能从空气中捕捉水份供给皮肤；与此相反，在极端干燥条件下，传统保湿剂(如山梨醇、聚乙二醇等)则会从皮肤吸水，可能加重皮肤缺水症状(Smejkalova等，Harry’s Cosmeticology 9th Edition Vol.2.Part 4.1.3,pp.605-622；Publisher:Chemical Publishing Company,2015)。
透明质酸的保湿效果与分子量呈正相关性，高分子量透明质酸具有更好的保湿、成膜及 润滑性能。当前护肤品中添加的高分子量透明质酸通常大于800kDa(80万道尔顿)，涂于皮肤表面不仅具有良好的保湿效果，同时还可起到提拉紧致的作用，可有效改善皮肤粗糙度，使肌肤更为细腻平滑(Biomatrix Inc.Hyaluronan Vol.2:Biomedical,Medical and Clinical Aspects,pp.285-288；Publisher:Woodhead Publishing Limited,2002)。
透明质酸的分子量分布很广泛，不同分子量具有不同的理化和生物性能。有研究表明分子量小于500KDa的中低分子量透明质酸可少量透过皮肤表皮浅表角质层，水合软化角质层，与表皮深层的内源性透明质酸之间建立桥梁通道，有利于营养物质的吸收和代谢废物的排出。透明质酸的透皮吸收性能与分子量呈负相关性，分子量的降低有助于透皮吸收。研究还表明小分子透明质酸(6～20个二糖重复单元的寡糖，亦即分子量为2.4KDa～8KDa)还可轻微改善血运(促进微小血管生成)、促进成纤细胞生长及内源性大分子透明质酸的分泌合成。因此，含有低分子量透明质酸的护肤品可在一定程度上起到修复滋养皮肤基底的效果(Farwick等，SOFW-Journal 2008,134:1-6；Smejkalova等，Harry’s Cosmeticology 9th Edition Vol.2.Part 4.1.3,pp.605-622；Publisher:Chemical Publishing Company,2015)。
因此，现有的透明质酸护肤品通常含有多种不同分子量的透明质酸，以起到综合的皮肤保护效果。CN 106109265B公开了一种透明质酸保湿组合物，分子量为1,100kDa的透明质酸或其盐的重量百分数为60-62％，分子量为400kDa的透明质酸或其盐的重量百分数为18-20％，分子量为6kDa的水解透明质酸或其盐的重量百分数为18-20％。CN 106137786B公开了一种透明质酸抗衰组合物，分子量为7KDa的水解透明质酸或其盐的重量百分含量为60％～62％，分子量为320KDa透明质酸或其盐的重量百分含量为18％～20％，分子量为1,100KDa的透明质酸或其盐的重量百分含量为18％～20％。CN 101450028A公开了一种高效保湿的透明质酸原液，由平均分子量大于2,600KDa的高分子量透明质酸钠和分子量小于10KDa以下的低分子量透明质酸钠配比组成。CN 101500535A公开了用于化妆品和医学用途的具有几种透明质酸级分的组合物，包含至少两种透明质酸级分或其盐，其中一种级分具有8KDa～100KDa的平均分子量，另一种级分具有100KDa～500KDa的平均分子量。
此外，透明质酸还可淬灭活性氧自由基，自身同时被破坏分解，具有弱的抗炎作用；然而，该等淬灭活性氧自由基的能力很弱，不足以有效清除活性氧自由基(Yamazaki等，Pathophysiology 2003,9:215-220.；Stern等，Biotechnology Advances 2007,25:537-557；Serban等，Biomaterials 2008,29:1388-1399；CN 106137786B)。
因此，当前的透明质酸护肤品虽然在一定程度上改善了皮肤老化的部分症状，但并不能有效清除活性氧自由基，未能有效消除导致皮肤老化的根源性因素。
发明内容
为了解决现有技术中透明质酸用于皮肤保护时不能有效清除活性氧自由基的问题，本申请提供了一种新型透明质酸皮肤保护组合物，不仅具有透明质酸保湿等护肤功能，同时还具有增强的抗氧化能力，可有效清除活性氧自由基，因而可更加有效地改善皮肤老化症状和更好解决皮肤老化问题。
本发明通过以下技术方案实现：
一种透明质酸皮肤保护组合物，含有：
1)透明质酸巯基化衍生物；和
2)至少一种分子量的透明质酸或其盐。
本发明所述的透明质酸或其盐包括钠盐、钾盐、锌盐、钙盐等，优选钠和钾盐，特别优选钠盐。
在本发明中，所采用的透明质酸巯基化衍生物含有巯基，可有效抑制由活性氧自由基直接引起的氧化应激，可防止脂质、蛋白质及DNA的损伤；此外，还可以通过与二价铁离子络合而间接抑制羟基自由基等的形成。
本发明所述的透明质酸巯基化衍生物是指含有巯基的透明质酸衍生物，可通过透明质酸或其盐的巯基化改性制备，也包括各种透明质酸衍生物进一步通过巯基化改性所制备的巯基化衍生物。透明质酸或其盐或其衍生物的侧链羧基、侧链羟基、以及还原性端基等通常是可进行巯基化改性的活性官能团，WO 2009/006780A1公开了多种透明质酸巯基化衍生物制备方法。
透明质酸侧链羧基的巯基化改性可采用氨基(酰肼基团)/碳化二亚胺偶合化学方法，碳化二亚胺包括1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐等。其通常的方式是侧链羧基在碳化二亚胺的活化下形成中间产物，含有二硫键的二氨或二酰肼亲核取代生成中间产物，最后二硫键还原为巯基即可得到透明质酸巯基化衍生物(Shu等，Biomacromolecules 2002,3:1304-1311；Aeschlimann等，US 7,196,180B1)；亦可采用含有巯基(或巯基保护)的伯胺代替含有二硫键的二氨或二酰肼，即可得到(或将所得到的中间产物脱除巯基保护基团)透明质酸巯基化衍生物(Gianolio等，Biocongjugate Chemistry 2005,16:1512-1518；Kafedjiiski等，International Journal of Pharmaceutics 2007,343:48-58)。侧链羧基的巯基化改性的另一种方法是直接与含有二硫键的碳二亚胺(如2,2’-二硫代双(N-乙基(N’-乙基碳二亚胺)))反应制备(Bulpitt等，US 6,884,788)。
透明质酸侧链羟基的巯基化改性可采用碱性条件下羟基与环硫乙烷的亲核开环反应制备(Serban等，Biomaterials 2008,29:1388-1399)。透明质酸侧链羟基的巯基化改性亦可采用间接的方式进行，如羟基首先在碱性条件进行羧甲基化，然后再进行羧基的巯基化改性(Prestwich等，WO 2005/056608A1)。
透明质酸的还原性端基也是可进行巯基化改性的活性基团，可通过化学方式引入末端巯基(Inoue等，Carbohydrate Research 1985,141:99-110)。此外，透明质酸衍生物，如CN 109206537A所公开的乙酰化透明质酸钠等，亦可采用上述巯基化改性方法得到本发明所述的透明质酸巯基化衍生物。
在本发明中，所采用的透明质酸巯基化衍生物中的巯基含量可通过Ellman’s试剂法或 1H NMR吸收谱图等方法测定(Shu等，Biomacromolecules 2002,3:1304-1311)，表示为每克透明质酸巯基化衍生物中含有的巯基的微摩尔数(μmol/g)。透明质酸巯基化衍生物的巯基化接枝度通常定义为每100个透明质酸二糖重复链段中所引入巯基数量的百分比。因为透明质酸巯基化衍生物二糖重复链段的分子量近似等于400Da，因此可通过巯基含量与巯基化接枝度可相互换算。例如当巯基含量为125μmol/g时，每100个二糖链段中引入的巯基数量为5个，亦即巯基化接枝度为5％。
在本发明中，各种巯基化接枝度的透明质酸巯基化衍生物均可用于制备本发明所述的透明质酸皮肤保护组合物，如Shu等报道的巯基化接枝度(巯基含量)为26.8％～66.8％(670μmol/g～1670μmol/g)的透明质酸巯基化衍生物(Shu等，Biomacromolecules 2002,3:1304-1311)，Serban等报道的巯基化接枝度(巯基含量)为4％～14％(100μmol/g～350μmol/g)的巯基化衍生物(Serban等，Biomaterials 2008,29:1388-1399)，Shu等所报道巯基化接枝度(巯基含量)≤4.5％(≤112.5μmol/g)的透明质酸巯基化衍生物(US 2013/0338099A1)，以及CN101622017B中所公开的多种不同巯基含量的透明质酸巯基化衍生物。
在本发明中，巯基含量是透明质酸巯基化衍生物抗氧化能力的决定性因素。与未改性透明质酸比较，本发明所采用透明质酸巯基化衍生物的抗氧化能力显著增强，其清除自由基的能力可增强至10～40倍，甚至更高。一方面，巯基含量的提高有助于增强活性氧自由基的清除能力；但在另一方面，当巯基含量较少时，有助于更好保持透明质酸的原始结构及透明质酸本身所具有的护肤功能。因此，本发明所采用的透明质酸巯基化衍生物综合了上述两方面因素，巯基含量通常小于500μmol/g，优选小于100μmol/g，特别优选在20μmol/g～50μmol/g之间。
在本发明中，巯基含量在很大程度上决定了透明质酸巯基化衍生物的抗氧化能力，分子 量对其抗氧化能力的影响很小。在本发明中，所采用的透明质酸巯基化衍生物的分子量通常在1KDa～10,000KDa之间，优选在1.6KDa～3,000KDa之间，特别优选在2.4KDa～1,500KDa之间。
在本发明中所述的分子量是指平均分子量。
在本发明所述的透明质酸皮肤保护组合物中，所含的透明质酸巯基化衍生物的浓度通常小于5mg/ml，优选小于2mg/ml，特别优选小于1mg/ml。
在本发明所述的透明质酸皮肤保护组合物中，也可以含有两种或两种以上透明质酸巯基化衍生物，这些巯基化衍生物可以具有不同的分子量和/或巯基含量。
在本发明所述的透明质酸皮肤保护组合物中，还同时含有至少一种分子量的透明质酸或其盐，其分子量通常在1KDa～10,000KDa之间，优选在1.6KDa～3,000KDa之间，特别优选在2.4KDa～2,800Kda之间。
在本发明所述的透明质酸皮肤保护组合物中，当含有两种或两种以上分子量的透明质酸或其盐时，优选其中至少一种透明质酸或其盐的分子量在1.6KDa～500KDa之间且其中至少一种透明质酸或其盐的分子量在800KDa～3,000KDa之间，特别优选其中至少一种透明质酸或其盐的分子量在2.4～8KDa之间且其中至少一种透明质酸或其盐的分子量在1,000KDa～2,800KDa之间。
在本发明所述的透明质酸皮肤保护组合物中，所含的透明质酸或其盐的浓度通常小于10mg/ml，优选小于8mg/ml，特别优选小于4mg/ml。
在本发明中，所述的透明质酸皮肤保护组合物中还可以添加维生素、氨基酸以及传统保湿剂(如山梨醇、聚乙二醇等)和抗氧化剂等物质。
本发明的透明质酸皮肤保护组合物通常可采用本领域技术人员所熟知的方式进行终端灭菌(如湿热灭菌)，可不添加防腐剂，可有效杜绝防腐剂所带来的危害。另一方面，如果需要，在本发明的透明质酸皮肤保护组合物中亦可适当加入本领域技术人员所熟知的防腐剂/抑菌剂。
与CN106137786B所公开的透明质酸抗衰组合物相比较，本发明组合物的自由基清除能力更为显著，自由基清除率可提高100％～1000％以上，因而本发明组合物用于皮肤保护时更具有独特的抗衰优势。
本发明的另一个目的是提供所述的透明质酸皮肤保护组合物的制备方法。所述的透明质酸皮肤保护组合物的制备方法，其特征在于各组分按照设定比例混合均匀即得。为了更好保持在本发明组合物中所含透明质酸巯基化衍生物的抗氧化能力，各组分混合均匀后亦可在惰 性气氛保护下灌装于密封容器。
本发明的再一个目的是提供所述的透明质酸皮肤保护组合物的应用。所述的透明质酸皮肤保护组合物的应用是指皮肤清洁、护理、化妆品中的应用，具有保湿和抗氧化的功效。
所述的透明质酸皮肤保护组合物不仅具有透明质酸本身的保湿等护肤功效，同时还具有强抗氧化功效，可有效清除活性氧自由基，因而可有效改善皮肤老化症状和较好解决皮肤老化问题；可用于所有形式的化妆品和清洁产品中，包括柔肤水、精华液、啫喱、乳液、膏霜、面膜、彩妆、香皂、洗面奶和沐浴露。
本发明的有益效果是：
本发明的组合物含有透明质酸巯基化衍生物，可有效淬灭活性氧自由基等活性氧自由基，抑制其直接引起的氧化应激，防止脂质、蛋白质及DNA的损伤，同时还可以间接抑制羟基自由基等活性盐自由基的形成。此外，本发明的组合物还含一种分子量或多种不同分子量的透明质酸或其盐，可形成不同分子量的组合，充分发挥不同分子量透明质酸的保湿、软化角质层及修复滋养皮肤基底等多重皮肤保护效果。
本发明组合物用于皮肤保护时具有独特的优势，不仅具有透明质酸本身的多重皮肤保护效果，同时还具有强抗氧化能力，可有效清除活性氧自由基，因而可在有效改善皮肤老化症状，如增加皮肤弹性及减轻皱纹等，具有良好的皮肤保护效果。
附图说明
图1.不同浓度及巯基含量的透明质酸巯基化巯基化衍生物对DPPH自由基的清除率。
图2.不同分子量透明质酸巯基化巯基化衍生物对DPPH自由基的清除率。
图3.透明质酸皮肤保护组合物(含有一种分子量的透明质酸钠)对DPPH自由基的清除率。
图4.透明质酸皮肤保护组合物(含有两种分子量的透明质酸钠)对DPPH自由基的清除率。
图5.透明质酸皮肤保护组合物的保湿性能。
图6.透明质酸皮肤保护组合物对皮肤弹性的影响。
图7.透明质酸皮肤保护组合物对皮肤皱纹深度的影响。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解， 下列实施例仅用于说明本发明，而不应视为限定本发明的范围。
实施例1：不同巯基含量透明质酸巯基化衍生物清除自由基的抗氧化作用
以1,1-二苯基-2-三硝基苯肼(DPPH)测试法评价本发明所采用的透明质酸巯基化衍生物的自由基清除性能。DPPH被广泛用于生物化学物质抗氧化能力的定量测定；此法是根据DPPH自由基有单电子，在波长为517nm处有一强吸收，其醇溶液呈紫色的特性；当有自由基清除剂存在时，由于与其单电子配对而使其吸收逐渐消失，其褪色程度与其接受的电子数量成定量关系，因而可用分光光度计进行快速的定量分析(Sharma等，Food Chemistry 2009,113:1202-1205)。
透明质酸巯基采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为18μmol/g、38μmol/g、57μmol/g、108μmol/g、266μmol/g、416μmol/g和530μmol/g。
透明质酸钠原料及上述透明质酸钠巯基化衍生物分别溶于磷酸盐缓冲溶剂(pH 7.4)，得到最终浓度为0.1mg/mL、0.5mg/mL或1mg/mL的测试样品。
用无水乙醇配置0.1mmol/L的DPPH溶液，避光保存。
将2mL测试样品溶液及2mL DPPH溶液加入到同一试管中，摇匀，室温下暗处静置30min后测定其在517nm处的吸光度(A1)；同时测定2mL DPPH溶液与2mL磷酸盐缓冲溶剂混合后在517nm处的吸光度(A0)以及2mL测试样品溶液与2mL无水乙醇混合后其在517nm处的吸光度(A2)。
自由基清除能力按如下公式计算：
Figure PCTCN2019127012-appb-000001
本实施例的结果参见图1。测试结果表明DPPH自由基的清除率与测试样品中的巯基数量呈正相关性，巯基化透明质酸钠衍生物的清除率比透明质酸钠原料提高了约10～40倍。
实施例2：不同分子量透明质酸巯基化衍生物清除自由基的抗氧化作用
透明质酸巯基化衍生物分别以180KDa、300KDa和1,500KDa的透明质酸钠为原料，采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为38μmol/g、33μmol/g和40μmol/g。
透明质酸钠原料及上述透明质酸钠巯基化衍生物分别溶于磷酸盐缓冲溶剂(pH 7.4)，得到下表所述测试样品溶液。
Figure PCTCN2019127012-appb-000002
按照实施例1所述的DPPH法测定上述样品溶液清除自由基的抗氧化作用。
本实施例的结果参见图2。测试样品1、3和5为不同分子量的透明质酸钠对照溶液，DPPH自由基清除率低；测试样品2、4和6为不同分子量的透明质酸巯基化衍生物的测试样品溶液，具有相同的巯基含量(38×10 -3μmol/mL)，DPPH自由基清除率相近，均比对照溶液增强约15倍。
实施例3：透明质酸皮肤保护组合物的制备(含有一种分子量的透明质酸钠)
所使用的透明质酸巯基化衍生物以180KDa的透明质酸钠为原料，采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为18μmol/g、38μmol/g、57μmol/g、108μmol/g、266μmol/g、416μmol/g和530μmol/g。
所使用的透明质酸钠的分子量为1.6KDa、3KDa、8KDa、50KDa、180KDa、500KDa、800KDa、1,500KDa、2,700KDa。
上述任意一种巯基含量的透明质酸巯基化衍生物溶于蒸馏水，得到6mg/mL的溶液；上述任意一种分子量的透明质酸钠溶于蒸馏水，得到12mg/mL的溶液；然后按照0.9:0.1～0.1:0.9的体积比混合均匀，即得到不同配比的透明质酸皮肤保护组合物。在该等组合物中，透明质酸巯基化衍生物的含量为5.4mg/mL～0.6mg/mL之间的任一浓度，透明质酸钠的含量为1.2mg/mL～10.8mg/mL之间的任一浓度。
在所得到的组合物中，按照1:0.08～0.08:1的体积比加入蒸馏水，混合均匀即可得到稀释后的组合物。在该稀释组合物中，透明质酸巯基化衍生物的含量为5.0mg/mL～0.04mg/mL之间的任一浓度，透明质酸钠的含量为0.09mg/mL～10.0mg/mL之间的任一浓度。
上述组合物在惰性气氛(氮气)保护下灌入玻璃安瓶，封口待用。
实施例4：透明质酸皮肤保护组合物(含有一种分子量的透明质酸钠)清除自由基的抗氧化作用
选取10种实施例3所制备的透明质酸保护组合物(其组成如下表所示)，根据实施例1 所描述的方法测定其清除自由基的抗氧化作用。
Figure PCTCN2019127012-appb-000003
以相同分子量的透明质酸钠原料替代各组合物中的透明质酸巯基化衍生物作为对照。
本实施例的结果参见图3。各配比组合物均具有显著的清除自由基抗氧化能力，比对照增强了3.4～26倍。
实施例5：透明质酸皮肤保护组合物的制备(含有两种分子量的透明质酸钠)
所使用的透明质酸巯基化衍生物以180KDa的透明质酸钠为原料，采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为18μmol/g、38μmol/g、57μmol/g、108μmol/g、266μmol/g、416μmol/g和530μmol/g。
所使用的透明质酸钠的分子量为1.6KDa、3KDa、8KDa、50KDa、180KDa、500KDa、800KDa、1,500KDa、2,700KDa。
上述任意一种巯基含量的透明质酸巯基化衍生物溶于蒸馏水，得到6mg/mL的溶液；上述任意两种分子量的透明质酸钠(任意重量比)溶于蒸馏水，得到总浓度为12mg/mL的溶液；然后按照0.9:0.1～0.1:0.9的体积比混合均匀，即得到不同配比的透明质酸皮肤保护组合物。在该等组合物中，透明质酸巯基化衍生物的含量为5.4mg/mL～0.6mg/mL之间的任一浓度，透明质酸钠的总浓度为1.2mg/mL～10.8mg/mL之间的任一浓度。
在所得到的组合物中，按照1:0.08～0.08:1的体积比加入蒸馏水，混合均匀即可得到稀释后的组合物。在该稀释组合物中，透明质酸巯基化衍生物的含量为5.0mg/mL～0.04mg/mL之间的任一浓度，透明质酸钠的总含量为0.09mg/mL～10.0mg/mL之间的任一浓度。
上述组合物在惰性气氛(氦气)保护下灌入玻璃安瓶，封口待用。
实施例6：透明质酸皮肤保护组合物(含有两种分子量的透明质酸钠)清除自由基的抗氧化作用
选取10种实施例5所制备的透明质酸保护组合物(其组成如下表所示)，根据实施例1 所描述的方法测定其清除自由基的抗氧化作用。
Figure PCTCN2019127012-appb-000004
以相同分子量的透明质酸钠原料替代各组合物中的透明质酸巯基化衍生物作为对照。
试验结果参见图4。各配比组合物均具有显著清除自由基的抗氧化能力，比对照增强了3.5～12倍。
实施例7：透明质酸皮肤保护组合物的制备(含有三种及以上分子量的透明质酸钠)
所使用的透明质酸巯基化衍生物以180KDa的透明质酸钠为原料，采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为18μmol/g、38μmol/g、57μmol/g、108μmol/g、266μmol/g、416μmol/g和530μmol/g。
所使用的透明质酸钠的分子量为1.6KDa、3KDa、8KDa、50KDa、180KDa、500KDa、800KDa、1,500KDa、2,700KDa。
上述任意一种巯基含量的透明质酸巯基化衍生物溶于蒸馏水，得到6mg/mL的溶液；上述任意三种及以上分子量的透明质酸钠(任意重量比)溶于蒸馏水，得到总浓度为12mg/mL溶液；然后按照0.9:0.1～0.1:0.9的体积比混合均匀，即得到不同配比的透明质酸皮肤保护组合物。在该等组合物中，透明质酸巯基化衍生物的含量为5.4mg/mL～0.6mg/mL之间的任一浓度，透明质酸钠的总含量为1.2mg/mL～10.8mg/mL之间的任一浓度。
在所得到的组合物中，按照1:0.08～0.08:1的体积比加入蒸馏水，混合均匀即可得到稀释后的组合物。在该稀释组合物中，透明质酸巯基化衍生物的含量为5.0mg/mL～0.04mg/mL之间的任一浓度，透明质酸钠的总含量为0.09mg/mL～10.0mg/mL之间的任一浓度。
上述组合物在惰性气氛(氮气)保护下灌入玻璃安瓶，封口待用。
实施例8：透明质酸皮肤保护组合物的制备(含有两种及以上透明质酸巯基化衍生物和 三种及以上分子量的透明质酸钠)
透明质酸巯基化衍生物分别以180KDa、300KDa和1,500KDa的透明质酸钠为原料，采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为38μmol/g、33μmol/g和40μmol/g。
所使用的透明质酸钠的分子量为1.6KDa、3KDa、8KDa、50KDa、180KDa、500KDa、800KDa、1,500KDa、2,700KDa。
上述任意两种及以上透明质酸巯基化衍生物(任意重量比)溶于蒸馏水，得到6mg/mL的溶液；上述任意三种及以上分子量的透明质酸钠(任意重量比)溶于蒸馏水，得到总浓度为12mg/mL溶液；然后按照0.9:0.1～0.1:0.9的体积比混合均匀，即得到不同配比的透明质酸皮肤保护组合物。在该等组合物中，透明质酸巯基化衍生物的含量为5.4mg/mL～0.6mg/mL之间的任一浓度，透明质酸钠的总含量为1.2mg/mL～10.8mg/mL之间的任一浓度。
在所得到的组合物中，按照1:0.08～0.08:1的体积比加入蒸馏水，混合均匀即可得到稀释后的组合物。在该稀释组合物中，透明质酸巯基化衍生物的含量为5.0mg/mL～0.04mg/mL之间的任一浓度，透明质酸钠的总含量为0.09mg/mL～10.0mg/mL之间的任一浓度。
上述组合物在惰性气氛(氮气)保护下灌入玻璃安瓶，封口待用。
实施例9：透明质酸皮肤保护组合物的保湿性能
选取实施例6中的透明质酸皮肤保护组合物(组合物编号5和6及其各自对照品)，将组合物0.5mL均匀涂布于在健康志愿者左右手臂内侧3×3cm 2的试验区域。在涂抹前及涂抹后1、2和4小时时测定受试区域皮肤的含水量(Cornrometer CM825)。
皮肤水分相对增加量按如下公式计算：
Figure PCTCN2019127012-appb-000005
试验结果参见图5。各配比组合物均具有良好的保湿能力，其保湿性能与对照相近，表明透明质酸巯基化衍生物不影响透明质酸的保湿性能。
实施例10：透明质酸皮肤保护组合物对皮肤弹性的影响
采用CN106137786B所公开的类似方法测定透明质酸组合物对皮肤弹性的影响。每组招募受试者6人，将透明质酸皮肤保护组合物(实施例6中组合物编号8和9及其各自对照品)涂抹在面颊部处，轻轻按摩至全部吸收。每天早晚净面后各一次，持续4周。分别于涂抹前 及涂抹后4周用皮肤弹性测定仪MPA580(Courage+Khazaka公司，德国)测定面颊部位(苹果肌)的弹性变化状况。
本测试的弹性指数包括初弹性、净弹性和生物弹性，以该等三种弹性指数的平均值来表征皮肤弹性；皮肤弹性相对增加率(％)按如下公式计算：
Figure PCTCN2019127012-appb-000006
试验结果参见图6。各配比组合物均明显增加了皮肤弹性，且比对照更为显著。
实施例11：透明质酸皮肤保护组合物对皮肤皱纹的影响
采用CN106137786B所公开的类似方法测定透明质酸组合物对皮肤皱纹的影响。每组招募受试者6人，将透明质酸皮肤保护组合物(实施例6中组合物编号8和9)涂抹在眼角处，轻轻按摩至全部吸收。每天早晚净面后各一次，持续4周。分别在使用前和使用后4周时用人体皮肤快速三维成像系统对受试者右眼皱纹深度进行评估。
皱纹平均深度相对减少值(％)按如下公式计算：
Figure PCTCN2019127012-appb-000007
试验结果参见图7。各配比组合物均明显减少了皱纹深度，且比对照更为显著。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受实施例的限制，其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式，都包含在本发明的保护范围之内。

Claims (25)

  1. 一种透明质酸皮肤保护组合物，其特征在于，该皮肤保护组合物含有：
    1)透明质酸巯基化衍生物；和
    2)至少一种分子量的透明质酸或其盐。
  2. 根据权力要求1所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的巯基含量小于500μmol/g。
  3. 根据权力要求2所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的巯基含量小于100μmol/g。
  4. 根据权力要求3所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的巯基含量在20μmol/g～50μmol/g之间。
  5. 根据权力要求1所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的分子量在1KDa～10,000KDa之间。
  6. 根据权力要求5所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的分子量在1.6KDa～3,000KDa之间。
  7. 根据权力要求6所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的分子量在2.4KDa～1,500Kda之间。
  8. 根据权力要求1所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的浓度小于5mg/ml。
  9. 根据权力要求8所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的浓度小于2mg/ml。
  10. 根据权力要求9所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸巯基化衍生物的浓度小于1mg/ml。
  11. 根据权力要求1所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸或其盐为钠盐。
  12. 根据权力要求1所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸或其盐的分子量在1KDa～10,000KDa之间。
  13. 根据权力要求13所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸或其盐分子量在1.6KDa～3,000KDa之间。
  14. 根据权力要求14所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸或其盐分子量在2.4KDa～2,800KDa之间。
  15. 根据权力要求1所述的透明质酸皮肤保护组合物，其特征在于，该皮肤保护组合物同时 含有两种或两种以上分子量的透明质酸或其盐。
  16. 根据权力要求15所述的透明质酸皮肤保护组合物，其特征在于，至少一种所述的透明质酸或其盐的分子量在1.6KDa～500KDa之间且至少一种所述的透明质酸或其盐的分子量在800KDa～3,000KDa之间。
  17. 根据权力要求16所述的透明质酸皮肤保护组合物，其特征在于，至少一种所述的透明质酸或其盐的分子量在2.4～8KDa之间且至少一种所述的透明质酸或其盐的分子量在1,000KDa～2,800KDa之间。
  18. 根据权力要求1所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸或其盐的浓度小于10mg/ml。
  19. 根据权力要求18所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸或其盐的浓度小于8mg/ml。
  20. 根据权力要求19所述的透明质酸皮肤保护组合物，其特征在于，所述的透明质酸或其盐的浓度小于4mg/ml。
  21. 一种如权利要求1-20中任一项所述的透明质酸皮肤保护组合物的制备方法，其特征在于，各组分混合均匀即得。
  22. 一种如权利要求21所述的透明质酸皮肤保护组合物的制备方法，其特征在于，各组分混合均匀后在惰性气氛保护下灌装于密封容器。
  23. 一种如权利要求1-20中任一项所述的透明质酸皮肤保护组合物在皮肤清洁、护理和化妆产品中的应用。
  24. 根据权利要求23所述的应用，其特征在于，所述的皮肤清洁、护理和化妆产品选自柔肤水、精华液、啫喱、乳液、膏霜、面膜、彩妆、香皂、洗面奶或沐浴露。
  25. 根据权利要求24所述的应用，其特征在于，所述的皮肤保护组合物在皮肤清洁、护理和化妆品中发挥保湿、清除自由基抗氧化、恢复皮肤弹性及减轻皮肤皱纹的功效。
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