1. A hyaluronic acid-superoxide dismutase conjugate, wherein hyaluronic acid and superoxide dismutase are linked by a covalent bond, and the conjugate has the following structural formula:

HA-(SOD)_n；

wherein n is a natural number of 1-200.

2. The hyaluronic acid-superoxide dismutase conjugate as claimed in claim 1, wherein the molecular weight of the hyaluronic acid is 2kDa to 150 kDa;

preferably, the molecular weight of the hyaluronic acid is 5kDa to 120 kDa;

further, the hyaluronic acid has a molecular weight of 5kDa, 50kDa or 120 kDa;

specifically, the molecular weight of the hyaluronic acid is 5kDa, and the number of superoxide dismutase is 1 or 6;

specifically, the molecular weight of the hyaluronic acid is 50kDa, and the number of superoxide dismutase is 13 or 65;

specifically, the molecular weight of the hyaluronic acid is 120kDa, and the number of superoxide dismutase is 31 or 158.

3. The method for preparing hyaluronic acid-superoxide dismutase conjugate according to claim 1 or 2, wherein the method comprises the steps of: hyaluronic acid, superoxide dismutase and sodium cyanoborohydride are put into a reaction system for crosslinking reaction.

4. The method of preparing a hyaluronic acid-superoxide dismutase conjugate as claimed in claim 3, wherein the sodium cyanoborohydride is recrystallized sodium cyanoborohydride in the following manner: reacting NaBH₃Dissolving CN in tetrahydrofuran to obtain NaBH₃CN solution, dripping the solution into dioxane with the volume of 1-3 times of that of the solution, and separating out crystals;

preferably, the volume of tetrahydrofuran is selected from the group consisting of dissolved NaBH₃Minimum solvent volume of CN, volume of dioxane reagent NaBH₃Twice the volume of the CN solution;

further, after the solution is dripped into dioxane, standing overnight at room temperature to filter out crystals, and cleaning and drying the obtained filtered crystals by using dioxane.

5. The method for preparing the conjugate of hyaluronic acid-superoxide dismutase as claimed in claim 3, wherein the reaction system is hexafluoroisopropanol and NaHCO₃The preparation method comprises the following steps: hexafluoroisopropanol was reacted with 0.1M NaHCO₃According to a volume ratio of 2-3: 1 to 2, and the pH value is 6.5 to 7.5.

6. The method for preparing a hyaluronic acid-superoxide dismutase conjugate as claimed in claim 3, wherein the preparation method further comprises a purification process of the product, the purification process comprising ultrafiltration, or ultrafiltration and measurement of the HA-SOD purity;

preferably, the ultrafiltration adopts an ultrafiltration tube, and the rotation speed is 2500-;

preferably, the HA-SOD purity detection method comprises the following steps: and detecting the mass ratio of HA in the concentrated liquid of the ultrafiltration tube by a carbazole sulfuric acid method, wherein when the mass ratio of HA is not reduced any more, the result shows that the uncrosslinked HA in the product is completely removed, and the crosslinking reaction is finished.

7. A pharmaceutical composition comprising the hyaluronic acid-superoxide dismutase conjugate according to claim 1 or 2.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is in the form of a formulation comprising: a solution, suspension, emulsion, cream, liniment, plaster, ointment, gel, foam, patch, plaster, powder or paste;

preferably, the pharmaceutical composition further comprises necessary carriers, including an oil phase, a water phase or an excipient;

further, the oil phase includes hydrocarbons, esters, animal and vegetable oils and fats, waxes, acerola oil, higher fatty acids, higher alcohols, silicone-based substances, sterols and resins, and the above-mentioned substances by enzymatic or chemical treatment;

further, the excipient is a compound including, but not limited to, a thickener, a gelling agent, a neutralizing agent, an occlusive agent, an antioxidant, a buffer, a pH adjuster, a filler, an emulsifier, a co-emulsifier, an emollient, a solvent, a stabilizer, a solubilizer, a hardening agent, a suspending agent, a binder, a tackifier, a penetration enhancer, a preservative, a chelating agent, a disintegrant, a plasticizer, a humectant, or a perfume.

9. Use of the hyaluronic acid-superoxide dismutase conjugate according to claim 1 or 2, the pharmaceutical composition according to claim 7 or 8 for the preparation of a skin care product.

10. Use of the hyaluronic acid-superoxide dismutase conjugate, pharmaceutical composition for the preparation of a skin care product, as claimed in claim 9, wherein the skin repair product comprises but is not limited to a medicament, a wash care product, a cosmetic or a dermatological care agent;

preferably, the medicament is a skin external preparation; further, the skin external preparation is used for inflammation, dryness, radiation-related skin symptoms including xeroderma syndrome, dermatitis, radiation dermatosis or sunburn;

preferably, the personal care products include, but are not limited to, facial or body cleansing products, care products; the cleaning product comprises shampoo, shower gel, facial cleanser, shaving cream, hand sanitizer and perfumed soap; the care products comprise face cream, hand cream and body milk.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

According to the aging free radical theory, free radical damage to the skin is an important factor in the aging process of the skin. Free radicals generated in vivo react with fatty acid to generate malondialdehyde, which reacts with proteins on cell membrane to form lipofuscin, which is deposited on skin to form various color spots. Free radicals can also break down collagen fibers in the epidermis, forming wrinkles. Therefore, the damage effect of free radicals to the skin is great, and measures can be taken to reduce the generation of free radicals of the skin, effectively remove the generated free radicals, keep the moisture of the skin and effectively slow down the aging of the skin.

Superoxide dismutase (SOD) is an effective scavenger of superoxide anion free radicals, and is applied to cosmetics at present, but the effect is not obvious because the SOD is easy to inactivate, so that a modifier for stabilizing the SOD is urgently needed.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a superoxide dismutase (SOD) conjugate modified by Hyaluronic Acid (HA), wherein the pH range of SOD adaptation can be enlarged after the SOD is modified by the hyaluronic acid, the hyaluronic acid crosslinked on the SOD can generate steric hindrance, proteolytic enzyme is blocked from approaching the SOD, and sensitive bonds of SOD molecules can be covered, so that the SOD is prevented from being damaged by the proteolytic enzyme. The invention obtains the conjugate of conjugate and superoxide dismutase by controlling different reaction conditions, and the conjugate has good moisture retention, antioxidant activity, anti-inflammatory activity and radiation protection activity.

Based on the technical effects, the invention provides the following technical scheme:

the invention firstly provides a hyaluronic acid-superoxide dismutase conjugate, wherein hyaluronic acid and superoxide dismutase are connected through a covalent bond, and the conjugate has the following structural formula:

HA-(SOD)_n；

wherein n is a natural number of 1-200.

Secondly, the present invention also provides a preparation method of the hyaluronic acid-superoxide dismutase conjugate, the preparation method comprising the steps of: mixing Hyaluronic Acid (HA), superoxide dismutase (SOD) and sodium cyanoborohydride (NaBH)₃CN) is put into a reaction system to carry out crosslinking reaction.

In the above reaction system, the cyano sodium borohydride (NaBH)₃CN) is used as a reducing agent in the reaction system, and plays a role in activating the hyaluronic acid in the reaction system. The research of the inventor finds that sodium cyanoborohydride (NaBH)₃CN) by adopting recrystallization operation, trace NaBH can be removed₄，NaBH₄Can reduce aldehyde group very quickly, and further prevent aldehyde group from participating in reductive amination reaction with slower speed. Therefore, the recrystallization operation can effectively improve the activation degree of the hyaluronic acid and the connection strength of the hyaluronic acid and the superoxide dismutase, thereby improving the moisturizing performance of the conjugate.

Based on the research results, the hyaluronic acid-superoxide dismutase conjugate provided by the invention can adjust the conjugation degree and molecular weight of SOD according to the use requirements, has good moisturizing, antioxidant, anti-inflammatory and radiation-proof effects, and can effectively play the effects of moisturizing, antioxidant and the like when being applied to the fields of functional cosmetics and skin external drugs.

The beneficial effects of one or more technical schemes are as follows:

1. the technical scheme of the invention prepares 6 HA-SOD conjugates in total by controlling the molecular weight and the reaction time of hyaluronic acid; according to the invention, the effect evaluation shows that the conjugation effect of HA and SOD is not simply and linearly related, and when the molecular weight of HA is 5kDa and the conjugation number of SOD is 6, the conjugate HAs better moisturizing effect and antioxidant effect.

2. In the research aiming at the preparation method, the invention provides a preparation method capable of adjusting the conjugation number of superoxide dismutase. In addition, the invention also discovers that the cross-linking effect of hyaluronic acid and superoxide dismutase can be effectively improved and the moisturizing performance can be improved by the recrystallization treatment of the reducing amine.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1: HA-SOD moisture retention test;

FIG. 2: HA-SOD was tested for DPPH free radical scavenging activity.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, superoxide dismutase is an effective scavenger of superoxide anion radicals, but its own poor stability is not ideal for cosmetic applications. In order to solve the above technical problems, the present invention proposes a hyaluronic acid-superoxide dismutase conjugate, wherein hyaluronic acid provides abundant binding sites for superoxide dismutase and effectively provides stability of superoxide dismutase.

In a first aspect of the present invention, there is provided a hyaluronic acid-superoxide dismutase conjugate, wherein hyaluronic acid and superoxide dismutase are linked by a covalent bond, and the conjugate has the following structural formula:

HA-(SOD)_n；

wherein n is a natural number of 1-200.

In a preferred embodiment of the first aspect, the molecular weight of the hyaluronic acid in the conjugate is 2kDa to 150 kDa. It is known in the art that hyaluronic acid is a disaccharide glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine, and hyaluronic acid with the above molecular weight can form a conjugate satisfying the technical effects of the present application, and as the molecular weight of hyaluronic acid increases, the sites on the surface of hyaluronic acid capable of binding superoxide dismutase also increase, i.e. the value of N in the above structural formula also increases.

In view of the above-mentioned use of the conjugate in the field of skin external preparations or biomedicines, the molecular weight of hyaluronic acid is more preferably in the range of 5kDa to 120kDa, and the molecular weight of commercially available hyaluronic acid products is generally a fixed number, and thus in some specific embodiments of the present invention, the molecular weight of hyaluronic acid is 5kDa, 50kDa, or 120 kDa.

In a more effective embodiment of the first aspect of the present invention, the molecular weight of the hyaluronic acid is 5kDa and the number of superoxide dismutase (SOD) is 1 or 6.

In yet another specific embodiment, the molecular weight of the hyaluronic acid is 50kDa and the number of superoxide dismutase is 13 or 65.

In yet another specific embodiment, the molecular weight of the hyaluronic acid is 120kDa and the number of superoxide dismutase is 31 or 158.

In a second aspect of the present invention, there is provided a method for preparing the hyaluronic acid-superoxide dismutase conjugate according to the first aspect, the method comprising the steps of: mixing Hyaluronic Acid (HA), superoxide dismutase (SOD) and sodium cyanoborohydride (NaBH)₃CN) is put into a reaction system to carry out crosslinking reaction.

Therefore, in a preferred embodiment of the second aspect, the sodium cyanoborohydride is recrystallized sodium cyanoborohydride (NaBH)₃CN), the recrystallization mode is as follows: reacting NaBH₃Dissolving CN in tetrahydrofuran to obtain NaBH₃And CN solution, namely dripping the solution into dioxane with the volume being 1-3 times that of the solution, and separating out crystals.

Further, the volume of the tetrahydrofuran is dissolved NaBH₃Minimum solvent volume of CN, the skilled person can apply dropwise addition until all NaBH₃CN can be dissolved to obtain the NaBH₃The volume of CN solution and dioxane reagent is preferably NaBH₃Twice the volume of the CN solution.

Further, after the solution is dripped into dioxane, standing overnight at room temperature to filter out crystals, and cleaning and drying the obtained filtered crystals by using dioxane.

Preferably, the reaction system is hexafluoroisopropanol and NaHCO₃The preparation method comprises the following steps: hexafluoroisopropanol was reacted with 0.1M NaHCO₃According to a volume ratio of 2-3: 1 to 2, and the pH value is 6.5 to 7.5.

Preferably, the temperature of the crosslinking reaction is 35-39 ℃, and the crosslinking reaction is stirred and mixed by magnetic force.

A specific implementation mode of the preparation method comprises the following steps: the molecular weight of the HA is 5kDa, and when the reaction time is 24 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 24 hours at 37 ℃ to obtain HA-SOD1 (the sum of SOD conjugates is 1).

In another specific embodiment, the preparation method comprises the following steps: the molecular weight of the HA is 5kDa, and when the reaction time is 48 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 48 hours at 37 ℃ to obtain HA-SOD2 (the sum of SOD conjugates is 6).

In another specific embodiment, the preparation method comprises the following steps: the molecular weight of the HA is 50kDa, and when the reaction time is 24 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 24 hours at 37 ℃ to obtain HA-SOD3 (the sum of SOD conjugates is 13).

In another specific embodiment, the preparation method comprises the following steps: the molecular weight of the HA is 50kDa, and when the reaction time is 48 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel and stirred by magnetic force,reacting at 37 ℃ for 48h to obtain HA-SOD4 (the sum of SOD conjugates is 65).

In another specific embodiment, the preparation method comprises the following steps: the molecular weight of the HA is 120kDa, and when the reaction time is 24 hours, the preparation method comprises the following steps:

and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted at 37 ℃ for 24h to obtain HA-SOD5 (the sum of SOD conjugates is 31).

In another specific embodiment, the preparation method comprises the following steps: the molecular weight of the HA is 120kDa, and when the activation time is 48 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 48 hours at 37 ℃ to obtain HA-SOD6 (the sum of SOD conjugates is 158).

Preferably, the preparation method further comprises a purification process of the product, wherein the purification process comprises ultrafiltration or ultrafiltration and detection of the HA-SOD purity.

Further, the ultrafiltration adopts an ultrafiltration tube, and the rotation speed is 2500-.

In the ultrafiltration process, the molecular weight cut-off of the ultrafiltration tube is adjusted according to the molecular weight of the product, the adjustment is based on the molecular weight of the SOD and HA which are not removed after the reaction, and the molecular weight cut-off can be determined by a person skilled in the art according to a conventional research idea.

Further, the HA-SOD purity detection method comprises the following steps: and detecting the mass ratio of HA in the concentrated liquid of the ultrafiltration tube by a carbazole sulfuric acid method, wherein when the mass ratio of HA is not reduced any more, the result shows that the uncrosslinked HA in the product is completely removed, and the crosslinking reaction is finished.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the hyaluronic acid-superoxide dismutase conjugate of the first aspect.

The pharmaceutical composition may be in the form of the following formulation: solutions, suspensions, emulsions, creams, liniments, plasters, ointments, gels, foams, patches, plasters, powders, pastes and the like. Preferably, the pharmaceutical composition further comprises an essential carrier. The "essential carrier" as referred to herein mainly means an auxiliary material necessary in the skin external product, and mainly includes an oil phase, a water phase, an excipient and the like.

Further, the oil phase includes, for example, hydrocarbons, esters, animal and vegetable oils and fats, waxes, acerola oil, higher fatty acids, higher alcohols, silicones, sterols and resins, and the above-mentioned materials treated by enzymes (e.g., hydrolysis and transesterification) or chemicals (e.g., transesterification and hydrogenation); specific examples are almond oil, nut oil, avocado oil, baobab oil, camellia oil, carrot oil, castor oil, citronella oil, coconut oil, cranberry oil, grape seed oil, hemp seed oil, jojoba oil, macadamia nut oil, meadow foam seed oil, oat emollient, raspberry seed oil, rose hip oil, soybean oil, rapeseed oil, corn oil, sesame seed oil, cottonseed oil, safflower oil, sunflower seed oil, peanut oil, germ oil, macadamia nut oil, garlic oil, camellia oil, palm oil, olive oil, linseed oil, eucalyptus oil, evening primrose oil, tortoise oil, mink oil, lard, tallow, horse oil, snake oil, fish oil, egg yolk oil; liquid paraffin, isoparaffin, vaseline, squalane, squalene, turpentine, polyethylene glycol, isopropyl myristate, isopalmitoyl myristate, 2-octyldodecyl myristate, hexadecyl 2-ethylhexanoate, tri-2-ethylglyceryl hexanoate, glyceryl tricaprylate, mixed fatty acid triglycerides of octanoic acid and decanoic acid, neopentyl glycol di-2-ethylhexanoate, diisostearyl malate, isononanoate (3,5, 5-trimethylhexyl-3 ', 5' -trimethylhexanoic acid), cholesterol 12-hydroxystearate, dipentaerythritol and mono-to hexaesters of isostearic acid and/or higher fatty acids, glycerol esters of p-methoxycinnamic acid and 2-hexanoic acid, isooctyl p-methoxycinnamate, isooctyl turpentine, polyethylene glycol, isopropyl myristate, 2-octyldodecyl myristate, cetyl 2-ethyldecyl 2-ethylhexanoate, cetyl 2-trimethylhexyl hexanoate, glycerol esters of p-methoxycinnamate and 2-hexanoic acid, and mixtures of these esters, Glyceryl tristearate, rosin, cholesterol, phytosterol, lanolin, myristic acid, palmitic acid, isopalmitic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, ricinoleic acid, 12-hydroxystearic acid, 10-hydroxystearic acid, behenic acid, erucic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, myristyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, lanolin alcohol, paraffin, microcrystalline wax, beeswax, shea butter, petrolatum, stearin, carnauba wax, candelilla wax, rice bran wax, sunflower wax, berry wax, bayberry wax, japan wax, shellac, dimethyl polysiloxane, methylphenyl polysiloxane.

The specific selection of the above components in the carrier depends on the preparation form of the pharmaceutical composition, and the oil phase component in solid form such as ointment and emulsion can be selected from one or more of vegetable oil, animal oil, butter and wax. The water-soluble ointment preparation at least comprises one polyethylene glycol component.

Further, the excipient is a compound including, but not limited to, a thickener, a gelling agent, a neutralizing agent, an occlusion agent, an antioxidant, a buffer, a pH adjuster, a filler, an emulsifier, a co-emulsifier, an emollient, a solvent, a stabilizer, a solubilizer, a hardening agent, a suspending agent, a binder, a tackifier, a penetration enhancer, a preservative, a chelating agent, a disintegrant, a plasticizer, a humectant, a perfume, and the like.

Examples of such antioxidants are ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, tocopherol (vitamin E), Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), carotene or panthenol (coenzyme Q).

Examples of such gelling agents are tragacanth, sodium alginate, pectin, silicates (e.g. fumed silica), gelatin, cellulose derivatives (e.g. methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose), carbomers, guar gum, polyvinyl alcohol clays and the like.

Examples of such penetration enhancers are oleic acid, lecithin, urea, clove oil, isopropyl myristate, menthol, carvacrol, linalool, limonene, geraniol, nerol, propylene glycol dipentaerythritol or cyclodextrins.

Examples of the antibacterial preservative are methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzoic acid, phenylmercuric nitrate, benzalkonium chloride, chlorhexidine acetate, benzyl alcohol or mercury.

Examples of such chelating agents are citric acid or maleic acid.

Examples of such humectants are polyethylene glycol, glycerol or sorbitol.

Examples of such fragrances are lavender oil, rose oil, lemon oil, lime oil, bergamot oil, orange oil, petgrarine oil, tangerine oil, chamomile oil, cinnamon oil, sage oil, cypress oil, geranium oil, ginger oil, juniper oil, marjoram oil, myrtle oil, neroli oil, pine oil, sandalwood oil, ylang-ylang oil, cedar wood oil, jasmine oil, frankincense oil, myrrh oil, patchouli oil, sandalwood oil or acanthopanax oil.

Examples of such emulsifiers are alkyl sulfates, soaps, dodecylbenzene sulfonates, lactates, sulfosuccinates, monoglyceride sulfonates, phosphate esters, silicones, taurates, and the like.

In a fourth aspect of the invention, there is provided a use of the hyaluronic acid-superoxide dismutase conjugate of the first aspect and the pharmaceutical composition of the third aspect for the preparation of a skin care product.

Preferably, the skin repair product includes, but is not limited to, a pharmaceutical, a wash care product, a cosmetic, or a leather care agent.

In the above technical solution, the medicament is preferably an external preparation for skin, and further, the external preparation for skin is used for treating inflammation, dryness and radiation-related skin symptoms, such as xeroderma syndrome, dermatitis, radiation dermatosis, sunburn and the like.

Such personal care products include, but are not limited to, facial or body cleansing products, care products; examples of such cleansing products are shampoos, body washes, facial cleansers, shaving creams, hand sanitizers, soaps, and the like; examples of such care products include, but are not limited to, face creams, hand creams, body milks, and the like.

In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.

Example 1

In the present embodiment, a method for preparing HA-SOD1(SOD conjugate number is 1) is provided:

s1 recrystallization of sodium cyanoborohydride

Reacting NaBH₃Dissolving CN in tetrahydrofuran, filtering, dropping tetrahydrofuran solution into dioxane of twice volume, mixing at room temperature overnight, filtering to obtain crystal, washing with dioxane, and vacuum drying.

S2 preparation of HA-SOD conjugate

The molecular weight of the HA is 5kDa, and the volume ratio of the HA to the HA is 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 24 hours at 37 ℃ to obtain HA-SOD1 (the sum of SOD conjugates is 1).

S3 purification of hyaluronic acid modified-superoxide dismutase conjugate

(1) And (3) ultrafiltration: performing ultrafiltration centrifugation by using a 15mL ultrafiltration tube with the molecular weight cutoff of 30kDa at the rotating speed of 2500-; each centrifugation process reduced the v-tube liquid from 5mL by about 0.5 mL.

(2) And (3) detecting the HA-SOD purity: and measuring the mass ratio of HA in the concentrated layer in the ultrafiltration tube by adopting a carbazole sulfuric acid method after each centrifugation until the mass ratio of HA is not reduced any more, and determining that the uncrosslinked HA in the product is completely removed.

Example 2

In the present example, a method for preparing HA-SOD2(SOD decorated sum is 6) is provided, which is different from example 1 as follows:

the molecular weight of the HA is 5kDa, and when the reaction time is 48 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 48 hours at 37 ℃ to obtain HA-SOD2 (the sum of SOD conjugates is 6).

Example 3

In the present example, a method for preparing HA-SOD3(SOD decorated sum is 13) is provided, which is different from example 1 in the following points:

the molecular weight of the HA is 50kDa, and when the reaction time is 24 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 24 hours at 37 ℃ to obtain HA-SOD3 (the sum of SOD conjugates is 13).

Example 4

In the present example, a method for preparing HA-SOD3(SOD decorated sum is 13) is provided, which is different from example 1 in the following points:

the molecular weight of the HA is 50kDa, and when the reaction time is 48 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 48 hours at 37 ℃ to obtain HA-SOD4 (the sum of SOD conjugates is 65).

Example 5

In this example, a method for preparing HA-SOD5(SOD conjugate number is 31) is provided, which is different from example 1 in the following points:

the molecular weight of the HA is 120kDa, and when the reaction time is 24 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted at 37 ℃ for 24h to obtain HA-SOD5 (the sum of SOD conjugates is 31).

Example 6

In this example, a method for preparing HA-SOD6(SOD decorated sum is 158) is provided, which is different from example 1 in the following points:

the molecular weight of the HA is 120kDa, and when the activation time is 48 hours, the preparation method comprises the following steps: and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; collecting 80mg HA, 80mg SOD, 30mg S1 and NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 48 hours at 37 ℃ to obtain HA-SOD6 (the sum of SOD conjugates is 158).

In this example, the moisturizing, antioxidant, anti-inflammatory and radioprotective effects of the above-mentioned combinations of hyaluronic acid-superoxide dismutase of different molecular weights were also evaluated:

(1) evaluation of moisture retention of HA-SOD: preparing 10mg/mL samples of six HA-SOD samples and glycerol, measuring at 20 deg.C, placing 200g dried allochroic silica gel at the bottom of a dryer, weighing about 3g solution, placing in a weighing dish with diameter of 3cm, placing in a silica gel dryer, and performing moisture retention experiment. The weighing dish was taken out and weighed every 24 hours, and the difference in mass of the sample before and after weighing was measured by continuously weighing 5 times. Calculating the moisture retention rate: the moisture retention rate (%) was Hn/H0 × 100% (in the formula, Hn represents the moisture content after leaving for n days, and H0 represents the moisture content before leaving).

(2) Evaluation of HA-SOD Total reducing power: weighing 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0mL of HA-SOD sample with a certain concentration into a test tube, adding PBS buffer (pH 6.60) to fix the volume to 2.5mL, adding 5mL of 1% potassium ferricyanide solution, uniformly mixing, putting into a water bath with 50 ℃ for heat preservation for 20min, then adding 5mL of 10% trichloroacetic acid, uniformly mixing, centrifuging at 5000r/min for 10min, transferring 2.5mL of supernatant into the test tube, adding 2.5mL of distilled water, 0.1% ferric trichloride, reacting at normal temperature for 5min, measuring the light absorption value at 700nm wavelength, wherein the larger the light absorption value is the stronger the reducing capability.

(3) Evaluation of HA-SOD on DPPH free radical scavenging Activity: taking HA-SOD samples with certain concentrations, wherein the HA-SOD samples are 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0mL, adding double distilled water to fix the volume to 2mL, respectively adding 2mL of 0.02% DPPH solution into each tube, replacing the samples with the double distilled water in a blank control group, and recording the light absorption values Ai and A0 of each group at 517 nm. The inhibition rate of DPPH radicals by the sample was calculated according to the formula (clearance (%) (a0-Ai)/a0 × 100%) (where Ai represents the sample group and a0 represents the blank control group).

(4) Evaluation of HA-SOD on hydroxyl radical scavenging Activity: sequentially adding 2mL of 6mmol/L FeSO4 solution, 2mL of sample solution with different concentrations and 2mL of 6mmol/L salicylic acid solution into a test tube, shaking up, standing at 37 ℃ for 15min, and adding 6mmol/L H₂O₂The reaction was started with 2mL of the solution, shaken well, left to stand at 37 ℃ for 15min, zeroed with distilled water, and the absorbance was measured at 510 nm. The hydroxyl radical inhibition rate of the sample was calculated according to the formula (clearance (%) - (a0-Ai)/a0 × 100%) (where Ai represents the sample group and a0 represents the blank control group).

(5) Evaluation of anti-inflammatory Activity of HA-SOD: the experiments were divided into the following 8 groups, each group comprising 4 parallel wells: normal control group (PBS is added); (II) HA-SOD (1-6); (III) SOD group; (IV) mixture group (SOD + HA); (V) HA group; (VI) model control group (plus PBS). The other experimental groups except the normal control group were simultaneously added with Lipopolysaccharide (LPS). According to grouping requirements, except a normal control group, in other 7 experimental groups, corresponding medicaments and LPS of each group are incubated for 24h mouse abdominal cavity macrophages, and supernatant liquid is taken and centrifuged. The level of each inflammatory factor is determined by ELISA kit of mouse TNF-alpha and IL-1 beta, and the operation process is strictly carried out according to the kit instruction.

(6) Evaluation of radiation protection of HA-SOD: cells in the logarithmic growth phase were plated at a concentration of 2000 cells per well. Cells were incubated at constant temperature for attachment. The next day, early doses were grouped as follows: normal control group; (II) HA-SOD (1-6) group; (III) SOD group; (IV) mixture group (SOD + HA); (V) HA group. After 24h of drug action, fresh medium was replaced for each well. The X-ray irradiation was carried out at an irradiation dose of 4 Gy. After irradiation, the cells were continuously placed in a constant temperature incubator for 48 hours for MTT detection.

Comparative example

In this example, NaBH is directed to sodium cyanoborohydride₃CN pretreatment was studied to determine whether NaBH needs to be performed in advance₃CN recrystallization was investigated, in this example, untreated NaBH was used directly₃CN is subjected to reductive amination:

and (2) mixing the components in a volume ratio of 3: 2 taking hexafluoroisopropanol and 0.1M NaHCO₃Putting 5mL (pH 7.0) in the reaction vessel, and mixing uniformly; 80mg of HA, 80mg of SOD and 30mg of S1 are taken as raw materials of NaBH₃CN is added into the reaction vessel, magnetically stirred and reacted for 24 hours at 37 ℃ to obtain HA-SOD 7.

Comparative results show that NaBH is absent₃CN is subjected to recrystallization treatment, HA cannot react with SOD, and preliminary conclusion is that NaBH₃CN recrystallization can be used to remove traces of NaBH₄，NaBH₄Can reduce aldehyde group very quickly, and further prevent aldehyde group from participating in reductive amination reaction with slower speed.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.