CN110841070A - Silk fibroin-based material loaded with active ingredients and preparation and application thereof - Google Patents

Silk fibroin-based material loaded with active ingredients and preparation and application thereof Download PDF

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CN110841070A
CN110841070A CN201911201961.8A CN201911201961A CN110841070A CN 110841070 A CN110841070 A CN 110841070A CN 201911201961 A CN201911201961 A CN 201911201961A CN 110841070 A CN110841070 A CN 110841070A
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silk fibroin
freeze
polyglutamic acid
based material
temperature
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王晓沁
郭文君
郑兆柱
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SUZHOU SIMEITE BIOTECHNOLOGY Co Ltd
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SUZHOU SIMEITE BIOTECHNOLOGY Co Ltd
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
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    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
    • A61K8/88—Polyamides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • A61Q19/08—Anti-ageing preparations
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    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying

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Abstract

The invention relates to the use of a composition comprising silk fibroin and polyglutamic acid as a freeze-drying excipient. The silk fibroin plays a main role of freeze-drying excipient, and the polyglutamic acid plays an auxiliary role of the freeze-drying excipient; the molecular weight of the silk fibroin is distributed below 10 ten thousand Da, and the content of the silk fibroin is not lower than 50 percent; the content of polyglutamic acid is not less than 5%. The invention also discloses a preparation method of the silk fibroin-based material loaded with the active ingredient, and the silk fibroin-based material loaded with the active ingredient has the effects of silkiness, compactness, moisture retention and anti-aging after being used as a skin care product.

Description

Silk fibroin-based material loaded with active ingredients and preparation and application thereof
Technical Field
The invention relates to the technical field of materials, in particular to a silk fibroin-based material loaded with active ingredients, and preparation and application thereof.
Background
In water, milk, cream and other aqueous cosmetics, antioxidants are added, and generally contain a plurality of active groups, and the active groups have different chemical instability tendencies, and chemical processes such as hydrolysis, enzymolysis and oxidation are frequently generated, so that the biological activity attribute of the antioxidants is lost. In addition, in water-based skin care products, in order to prevent the growth of microorganisms and to reasonably extend the shelf life of the product, preservatives are often added to the skin care products to ensure that the product does not deteriorate during use due to various contaminations. However, the preservative often has certain irritation, and long-term use of the skin care product containing the preservative can cause skin to be easily aged, reduce the oxidation resistance and the like, even cause allergy and lose the skin care function.
In recent years, a novel skin care product which is separated from dry skin and wet skin and represented by soluble collagen essence balls in Japan, longevity and nai, which not only maintains the safety and functionality of raw materials, but also improves the use convenience, appears on the market. But excipients were used in the preparation. Excipients often refer to binders and matrix supports added prior to pre-lyophilization to allow the sample to retain the shape of the molding die after lyophilization. The technology is widely applied to medicine freeze-drying, but is rarely applied to skin care products. The freeze-drying binder is generally artificial or natural high molecular polymer, inorganic substance gel, cellulose ethers, modified starch, dextran, chitosan, sodium alginate, PVP, PVA, polyethylene glycol, agar, polyamino acid, glycan or their combination. The components are not skin care functional components, so that the cost of the skin care product is increased and the safety is reduced. The Chinese patent CN 104198717B can obtain a spherical particle freeze-drying reagent for glucose detection by adding a freeze-drying protective agent and a plasticizer, and has the advantages of realization of stable storage at room temperature, transportation and the like compared with the existing liquid detection reagent. Chinese patent CN 105078783A uses 10-15% of trehalose, 10-15% of pullulan and 30-56% of mannitol as excipients to obtain a quick-dissolving collagen freeze-dried ball, and solves the problem of quick re-dissolving of collagen freeze-dried products.
The composition of Silk fibroin is similar to that of human keratin, the Silk fibroin HAs extremely strong affinity, safety and good biocompatibility with human skin, the Silk fibroin contains 18 amino acids, 11 of the components are required by human body, the activity of skin cells can be improved, skin aging is prevented and metabolism is promoted by effective supplement, the Silk fibroin HAs three different structural forms, namely Silk I, Silk II and Silk III, Silk I mainly comprise a-helical region mixed with a random coil and β -corner structure, the Silk fibroin is in a metastable state (a-helix domain, mixed with random coil and protein oil and β -turn structure, and water-soluble Silk II mainly comprises β -folded composition, HAs high stability, water-insoluble microsphere, the Silk III refers to an air-liquid interface and oil-water interface protein, CN 281A is added with a protective agent, 20-40% of a Silk fibroin, the Silk fibroin is a composite freeze-drying powder with a hydrophobic microcapsule, vitamin C is a composite freeze-drying powder with a hydrophobic protein microcapsule, vitamin C, a vitamin C is a composite freeze-drying powder with a hydrophobic microcapsule, a skin-protecting microcapsule, a vitamin C.
Therefore, how to prepare a skin care function by utilizing sufficient silk fibroin and prepare a freeze-dried material with a certain shape, particularly essence without using a freeze-dried excipient so that the freeze-dried material can be instantly dissolved in a water system to obtain an aqueous solution and can play a role of an active ingredient after being used is a technical problem to be solved urgently.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide the active ingredient loaded silk fibroin-based material and the preparation and application thereof.
The first objective of the present invention is to disclose the use of a composition comprising silk fibroin and polyglutamic acid as a freeze-drying excipient.
Further, in the composition, the mass fraction of the silk fibroin is not less than 50%, and the main molecular weight distribution of the silk fibroin is below 100 kDa.
Since the molecular weight distribution is wide, in order to describe the molecular weight range more precisely, in the present invention, the "molecular weight bulk distribution" refers to the "core region of the electrophoretic band", i.e., the molecular weight occupying more than 90% of the total silk fibroin fraction.
Further, in the composition, the mass fraction of the polyglutamic acid is not less than 5%, and the molecular weight of the polyglutamic acid is less than 100kDa, preferably 5-100 kDa.
In the composition, the silk fibroin plays a main role in freeze-drying excipient, the polyglutamic acid plays an auxiliary role in freeze-drying excipient, and meanwhile, the polyglutamic acid is beneficial to the quick dissolution of freeze-dried materials in various solutions.
The second purpose of the invention is to disclose a preparation method of silk fibroin-based material loaded with active ingredients, which comprises the following steps:
pouring a mixed solution containing polyglutamic acid, silk fibroin and bioactive components into a mould, and pre-freezing and forming, wherein the pre-freezing and forming conditions comprise that:
pre-freezing at-196 deg.C for more than 1 min; or
Pre-freezing for more than 2 hours at one or a combination of a plurality of temperatures of-5 to 80 ℃;
and then freeze-drying the pre-frozen and molded product with a certain geometric shape (non-powder shape) to obtain the silk fibroin-based material loaded with the active ingredient.
Further, the preparation method of the mixed solution containing polyglutamic acid, silk fibroin and bioactive components comprises the following steps:
respectively preparing polyglutamic acid aqueous solution and bioactive component solution, and then adding the polyglutamic acid aqueous solution and the bioactive component solution into the silk fibroin aqueous solution.
Further, in the mixed solution, the mass fraction of the silk fibroin is not less than 50%, and the main molecular weight distribution of the silk fibroin is below 100 kDa. The molecular weight of the silk fibroin is controlled by the conditions of degumming time, silk dissolving temperature and the like.
Further, in the mixed solution, the mass fraction of the polyglutamic acid is not less than 5%, and the molecular weight of the polyglutamic acid is less than 100kDa, preferably 5-100 kDa.
Further, the silk fibroin-based material loaded with active ingredients is essence, and the bioactive ingredients comprise moisturizing active ingredients except polyglutamic acid and an antioxidant; in the mixed solution, the mass fraction of the moisture-keeping active component is 0.01-20%, and the mass fraction of the antioxidant is 0.01-30%. The material containing moisture-keeping active ingredients and antioxidants has silky, compact, moisture-keeping and anti-aging effects after being used as a skin care product.
Preferably, the moisture-retaining active ingredient comprises one or more of hyaluronic acid, ceramide, synthetic polymer, collagen, glycerol and the like in any proportion.
Preferably, the antioxidant includes a synthetic antioxidant and a natural antioxidant. Artificial synthetic antioxidants such as BHA, BHT, PG, etc., and natural antioxidants such as tea polyphenols, phytic acid, SOD, glutathione, vitamin C, vitamin E, vitamin A, EGCG, coenzyme Q-10, etc.
Furthermore, the bioactive component also comprises one or a mixture of several of arbutin, tartaric acid, amino acid, polypeptide substances and the like in any proportion.
Preferably, the bioactive ingredient further comprises vitamin C.
Further, the mixed solution also comprises other auxiliary materials, including one or more of skin feel modifier, flavoring agent, essential oil, essence, trans-mucous membrane, transdermal absorption enhancer, pH regulator and the like.
Furthermore, because the pre-frozen product of the mixed solution is non-powdery, the silk fibroin-based material loaded with the active ingredients after freeze drying is not powdery in appearance, but has a certain geometric shape. These shapes include, but are not limited to, spherical, ellipsoidal, square, star, pentagram, circular, semi-circular, etc., and even combinations between different geometries.
Preferably, the prefreezing conditions include: directly dropping the mixed solution into liquid nitrogen (-196 ℃) for forming, wherein the pre-freezing time is not less than 1 minute; placing the mixed solution in a mould and then in a liquid nitrogen steam environment at (-196 ℃) for forming, wherein the pre-freezing time is not less than 1 minute; standing the mixed solution at a single constant temperature of-5 to-80 ℃ or in any combination environment among different temperatures for pre-freezing and forming, wherein the pre-freezing time at each temperature is not less than 2 hours; the mixed solutions are respectively stood in a freeze dryer with program temperature control, and the sample temperature is controlled by the freeze dryer program to be between-5 ℃ and-80 ℃ to be pre-frozen and formed in any combination environment. The pre-freezing step may also include a pre-cooling step at 2-8 deg.C for not less than 2 hr.
More preferably, the microdroplets are prepared by using a precisely quantifiable liquid separation system, and the microdroplets are dropped into liquid nitrogen and cooled by the liquid nitrogen to form the frozen reagent microspheres.
More preferably, the mixed solution is poured into a cavity of the forming die and cooled by liquid nitrogen to form a non-powdery solid with a certain geometric shape. The pre-freezing step may also include a pre-cooling step at 2-8 deg.C for not less than 2 hr.
More preferably, the mixed solution is poured into a forming mold cavity, and is cooled by liquid nitrogen steam (the temperature is-196 ℃) to form non-powdery solid with a certain geometric shape. The pre-freezing step may also comprise a pre-cooling step at 2-8 deg.C for not less than 2 hr
More preferably, the mixed solution is poured into a cavity of the forming die, and is cooled by a refrigerator (with a constant temperature of-5 to-80 ℃ or any combination of different temperatures) to form a non-powdery solid with a certain geometric shape. The pre-freezing step may also comprise a pre-cooling step at 2-8 deg.C for not less than 2 hr
More preferably, the mixed solution is poured into a cavity of the forming mold, and is subjected to gradient cooling by a pre-freezing program of a freeze dryer to form a non-powdery solid with a certain geometric shape. Freeze dryer prefreezing procedure the gradient cooling procedure was as follows: the mixed solutions are respectively stood in a freeze dryer with program temperature control, and the sample temperature is controlled by the freeze dryer program to be between-5 ℃ and-80 ℃ to be pre-frozen and formed in any combination environment. There is no vacuum. The pre-freezing step may also include a pre-cooling step at 2-8 deg.C for not less than 2 hr.
Further, before pre-freezing and forming, the method also comprises the step of pre-cooling for more than 2 hours at the temperature of 2-8 ℃.
Further, the freeze-drying mode comprises a non-temperature-control freeze-drying mode or a program freeze-drying mode, wherein the cold well temperature of the non-temperature-control freeze-drying mode is as follows: -45-80 ℃ and vacuum degree: 0.02-600 mBar, wherein the sample temperature is room temperature; the procedural freeze-drying mode comprises the following steps: -45 to-80 ℃, the sample temperature is-50 to 40 ℃, and the vacuum degree is 0.02 to 600 mBar. The sample temperature and vacuum were automatically controlled by the lyophilizer program. The freeze-drying time is comprehensively set according to the unit time deicing amount of the freeze dryer and the machine loading amount of the sample.
The third purpose of the invention is to protect the silk fibroin-based material loaded with active ingredients prepared by the preparation method.
The silk fibroin-based material loaded with active ingredients prepared by the method of the present invention is preferably essence which can be dissolved in face wash, bath lotion, shampoo, lotion, perfume, skin cleanser, makeup remover, essence, stock solution, sterile water, honey emulsion, milk emulsion, hair care emulsion, essence emulsion, moisturizing cream, foundation cream, shampoo, concealer, hair treatment cream, essence cream, pre-makeup cream, etc.
The invention also discloses application of the silk fibroin-based material loaded with the active ingredients in the field of preparation of skin care products, health care products, foods, medicines or medical appliances.
Furthermore, the silk fibroin-based material loaded with the active ingredients has the effects of silkiness, compactness, moisture retention and anti-aging after being used as a skin care product.
By the scheme, the invention at least has the following advantages:
the invention discloses a novel freeze-dried excipient composition, which comprises silk fibroin and polyglutamic acid. The silk fibroin-based material loaded with active ingredients can be prepared by utilizing the two materials, is non-powdery and easily soluble in water, can quickly release the active ingredients in the material, and has wide application prospect in the fields of skin care products, health care products, foods, medicines or medical devices.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of silk fibroin;
FIG. 2 is a graph showing the effect of reconstitution of lyophilized silk fibroin essence in water;
fig. 3 is an external view of freeze-dried silk fibroin essences of various shapes prepared in examples 1 to 4 of the present invention;
fig. 4 is a graph showing rapid dissolution of active ingredient-loaded silk fibroin lyophilized material in a cream-type daily chemical product;
fig. 5 is a topography of different silk fibroin lyophilized materials before and after water vapor treatment.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: preparation of freeze-dried silk fibroin essence
(1) Weighing 250 g of raw silk of domestic silkworm, cutting silk fiber, and the length of each section is about 15 cm. Heating 100 liters of pure water by using an electric heating tank, slowly adding 212 grams of anhydrous sodium carbonate when the pure water is heated to be nearly boiled, uniformly stirring, continuously heating until the pure water is boiled, adding raw silk, starting timing when the raw silk is completely immersed in water, stirring once every 7 minutes, respectively boiling for 30 minutes, 45 minutes, 60 minutes, 90 minutes and 120 minutes, and then taking out. Placing degummed silk into a washing machine, setting a rinsing program, washing twice by using pure water, and placing the dehydrated degummed silk into a forced air drying oven to be dried for 12-18 hours.
(2) Preparing 9.3M lithium bromide solution, adding degummed silk according to the bath ratio of the degummed silk to the lithium bromide solution 1/4, dissolving for 4 hours in a drying box with the temperature of 60 ℃, taking out, cooling to room temperature, filling the dissolved silk fibroin solution into a dialysis bag with the molecular weight cutoff of 3500, dialyzing for 48 hours with pure water, and changing water for 6 times. And filtering the dialyzed silk fibroin solution by using 5 layers of gauze to remove impurities, and placing the silk fibroin solution in a refrigerated cabinet at the temperature of 2-8 ℃ for later use. And simultaneously determining the mass fraction of the silk fibroin by using a drying and weighing method. The molecular weight of the silk fibroin processed in the step (2) is determined by SDS-PAGE, the molecular weight of the silk fibroin indicated by degumming time of 90min is listed in figure 1, and the main body of the silk fibroin is distributed below 100 kDa.
(3) And (3) further preparing the silk fibroin processed in the step (2) into a 5% (wt/V) silk fibroin solution by using deionized water.
(4) Preparing 0.36g/mL vitamin C water solution.
(5) An aqueous solution of polyglutamic acid having a molecular weight of about 100kDa at a concentration of 0.24g/mL was prepared.
(6) Mixing the vitamin C aqueous solution and the polyglutamic acid aqueous solution prepared in the steps (4) and (5) with the silk fibroin solution according to the volume ratio of 1: 1: 10, mixing and stirring uniformly.
(7) The liquid separation system capable of accurately quantifying is utilized to prepare micro-droplets, the shapes of the droplets are various, the droplets are dripped into liquid nitrogen, and the frozen reagent microspheres are formed after the droplets are cooled by the liquid nitrogen.
(8) The frozen reagent microspheres were non-lyophilized. And (3) obtaining the freeze-dried silk fibroin essence by cold trap temperature of not higher than-55 ℃ and vacuum degree of not higher than 0.05 mBar.
(9) The subjects respectively take 25 mg of freeze-dried silk fibroin essence to the palms and add 1.5mL of sterile water. As a result, as shown in fig. 2, it can be seen that the lyophilized silk fibroin essence of the present invention can be reconstituted in water, and the solution is clear. The essence aqueous solution was uniformly wiped on the face, and the subjects continuously used for 8 weeks for moisture retention and anti-aging effect evaluation.
A subject: 100 women without skin sensitivity history and aged 25-40 years, wherein the test product is randomly used by a volunteer on one half face and the other half face is blank during the test period; and randomly and evenly distributing freeze-dried silk fibroin essence prepared from silk fibroin with degumming time of 30min, 45min, 60min, 90min and 120min to 100 subjects, namely 20 subjects in each composite microsphere freeze-dried powder.
The using method comprises the following steps: the freeze-dried silk fibroin essence prepared from silk fibroin with different degumming time is redissolved to obtain a solution, and the solution is uniformly smeared on the face and is used once a day in the morning and at night. The application is continued for 8 weeks.
And (4) recording return visits: the evaluation statistics of the compact and silky effects are shown in tables 1 and 2, wherein the test subjects are revisited after 8 weeks, and the test subjects are queried and recorded for the effect of the use.
Table 1 evaluation of tightening effect of lyophilized silk fibroin essence prepared from silk fibroin having different molecular weights
Figure BDA0002296095320000071
Table 2 evaluation of silky slipping effect of lyophilized silk fibroin essence prepared from silk fibroin having different molecular weights
Figure BDA0002296095320000072
Example 2: preparation of freeze-dried silk fibroin essence
(1) The silk fibroin solution was prepared according to the method of example 1, steps (1) to (3), wherein the degumming time was 90 min.
(2) A mixed solution of vitamin C, polyglutamic acid and silk fibroin was prepared according to the method of steps (4) to (6) of example 1.
(3) Pouring the mixed solution into a cylindrical, heart-shaped, disc, semicircle or square die, and pre-freezing and forming by liquid nitrogen or nitrogen (the temperature is-196 ℃).
(4) And (5) freeze-drying the program. A main drying stage: the temperature is increased to minus 20 ℃, the temperature is 0.2 ℃ per minute, the vacuum degree is 100mTorr, and the temperature is maintained for 2400 minutes; final drying stage: the temperature was raised to 4 deg.C, 0.2 deg.C per minute, and the vacuum was maintained at 100mTorr for 620 minutes.
Example 3: preparation of freeze-dried silk fibroin essence
Lyophilized silk fibroin essence was prepared according to the method of example 2, except that, in the step (3), pre-freezing molding was performed using a refrigerator, the freezing temperature of which was-80 ℃.
Example 4: preparation of freeze-dried silk fibroin essence by programmed pre-freezing method
The lyophilized silk fibroin essence was prepared according to the method of example 2, except that in the step (3), a programmed pre-freezing method was adopted, and the specific steps were as follows: the mixed solution is poured into a cylinder, a heart-shaped mold, a disc, a semicircle and a square mold, the mold is placed in a freeze dryer, and the pre-freezing program is formed by gradient cooling: the temperature is changed from room temperature to-45 ℃, the temperature is quickly recorded at 0.1 ℃ per minute, and the maintaining time is 480 minutes.
Fig. 3 is a photograph of lyophilized silk fibroin essences of various shapes, which can be cylindrical, circular, spheral, square, heart-shaped, etc., prepared in examples 1 to 4 of the present invention.
Example 5: preparation of freeze-dried silk fibroin essence carrying active ingredients
(1) The silk fibroin solution was prepared according to the method of example 1, steps (1) to (3), wherein the degumming time was 90 min.
(2) Vitamin C and polyglutamic acid solutions were prepared in the same manner as in steps (4) to (5) of example 1. In addition, a solution of bioactive components is prepared, such as synthetic antioxidants (e.g. BHA, BHT, PG, etc.), natural antioxidants (e.g. tea polyphenols, phytic acid, SOD, glutathione, vitamin C, vitamin E, vitamin A, EGCG, coenzyme Q-10, etc.), arbutin, tartaric acid, amino acid solution. Preparing moisture-keeping active ingredient solution, such as hyaluronic acid, ceramide, synthetic polymer, collagen protein, and glycerol solution.
(3) Adding polyglutamic acid solution, bioactive component solution, and moisture keeping active component solution into silk fibroin solution, adding skin feeling modifier, correctant, essential oil, essence, trans-mucosa, transdermal absorption enhancer and pH regulator, and mixing. Wherein the silk fibroin accounts for 50% of the total dry weight of the raw materials of the mixture, the polyglutamic acid accounts for 20% of the total dry weight of the raw materials of the mixture, the total sum of the bioactive components accounts for 20% of the total dry weight of the raw materials of the mixture, the total sum of the moisture-preserving active components accounts for 9% of the total dry weight of the raw materials of the mixture, and the total sum of the skin feel modifier, the flavoring agent, the essential oil and the essence, the trans-mucous membrane, the transdermal absorption.
(4) The mixed solution is poured into a cylinder, a heart-shaped mold, a disc, a semicircle and a square mold, the mold is placed in a freeze dryer, and the pre-freezing program is formed by gradient cooling: the temperature is changed from room temperature to-45 ℃, the temperature is quickly recorded at 0.1 ℃ per minute, and the maintaining time is 480 minutes.
(5) And (5) freeze-drying the program. A main drying stage: the temperature is increased to minus 20 ℃, the temperature is 0.2 ℃ per minute, the vacuum degree is 100mTorr, and the temperature is maintained for 2400 minutes; final drying stage: the temperature is raised to 4 ℃, the temperature is 0.2 ℃ per minute, the vacuum degree is 100mTorr, and the temperature is maintained for 620 minutes, so that the freeze-dried silk fibroin essence carrying the active ingredients is obtained. The obtained product is integrated, does not drop slag, does not have scraps, and has a full shape and a smooth surface.
The prepared freeze-dried silk fibroin essence carrying the active ingredients can be respectively dissolved in liquid: face washing lotion, bath lotion, shampoo, cosmetic water, perfume, skin cleaning water, makeup remover, essence, stock solution, sterile water, etc.; emulsion: honey, milk, hair care cream, essence cream; creams and ointments: moisturizing cream, foundation cream, shampoo, concealer, hair treatment cream, essence cream, pre-makeup cream, etc. Fig. 4 is a diagram showing that the freeze-dried silk fibroin essence carrying the active ingredient can be rapidly dissolved in the cream type daily chemical products, and the freeze-dried silk fibroin essence carrying the active ingredient can be dissolved in the cream type daily chemical products within 1 minute.
The prepared freeze-dried silk fibroin essence carrying the active ingredient is added into 10mL of sterile water, and the obtained freeze-dried silk fibroin essence is rapidly disintegrated, completely dissolved, free of particles and free of agglomeration.
Placing the prepared freeze-dried silk fibroin essence carrying the active ingredient in a palm, adding about 1mL of sterile water, gently kneading uniformly, then applying the obtained product on the face, drying, and then ensuring smooth and firm feeling without residue or debris.
Example 6: preparation of freeze-dried silk fibroin essence carrying active ingredients
(1) A Silk fibroin (Silk) solution was prepared according to the method of steps (1) to (3) of example 1, wherein the degumming time was 90 min.
(2) A50 mg/mL Vitamin C (VC) solution and a 50mg/mL polyglutamic acid (PGA) solution were prepared.
(6) The silk fibroin solution, the vitamin C solution and the polyglutamic acid solution are mixed according to different proportions, and the statistics of the shaping effect, the solubility, the shaping effect after water vapor treatment, the skin feel grade evaluation and the like of the freeze-dried essence corresponding to the final proportions of the silk fibroin solution, the vitamin C solution and the polyglutamic acid solution are shown in table 3.
In table 3, the dry state shaping effect is classified into 1, 2, and 3: wherein the grade 1 is a whole body after freeze-drying, has no scraps and broken slag, is full in shape and has a smooth surface; the 2 level is that partial chip falling or slag crushing phenomenon exists; the grade 3 shows that the chips or the slag are much, the shape is shrivelled and the shape is basically not formed. The solubility is divided into 1, 2 and 3 grades: wherein the 1 grade is that the complete dissolution time is within 1 minute; the 2 grade is that the complete dissolution time is more than 1 minute and less than 15 minutes; the complete dissolution time of grade 3 is 15 minutes or more. The shaping effect after the water vapor treatment is divided into 1, 2 and 3 grades: wherein, the grade 1 is that the original shape is still kept after the water vapor treatment; the 2-stage is that the original shape is basically kept after the water vapor treatment, and the shrinkage is slight; the grade 3 is that the original shape can not be maintained at all, and the shrinkage phenomenon is serious. The skin feel grades are comprehensively evaluated in combination with the silky and compact effects and are divided into 1, 2 and 3 grades: wherein level 1 is very comfortable; level 2 is more comfortable; level 3 is uncomfortable; "-" indicates no statistics.
TABLE 3 statistics of the shaping effect and skin feel of lyophilized silk fibroin essence with different components
Figure BDA0002296095320000091
As shown in Table 3, silk fibroin occupies 50% or more of the mass of the mixture to have a good shaping effect, and the shaping effect can be still maintained after steam treatment when the content of polyglutamic acid is more than 5%, and the skin feel is the best when the content of silk fibroin is 50% -70%, the content of polyglutamic acid is more than 5%, and the content of VC is less than 30%, and the skin feel is not counted because the formability is poor in the rest addition ratios.
In order to prove the auxiliary shaping effect of the polyglutamic acid, the appearance of the essence ball after water vapor treatment is used as an evaluation item of the shaping effect. Fig. 5 is a topography of the essence balls No. 1 and No. 3 in table 3 before and after water vapor treatment, respectively, and the results show that the appearance of the pure silk fibroin (fig. 5a) and the appearance of the essence ball added with polyglutamic acid (fig. 5b) after freeze-drying are not obviously different, but the pure silk fibroin ball is reduced after water vapor treatment, the overall shape is still spherical, but a large number of pointed structures appear on the edge (fig. 5 c); the whole ball is still spherical after the polyglutamic acid essence ball is added, the edge is clear, and the ball is complete (figure 5 d).
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims (10)

1. Use of a composition comprising silk fibroin and polyglutamic acid as a freeze-drying excipient.
2. Use according to claim 1, characterized in that: in the composition, the mass fraction of the silk fibroin is not less than 50%, and the main molecular weight distribution of the silk fibroin is below 100 kDa.
3. Use according to claim 1, characterized in that: in the composition, the mass fraction of the polyglutamic acid is not less than 5%, and the molecular weight of the polyglutamic acid is less than 100 kDa.
4. A preparation method of a silk fibroin-based material loaded with active ingredients is characterized by comprising the following steps:
pre-freezing and forming a mixed solution containing polyglutamic acid, silk fibroin and bioactive components, wherein the pre-freezing and forming conditions comprise that:
pre-freezing at-196 deg.C for more than 1 min; or
Pre-freezing for more than 2 hours at one or a combination of a plurality of temperatures of-5 to 80 ℃;
and then freeze-drying the non-powdery pre-frozen molded product to obtain the silk fibroin-based material loaded with the active ingredient.
5. The method of claim 4, wherein: in the mixed solution, the mass fraction of silk fibroin is not less than 50%, and the main molecular weight distribution of the silk fibroin is below 100 kDa.
6. The method of claim 4, wherein: in the mixed solution, the mass fraction of the polyglutamic acid is not less than 5%, and the molecular weight of the polyglutamic acid is less than 100 kDa.
7. The method of claim 4, wherein: the silk fibroin-based material loaded with the active ingredients is essence, and the bioactive ingredients comprise moisturizing active ingredients except polyglutamic acid and an antioxidant; in the mixed solution, the mass fraction of the moisture-keeping active component is 0.01-20%, and the mass fraction of the antioxidant is 0.01-30%.
8. The method of claim 4, wherein: the freeze-drying mode comprises a non-temperature-control freeze-drying mode or a program freeze-drying mode, wherein the cold well temperature of the non-temperature-control freeze-drying mode is as follows: -45-80 ℃ and vacuum degree: 0.02-600 mBar, wherein the sample temperature is room temperature; the procedural freeze-drying mode comprises the following steps: -45 to-80 ℃, the sample temperature is-50 to 40 ℃, and the vacuum degree is 0.02 to 600 mBar.
9. An active ingredient-loaded silk fibroin-based material prepared by the preparation method of any one of claims 1-8.
10. Use of the active ingredient loaded silk fibroin-based material of claim 9 in the field of preparation of skin care products, health products, foods, pharmaceuticals or medical devices.
CN201911201961.8A 2019-11-29 2019-11-29 Silk fibroin-based material loaded with active ingredients and preparation and application thereof Pending CN110841070A (en)

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CN113576944A (en) * 2021-08-09 2021-11-02 中山中研化妆品有限公司 Freeze-dried ball and preparation method thereof and skin care product
CN114903809A (en) * 2021-02-08 2022-08-16 李和伟 Single-matrix freeze-dried excipient, preparation method thereof and freeze-dried preparation for beautifying and protecting skin

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