CN115778886B - Glabridin plant source microcapsule inclusion and preparation method and application thereof - Google Patents
Glabridin plant source microcapsule inclusion and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a glabridin plant source microcapsule inclusion and a preparation method and application thereof, belonging to the technical field of cosmetics. The glabridin plant source microcapsule inclusion comprises the following components in percentage by weight: 0.1-10% of licorice root extract, 0.1-10% of apple extract, 0.1-10% of rice bran extract, 1-20% of grease, 1-20% of emulsifying agent, 5-40% of auxiliary emulsifying agent and the balance of water; the particle size of the glabridin plant source microcapsule inclusion is 10-200nm. According to the invention, the specific components are selected for matching, and the components perform whitening and freckle removal by different action mechanisms, so that the whitening and freckle removal effects of the glabridin plant source microcapsule inclusion can be synergistically enhanced; the plant extract is wrapped in the form of microcapsule inclusion, so that the bioavailability can be improved, and the whitening and freckle removing effects of the inclusion are further enhanced.
Description
Technical Field
The invention relates to the technical field of cosmetics, in particular to a glabridin plant source microcapsule inclusion and a preparation method and application thereof.
Background
With the pursuit of whitening and beautifying skin by modern females, scientists have been intensively studied on whitening mechanisms. The research of scientists shows that in melanosomes in skin epidermal cells, melanin generated by tyrosinase catalysis has a transfer and metabolism process closely related to skin color, and the more the generated melanin content is, the higher the accumulated melanin content in the epidermis stratum corneum is, the more the skin is blackened; and excessive melanin deposition can also lead to stain formation. Therefore, whitening skin is deep in terms of inhibiting the transmission of melanin production signals, inhibiting the production of melanin, inhibiting the transfer of melanin, and promoting the metabolism of the stratum corneum.
The whitening active ingredients on the market at present are endlessly layered, mainly take tyrosinase inhibitors as main ingredients, and reduce melanin generation by inhibiting enzyme activity; part of active ingredients such as resorcinol derivatives, vitamin A and derivatives thereof, salicylic acid, nicotinamide and the like have remarkable whitening effect, but have strong skin irritation and even phototoxicity, and are not suitable for people with thinner horny layer, sensitive muscles and the like. Therefore, the plant active whitening cosmetics which are natural in source, safe and free of stimulation are deeply favored by people with sensitive muscles and the like; however, the natural plant whitening active ingredients of the current stage still have some defects in the use process: (1) Most of plant whitening extracts extracted by water or alcohol have low concentration of active ingredients, complex composition of the active ingredients, undefined whitening mechanism and very little whitening effect; (2) The high-purity plant whitening active ingredients such as phloretin, ferulic acid and the like have poor stability, are sensitive to light, heat and oxygen, are easy to oxidize and degrade after being directly added into the skin care product for a period of time, and not only lead to the reduction of the efficacy of the skin care product, but also lead to skin irritation; (3) Part of plant whitening components have poor solubility and are difficult to be applied to water aqua skin care products; (4) Most importantly, the melanocytes exist in the basal layer of the epidermis, and the horny layer, the transparent layer, the granular layer and the thorn layer also exist on the upper part of the basal layer of the epidermis, so that the active ingredients hardly penetrate the horny layer to enter the basal layer due to the self barrier function of the skin, cannot act on the melanocyte target cells, and have low bioavailability, so that the skin care effect is poor.
Aiming at the defects of the prior whitening products, a glabridin plant source microcapsule inclusion which can fully exert the whitening and freckle removing effects and has green and safe components is developed and is a hot spot of the prior research.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a glabridin plant source microcapsule inclusion with good whitening and freckle removing effects and high bioavailability, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a glabridin plant source microcapsule inclusion, which comprises the following components in percentage by weight: 0.1-10% of licorice root extract, 0.1-10% of apple extract, 0.1-10% of rice bran extract, 1-20% of grease, 1-20% of emulsifying agent, 5-40% of auxiliary emulsifying agent and the balance of water; the particle size of the glabridin plant source microcapsule inclusion is 10-200nm.
The glabridin in the glabridin plant source microcapsule inclusion can competitively inhibit the activity of tyrosinase and prevent the combination of a substrate and tyrosinase, so that the synthesis of melanin is inhibited; the phloretin extracted from apple peel and root bark can be inserted into the active center of tyrosinase, occupy the active center site, and prevent the combination of substrate and tyrosinase, so that the catalytic activity of tyrosinase is reduced, and meanwhile, the phloretin can also be used as a transdermal penetration enhancer to increase the absorption and utilization of functional factors in inclusion; the molecular structure of ferulic acid extracted from active phenolic acid in the rice bran extract is similar to that of a tyrosinase substrate, so that tyrosinase activity can be competitively inhibited, melanin generation is inhibited, the maximum ultraviolet absorption peak is 236nm and 322nm, and the skin injury caused by ultraviolet rays in the wavelength range can be prevented or reduced, and the blackening effect of the ultraviolet rays on the skin is prevented; according to the invention, the specific components are selected for matching, and different action mechanisms are used for whitening and removing the spots, so that the whitening and removing effects of the glabridin plant source microcapsule inclusion can be synergistically enhanced.
The inventors have unexpectedly found that glabridin in the glabra extract can also inhibit the activities of dopachrome tautomerase and DHICA oxidase, and has strong anti-inflammatory, antioxidant and antiallergic effects; phloretin in apple extract has anti-inflammatory and antiallergic effects, can inhibit excessive secretion of sebum, and has good moisture keeping effect; the two components are matched for use, so that the skin whitening and freckle removing effects can be enhanced, the skin whitening and freckle removing effects can be achieved, the skin whitening and soothing effects can be achieved, the effects of inhibiting sebum secretion and moisturizing the skin can be achieved, and the skin whitening and freckle removing cream is particularly suitable for oily skin.
The glycyrrhiza glabra extract, the apple extract and the rice bran extract are all natural plant extracts with high purity, the components are natural in source, the action mechanism is clear, the efficacy is obvious, and the glycyrrhiza glabra extract, the apple extract and the rice bran extract are especially suitable for sensitive skin.
The inventor also found that when the particle size of the glabridin plant source microcapsule inclusion is in the range, the glabridin plant source microcapsule inclusion can meet the practical application requirement, has higher stability, does not change greatly after being placed for 3 months under different temperatures and ultraviolet irradiation, and does not have the phenomena of layering, precipitation and the like.
The inventor also found that when each component in the glabridin plant source microcapsule inclusion is in the weight percentage range, the prepared glabridin plant source microcapsule inclusion has good water dispersibility and stability.
As a preferred embodiment of the glabridin plant-derived microcapsule inclusion according to the present invention, the glabridin plant-derived microcapsule inclusion comprises the following components in percentage by weight: 0.5 to 8 percent of licorice root extract, 0.5 to 8 percent of apple extract, 0.5 to 8 percent of rice bran extract, 3 to 15 percent of grease, 5 to 18 percent of emulsifying agent, 10 to 35 percent of auxiliary emulsifying agent and the balance of water; the inventor finds through experiments that when each component in the glabridin plant source microcapsule inclusion is in the weight percentage range, the glabridin plant source microcapsule inclusion has good water dispersibility and stability, and meanwhile, the particle size is distributed within 10-200 nm.
As a preferred embodiment of the glabridin plant-derived microcapsule inclusion according to the present invention, the glabridin plant-derived microcapsule inclusion comprises the following components in percentage by weight: 1-5% of licorice root extract, 1-5% of apple extract, 1-5% of rice bran extract, 5-10% of grease, 8-16% of emulsifying agent, 15-30% of auxiliary emulsifying agent and the balance of water; the inventor finds through experiments that when each component in the glabridin plant source microcapsule inclusion is in the weight percentage range, the particle size of the glabridin plant source microcapsule inclusion can be further reduced, and the uniformity of the particle size distribution is improved.
As a preferred embodiment of the glabridin plant-derived microcapsule inclusion according to the present invention, at least one of the following (I) to (III) is used:
the grease comprises at least one of caprylic/capric triglyceride, palmitic triglyceride, diisopropyl sebacate, isononyl isononanoate, diisostearyl malate, diglyceryl stearate malate, isostearyl lactate, glycerol tri (ethylhexanoate), squalane, isodecyl laurate, isopropyl myristate and ethylhexyl cocoate;
(ii) the emulsifier comprises at least one of soybean lecithin, polyglycerol-10 oleate, polyglycerol-10 myristate, polyglycerol-10 diisostearate, olive oil polyglycerol-6 esters, polyglycerol-3 ricinoleate, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, PEG-10 oleate, PEG-8 caprylic/capric glycerides, PEG-12 distearate, xylitol-based glucoside, myristyl glucoside, cholesterol;
(iii) the co-emulsifier comprises at least one of octyldodecanol, PPG-26-butanol polyether-26, trideceth-12, ethoxydiglycol, beheneth-20, sorbitol polyether-30, 1, 3-propanediol, 1, 2-hexanediol, 1, 2-pentanediol, dipropylene glycol, glycerol, butylene glycol, octylglycol, propylene glycol, sorbitol.
Experiments show that when the oil, the emulsifier and the auxiliary emulsifier are in the preferable ranges, the stability of the glabridin plant source microcapsule inclusion is better, the glabridin plant source microcapsule inclusion is not separated out or layered after being placed for 3 months under different temperatures and ultraviolet irradiation, and the particle size is between 10 and 200nm, so that the practical application requirements are met.
As a more preferable embodiment of the glabridin plant-derived microcapsule inclusion in the present invention, at least one of the following (IV) to (VI) is employed:
(iv) the oil and fat comprises at least one of caprylic/capric triglyceride, palmitic triglyceride, diisostearyl malate, tri (ethylhexanoate) glycerol, squalane, isopropyl myristate, and ethylhexyl cocoate;
(v) the emulsifier comprises at least one of soybean lecithin, polyglycerol-10 oleate, polyglycerol-10 myristate, polyglycerol-10 diisostearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, PEG-10 oleate, PEG-8 caprylic/capric glycerides, cholesterol;
the auxiliary emulsifier comprises at least one of octyl dodecanol, PPG-26-butanol polyether-26, 1, 3-propanediol, 1, 2-hexanediol, 1, 2-pentanediol, dipropylene glycol, glycerol and butanediol;
Experiments show that when the oil, the emulsifier and the auxiliary emulsifier are in the preferable ranges, the stability of the glabridin plant source microcapsule inclusion is better, the glabridin plant source microcapsule inclusion does not precipitate or delaminate after being placed for 3 months under different temperatures and ultraviolet irradiation, and the particle size is less than or equal to 50nm, so that the glabridin plant source microcapsule inclusion meets the practical application requirements.
In a second aspect, the invention provides a preparation method of the glabridin plant source microcapsule inclusion, which comprises the following steps:
s1, mixing a licorice root extract, an apple extract, a rice bran extract, grease, an emulsifying agent and a part of auxiliary emulsifying agent to obtain an oil phase; mixing the rest auxiliary emulsifier with water to obtain a water phase;
s2, adding the water phase in the step S1 into the oil phase under the stirring condition, and fully mixing, and carrying out micron treatment to obtain micron-sized fraction;
and S3, carrying out nanocrystallization treatment on the micron-sized particles in the step S2 to obtain the glabridin plant source microcapsule inclusion.
As a preferred embodiment of the preparation method of the glabridin plant source microcapsule inclusion, the micronization treatment is carried out by adopting a shearing mixing mode, and the parameters of the shearing mixing are as follows: the rotation speed is 4000-16000rpm, and the time is 1-10min.
As a preferred implementation mode of the preparation method of the glabridin plant source microcapsule inclusion, high-pressure homogenization treatment or high-pressure microjet treatment is adopted for nanocrystallization, wherein the pressure of the high-pressure homogenization treatment is 300-1600bar, the temperature is 20-70 ℃, and the cycle time is 1-10 times; the pressure of the high-pressure micro-jet treatment is 3000-16000psi, the temperature is 20-70 ℃, and the cycle times are 1-10.
According to the preparation method, the plant active ingredients are wrapped in the nano-microcapsules, and the nano-microcapsules have the advantages of small particle size, large specific surface area and stronger adhesiveness and biocompatibility with cells, so that the plant active ingredients can penetrate into the skin cuticle to reach the basal layer of the skin more efficiently and directly act on the target cells of the myobasal melanin and stay in the target cells of the melanin for a long time, are slowly released and are controlled to release, and compared with the traditional penetration mode, the bioavailability of the active ingredients is greatly improved, and the whitening and freckle removing effects of the active ingredients are further enhanced; the microcapsule coating prepared by the invention can effectively protect the coated plant whitening active ingredient, improve the light and heat stability of the active ingredient, and further improve the whitening effect of the active ingredient and the quality guarantee period of the product.
In a third aspect, the invention provides an application of the glabridin plant source microcapsule inclusion in preparation of cosmetics.
In a fourth aspect, the invention provides a whitening and freckle-removing cosmetic, which is prepared from the following raw materials in percentage by weight.
As a preferred implementation mode of the whitening and freckle-removing cosmetic, the mass percentage of glabridin plant source microcapsule inclusion in the whitening and freckle-removing cosmetic is 0.1-30%.
As a preferred embodiment of the whitening and freckle-removing cosmetic, the whitening and freckle-removing cosmetic is toner, cream, emulsion, essence or gel.
Compared with the prior art, the invention has the beneficial effects that:
(1) The glabridin in the glabridin plant source microcapsule inclusion, the phloretin extracted from the glabridin extract, the phloretin extracted from the apple peel and the root bark, and the ferulic acid extracted from the active phenolic acid extracted from the rice bran can whiten and remove spots from different mechanisms, so that the whitening and spot removing effects of the glabridin plant source microcapsule inclusion are synergistically enhanced.
(2) The glabridin in the glabridin plant source microcapsule inclusion and the phloretin in the apple extract are matched for use, so that the effects of whitening and removing freckles can be enhanced, the anti-aging, anti-inflammatory and antiallergic functions of the skin can be realized, the whitening effect on sensitive skin can be realized, the sedation and relaxation can be realized, the effects of inhibiting sebum secretion and moisturizing the skin can be realized, and the glabridin plant source microcapsule inclusion is especially suitable for oily skin.
(3) The glycyrrhiza glabra extract, the apple extract and the rice bran extract are all natural plant extracts with high purity, the components are natural in source, the action mechanism is clear, the efficacy is obvious, and the glycyrrhiza glabra extract, the apple extract and the rice bran extract are especially suitable for sensitive skin.
(4) The preparation method of wrapping the plant active ingredient in the nano microcapsule is adopted, so that the plant active ingredient can more efficiently penetrate into the skin cuticle to reach the basal layer of the skin, directly act on the target cells of the myobasal melanin, and stay in the target cells of the myobasal melanin for a long time, and are slowly released and controlled, the bioavailability of the active ingredient is greatly improved, and the whitening and freckle removing effects of the active ingredient and the quality guarantee period of cosmetics are further enhanced.
Drawings
FIG. 1 shows the results of evaluation of safety of glabridin plant-derived microcapsule inclusion on HaCaT cells according to an embodiment of the present invention;
FIG. 2 shows the results of HSF cell safety evaluation of glabridin plant-derived microcapsule inclusion according to an embodiment of the present invention;
FIG. 3 is a graph showing experimental evaluation of the irritation of glabridin plant-derived microcapsule inclusion to chick embryo allantois according to an embodiment of the present invention;
FIG. 4 shows the cumulative skin permeation and skin retention per unit area of glabridin, ferulic acid and phlorizin contained in a whitening and freckle-removing cosmetic according to one embodiment of the present invention;
FIG. 5 shows the skin permeation behavior of the whitening and freckle-removing cosmetic according to one embodiment of the present invention;
FIG. 6 shows the results of uptake behavior of FITC-labeled nanocarriers prepared from glabridin plant-derived microcapsule packages according to one embodiment of the present invention by B16F10 cells observed by a laser confocal microscope;
FIG. 7 shows the results of detecting the uptake behavior of FITC-labeled nanocarriers by B16F10 cells via a flow cytometer using a glabridin plant-derived microcapsule encapsulation body according to an embodiment of the present invention;
FIG. 8 shows the effect of the whitening and freckle-removing cosmetic on tyrosinase activity in B16F10 cells according to one embodiment of the present invention;
FIG. 9 is a graph showing the effect of melanin production in the inclusion of glabridin plant-derived microcapsules in accordance with one embodiment of the present invention;
FIG. 10 shows the effect of glabridin plant-derived microcapsule inclusion on oxidative damage HaCaT cells according to one embodiment of the present invention;
FIG. 11 is a graph showing the effect of glabridin plant-derived microcapsule inclusion on apparent chromaticity of a 3D skin melanin model according to an embodiment of the present invention;
FIG. 12 is a graph showing the effect of plant glabridin plant source microcapsule inclusion on apparent brightness and melanin content of a 3D skin melanin model according to an embodiment of the present invention.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
The particle sizes of the glabridin plant source microcapsule inclusion are detected by using a Zetasizer Nano-ZS90 laser particle sizer; other materials, reagents, etc. used, unless otherwise specified, are commercially available.
In the following examples, the percentages mentioned are all percentages by weight of the corresponding components in the glabridin plant source microcapsule inclusion unless otherwise specified.
Example 1
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 1% of licorice extract, 1% of apple extract, 1% of rice bran extract, 6% of caprylic/capric triglyceride, 8% of polyglycerol-10 diisostearate and 5% of PPG-26-butanol polyether-26 at 20 ℃ to obtain an oil phase; adding 10% of 1, 3-propylene glycol into 68% of pure water, and heating and dissolving in a water bath at 20 ℃ to obtain a water phase;
s2, adding the water phase into the oil phase at a speed of 15 drops/S, stirring and mixing at a temperature of 20 ℃ and a speed of 300rpm, and carrying out high-speed shearing treatment for 1min at a speed of 4000rpm after the mixing is completed, so as to obtain the micro-sized fraction;
S3, carrying out high-pressure homogenization treatment on the micron-sized particles at 20 ℃ and 300bar pressure, and circulating for 1 time to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, so that the microcapsule inclusion particle size is 13.6nm, and the PDI value is 0.22.
Example 2
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 1% of licorice extract, 1% of apple extract, 5% of rice bran extract, 4% of palmitic acid triglyceride, 2% of isononyl isononanoate, 5% of polyglycerol-10 myristate and 8% of octyl dodecanol at 30 ℃ to obtain an oil phase; adding 2% of 1, 2-pentanediol into 72% of pure water, and heating and dissolving in a water bath at 30 ℃ to obtain a water phase;
s2, adding the water phase into the oil phase at a speed of 13 drops/S, stirring and mixing at a temperature of 30 ℃ and a speed of 500rpm, and carrying out high-speed shearing treatment for 4min at a speed of 8000rpm after the mixing is completed, so as to obtain the micro-scale micro-particles;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 30 ℃ and 600bar pressure, and circulating for 2 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, and the microcapsule inclusion particle size is 107.4nm and the PDI value is 0.47.
Example 3
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 1.5% of licorice extract, 1.5% of apple extract, 3% of rice bran extract, 5% of ethylhexyl cocoate, 5% of squalane, 8% of polyglycerol-3 ricinoleate and 4% of PPG-26-butanol polyether-26 at 40 ℃ to obtain an oil phase; adding 5% glycerol and 5% butanediol into 67% pure water, heating and dissolving in water bath at 40deg.C to obtain water phase;
s2, adding the water phase into the oil phase at a speed of 13 drops/S, stirring and mixing at 40 ℃ and 700rpm, and carrying out high-speed shearing treatment at a rotating speed of 7000rpm for 10min after the mixing is finished to obtain the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 40 ℃ and 600bar pressure, and circulating for 2 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, so that the microcapsule inclusion particle size is 75.1nm, and the PDI value is 0.47.
Example 4
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
S1, heating and mixing 2% of licorice extract, 2% of apple extract, 2% of rice bran extract, 8% of caprylic/capric triglyceride, 5% of polyoxyethylene hydrogenated castor oil, 5% of polyglycerol-10 oleate, 4% of soybean lecithin and 1% of cholesterol at 45 ℃ to obtain an oil phase; adding 10% of 1, 2-hexanediol into 61% of pure water, and heating and dissolving in a water bath at 45 ℃ to obtain a water phase;
s2, adding the water phase into the oil phase at the speed of 10 drops/S, stirring and mixing at the temperature of 45 ℃ and at the speed of 600rpm, and carrying out high-speed shearing treatment for 3min at the speed of 6000rpm after the mixing is finished, thus obtaining the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 45 ℃ and 500bar pressure, and circulating for 3 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, so that the microcapsule inclusion particle size is 22.8nm, and the PDI value is 0.15.
Example 5
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 3% of licorice extract, 3% of apple extract, 3% of rice bran extract, 5% of triglyceride (ethylhexanoate), 5% of diisopropyl sebacate, 2% of olive oil polyglycerol-6 esters, 8% of polyoxyethylene sorbitan fatty acid ester, 10% of tridecyl alcohol polyether-12 and 10% of behenyl alcohol polyether-20 at 50 ℃ to obtain an oil phase; adding 5% dipropylene glycol into 46% pure water, heating and dissolving in water bath at 50deg.C to obtain water phase;
S2, adding the water phase into the oil phase at the speed of 8 drops/S, stirring and mixing at the temperature of 50 ℃ and at the speed of 800rpm, and carrying out high-speed shearing treatment for 5min at the speed of 10000rpm after the mixing is finished, thus obtaining the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 50 ℃ and 800bar pressure, and circulating for 4 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, and the microcapsule inclusion particle size is 65.5nm, and the PDI value is 0.27.
Example 6
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 5% of licorice extract, 8% of apple extract, 10% of rice bran extract, 10% of diisostearyl malate, 5% of isostearyl lactate, 5% of squalane, 7% of polyglycerol-3 ricinoleate, 8% of PEG-10 oleate, 5% of myristyl glucoside, 10% of sorbitol polyether-30 and 10% of sorbitol at 70 ℃ to obtain an oil phase; adding 10% butanediol into 7% pure water, and heating in water bath at 70deg.C to obtain water phase;
s2, adding the water phase into the oil phase at the speed of 2 drops/S, stirring and mixing at the temperature of 70 ℃ and the speed of 1200rpm, and carrying out high-speed shearing treatment for 10min at the rotating speed of 16000rpm after the mixing is finished, thus obtaining the micro-sized fraction;
S3, carrying out high-pressure homogenization treatment on the micron-sized particles at 70 ℃ and 1600bar pressure, and circulating for 10 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, so that the microcapsule inclusion particle size is 153.8nm, and the PDI value is 0.53.
Example 7
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 6% of licorice extract, 5% of apple extract, 2% of rice bran extract, 8% of ethylhexyl cocoate, 3% of diglyceryl stearate malate, 2% of isopropyl myristate, 4% of behenyl polyether-20, 4% of sorbitol polyether-30, 2% of ethoxydiglycol and 5% of 1, 2-hexanediol at 75 ℃ to obtain an oil phase; adding 5% propylene glycol and 5% sorbitol into 49% pure water, and heating in 75deg.C water bath to obtain water phase;
s2, adding the water phase into the oil phase at the speed of 9 drops/S, stirring and mixing at the temperature of 75 ℃ and the speed of 1000rpm, and carrying out high-speed shearing treatment for 3min at the speed of 15000rpm after the mixing is finished, thus obtaining the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at the temperature of 75 ℃ and the pressure of 800bar, and circulating for 6 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, so that the microcapsule inclusion particle size is 72.8nm, and the PDI value is 0.44.
Example 8
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 6% of licorice extract, 5% of apple extract, 6% of rice bran extract, 10% of diglyceryl stearate malate, 3% of isodecyl laurate, 6% of PEG-8 caprylic/capric glyceride, 6% of xylitol-based glucoside, 10% of PPG-26-butanol polyether-26, 10% of ethoxydiglycol and 10% of 1, 2-hexanediol at 40 ℃ to obtain an oil phase; adding 10% propylene glycol into 18% pure water, and heating in water bath at 40deg.C to obtain water phase;
s2, adding the water phase into the oil phase at the speed of 6 drops/S, stirring and mixing at the temperature of 40 ℃ and at the speed of 900rpm, and carrying out high-speed shearing treatment for 6min at the speed of 12000rpm after the mixing is finished, thus obtaining the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 40 ℃ and 1000bar pressure, and circulating for 5 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, and the microcapsule inclusion particle size is 78.3nm and the PDI value is 0.49.
Example 9
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 8.3% of licorice extract, 7.7% of apple extract, 1.9% of rice bran extract, 7% of diisopropyl sebacate, 2% of triglyceride (ethylhexanoate), 1% of isodecyl laurate, 3% of PEG-8 caprylic/capric glyceride, 6% of xylitol-based glucoside, 4% of polyglycerol-3 ricinoleate, 6% of behenyl polyether-20 and 7% of butanediol at 50 ℃ to obtain an oil phase; adding 4% glycerol and 4% octanediol into 34.1% pure water, and heating and dissolving in water bath at 50deg.C to obtain water phase;
s2, adding the water phase into the oil phase at a speed of 20 drops/S, stirring and mixing at 50 ℃ and 2000rpm, and carrying out high-speed shearing treatment at a rotating speed of 10000rpm for 3min after the mixing is completed, thus obtaining the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 50 ℃ and 800bar pressure, and circulating for 4 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, and the microcapsule inclusion particle size is 62.5nm, and the PDI value is 0.42.
Example 10
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 8% of licorice extract, 6% of apple extract, 8% of rice bran extract, 8% of isopropyl myristate, 10% of caprylic/capric triglyceride, 4% of PEG-12 distearate, 4% of soybean lecithin, 10% of polyoxyethylene hydrogenated castor oil, 10% of octyl dodecanol, 10% of PPG-26-butanol polyether-26 and 10% of 1, 2-pentanediol at 60 ℃ to obtain an oil phase; adding 8% octanediol into 4% pure water, heating and dissolving in water bath at 60deg.C to obtain water phase;
s2, adding the water phase into the oil phase at the speed of 3 drops/S, stirring and mixing at the temperature of 60 ℃ and at the speed of 1100rpm, and carrying out high-speed shearing treatment for 8min at the speed of 14000rpm after the mixing is finished, thus obtaining the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 60 ℃ and 1400bar pressure, and circulating for 8 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, and the microcapsule inclusion particle size is 102.7nm and the PDI value is 0.35.
Example 11
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
S1, heating and mixing 9% of licorice extract, 8% of apple extract, 5% of rice bran extract, 6% of palmitic acid triglyceride, 5% of isononyl isononanoate, 3% of diglyceryl stearate malate, 6% of polyglycerol-3 ricinoleate, 5% of octyl dodecanol, 8% of sorbitol polyether-30 and 4% of dipropylene glycol at 65 ℃ to obtain an oil phase; adding 7% of 1, 3-propanediol and 3% of 1, 2-hexanediol into 31% of pure water, and heating and dissolving in a water bath at 65 ℃ to obtain a water phase;
s2, adding the water phase into the oil phase at a speed of 15 drops/S, stirring and mixing at a temperature of 65 ℃ and a speed of 540rpm, and carrying out high-speed shearing treatment for 6min at a rotating speed of 9000rpm after the mixing is finished, thus obtaining the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 65 ℃ and 1500bar pressure, and circulating for 5 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, and the microcapsule inclusion particle size is 89.6nm and the PDI value is 0.41.
Example 12
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, heating and mixing 5% of licorice extract, 5% of apple extract, 5% of rice bran extract, 10% of caprylic/capric triglyceride, 5% of ethylhexyl cocoate, 10% of polyoxyethylene hydrogenated castor oil, 6% of polyglycerol-10 oleate, 5% of PPG-26-butanol polyether-26, 8% of ethoxydiglycol and 8% of 1, 2-pentanediol at 50 ℃ to obtain an oil phase; adding 5% glycerol into 28% pure water, and heating in 50deg.C water bath to dissolve to obtain water phase;
S2, adding the water phase into the oil phase at a speed of 5 drops/S, stirring and mixing at 50 ℃ and 1000rpm, and carrying out high-speed shearing treatment at a rotating speed of 13000rpm for 7min after the mixing is completed to obtain the micro-sized fraction;
s3, carrying out high-pressure homogenization treatment on the micron-sized particles at 50 ℃ and 1200bar, and circulating for 6 times to obtain the glabridin plant source microcapsule inclusion.
The particle size of the microcapsule inclusion is detected, and the microcapsule inclusion particle size is 40.7nm and the PDI value is 0.11.
Comparative example 1
Preparing a free plant extract solution: adding 6% Glycyrrhiza glabra extract, 5% apple extract, 2% rice bran extract and 2% dimethyl sulfoxide into 85% purified water, and dissolving by ultrasonic dispersion at 45deg.C to obtain free plant extract solution with main effective components accounting for 13% of total amount.
Comparative example 2
Preparing a solution of single free licorice extract: adding 13% Glycyrrhiza glabra extract and 2% dimethyl sulfoxide into 85% purified water, and dissolving at 45deg.C by ultrasonic dispersion to obtain single free Glycyrrhiza glabra extract solution with total content of main effective components of 13%.
Comparative example 3
Preparing a single free apple extract solution: adding 13% apple extract and 2% dimethyl sulfoxide into 85% purified water, and dissolving by ultrasonic dispersion at 45deg.C to obtain single free apple extract solution with main effective components total amount of 13%.
Comparative example 4
Preparation of single free rice bran extract solution: adding 13% of rice bran extract and 2% of dimethyl sulfoxide into 85% of purified water, and dissolving by ultrasonic dispersion at 45 ℃ to obtain a single free rice bran extract solution with main effective components accounting for 13%.
Comparative example 5
Preparing a combination solution of free licorice and apple extract: adding 7.1% of Glycyrrhiza glabra extract, 5.9% of apple extract and 2% of dimethyl sulfoxide into 85% of purified water, and dissolving by ultrasonic dispersion at 45 ℃ to obtain a combined solution of free Glycyrrhiza glabra and apple extract with main effective components accounting for 13% of the total amount.
Comparative example 6
Preparing a combined solution of free apples and rice bran extracts: adding 9.3% apple extract, 3.7% rice bran extract and 2% dimethyl sulfoxide into 85% purified water, and dissolving by ultrasonic dispersion at 45deg.C to obtain a combined solution of free apple and rice bran extract with total content of main effective components of 13%.
Comparative example 7
Preparing a combined solution of free licorice and rice bran extract: adding 9.75% of Glycyrrhiza glabra extract, 3.25% of rice bran extract and 2% of dimethyl sulfoxide into 85% of purified water, and dissolving by ultrasonic dispersion at 45deg.C to obtain a free Glycyrrhiza glabra and rice bran extract combined solution with main effective components accounting for 13% of the total amount.
Comparative example 8
Preparation of aloin-containing free plant extract I: the glabra licorice extract of comparative example 1 was replaced with aloin, and the other conditions were the same as those of comparative example 1, to prepare an aloin-containing free plant extract I.
Comparative example 9
Preparing a plant extract II containing aloin free: the apple extract of comparative example 1 was replaced with aloin, and the remaining conditions were the same as in comparative example 1, to prepare an aloin-containing free plant extract II.
Comparative example 10
Preparing a free plant extract III containing aloin: the rice bran extract of comparative example 1 was replaced with aloin to prepare an aloin-containing free plant extract III.
Comparative example 11
This comparative example differs from example 1 only in that: the glabridin plant source microcapsule inclusion comprises the following components in percentage by weight: 15% of licorice extract, 15% of apple extract, 15% of rice bran extract, and the rest of components and preparation methods are the same as in example 1.
Comparative example 12
This comparative example differs from example 1 only in that: the glabridin plant source microcapsule inclusion comprises the following components in percentage by weight: the components and preparation methods of Glycyrrhiza glabra extract 0.05%, apple extract 0.05%, rice bran extract 0.05% were the same as in example 1.
Comparative example 13
This comparative example differs from example 1 only in that: the grease in the inclusion was dimethicone, and the remainder was the same as in example 1.
Comparative example 14
This comparative example differs from example 1 only in that: the emulsifier in the inclusion was polyethylene glycol stearate, the remainder being the same as in example 1.
Test example 1
1. Stability test
The glabridin plant source microcapsule inclusions described in examples 1 to 12 and comparative examples 11 to 14 were placed in a closed container, and the microcapsule inclusions were subjected to the respective conditions of room temperature, 4 ℃, 45 ℃ and 10 ℃ ultraviolet irradiation (illuminance 4000 lux) for 3 months, and the particle diameters under the respective conditions were measured, and the results are shown in table 1; and the appearance of the different microcapsule inclusion bodies before and after the placement was checked, and the results are shown in table 2; the stability of the microcapsule inclusion was evaluated comprehensively according to tables 1 and 2.
TABLE 1
| | Room temperature | 4 |
45℃ | Ultraviolet radiation | ||
| Example 1 | 13.6 | 14.2 | 14.4 | 13.8 | 14.0 | |
| Example 2 | 107.4 | 110.3 | 108.5 | 114.9 | 118.7 | |
| Example 3 | 75.1 | 75.3 | 75.4 | 76.5 | 77.8 | |
| Example 4 | 22.8 | 23.4 | 23.7 | 23.5 | 23.9 | |
| Example 5 | 65.5 | 66.8 | 66.4 | 67.2 | 66.5 | |
| Example 6 | 153.8 | 155.8 | 156.4 | 156.7 | 154.9 | |
| Example 7 | 72.8 | 74.3 | 75.2 | 76.1 | 76.8 | |
| Example 8 | 78.3 | 78.9 | 79.5 | 79.8 | 79.2 | |
| Example 9 | 62.5 | 63.9 | 64.5 | 64.9 | 65.3 | |
| Example 10 | 102.7 | 104.5 | 104.8 | 105.6 | 103.7 | |
| Example 11 | 89.6 | 90.5 | 90.3 | 90.8 | 91.4 | |
| Example 12 | 40.7 | 42.6 | 42.7 | 43.8 | 44.0 | |
| Comparative example 11 | 3150.7 | - | - | - | - | |
| Comparative example 12 | 130.06 | 138.73 | 159.54 | 153.84 | 147.01 | |
| Comparative example 13 | 1892.99 | - | - | - | - | |
| Comparative example 14 | 2851.2 | - | - | - | - |
TABLE 2
As can be seen from tables 1 and 2, the particle size of the glabridin plant source microcapsule inclusion prepared by adopting the formula of the invention is between 10 and 200nm, meets the practical application requirements, and has no obvious change in particle size after being placed for 3 months under the conditions of normal temperature, 4 ℃, 45 ℃ and 10 ℃ ultraviolet irradiation, and no phenomena of precipitation, layering and the like appear in appearance; wherein, the particle size of the glabridin plant source microcapsule inclusion in examples 1, 4 and 12 is less than 50nm, so that the effect of the inclusion can be exerted more deeply; comparative examples 11 to 12 since the weight percentage of the plant extract in the inclusion is not within the range provided by the present invention, the stability of the inclusion in comparative example 11 is lowered, and the particle size data after placement cannot be accurately measured, and the whitening and freckle removing effects of the inclusion in comparative example 12 are weak or ineffective; in comparative examples 13 to 14, since a part of the components are selected to deviate from the range provided by the present invention, the inclusion is finally unstable such as delamination or precipitation, and accurate particle diameter data cannot be measured; in addition, the appearance and the particle size of the inclusion still meet the requirements under the condition of higher content of active ingredients, which indicates that the glabridin plant source microcapsule inclusion has good stability.
2. Evaluation of cell safety
Cell safety evaluation was performed on glabridin plant-derived microcapsule inclusions described in examples 1 to 12 of the present invention and the free plant extract solution described in comparative example 1.
The testing method comprises the following steps: HSF cells (human skin fibroblasts) and HaCaT cells (human keratinocytes) were individually seeded in 96-well plates at 5% CO using the CCK-8 assay 2 After culturing at 37℃for 24 hours, 100. Mu.L of the DMEM complete medium (prepared at concentrations of 1.25, 2.5, 5, 10 and 20. Mu.g/mL) containing the free plant extract solution described in comparative example 1 and the glabridin plant source microcapsule inclusion described in examples 1 to 12 was added to each of the 2 dishes, and the culture was continued for 24 hours, and then the cell viability was measured by CCK-8 method, and the control was 100. Mu.L of DMEM complete medium alone.
The final results of examples 1-12 of the present invention are similar, and the test results of example 8 are represented by the test results of example 8, and the test results of example 8 and comparative example 1 are shown in FIGS. 1 and 2; as can be seen from FIGS. 1 and 2, when the concentration of glabridin is in the range of 1.25-10 μg/mL, the free plant extract solution and the glabridin plant source microcapsule inclusion in the invention are nontoxic to HaCaT and HSF cells; however, when the concentration of glabridin is 20 mug/mL, the activity of the free plant extract solution on HaCaT and HSF cells is obviously reduced compared with that of a control group, and the cell survival rate is less than 80 percent, but the glabridin plant source microcapsule inclusion bodies disclosed by the invention are nontoxic on the HaCaT and HSF cells, so that the whitening spot-fading plant microcapsule inclusion bodies disclosed by the invention have better safety on the cells.
3. Evaluation of chick embryo allantoic membrane irritation
The glabridin plant-derived microcapsule inclusion described in examples 1 to 12 was diluted 10 times with physiological saline, 0.2mL of the sample was sucked and dropped on the surface of chorioallantoic membrane, blood vessel changes were observed within 5 minutes and the initial time of the occurrence of congestion, bleeding and coagulation of chorioallantoic membrane blood vessel was recorded, the stimulation score IS calculated according to the following formula (i), and the stimulation classification was performed according to the average value, wherein 0 to 0.9, 1.0 to 4.9, 5.0 to 8.9 and 9 to 21.0 were classified as no stimulation, slight stimulation, moderate stimulation, severe stimulation, respectively, the final results of examples 1 to 12 of the present invention were similar, and the results of example 6 are represented by example 6, and see fig. 2.
As can be seen from fig. 3, when the 10-fold diluted glabridin plant source microcapsule inclusion was in contact with chick embryo allantoic membrane for 300s, capillary vessel had no bleeding, no vascular thawing and no coagulation phenomenon, and the reaction integral was 0.7, which indicates that the 10-fold diluted glabridin plant source microcapsule inclusion has good safety and no irritation.
IS＝[(301-secH)×5+(301-secL)×7+(301-secC)×9]/300
(I)
In the formula I, secH is the initial time of superficial congestion, secL is the initial time of superficial hemorrhage, secC is the initial time of superficial coagulation, and the units are s.
4. Product usage evaluation
30 subjects were selected, and a filter paper sheet containing glabridin plant source microcapsule inclusion diluted 10 times as described in examples 1 to 12 was placed in a patch tester, and a normal hole was used as a blank control group, and both the sample and the blank were applied to the forearm curved side of the subject, and applied to the skin uniformly by palm light pressure for 24 hours, and after removing the patch tester, the skin reaction was observed after the indentation disappeared, and after removing the patch tester for 24 hours, 48 hours, the skin reaction was observed again.
The results show that 30 subjects do not have erythema pallidum, erythema erythrorum, edema erythema, significant redness, infiltration or pimple, pimple or bleb, etc., indicating that the glabridin plant-derived microcapsule inclusion bodies of the invention are not irritating to human skin.
Test example 2
Blank essence and plant extracts of example 6, example 7 and comparative examples 1-10 were combined as a blank essence: (whitening spot-lightening microcapsule inclusion) plant extract = 9:1 to obtain essence with the total whitening and freckle-removing functional components of 2.3% or 1.3% by mass, and numbering the essence as 1-12 according to the sequence of the examples 6, 7 and the comparative examples 1-10, and performing the following test.
The preparation method of the blank essence comprises the following steps: 1.0% glycerol, 0.5%1, 3-butanediol, 0.3% polydimethylsiloxane, 2.0% xanthan gum, 1.0% cetyl alcohol, 1.3% PEG-100 stearate and 90.95% purified water were stirred and dissolved in a 75℃water bath to obtain a blank concentrate.
1. In vitro transdermal experiments
Performing an isolated pig skin transdermal experiment by adopting a vertical Franz diffusion cell method, preparing the free plant extract solution, the essence 2 and the essence 3 in the comparative example 1, ensuring that the content of the effective components of the free plant extract solution, the essence 2 and the essence 3 is consistent, placing the mixture in a supply chamber, taking PBS as a receiving solution, stirring and diffusing the mixture at 37 ℃, taking 0.5mL of the receiving solution at 4, 8, 12 and 24 hours, calculating the accumulated permeation quantity of specific medicine unit areas at different times through HPLC analysis, taking down the skin after 24 hours, grinding the skin into homogenate, centrifuging, taking supernatant, performing HPLC analysis, and calculating the skin storage quantity of active substances in unit area; the functional components measured in the experiment are glabridin, ferulic acid and phloretin.
Based on the concentrations of glabridin, ferulic acid and phloretin analyzed by HPLC, the skin cumulative permeation amounts of glabridin, ferulic acid and phloretin at different sampling times were calculated according to the following formula II.
Wherein Q is n For the accumulated penetration of the drug, C n For the nth measured drug concentration, C i Drug concentration measured for the ith point, V 0 For the volume of the diffusion cell, i.eThe amount of release medium added, V i For each sampling amount, the integrated permeation amount per unit area q=q n S, wherein S is the area of the diffusion cell of 2.27cm 2 The final test results are shown in FIG. 3.
FIG. 4 shows that the cumulative skin permeation amounts of glabridin 24h per unit area in the free plant extract solution, the essence 3 free composition complex essence and the essence 2 nano-carrier complex essence of comparative example 1 are 0.77 μg/cm, respectively 2 、0.87μg/cm 2 And 1.99. Mu.g/cm 2 The skin storage amounts were 8.63. Mu.g/cm, respectively 2 、13.01μg/cm 2 And 48.77. Mu.g/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The cumulative skin permeation rate of 24h unit area of ferulic acid is 2.66 mug/cm respectively 2 、2.79μg/cm 2 And 4.05. Mu.g/cm 2 The skin storage amounts were 17.85. Mu.g/cm, respectively 2 、21.06μg/cm 2 And 58.67. Mu.g/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The cumulative skin permeation rate of phloretin per 24h unit area is 1.59 μg/cm 2 、2.33μg/cm 2 And 4.11. Mu.g/cm 2 The skin storage amounts were 21.39. Mu.g/cm, respectively 2 、29.56μg/cm 2 And 105.31. Mu.g/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the Compared with the free plant extract solution, the unit enzymolysis skin accumulation and the skin storage amount of the glabridin, the ferulic acid and the phlorizin are slightly improved after the free composition is compounded with the essence, and compared with the free plant extract solution compounded with the essence, the unit area skin accumulation and the skin permeation of the glabridin, the ferulic acid and the phlorizin in the glabridin plant source microcapsule inclusion are respectively improved by 128.7 percent, 45.16 percent and 76.39 percent, and the skin storage amount is respectively improved by 274.86 percent, 178.58 percent and 256.25 percent, which indicates that the transdermal absorption and the skin storage amount of the plant extract active ingredient can be effectively promoted after the plant extract active ingredient is wrapped by the nano carrier, and the skin bioavailability is improved.
2. Skin penetration behavior observation
The test experimental device is the same as the transdermal experimental device, rhodamine B (RhoB) marked essence 1 is taken in a supply room, PBS is taken as receiving liquid, the PBS is stirred and diffused at 37 ℃, residual samples on the skin are gently wiped off after 15min, 30min, 60min and 120min, the skin in a target area is taken down, the skin is rinsed again, residual moisture is wiped off after thorough cleaning, the sample is frozen and sliced, and the slice is observed by a laser confocal microscope, and the result is shown in figure 5.
As can be seen from fig. 5, with the extension of time, the fluorescence penetration depth of the skin increases, the fluorescence intensity of the microcapsule inclusion essence of the present invention in the skin is obviously stronger than that of free RhoB, and the microcapsule inclusion essence penetrates into deep skin tissues, which indicates that the nano-carrier can promote the rapid penetration of the entrapped active ingredient into the deep skin tissues, thereby improving the skin bioavailability of the active ingredient.
3. Laser confocal observation of cell uptake behavior
FITC labeled nano-carriers were prepared according to the preparation method described in example 7 by using Fluorescein Isothiocyanate (FITC) instead of all functional components as fluorescent markers, free FITC with equal concentration was prepared according to the preparation method described in comparative example 1, and after mouse skin melanoma cells (B16F 10) were inoculated in a 35mm confocal dish for 24 hours of culture, DMEM medium containing free FITC and FITC labeled nano-carriers was added for 2 hours and 4 hours of incubation, respectively, and each treatment was sequentially performed with RhoB solution staining, 4% paraformaldehyde fixation and DAPI solution staining for 15 minutes, and photographs were observed under a 60-fold objective lens using a laser confocal microscope, and the results are shown in FIG. 5.
FIG. 6 shows that with prolonged incubation time, the intracellular fluorescence intensity is obviously enhanced, the intracellular fluorescence intensity of the free FITC group is weak when incubated for 2 hours, while FITC-labeled nano-carrier fluorescence enters cells, the fluorescence intensity is obviously stronger than that of the free FITC, and the fluorescence intensity of the FITC-labeled nano-carrier is further enhanced when incubated for 4 hours; experimental results show that compared with free FITC, the glabridin plant source microcapsule coating with the FITC can be more ingested by B16F10 cells, and the coated active substance can be effectively delivered into skin target cells to play a role.
4. Flow cytometry to detect cell uptake behavior
B16F10 cells were seeded in 6-well plates, free FITC and FITC-labeled nanocarriers of the same concentration as those in the above test 3 were added to each well after adherence, the cells without any treatment were used as negative controls, and after further culturing for 2 hours and 4 hours, the cells were collected and the fluorescence intensity in the cells was measured by flow cytometry, and the results are shown in fig. 7.
FIG. 7 shows that after FITC-labeled nanocarriers are treated for 2h and 4h, the average fluorescence intensities of cells are 3981 and 9314 respectively, and compared with the free FITC group, the fluorescence intensities are respectively improved by 64.98% and 131.06%, which indicates that the nanocarriers of the invention can significantly improve the cell uptake and intracellular accumulation capacity of B16F10 cells on the entrapped substances.
5. Intracellular tyrosinase activity assay
The activity of tyrosinase in cells was measured by L-Dopa oxidation, B16F10 was inoculated into 24-well plates, cultured for 24 hours, 100 nM. Alpha. -MSH (melanotropin) was added for induction (except for normal control), each of the test samples in Table 3 was added to give glabridin final concentrations of 2.5, 5 and 10. Mu.g/mL in example 7, the amount of the other samples was determined with reference to example 7, cells were lysed after 48 hours of culture, 0.1% L-Dopa was added to the supernatant, incubated at 37℃for 1 hour, and the absorbance of each well was measured at 490nM, and the test results are shown in FIG. 8.
TABLE 3 Table 3
FIG. 8 shows that niacinamide and vitamin C at concentrations up to 10 μg/mL showed reduced tyrosinase activity compared to the model group, whereas the comparative example 1 free plant extract showed higher tyrosinase inhibitory activity than the positive control group at the same dose, reduced tyrosinase activity to 72.62% at a concentration of 2.5 μg/mL, showing significant inhibition activity (P <0.05 or P < 0.01), and comparative examples 5-7 were the same dose compositions of any two of the functional ingredients of comparative example 1, respectively, wherein the comparative example 6 free apple and rice bran extract combination solution had the relatively highest tyrosinase inhibitory activity, and the intracellular tyrosinase activity was 63.47% at a concentration of 10 μg/mL, but the effect was worse than the comparative example 1 free plant extract; the combination of the licorice extract, the apple extract and the rice bran extract has stronger tyrosinase activity than any two extracts under the same efficacy content, and the three components have synergistic effect in cooperation; compared with the free plant extract, the glabridin plant source microcapsule inclusion in the embodiment 7 provided by the invention has a further improvement on the inhibition effect of B16F10 cell tyrosinase activity, wherein the tyrosinase activity is 25.13% at the minimum, and the nano delivery system is proved to be capable of promoting the effect of the functional components (P < 0.01).
6. Influence on melanogenesis in cells
Measurement of the intracellular melanin content by NaOH lysis method, cell grouping and model induction were carried out in the same manner as in test example 5, after 48 hours of cell culture, 300. Mu.L of 1.0mmol/L NaOH solution (containing 10% DMSO) was added to each well, the cells were fully lysed at 80℃for 1 hour, and the absorbance of each well was measured at 405nm, and the results are shown in FIG. 9.
FIG. 9 shows that compared with the model group, the concentration of nicotinamide and vitamin C positive control group is 2.5 mug/mL, the content of cytomelanin is reduced to a certain extent but the effect is not obvious, and the content of cytomelanin is obviously reduced to 62.21% after the free plant extract of comparative example 1 is treated at the same dosage; the cytomelanin content reducing effect of the free plant extract is also better than that of the positive control (P <0.05 or P < 0.01) at 10 mug/mL; comparative examples 5-7 are equivalent dosage compositions of any two of the functional ingredients of comparative example 1, respectively, wherein the comparative example 6 free licorice and apple extract combination solution has a relatively minimal cytomelanin content. At a concentration of 10. Mu.g/mL, the cytomelanin content was 57.42%, which was weaker than the free plant extract of comparative example 1 (melanin content 53.01%). The combination of the licorice extract, the apple extract and the rice bran extract has stronger cytomelanin content reducing effect than any two extracts under the same effect content, and the three components are matched to have a synergistic effect. Example 7 glabridin plant source microcapsule inclusion at concentration of 2.5, 5 and 10 μg/mL has stronger inhibition effect on B16F10 cell melanogenesis than the free plant extract (P < 0.01) of the same dose comparative example 1, and the nano delivery system is proved to be capable of promoting the effect of the functional components.
7. Cell antioxidant study
HaCaT cells (human keratinocytes) were inoculated in 24-well plates for culture, control group was supplemented with DMEM complete medium, model group was supplemented with 0.6mmol/L H 2 O 2 DMEM complete medium containing 0.6mmol/L H was added to each of the drug administration groups 2 O 2 And the free plant extract of comparative example 1 and the DMEM complete medium of the plant glabridin plant source microcapsule inclusion of example 7 (corresponding to glabridin concentration of 2.5, 5, 10. Mu.g/mL) were co-cultured for 24, and then incubated with DMEM containing 20. Mu.M active oxygen fluorescent probe (DCFH-DA) for 20min, and the fluorescence intensity was observed with a fluorescence microscope, as shown in FIG. 10.
FIG. 10 shows that the fluorescent intensity of model group cells is strongest after labeling with ROS probe. The fluorescence intensity of the free plant extract group and the plant glabridin plant source microcapsule inclusion group is weakened, which shows that the free plant extract group and the plant glabridin plant source microcapsule inclusion group can obviously inhibit the generation of intracellular ROS and play a role in protecting oxidative damage cells. The fluorescence intensity of the cells treated by the plant glabridin plant source microcapsule inclusion is weaker than that of the free plant extract, which means that the intracellular ROS content is lower, and the glabridin plant source microcapsule inclusion has better antioxidation.
8. Whitening efficacy evaluation of 3D skin melanin model
The 3D melanin skin model was placed in a 6-well plate, CO 2 Culturing in an incubator. Negative Control (NC), positive control (PC, kojic acid 500. Mu.g/mL) and sample group (the plant glabridin plant source microcapsule inclusion as described in example 6, corresponding to glabridin content of 100. Mu.g/mL; UVB irradiation treatment (50 mJ/cm 2) was performed every day, and Blank Control (BC) was not subjected to UVB irradiation. The positive control group and the sample group were administered twice on day 3 and 5, respectively, in a volume of 10. Mu.L. After 7 days of continuous model culture, the 3D skin melanin model was tested for apparent chromaticity, apparent brightness (L values) and melanin content. The results are shown in FIG. 11 and FIG. 12.
Fig. 11 shows that the apparent chromaticity of each group was significantly blackened by UVB irradiation treatment compared to the blank group, and that the apparent chromaticity of the skin model was significantly whitened by treatment with plant glabridin plant-derived microcapsule inclusion compared to the negative control group, and the whitening effect was comparable to that of high dose of kojic acid (PC 500 μg/mL).
Fig. 12 shows that each group showed a significant decrease in apparent brightness (P <0.05 or P < 0.01) after UVB irradiation treatment compared to the blank group, and a significant increase in apparent brightness (P < 0.05) of the skin model after plant glabridin plant source microcapsule inclusion treatment compared to the negative control group, with an effect comparable to that of high dose kojic acid (PC 500 μg/mL). The melanin content of each group was significantly increased (P < 0.01) after UVB irradiation treatment compared to the blank group, and the melanin content of the skin model was significantly decreased (P < 0.05) after plant glabridin plant source microcapsule inclusion treatment compared to the negative control group.
9. Volunteer whitening efficacy evaluation
Performing whitening and freckle removing efficacy test on the blank essence and the essence 2-12, selecting 60 18-40-year-old male or female volunteers, regularly using one of the above-mentioned essence twice daily on the facial skin for 28 days, and keeping regular diet and rest without using other whitening and freckle removing products or medicines in the middle of the test; the volunteers were asked to truly and accurately respond to the skin condition and feeling after use, and the reduction rate P of the melanin content of the skin was recorded weekly A (%) and skin brightness increase rate P B (%) and the results are shown in Table 4.
TABLE 4 Table 4
As can be seen from table 4, the skin melanin content and skin brightness did not change significantly before and after the volunteer used the blank essence (comparative example 2); after the single free licorice extract essence, the single free apple extract essence and the single free rice bran extract essence (essence 4-6) are used for one week, the facial skin pigment content of the volunteer is obviously reduced, wherein the reduction rate of the apple extract essence to melanin is 15.02%, and the skin brightness is increased by 9.05%; after 4 weeks of using the single free extract essence, the skin melanin content is reduced by at most 25.72%, and the brightness is increased by at most 18.29%; in the essence 7-9, the melanin reduction rate and the brightness increase rate of the essence 9-licorice and rice bran extract combined essence are respectively highest and are respectively 26.69 percent and 21.43 percent, the melanin reduction rate of the essence 7-licorice and apple extract combined essence is 22.28 percent, the brightness increase rate is 18.54 percent, after three different action mechanisms are matched, the whitening and freckle-removing plant extracts are compounded, the whitening and freckle-removing effects can be enhanced, after a volunteer uses the free composition for 4 weeks, the melanin reduction rate reaches 46.21 percent, the brightness increase rate reaches 34.89 percent, and the free composition is higher than that of the independent or two combined extract essences, so that the three extract collocations have a synergistic effect. In order to further verify the synergistic effect of the combination, the essence 10-12 respectively adopts aloin to replace one extract component in the whitening and freckle-removing plant extract, and the result shows that the essence 12 has relatively optimal action effect, the melanin reduction rate is 33.23%, the brightness increase rate is 28.64%, but the action effect is far lower than the combination of the glycyrrhiza glabra extract, the apple extract and the rice bran extract, and the rationality and the synergistic effect of the collocation extraction composition are proved. Finally, compared with the free composition compound essence, the nano-carrier compound essence shows more excellent whitening and freckle removing effects, the melanin content reducing effect is improved by 33.72%, the skin brightness increasing effect is improved by 12.32%, and the whitening and freckle removing effect is enhanced due to the improvement of the transdermal property, the skin absorption and storage quantity and the bioavailability of the whitening and freckle removing plant extract composition wrapped by the nano-carrier.
In conclusion, the glabridin plant source microcapsule inclusion provided by the invention has excellent stability, the particle size of the microcapsule inclusion is not remarkably changed after being placed for 3 months under the irradiation of ultraviolet rays at normal temperature, 4 ℃ and 45 ℃ and 10 ℃ under the condition of high active ingredient content, the phenomena of precipitation, layering and the like are not generated in the appearance, and the particle size is between 10 nm and 200nm, so that the practical application requirements are met. Cell experiments prove that the microcapsule inclusion has high safety, and has no toxicity to HaCaT and HSF cells when the glabridin concentration is 20 mug/mL; the chick embryo chorioallantoic membrane and human experiments show that the microcapsule inclusion has no irritation; the microcapsule coating can effectively promote deep penetration and absorption of plant whitening functional components in skin, and remarkably improve the storage quantity of the plant whitening functional components in the skin; meanwhile, the microcapsule inclusion can enhance the uptake and intracellular accumulation of plant whitening functional components by target cells, improve the bioavailability and enable the functional components to better play a whitening role; the glabridin plant source microcapsule inclusion contains various whitening efficacy components, can obviously inhibit tyrosinase activity, reduce melanin generation, and has the function of protecting cells from oxidative damage; the plant glabridin plant source microcapsule inclusion can reduce the content of skin melanin, obviously improve the apparent chromaticity of the skin and obviously improve the apparent brightness.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (7)
1. The glabridin plant source microcapsule inclusion is characterized by comprising the following components in percentage by weight: 0.1-10% of licorice root extract, 0.1-10% of apple extract, 0.1-10% of rice bran extract, 1-20% of grease, 1-20% of emulsifying agent, 5-40% of auxiliary emulsifying agent and the balance of water; the particle size of the glabridin plant source microcapsule inclusion is 10-200nm; the oil comprises at least one of caprylic/capric triglyceride, palmitic triglyceride, diisopropyl sebacate, isononyl isononanoate, diisostearyl malate, diglyceryl stearate malate, isostearyl lactate, triethylhexanoate glycerol, squalane, isodecyl laurate, isopropyl myristate and ethylhexyl cocoate; the emulsifier comprises at least one of soybean lecithin, polyglycerol-10 oleate, polyglycerol-10 myristate, polyglycerol-10 diisostearate, olive oil polyglycerol-6 esters, polyglycerol-3 ricinoleate, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, PEG-10 oleate, PEG-8 caprylic/capric glyceride, PEG-12 distearate, xylitol-based glucoside, myristyl glucoside, cholesterol; the auxiliary emulsifier comprises at least one of octyl dodecanol, PPG-26-butanol polyether-26, tridecyl alcohol polyether-12, ethoxydiglycol, behenyl alcohol polyether-20, sorbitol polyether-30, 1, 2-hexanediol, 1, 2-pentanediol, dipropylene glycol, glycerol, butanediol, octanyl glycol, propylene glycol and sorbitol;
The preparation method of the glabridin plant source microcapsule inclusion comprises the following steps:
s1, mixing a licorice root extract, an apple extract, a rice bran extract, grease, an emulsifying agent and a part of auxiliary emulsifying agent to obtain an oil phase; mixing the rest auxiliary emulsifier with water to obtain a water phase;
s2, adding the water phase in the step S1 into the oil phase under the stirring condition, and then carrying out micron treatment to obtain micron-sized particles, wherein the micron treatment is carried out in a shearing mixing mode, and the parameters of the shearing mixing are as follows: the rotating speed is 4000-16000 rpm, and the time is 1-10 min;
s3, carrying out nanocrystallization on the micron-sized particles obtained in the step S2 to obtain the glabridin plant source microcapsule inclusion; the nanocrystallization is carried out by adopting a high-pressure homogenization or high-pressure microjet mode, the pressure of the high-pressure homogenization is 300-1600 bar, the temperature is 20-70 ℃, and the cycle times are 1-10 times; the pressure of the high-pressure micro-jet treatment is 3000-16000 psi, the temperature is 20-70 ℃, and the cycle times are 1-10.
2. The glabridin plant-derived microcapsule inclusion according to claim 1, wherein the glabridin plant-derived microcapsule inclusion comprises the following components in percentage by weight: 0.5 to 8 percent of licorice root extract, 0.5 to 8 percent of apple extract, 0.5 to 8 percent of rice bran extract, 3 to 15 percent of grease, 5 to 18 percent of emulsifying agent, 10 to 35 percent of auxiliary emulsifying agent and the balance of water.
3. The glabridin plant-derived microcapsule inclusion according to claim 1, wherein the glabridin plant-derived microcapsule inclusion comprises the following components in percentage by weight: 1-5% of licorice root extract, 1-5% of apple extract, 1-5% of rice bran extract, 5-10% of grease, 8-16% of emulsifying agent, 15-30% of auxiliary emulsifying agent and the balance of water.
4. The glabridin plant-derived microcapsule inclusion according to claim 3, wherein at least one of the following (iv) to (vi) is employed:
(iv) the oil comprises at least one of caprylic/capric triglyceride, palmitic triglyceride, diisostearyl malate, triethylhexanoin, squalane, isopropyl myristate, and ethylhexyl cocoate;
(v) the emulsifier comprises at least one of soybean lecithin, polyglycerol-10 oleate, polyglycerol-10 myristate, polyglycerol-10 diisostearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, PEG-10 oleate, PEG-8 caprylic/capric glycerides, cholesterol;
the auxiliary emulsifier in (VI) comprises at least one of octyl dodecanol, PPG-26-butanol polyether-26, 1, 3-propanediol, 1, 2-hexanediol, 1, 2-pentanediol, dipropylene glycol, glycerol and butanediol.
5. Use of glabridin plant-derived microcapsule inclusion according to any of claims 1-4 in the preparation of cosmetics.
6. A whitening and freckle-removing cosmetic, characterized in that the raw materials for preparing the whitening and freckle-removing cosmetic comprise 0.1-30% of glabridin plant-derived microcapsule inclusion according to any one of claims 1-4 in percentage by mass of the cosmetic.
7. The whitening and freckle removing cosmetic according to claim 6, wherein the whitening and freckle removing cosmetic is a toner, cream, emulsion, essence or gel.
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