Levo-vitamin C hyaluronic acid ester derivative and preparation method and application thereof
阅读说明：本技术 一种左旋维生素c透明质酸酯衍生物及其制备方法和应用 (Levo-vitamin C hyaluronic acid ester derivative and preparation method and application thereof ) 是由 姬胜利 郭凯 殷金岗 王盈盈 于 2021-07-14 设计创作，主要内容包括：本发明提供一种左旋维生素C透明质酸酯衍生物及其制备方法和应用,一种左旋维生素C透明质酸酯衍生物,其特征在于,其为由维生素C结构和透明质酸结构共价键合的结构,所述维生素C结构如式II所示,其中式II通过处与透明质酸结构键合,其中,k选自2-4。本发明的衍生物兼具了透明质酸和左旋维生素C的功能活性,其具有优异的保湿、祛皱和美白的效果,同时具有延长的功效,应用范围广,可应用于化妆品、药品以及食品领域。(The invention provides a levorotatory vitamin C hyaluronic acid ester derivative and a preparation method and application thereof, and the levorotatory vitamin C hyaluronic acid ester derivative is characterized in that the levorotatory vitamin C hyaluronic acid ester derivative is a structure formed by covalently bonding a vitamin C structure and a hyaluronic acid structure, wherein the vitamin C structure is shown as a formula II, wherein the formula II is formed by bonding a vitamin C structure and a hyaluronic acid structure in a covalent manner Is bonded with the hyaluronic acid structure, wherein k is selected from 2-4. The derivative has the functional activities of hyaluronic acid and levorotatory vitamin C, has excellent effects of moisturizing, wrinkle removing and whitening, has a prolonged effect, is wide in application range, and can be applied to the fields of cosmetics, medicines and foods.)
1. The levo-vitamin C hyaluronic acid ester derivative is characterized by having a structure formed by covalently bonding a vitamin C structure and a hyaluronic acid structure, wherein the vitamin C structure is shown as a formula II, and the formula II is formed byIs bonded with the hyaluronic acid structure,
wherein k is selected from 2-4.
2. The L-vitamin C hyaluronic acid ester derivative according to claim 1, wherein the hyaluronic acid structure is hyaluronic acid acetylated or not acetylated;
preferably, the acetylated hyaluronic acid has an acetyl group content of 23-29%.
3. The L-vitamin C hyaluronic acid ester derivative according to claim 1 or 2, which has a structure represented by formula I:
wherein R is1、R2、R3、R4Each independently selected from hydrogen or acetyl, m is more than or equal to 1, n is more than or equal to 0, and k is selected from 2-4;
preferably, the average molecular weight of the structure of formula I is less than 200 kDa; preferably 10kDa to 100 kDa;
preferably, the degree of substitution of the vitamin C structure in the structure of formula I is from 0.04 to 1.0; preferably 0.1 to 0.4.
4. The L-ascorbic acid hyaluronic acid ester derivative of claim 3, wherein R is1、R2、R3、R4Are both H or at least one is not H.
5. A process for the preparation of a hyaluronic acid ester derivative of l-vitamin C according to any of claims 1 to 4, comprising the preparation of a compound of formula I starting from a compound of formula III or as an intermediate;
wherein, in the compound shown in the formula III, X is a leaving group, preferably halogen, and k is an integer of 2-4;
preferably, the halogen is bromine or chlorine.
6. The method of claim 5, wherein the method comprises: taking a compound shown in a formula III as a starting material, carrying out coupling reaction on the compound shown in the formula III and hyaluronic acid-CTA salt, and then carrying out salt conversion to prepare a compound shown in a formula I;
or, the method comprises using vitamin C as the starting material, and mixing vitamin C and vitamin CCarrying out Mitsunobu reaction to obtain a compound shown in a formula III, carrying out coupling reaction on the compound shown in the formula III serving as an intermediate and hyaluronic acid-CTA salt, and then carrying out salt conversion to prepare a compound shown in the formula I, wherein X is halogen, and k is an integer of 2-4;
preferably, the vitamin C is levovitamin C;
preferably, the process for preparing the compound of formula III comprises: mixing vitamin C,Reacting 1, 2-diiodoethane with triphenylphosphine in tetrahydrofuran at room temperature;
preferably, the method for preparing the hyaluronic acid-CTA salt comprises: dissolving sodium hyaluronate or sodium acetylhyaluronate in water, adding CTA, stirring, filtering, and drying to obtain hyaluronic acid-CTA salt, wherein X is halogen and k is an integer of 2-4;
preferably, the method for preparing the levorotatory vitamin C hyaluronic acid ester derivative comprises the following steps: performing coupling reaction on a compound shown in a formula III, hyaluronic acid-CTA salt and sodium carbonate in dimethyl sulfoxide, adding the obtained product into water after the reaction is finished, and performing salt conversion on the obtained product through cation exchange resin after filtering, ultrafiltration and washing to obtain a compound shown in a formula I;
preferably, the salt conversion is completed and a lyophilization operation may be performed to obtain a lyophilized compound of formula I.
7. A compound represented by formula III:
wherein X is halogen and k is an integer of 2 to 4.
8. Use of a compound of formula III according to claim 7 for the preparation of a derivative of l-vitamin C hyaluronate according to claim 1.
9. A composition comprising or starting from a combination of one or more of the l-vitamin C hyaluronate derivatives of claim 1 or 2;
preferably, the composition is a pharmaceutical, cosmetic or food composition;
preferably, the composition may further comprise a pharmaceutically active compound, and optionally a pharmaceutically acceptable carrier;
preferably, the composition is orally, topically or parenterally applicable.
10. Use of the levovitamin C hyaluronic acid ester derivative of claim 1 or 2 or the composition of claim 9 for the preparation of a medicament or a cosmetic for treating a skin condition, or for use in a food product;
preferably, the medicament or cosmetic for treating a skin condition comprises a surgical implant, a skin external agent, a dermal filler.
Technical Field
The invention relates to the field of medicines and daily necessities, in particular to a levorotatory vitamin C hyaluronic acid ester derivative and a preparation method and application thereof.
Background
The information disclosed in this background of the invention is intended to enhance an understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known as prior art to a person skilled in the art.
The development of functional cosmetics has become the subject of the development of the cosmetic industry, wherein the added bioactive components have important regulation and control effects on the growth and metabolism of cells. The hyaluronic acid molecular structure is a repetitive structure of a disaccharide unit consisting of D-glucuronic acid and N-acetylglucosamine, has multiple important physiological functions, including prevention and repair of skin injury, promotion of wound healing and the like, can lock 1000 times of moisture of the skin per se, prevent the loss of the moisture of the skin through the epidermis, improve the metabolism of the skin, enable the skin to become smooth and elastic, is also a very good transdermal absorption promoter, and is widely used for production of beauty skin care products. The effect of the macromolecular hyaluronic acid applied to the cosmetics is mainly represented by external moisture preservation, difficult penetration into the skin and incapability of carrying out nutrition and repair on the skin, the low-molecular hyaluronic acid is easy to penetrate through cell membranes to reach the dermis layer and rebuilt macromolecules to penetrate through the skin surface layer, the viscosity is low, the usability is greatly enhanced, and the special functions which are not possessed by the common hyaluronic acid are represented, such as biological activities of promoting angiogenesis, promoting wound healing, removing wrinkles, resisting inflammation, resisting tumors, regulating immunity and the like. The low molecular hyaluronic acid can promote proliferation and differentiation of epidermal cells and remove oxygen free radicals, thereby achieving the functions of removing wrinkles and enhancing skin elasticity. The hyaluronan is a derivative of hyaluronan modified by acetylation, is a novel and efficient skin softening factor, has hydrophilicity and lipophilicity, can highly soften cuticle, has strong affinity to skin, has the water retention capacity 2 times higher than that of the hyaluronan, and has better skin care effect than the hyaluronan. The functional cosmetic containing hyaluronic acid and derivatives thereof has the advantages of safety, stability, easy absorption and good effect, and is favored by consumers.
Propylene glycol, butylene glycol, pentylene glycol, commonly known as polyols, are targeted as small molecule humectants. The cosmetic is mainly used as a moisturizing agent and a smoothing agent, and can temporarily eliminate fine wrinkles of the skin. Can enter into the keratinocyte gap deeply to achieve the effect of moisturizing the muscle base.
The levorotatory vitamin C and the derivative thereof have the capacity of inhibiting melanin formation and reducing melanin, are widely applied to whitening products, and can help skin resist ultraviolet ray damage and avoid black spots and freckles. The levorotatory vitamin C has whitening effect, and also has effects of shrinking pores and regulating skin texture. Preventing skin from being damaged after sun exposure in summer, promoting metabolism, removing melanin, and removing speckle. In addition, the levo-vitamin C has the effects of promoting collagen synthesis, promoting fibroblast proliferation, helping wound healing, removing wrinkles, resisting inflammation and oxidation and the like.
However, the permeability of the levorotatory vitamin C is poor, in a water-based formula, in order to achieve the optimal permeation effect, the pH value of the formula needs to be adjusted to be lower than the pKa 4.2 of the levorotatory vitamin C so as to reduce the charge density of molecules, the pH value applied to a skin care product is generally not more than 3.5, and skin irritation is easily caused. In addition, the structure of the levovitamin C is unstable and is easy to be oxidized and deteriorated. Factors that influence the degradation rate of levovitamin C include: solution pH, temperature, light, dissolved oxygen, and metal ions, among others, are factors that are difficult to avoid during development, use, and storage. The degradation of the levo-vitamin C is also accompanied with the yellowing and darkening of the color of the levo-vitamin C, and the appearance and the using effect of the product are greatly influenced. Measures to prevent degradation are: in the process of formulation, packaging and storage, oxygen and light are isolated, the pH value of the formulation is reduced, water and other antioxidants are used to the minimum extent, and iron and copper appliances cannot be used. Even so, the stability cannot be effectively guaranteed, and the cost and difficulty of the formula are increased.
The development of the product is limited by the defects of unstable structure, poor permeability, strong irritation, easy yellowing and the like of the levorotatory vitamin C. To avoid the above-mentioned drawbacks of levovitamin C, a series of derivatives were designed, as shown in the following table:
the comparison of the derivatives shows that the vitamin C sugar derivative (AA-2G) has outstanding advantages in the aspects of stability, transdermal property, ultraviolet injury protection, collagen synthesis promotion, skin pigmentation reduction and the like. In recent years, hyaluronic acid has been widely used as a polysaccharide, and vitamin C derivatives thereof have been proved to be better vitamin C sugar derivatives, and the products have the efficacy of hyaluronic acid. Korean CELLINBIO corporation, patent No. US20120283428a1, discloses a method for preparing hyaluronic acid vitamin C derivatives and their use for treating infantile eczema (i.e., atopic dermatitis, AD), which can also be used in cosmetics for skin injury and regeneration, but the structure is not protected from easily oxidized hydroxyl groups and does not have the effect of preventing the oxidation of vitamin C. Patent CN107412002A discloses an injectable hydrogel composition crosslinked by sodium hyaluronate, vitamin C and Star PEG applied to dermal filler, and the external effect is not mentioned, nor the preparation method is disclosed; CN110467689A discloses an application of hyaluronic acid derivatives in cosmetics or foods, wherein the hyaluronic acid derivatives can be applied in cosmetics as whitening, antioxidant and moisturizing components, and can be applied in foods as antioxidant, cosmetic and joint protecting components, and discloses a preparation method of hyaluronic acid vitamin C derivatives, but the preparation process is complex, the conditions are harsh, and the environmental protection is poor.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a levovitamin C hyaluronic acid ester derivative, and a preparation method and application thereof. The levorotatory vitamin C hyaluronic acid ester derivative has good stability and good sustained-release transdermal effect, has excellent functions of moisturizing, wrinkle removing and whitening, overcomes the defects of unstable structure, poor permeability, strong irritation, easy yellowing and the like of levorotatory vitamin C in the prior art, has the effect of hyaluronic acid, and also solves the technical problems that hyaluronic acid ester and vitamin C in functional cosmetics need to be added respectively, are easy to inactivate due to environmental influence, have short action time, reduce the activity of partial vitamin C derivatives and the like. According to the invention, the levorotatory vitamin C and the hyaluronic acid are connected through the flexible Linker, so that the reaction site is clear, the product quality is easy to control, and the stability of the levorotatory vitamin C is ensured. For the salt conversion operation of hyaluronic acid, the operation of ion exchange resin and the like is avoided, and the method is environment-friendly. The invention adopts the latest Mitsunobu reaction to carry out site-directed etherification on the levorotary vitamin C, avoids the reaction on the protecting group on the levorotary vitamin C, and has the advantages of high atom economy, high yield and environmental protection.
Specifically, the present invention provides the following technical features, and one or a combination of the following technical features constitutes the technical solution of the present invention.
In a first aspect of the invention, the invention provides a levo-vitamin C hyaluronate derivative which is formed by the aggregation of vitamin CStructure and hyaluronic acid structure are covalently bonded, the structure of the vitamin C is shown as formula II, wherein the formula II is formed byIs bonded with the hyaluronic acid structure,
wherein k is selected from 2-4.
In some embodiments of the invention, the hyaluronic acid structure is sodium hyaluronate, acetylated or not acetylated; wherein, the content of acetyl in the acetylated sodium hyaluronate is 23-29%.
In some embodiments of the invention, the structure of formula II is covalently bonded to a carboxyl group in a hyaluronic acid structure, and the bond forms an ester bond to form a levovitamin C hyaluronate derivative having the structure of formula I:
wherein R is1、R2、R3、R4Each independently selected from hydrogen or acetyl, m is more than or equal to 1, n is more than or equal to 0, k is selected from 2-4 and can be 2, 3 or 4.
In some embodiments of the invention, the average molecular weight of the derivative structure of formula I is less than 200 kDa; preferably 10kDa to 100 kDa.
Preferably, the degree of substitution of the vitamin C structure in the structure of formula I is from 0.04 to 1.0; preferably 0.1 to 0.4.
In some embodiments of the invention, R1、R2、R3、R4Are both H or at least one is not H.
For example, in some embodiments of the invention, R1、R2、R3、R4When all are H, k is 2, 3 or 4, the derivatives of the invention may have formula I1I2 or formula I3:
wherein in the compounds, m is more than or equal to 1, n is more than or equal to 0, and the average molecular weight is less than 200 kDa; preferably 10kDa to 100 kDa; the substitution degree of the levorotatory vitamin C is 0.04-1.0; preferably 0.1 to 0.4.
As another example, in yet other embodiments, R1、R2、R3、R41 or more of them are acetyl groups, and the content of acetyl groups is 23-29%.
The levorotatory vitamin C hyaluronic acid ester derivative has the functional activities of hyaluronic acid and levorotatory vitamin C, the moisturizing, wrinkle removing and whitening effects of the levorotatory vitamin C hyaluronic acid ester derivative are obviously superior to the effects of separate use and combined use of sodium hyaluronate and levorotatory vitamin C, the effects of the levorotatory vitamin C hyaluronic acid ester derivative are prolonged, and the use effect of functional cosmetics is enhanced.
In a second aspect of the present invention, the present invention provides a process for preparing the hyaluronic acid ester derivative of levovitamin C (formula I) of the first aspect, which comprises preparing a compound of formula I from a compound of formula III as a starting material or as an intermediate;
wherein, in the compound of formula III, X is a leaving group, such as halogen, and k is an integer of 2-4; preferably, the halogen is bromine or chlorine.
In some embodiments of the invention, the method comprises: taking a compound shown in a formula III as a starting material, carrying out coupling reaction on the compound shown in the formula III and hyaluronic acid-CTA salt, and then carrying out salt conversion to prepare a compound shown in a formula I;
or, the method comprises using vitamin C as the starting material, and mixing vitamin C and vitamin CCarrying out Mitsunobu reaction to obtain a compound shown in a formula III, carrying out coupling reaction on the compound shown in the formula III serving as an intermediate and hyaluronic acid-CTA salt, and then carrying out salt conversion to prepare a compound shown in the formula I; wherein k is selected from 2-4.
The vitamin C of the present invention is L-vitamin C unless otherwise specified.
The reaction for preparing the compound of formula I from the compound of formula III comprises:
wherein R is hydrogen or acetyl, m is more than or equal to 1, n is more than or equal to 0, k is selected from 2-4, and X is halogen.
In some embodiments of the invention, the method comprises: performing coupling reaction on a compound shown in a formula III, hyaluronic acid-CTA salt and sodium carbonate in dimethyl sulfoxide, adding the obtained product into water after the reaction is finished, and performing salt conversion on the obtained product through cation exchange resin after filtering, ultrafiltration and washing to obtain a compound shown in a formula I;
preferably, the salt conversion is completed and a lyophilization operation may be performed to obtain a lyophilized compound of formula I.
In some embodiments of the invention, ultrafiltration may be performed using a nanofiltration membrane having a molecular weight cut-off of 1000 Da. In some embodiments of the invention, the salt-converting is carried out by passing the CTA in the product of the coupling reaction through a cation exchange resin+Conversion of salts to Na+. The cation exchange resin may be, for example, a 732 type cation exchange resin.
In some embodiments of the present invention, the present invention provides a process for preparing a compound of formula III, comprising: mixing vitamin C,1, 2-diiodoethane and triphenylphosphine were reacted in tetrahydrofuran at room temperature.
The reaction for the preparation of the compound of formula III is shown below:
wherein X is halogen and k is an integer of 2 to 4.
In some embodiments of the invention, a method of preparing a hyaluronic acid-CTA salt comprises: dissolving sodium hyaluronate or hyaluronan in water, adding CTAC, stirring, filtering, and oven drying to obtain hyaluronic acid-CTA salt or hyaluronan-CTA salt.
In some embodiments of the invention, the CTAC of the invention is cetyltrimethylammonium chloride.
The reaction for the preparation of hyaluronic acid-CTA salt is shown below:
wherein, R is hydrogen or acetyl.
In a third aspect of the invention, the invention provides a compound of formula III:
wherein X is halogen or H, the halogen is preferably bromine or chlorine, and k is an integer of 2-4.
And the use of a compound of formula III for the preparation of a levo-vitamin C hyaluronate derivative as described in the first aspect above.
In a fourth aspect of the present invention, the present invention provides a composition comprising the above-mentioned hyaluronic acid ester derivative of levovitamin C in the first aspect or the above-mentioned hyaluronic acid ester derivative of levovitamin C in the first aspect as a raw material; in some embodiments of the present invention, the composition may contain or be prepared from one or more of the derivatives of the present invention. For example, any one, two or three of formulas I1-I3 may be included in the composition.
In some embodiments of the invention, the composition is a pharmaceutical composition, a cosmetic composition (including general cosmetics and functional cosmetics), or a food composition.
Further, in some embodiments of the present invention, when the composition is a pharmaceutical composition, the composition may further comprise other pharmaceutically active compounds, and optionally a pharmaceutically acceptable carrier. And when the composition is a cosmetic composition, other functional components and necessary auxiliary materials can be further included.
The compositions of the present invention are orally administrable, topically applicable or injectable, and those skilled in the art can prepare the compositions of the present invention into appropriate dosage forms or use forms by adding appropriate adjuvants according to conventional methods, unless otherwise specified. Such as injection, ointment, powder injection, liniment, dressing, liquid preparation, etc. As a cosmetic, the composition may include, but is not limited to, lotions, milky lotions, essences, gels, foundations, cosmetic powders and powders, lipsticks, shampoos, body washes, toothpastes, creams, and masks, etc., and the cosmetic may be applied to a range of applications including, but not limited to, head care, face care, body care, and the like. The levorotatory vitamin C hyaluronic acid ester derivative can be used for preparing cosmetics with the functions of sun screening, moisture preservation, skin nutrition supplementation, acne removal, oxidation resistance, wrinkle removal, alopecia prevention, freckle removal, skin cleaning, skin whitening and the like.
For example, in some embodiments of the present invention, there is provided a cosmetic stock solution for external use comprising the hyaluronic acid ester derivative of levovitamin C according to the above first aspect of the present invention and a base. In some embodiments of the invention, the matrix may be functional or merely an adjuvant, for example the matrix may be 5% butanediol and 0.5% PE9010 by mass fraction.
In particular, in the embodiment of the present invention, when the addition amount of the levovitamin C hyaluronic acid ester derivative is 0.001% to 25% (w/w), the moisturizing, wrinkle-removing, and whitening effects are most significant, and particularly, the addition amount (mass fraction) of the levovitamin C hyaluronic acid ester derivative may be small, for example, 0.001% to 0.5%, and a suitable amount of the levovitamin C hyaluronic acid ester derivative may be 0.5% to 5%, 0.5% to 10%, or 5% to 10%, or a relatively large amount, for example, 10% to 25%. If on the basis, the content is lower, the effect is not obvious, the content is higher, and the continuous improvement of the effect is limited.
In a fifth aspect of the invention, the invention provides the use of the hyaluronic acid ester derivative of levovitamin C as described in the first aspect above or the composition as described in the fourth aspect above for the preparation of a medicament, a medical device or a cosmetic for the treatment of a skin condition, or for use in a food product.
In embodiments of the present invention, the pharmaceutical, medical device or cosmetic for treating a skin condition includes, but is not limited to, surgical implants, skin external agents, dermal fillers, and the like.
Any one or combination of derivatives described in the above first aspect of the present invention may be applied to cosmetics as an ingredient having whitening and/or wrinkle-removing and/or water-replenishing and/or repairing effects, and may be applied to foods as an ingredient having antioxidant and/or cosmetic and/or joint-protecting effects.
Compared with the prior art, the invention has the advantages that:
in the technical scheme of the invention, in the structure of the derivative, the levovitamin C diol hyaluronic acid ester or the levovitamin C diol hyaluronan ester combines hyaluronic acid or hyaluronan and levovitamin C in a covalent bond mode. The levo-vitamin C dihydric alcohol hyaluronic acid ester or levo-vitamin C dihydric alcohol acetyl hyaluronic acid ester ensures the stability of the levo-vitamin C in the manufacturing and the preservation of the skin care product by a unique ester bond and an ether bond.
The derivative has a unique transdermal absorption mechanism, and ester bonds and ether bonds are opened under the action of hydrolase in body fluid, so that the levo-vitamin C and the hyaluronic acid or the hyaluronan are stably delivered to the dermis layer of the skin. An aqueous hyaluronic acid film is formed on the surface, and a sandwich structure is formed on two sides of the horny layer together with endogenous hyaluronic acid. The water content of the horny layer is rapidly increased to form a semipermeable membrane, the levorotatory vitamin C can easily pass through the semipermeable membrane by utilizing the osmotic pressure principle, and the levorotatory vitamin C can be slowly released, so that the skin care effects of promoting the generation of collagen, improving the immune function, resisting oxidation, whitening and the like are achieved. Besides maintaining the function of the L-vitamin C completely, the product also has the efficacy of hyaluronic acid or hyaluronan.
According to the invention, the levorotatory vitamin C and the hyaluronic acid are connected through the flexible Linker, unnecessary coupling can be avoided without a protective group in the reaction process, the reaction site is very clear, and the product quality is easy to control, so that the stability of the levorotatory vitamin C is ensured. Vitamin C has a plurality of hydroxyl groups, can be subjected to esterification reaction with carboxyl on hyaluronic acid, but the connection sites are uncertain due to direct esterification, so that the esterified product is a mixture, wherein the esterification reaction of the oxidized hydroxyl groups is difficult, the quality cannot be controlled, and the easy-oxidized sites of the levorotatory vitamin C cannot be protected. For the salt conversion operation of hyaluronic acid, the operation of ion exchange resin and the like is avoided, and the method is environment-friendly.
The invention adopts the latest Mitsunobu reaction to carry out site-directed etherification on the levorotary vitamin C, avoids the reaction of a protective group on the levorotary vitamin C, and has the advantages of high atom economy, high yield and environmental friendliness.
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. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
figure 1 is a nuclear magnetic spectrum of intermediate 1 prepared in example 2.
Fig. 2 is a nuclear magnetic spectrum of levovitamin C propylene glycol hyaluronate prepared in example 5.
FIG. 3 is a graph comparing the effect of water addition (average moisture content increase rate) in different experimental groups of example 9.
Fig. 4 is a graph comparing the wrinkle removing effects (reduction of the wrinkle area of the skin) of different experimental groups in example 9.
Fig. 5 is a graph comparing the whitening effects (skin melanin reduction amounts) of different experimental groups in example 9.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
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. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
The invention provides a levorotatory vitamin C hyaluronic acid ester derivative, which has a structure shown in a formula I:
wherein R is1、R2、R3、R4Each independently selected from hydrogen or acetyl, m is more than or equal to 1, n is more than or equal to 0, and k is selected from 2 to 4.
It can be prepared by the following reaction scheme:
in some embodiments of the invention, bromoalcohols with k of 2-4, namely bromopropanol, bromobutanol and bromopentanol, are first prepared with levovitamin C to obtain intermediate 1, intermediate 2 and intermediate 3 (structure)As follows), and then any one of the intermediates 1 to 3 and hyaluronic acid-CTA salt are used as raw materials (namely R)1、R2、R3、R4All H) to produce a product having the structure of formula I1, formula I2, and formula I3 (structures below).
Similarly, according to the same method, when acetylated hyaluronic acid-CTA salt is used as a raw material (the content of acetyl is 23-29%), the raw material and any 1 of the intermediates 1-3 are respectively subjected to coupling reaction to prepare a series of L-vitamin C acetylated hyaluronic acid ester derivatives (namely R in the structure of the formula I)1、R2、R3、R4One or more of which is acetyl).
Specifically, the preparation and properties of the derivatives of the present invention will be schematically illustrated in the following examples, it should be understood that the setting of the examples is merely illustrative and is not exhaustive, and those skilled in the art can easily prepare derivatives having the structures of the present invention or other derivatives having structures similar to the structures of the present invention according to the description of the present invention, and perform structure verification by nuclear magnetic spectroscopy.
EXAMPLE 1 preparation of hyaluronic acid-CTA salt
300g of sodium hyaluronate with a molecular weight of 10kDa to 100kDa is dissolved in 10L of deionized water for later use. 300g of cetyltrimethylammonium chloride was dissolved in 6L of deionized water, and then a sodium hyaluronate solution was added dropwise. After 16 hours at 40 ℃, a white solid precipitated. Filtering, and drying the solid at 50-55 ℃ under reduced pressure to obtain a product (the yield is 486 g).
EXAMPLE 2 etherification of L-vitamin C with Bromopropanol
211g of triphenylphosphine are added to 500mL of tetrahydrofuran under nitrogen protection, stirred until the solution is completely dissolved, and the temperature of the brine is reduced to about 0 ℃ in an ice bath. 212g of 1, 2-diiodoethane are dissolved in 200mL of tetrahydrofuran and added dropwise to the triphenylphosphine solution. After stirring for 30 minutes, a solid precipitated. Under the protection of nitrogen, 50g of L-vitamin C and 50g of 3-bromo-1-propanol were dissolved in 300mL of tetrahydrofuran and added dropwise to the reaction system. After the dropwise addition, the temperature is raised to 20-30 ℃ and stirred for 16 hours. The reaction temperature was then raised to 40 ℃ and stirred for an additional 3 hours and the completion of the reaction was detected by thin layer chromatography (ethyl acetate: ethanol: water: 20:10: 1). The reaction was concentrated until no solvent was dropped off to give a white solid. The obtained white solid was dissolved in dichloromethane and purified by column chromatography (ethyl acetate/ethanol as developing solvent) to obtain a colorless oil as intermediate 1 (yield 52g), and its nuclear magnetic spectrum is shown in fig. 1.
EXAMPLE 3 etherification of L-vitamin C with Bromobutanol
Under nitrogen protection, 230g of triphenylphosphine was added to 540mL of tetrahydrofuran, stirred to be completely dissolved, and the temperature of the brine was reduced to about 0 ℃ in an ice bath. 230g of 1, 2-diiodoethane are dissolved in 250mL of tetrahydrofuran and added dropwise to the triphenylphosphine solution. After stirring for 30 minutes, a solid precipitated. Under the protection of nitrogen, 55g of levovitamin C and 60g of 4-bromo-1-butanol are dissolved in 300mL of tetrahydrofuran and added dropwise to the reaction system. After the dropwise addition, the temperature is raised to 20-30 ℃ and stirred for 16 hours. The reaction temperature was then raised to 40 ℃ and stirred for an additional 3 hours and the completion of the reaction was detected by thin layer chromatography (ethyl acetate: ethanol: water: 20:9: 1). The reaction was concentrated until no solvent was dropped off to give a white solid. The resulting white solid was dissolved in dichloromethane and purified by column chromatography (ethyl acetate/ethanol as developing solvent) to give intermediate 2 as a colorless oil (yield 59 g).
EXAMPLE 4L-ascorbic acid Bromopentanol etherification
243g of triphenylphosphine was added to 560mL of tetrahydrofuran under nitrogen, stirred to full solution, and the brine was cooled to about 0 ℃ in an ice bath. 244g of 1, 2-diiodoethane was dissolved in 220mL of tetrahydrofuran and added dropwise to the triphenylphosphine solution. After stirring for 30 minutes, a solid precipitated. Under nitrogen protection, 57g of L-vitamin C and 70g of 5-bromo-1-pentanol were dissolved in 420mL of tetrahydrofuran and added dropwise to the reaction system. After the dropwise addition, the temperature is raised to 20-30 ℃ and stirred for 16 hours. The reaction temperature was then raised to 40 ℃ and stirred for an additional 3 hours and the completion of the reaction was detected by thin layer chromatography (ethyl acetate: ethanol: water: 20:7: 1). The reaction was concentrated until no solvent was dropped off to give a white solid. The resulting white solid was dissolved in dichloromethane and purified by column chromatography (ethyl acetate/ethanol as developing solvent) to give intermediate 3 as a colorless oil (yield 60 g).
EXAMPLE 5 preparation of L-vitamin C propylene glycol hyaluronate (formula I1)
50g of the intermediate 1 prepared in the example 2, 110g of hyaluronic acid-CTA salt and 35g of sodium carbonate are placed in 500mL of dimethyl sulfoxide and stirred at 20-30 ℃ for 8 hours, the reaction system is a clear solution, then stirring is continued for 40 hours, after the reaction is finished, the clear solution is added into 6L of water, filtering is carried out, ultrafiltration and washing are carried out through a nanofiltration membrane (with the molecular weight cutoff of 1000Da), and then the clear solution is converted into Na through 732 type cation exchange resin+And (3) freeze-drying the salt to obtain the levovitamin C propylene glycol hyaluronate, wherein the substitution degree of the vitamin C is 0.36, and the nuclear magnetic spectrum of the levovitamin C propylene glycol hyaluronate is shown in figure 2.
EXAMPLE 6 preparation of L-ascorbic acid butanediol hyaluronate (formula I2)
54g of the intermediate 2 prepared in the example 3, 110g of hyaluronic acid-CTA salt and 35g of sodium carbonate are placed in 500mL of dimethyl sulfoxide and stirred at 20-30 ℃ for 8 hours, the reaction system is a clear solution, then stirring is continued for 40 hours, after the reaction is finished, the clear solution is added into 6L of water, filtering is carried out, ultrafiltration and washing are carried out through a nanofiltration membrane (with the molecular weight cutoff of 1000Da), and then the clear solution is converted into Na through 732 type cation exchange resin+And (4) freeze-drying the salt to obtain the levovitamin C butanediol hyaluronate, wherein the substitution degree of the vitamin C is 0.32.
EXAMPLE 7 preparation of L-vitamin C pentanediol hyaluronate (formula I3)
56g of the intermediate 3 prepared in example 4, 110g of hyaluronic acid-CTA salt and 35g of sodium carbonate are placed in 500mL of dimethyl sulfoxide and stirred at 20-30 ℃ for 8 hours, the reaction system is a clear solution, then stirring is continued for 40 hours, after the reaction is finished, the clear solution is added into 6L of water, and the mixture is filtered and passes through a nanofiltration membrane (a cut-off nanofiltration membrane)Retaining molecular weight 1000Da), ultrafiltering, washing, and converting into Na with 732 type cation exchange resin+And (4) freeze-drying to obtain the levovitamin C pentanediol hyaluronate, wherein the substitution degree of the vitamin C is 0.31.
According to the method of the embodiments 1 to 7, the sodium hyaluronate in the preparation process is replaced by acetylated sodium hyaluronate (the content of acetyl is 25%), and the intermediates 1,2 and 3 are respectively adopted to prepare I4, I5 and I6, and the degrees of substitution of vitamin C are respectively 0.38, 0.37 and 0.33.
Example 8 stability test comparison
The compounds I1-I6 prepared in the above examples are subjected to light, heat and humidity stability experiments, and compared with levorotary vitamin C, the results show that the stability of the compounds is remarkably superior to that of the levorotary vitamin C; the test method and the results are shown as follows:
strong light stability test: levo-vitamin C and I1-I6 prepared in the above examples were placed in a test chamber at the same time, and placed under the condition of an illumination of 4500lx + -500 lx for 0 days, 5 days, and 10 days. The samples were taken to examine the degree of decrease in the amount of L-ascorbic acid and the degree of decrease in the amount of L-ascorbic acid in formula I1-I6, and the results are shown in Table 1.
TABLE 1
Thermal stability test: and simultaneously placing the levorotatory vitamin C and the I1-I6 prepared in the embodiment in a vacuum drying oven at 85-90 ℃ for 0h, 0.5h and 1.0 h. The samples were taken to examine the degree of decrease in the amount of L-ascorbic acid and the degree of decrease in the amount of L-ascorbic acid in formula I1-I6, and the results are shown in Table 2.
TABLE 2
And (3) testing the wet heat stability: the levorotatory vitamin C and the I1-I6 prepared in the above examples are stored for 15 days under the conditions of high temperature (37-40 ℃), high humidity (RH 75%) and iron ions at the same time. The samples were taken to determine the amount of L-ascorbic acid and the amount of L-ascorbic acid in formula I1-I6, and the results are shown in Table 3.
TABLE 3
Note: ND is not detected.
Through the comparison of the stability tests, the stability of the L-vitamin C hyaluronic acid ester derivative I1-I6 in light, heat and humid heat environments is far higher than that of the L-vitamin C.
Example 9 examination of moisturizing, wrinkle-removing and whitening effects
The moisturizing, wrinkle-removing and whitening cosmetic stock solution prepared by the above example and using the I1-I6 as a component is prepared and tested for moisturizing, wrinkle-removing and whitening effects, and the results show that the I1-I6 shows more excellent moisturizing, wrinkle-removing and whitening effects compared with sodium hyaluronate, sodium hyaluronan, levovitamin C and sodium hyaluronate or a mixture of sodium hyaluronate and levovitamin C. The test method is described below by taking I1-I3 as examples.
At room temperature, 5 mass percent of butanediol and 0.5 mass percent of PE9010 are respectively added into 7 parts of equal amount of sterile water as substrates and are respectively used for making experiment groups 1,2, 3, 4, 5, 6 and 7 for marking, sodium hyaluronate is added into the experiment group 1, levovitamin C is added into the experiment group 2, a mixture (1:1) of sodium hyaluronate and levovitamin C is added into the experiment group 3, levovitamin C propylene glycol hyaluronate (I1), levovitamin C butylene glycol hyaluronate (I2) and levovitamin C pentanediol hyaluronate (I3) prepared in examples 5, 6 and 7 are respectively added into the experiment groups 4, 5 and 6, sterile water with the same mass as that of the other groups is added into the experiment group 7 as a blank group, the experiment groups 1 to 7 are all stirred until the experiment groups 1 to 7 are completely dissolved, and adjusting the pH value to 5-7 to be used as a stock solution to be tested, wherein the stock solution to be tested is used as a test sample to be tested.
The properties of the stock solution to be tested are further illustrated by the following experimental reports.
(1) Detecting items:
and detecting the moisturizing, wrinkle removing and whitening effects of the stock solution to be detected.
(2) And (3) testing a sample:
stock solutions to be tested of the above experimental groups 1-7.
(3) And (3) measuring the using effect:
1. the moisturizing effect is as follows:
1.1 subject: 70 volunteers are selected, aged 35-40 years and tested on the skin of the forearm.
1.2 test samples: taking the stock solution to be tested, namely experimental groups 1-7 (wherein the experimental group 7 is a blank group), as a test object;
1.3 before the test: the examinee needs to wash the inner sides of the forearms of both hands uniformly with clear water, and measurement marks are made on the inner sides of the forearms of both hands of the examinee after the washing. The left and right forearms in this experiment were each marked with two test areas.
1.4 in the test: the arm of each tester is divided into 7 test areas in the marked test area, the areas are separated by 1cm, and each test area is 3 multiplied by 3cm2The amount of the test sample was 0.2 g. The subjects were each coated with one test substance in 7 test areas, and the stock solutions of the experimental groups 1 to 7 were applied to each test area. Before the stock solution is applied, the test subject sits still in a constant environment (the measurement environment temperature is 20 ℃ and the relative humidity is 50%) for 30min, and then a Corneometer CM 825 moisture tester is used for measuring blank values of the tested part, and 5 points are fixedly measured in each area according to a certain sequence to obtain an average value. Then the special person is responsible for smearing the sample, timing is started, and the MMV value change is measured at each time according to the design of the test. Average moisture content growth rate%t-MMV0)/MMV0X 100%, wherein: MMV0MMV of the skin before applicationt-MMV of the skin t time after application.
The results of using the Corneometer CM 825 moisture tester to test the stock solution 4h of experimental group 1 to experimental group 7, and the results of statistically calculating the change in the MMV value of the skin of each test article are shown in table 4 and fig. 3.
TABLE 4 change in MMV value of skin (%)
Group of
15min
30min
60min
90min
120min
180min
240min
Experimental group 1
39.2
35.5
33.6
30.3
26.3
22.6
20.2
Experimental group 2
22.7
20.4
15.5
8.3
5.5
3.1
1.2
Experimental group 3
42.3
37.1
34.5
31.8
28.7
23.7
21.6
Experimental group 4
50.8
46.6
44.3
43.2
41.3
37.6
32.4
Experimental group 5
48.5
45.2
43.3
40.5
39.2
35.4
29.8
Experimental group 6
49.9
46.7
43.7
41.8
40.4
36.1
31.6
Experimental group 7
20.7
18.8
14.5
9.4
4.3
2.1
0.8
As can be seen from table 4 and fig. 3, the change of MMV value of the skin is gradually reduced along with the increase of time, the moisturizing effect of the experimental group 7 (blank group) is the worst, and it is fully proved that the human skin cannot effectively moisturize and lock water when moisturizing substance components are not added and only a sterile water matrix is given; the water replenishing effect of the stock solutions of the experimental group 4 to the experimental group 6 is obviously higher than that of the stock solution of sodium hyaluronate (the experimental group 1) and the stock solution of levovitamin C (the experimental group 2), and simultaneously is also obviously higher than that of the mixed stock solution of sodium hyaluronate and levovitamin C (the experimental group 3), so that the obvious water replenishing effect of the levovitamin C hyaluronic acid ester derivative prepared by the invention is fully proved, and the excellent water replenishing effect of the levovitamin C hyaluronic acid ester derivative is not the result of simply mixing sodium hyaluronate and levovitamin C, but the special structure of the derivative provided by the invention is ensured. According to the invention, the L-vitamin C and the hyaluronic acid are generated in a covalent bond coupling manner through a flexible Linker, and the better characteristics are fundamentally endowed through chemical modification, so that the stability of the sodium hyaluronate and the L-vitamin C is improved, the half-life period is prolonged, the action time is prolonged, the moisturizing effect is obviously improved, and the long-term moisturizing effect is achieved.
2. Wrinkle removing effect:
2.1 subject: selecting 70 volunteers, wherein the age is 35-55 years old, dividing the volunteers into 7 groups, and carrying out skin experiments on 10 persons in each group;
2.2 test samples and methods of use thereof: the stock solutions of experiment groups 1-7, 0.5g each, were applied to the facial skin of volunteers, once a day, in the morning and evening, and continuously for 4 weeks.
Before the experiment, firstly, the facial skin wrinkle area S of the tested person before the experiment is measured by a Visoline VL 650 wrinkle tester0: the change of the skin wrinkles and the change of the facial skin wrinkle area were obtained by software analysis.
In the test, the volunteers used the test samples according to the method of use, and the wrinkle area S of the facial skin was measured by Visoline VL 650 wrinkle tester at 2 pm (ambient temperature 20 ℃ C., relative humidity 50%) every dayt: the change of the skin wrinkles and the change of the facial skin wrinkle area were obtained by software analysis.
Finally, the skin wrinkle area reduction (%) is calculated as (skin wrinkle area before test-average skin wrinkle area after test)/skin wrinkle area before test × 100%.
The results of the reduction of the wrinkle area of each group are shown in Table 5 and FIG. 4.
Table 5 wrinkle area reduction amount (%)
Group of
1 week
2 weeks
3 weeks
4 weeks
Experimental group 1
8.1
9.5
10.3
11.2
Experimental group 2
1.8
2.6
2.9
3.2
Experimental group 3
8.6
9.3
10.7
12.3
Experimental group 4
11.5
14.2
16.1
17.6
Experimental group 5
13.1
15.6
18.5
20.1
Experimental group 6
12.1
14.5
16.8
18.5
Experimental group 7
0.2
0.1
0.1
0.1
As can be seen from table 5 and fig. 4, the skin wrinkle area of the experimental group 7 (blank group) was almost unchanged (almost coincident with the abscissa axis in fig. 4), demonstrating that the human facial skin wrinkles did not disappear by themselves after forming their own wrinkles when the wrinkle-removing product was not applied; the wrinkle removing effect of the stock solutions of the experimental group 4 and the experimental group 6 is obviously higher than that of a sodium hyaluronate stock solution (the experimental group 1) and a levorotatory vitamin C stock solution (the experimental group 2) and also obviously higher than that of a mixed stock solution of sodium hyaluronate and levorotatory vitamin C (the experimental group 3), and the obvious wrinkle removing effect of the levorotatory vitamin C hyaluronic acid ester derivative prepared by the invention is fully proved to be generated by simply mixing sodium hyaluronate and levorotatory vitamin C, but the structure of the derivative is the same. The chemical modification endows the hyaluronic acid ester derivative with more excellent characteristics fundamentally, improves the stability of the sodium hyaluronate and the levorotatory vitamin C, prolongs the half-life period, and enhances the action time.
3. Whitening effect:
3.1 subject: 70 volunteers are selected, the age is 35-55 years old, the selected volunteers are divided into 7 groups, and 10 persons in each group are subjected to skin experiments.
3.2 test samples and methods of use thereof: the stock solutions of experiment groups 1-7, 0.5g each, were applied to the facial skin of volunteers, once a day, in the morning and evening, and continuously for 4 weeks.
The test conditions are as follows: (1) an indoor environment; (2) no strong sunlight or direct light; (3) the ambient temperature is 20 +/-2 ℃, and the ambient humidity is 40-60%. Face testing: whitening products (cosmetics, external medicines or oral health products) cannot be used for 2-3 days before the face.
Before the test, the subject needs to uniformly clean the facial skin and then wipe it clean with a dry facial tissue. The subject was allowed to sit still in a standard room for at least 30min with facial skin exposed, unable to rub, and kept relaxed. The melanin test was performed on the face of the subject by means of a Mexameter MX18 tester (test areas: including the frontal area 2cm above the center of the eyebrow, the chin area 2cm below the center of the lower lip, and the left and right cheek areas at the intersection of the tip of the nose and the pupil, the test environment temperature was 20 ℃ and the relative humidity was 50%), and pre-test values were obtained. The amount of melanin was then tested at the same location in the experiment at the same time point per week.
Finally, the skin melanin reduction amount (%) (skin melanin amount before test-skin melanin amount after test)/skin melanin amount before test × 100% was calculated.
The results of the reduction of melanin in each group of skin are shown in Table 6 and FIG. 5.
Table 6 skin melanin reduction amount (%)
Group of
1 week
2 weeks
3 weeks
4 weeks
Experimental group 1
8.1
10.9
13.3
14.9
Experimental group 2
12.2
14.1
17.3
19.2
Experimental group 3
15.8
18.7
20.5
22.1
Experimental group 4
20.7
29.4
35.2
38.6
Experimental group 5
19.4
28.2
33.6
35.7
Experimental group 6
18.8
29.5
34.2
37.4
Experimental group 7
0.1
0.1
0.2
0.1
As can be seen from tables 6 and 5, the skin melanin reduction amount of the experiment group 7 (blank group) is almost unchanged (almost coincides with the abscissa axis in fig. 5), and the whitening effect of the experiment group 4-experiment group 6 stock solution is significantly higher than that of the sodium hyaluronate stock solution (experiment group 1) and the levorotary vitamin C stock solution (experiment group 2), and is also significantly higher than that of the mixed stock solution of sodium hyaluronate and levorotary vitamin C (experiment group 3). The invention endows the hyaluronic acid with more excellent characteristics through chemical modification, improves the stability of the sodium hyaluronate and the levorotatory vitamin C, enhances the absorption and combination of the skin to the levorotatory vitamin C, and increases the using effect of the levorotatory vitamin C.
The L-vitamin C hyaluronic acid ester derivative has the targeting property of sodium hyaluronate, effectively promotes L-vitamin C to penetrate through skin barriers, enhances the absorption and combination of the skin on the L-vitamin C, increases the using effect of the L-vitamin C, and obviously improves the whitening effect. In the above experiment, the addition amount of the levovitamin C glycol hyaluronic acid ester of the present invention is 0.5% (w/w), and at this concentration, excellent moisturizing, wrinkle-removing, and whitening effects are exhibited. In the embodiment of the invention, other dosage conditions are tested, and as a result, the addition amount of the levorotatory vitamin C diol hyaluronic acid ester is 0.001-25% (w/w), so that the effects of moisturizing, wrinkle removing and whitening are most remarkable.
The levo-vitamin C hyaluronic acid ester derivative creatively overcomes the instability of vitamin C in the preparation and storage of skin care products, has the characteristics of compound activity, strong product efficacy and long efficacy duration, and has the using effect superior to the effect of single use of sodium hyaluronate or levo-vitamin C and the effect of combined use of sodium hyaluronate and levo-vitamin C. Also provides an application method of the composition in cosmetics and beauty products.
In conclusion, aiming at the defects of unstable structures of sodium hyaluronate and levorotatory vitamin C substances, the invention solves the technical problems that the levorotatory vitamin C has unstable structure, poor permeability, strong irritation, easy yellowing and the like, hyaluronic acid ester and vitamin C in functional cosmetics need to be added respectively, the hyaluronic acid ester and the vitamin C are easy to inactivate under the influence of environment, the action time is short, the activity of partial vitamin C derivatives is reduced and the like; the series of L-vitamin C hyaluronic acid ester derivatives prepared by the invention have the functional activities of sodium hyaluronate and L-vitamin C, the moisturizing, wrinkle removing and whitening effects of the L-vitamin C hyaluronic acid ester derivatives are obviously superior to the effects of separate use and combined use of the sodium hyaluronate and the L-vitamin C, the effects of the L-vitamin C hyaluronic acid ester derivatives are prolonged, the use effect of functional cosmetics is enhanced, the use satisfaction of users is improved, and the L-vitamin C hyaluronic acid ester derivatives can be widely applied.
The application range of the levo-vitamin C hyaluronic acid ester derivative is as follows: can be used for preparing cosmetics with skin repairing functions of sun screening, moisturizing, skin nutrition supplementing, acne removing, oxidation resisting, wrinkle removing, alopecia preventing, freckle removing, cleaning, whitening and the like; the cosmetic is in the form of aqueous solution, lotion, essence, gel, foundation, cream, and pack; the application range of the cosmetics comprises head washing, face washing, body washing and the like, and the application method of the levorotatory vitamin C hyaluronic acid ester derivative can be realized.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. 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.