CN117797040B

CN117797040B - Whitening nanoemulsion containing 4-butylresorcinol and preparation method thereof

Info

Publication number: CN117797040B
Application number: CN202311871201.4A
Authority: CN
Inventors: 薛建军; 王思勇; 武之阔; 马永康; 韩斌; 孙德帝; 李祖银
Original assignee: Jining Huanju Pharmaceutical Technology Co ltd
Current assignee: Jining Huanju Pharmaceutical Technology Co ltd
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-06-04
Anticipated expiration: 2043-12-29
Also published as: CN117797040A

Abstract

The invention discloses a whitening nanoemulsion containing 4-butylresorcinol and a preparation method thereof, and aims to provide a whitening nanoemulsion with the effects of inhibiting melanin synthesis and accelerating melanin metabolic degradation; the technical scheme comprises the following components in percentage by mass: 13.08 to 29.12 percent of active factor, 4 to 40.5 percent of emulsifier phase, 2.2 to 41.8 percent of organic phase, 0.1 to 1 percent of preservative and a proper amount of water; the active factors comprise 4-butyl resorcinol, nonapeptide-1, decarboxylated carnosine hydrochloride, 3-o-ethyl ascorbic acid and tocopheryl acetate; the emulsifier comprises poloxamer, polyvinylpyrrolidone and cetostearyl alcohol polyether-20; belongs to the technical field of cosmetics.

Description

Whitening nanoemulsion containing 4-butylresorcinol and preparation method thereof

Technical Field

The invention discloses a whitening nanoemulsion, in particular a 4-butylresorcinol-containing whitening nanoemulsion, and also discloses a preparation method of the whitening nanoemulsion, belonging to the technical field of cosmetics.

Background

With the development of social economy and the improvement of living standard of people, the pursuit of beauty has become an important part of people's daily life. As the whitening effect is more and more sought by the aesthetic concept of "whitening, more and more loving people, especially many women, are also pursuing, various whitening products are becoming hot spots of research.

Medical research shows that the difference of the skin color of a human body mainly depends on the content of melanin in basal layers of the skin, wherein the melanin is black or brown biological pigment generated by melanocytes of the skin, is mainly generated by the melanocytes in the skin, is transferred to keratinocytes of epidermis of the skin, and is finally presented on the skin.

Tyrosine in melanocytes is hydroxylated to dopa under the action of tyrosinase, dopa is oxidized to dopa quinone under the action of tyrosinase, and melanin particles are formed, and melanin is gradually transferred from cells to basal layers and precipitated in the basal layers, so that pigmentation is caused. The more melanin is produced, the darker the skin; on the contrary, the more fair the skin is.

In addition, melanin production is affected by external environmental factors and in-vivo metabolic disorders, such as ultraviolet rays, air pollution, in-vivo oxidation and oxidation resistance system imbalance, microecological balance, abnormal trace element content of organisms and the like, besides genetic factors.

Therefore, skin whitening is mainly required to achieve a whitening effect by suppressing the generation and deposition of melanin in the basal layer, and improving the brightness of the skin. Whitening cosmetics can be classified into the following categories according to the action mechanism of whitening components: (1) Reducing melanin production by inhibiting the activity of tyrosinase and other enzymes; (2) Reducing melanin deposition on the top layer of the skin by inhibiting transfer of melanosomes; (3) reducing melanin production by scavenging free radicals; (4) By promoting metabolism of the stratum corneum, melanin excretion is accelerated.

4-Butyl resorcinol is a novel antioxidant capable of inhibiting browning, is the strongest tyrosinase inhibitor discovered so far, and has super-strong whitening effect: in vitro experiments also prove that the whitening effect is 500 times stronger than that of arbutin, and can effectively inhibit melanin synthesis, melanin pigmentation, chloasma and freckle; however, 4-butyl resorcinol has certain irritation, has the phenomenon of intolerance to skin, and is easy to cause allergic phenomena such as redness, swelling and pain and the like of the skin; in addition, 4-butylresorcinol is poorly soluble in water, is easily oxidized and is easily discolored, thus limiting its use.

CN202310858296.X discloses a whitening 4-butyl resorcinol gel bead and a preparation method thereof, wherein the scheme is prepared from the following raw materials in percentage by mass: agarose 0-10%, agar 0-10%, xanthan gum 0-5%, guar gum 0-5%, sodium alginate 0-20%, calcium chloride 0-20%, water 10-98%, 4-butylresorcinol 0.001-10%, other oil-soluble functional components 0-10%, other water-soluble functional substances 0-10%, emulsifying agent 5-60%, auxiliary emulsifying agent 1-40% and oily substances with moisturizing and sliding effects 0-50%, wherein the weight percentages of the components are the weight percentages of the whitening 4-butylresorcinol gel beads. 0-10% of other oil-soluble functional components including 0-10% of 3-o-ethyl ascorbic acid and 0-10% of glycyrrhetinic acid; other water-soluble functional components comprise 0-10% of nicotinamide and 0-10% of tranexamic acid. The emulsifier comprises 800 to 40 percent of tween, 400 to 40 percent of tween, 800 to 40 percent of span, 200 to 40 percent of span, 0 to 50 percent of PEG-40 hydrogenated castor oil, 0 to 50 percent of polyglycerol-10 myristate, 0 to 40 percent of polyglycerol-6 caprylate and 0 to 20 percent of poloxamer. The auxiliary emulsifier comprises 0-30% of propylene glycol, 0-30% of 1, 2-hexanediol, 0-30% of 1, 2-pentanediol, 0-45% of ethoxydiglycol and 0-30% of n-butanol. The oily substances with the moisturizing and sliding effects comprise 0-20% of caprylic triglyceride, 0-20% of isopropyl palmitate, 0-20% of tocopheryl acetate and 0-20% of ceramide.

Therefore, development of a composition for inhibiting melanin synthesis and accelerating melanin metabolism and degradation is particularly necessary.

Disclosure of Invention

In view of the above-mentioned shortcomings, the present invention aims to provide a whitening nanoemulsion with effects of inhibiting melanin synthesis and accelerating melanin metabolic degradation; the whitening nanoemulsion system is stable, is not easy to oxidize, has small irritation to skin and is easy to be absorbed by skin.

The invention also discloses a preparation method of the whitening nanoemulsion.

For this purpose, the first technical solution provided by the present invention is as follows:

The whitening nanoemulsion containing 4-butylresorcinol comprises the following components in percentage by mass: 13.08 to 29.12 percent of active factor, 4 to 40.5 percent of emulsifier phase, 2.2 to 41.8 percent of organic phase, 0.1 to 1 percent of preservative and a proper amount of water;

The active factors comprise 4-butyl resorcinol, nonapeptide-1, decarboxylated carnosine hydrochloride, 3-o-ethyl ascorbic acid and tocopheryl acetate;

The emulsifier comprises poloxamer, polyvinylpyrrolidone and cetostearyl alcohol polyether-20;

Furthermore, the active factors of the whitening nanoemulsion containing 4-butylresorcinol also comprise costustoot extract, hop extract and apple extract.

Further, the weight ratio of the 4-butylresorcinol, the nonapeptide-1, the decarboxylated carnosine hydrochloride, the 3-o-ethyl ascorbic acid and the tocopheryl acetate is as follows: 4-8:0.08-0.12:4-8:4-8:1-5.

Further, the whitening nanoemulsion containing 4-butylresorcinol, wherein the mass ratio of the 4-butylresorcinol, the nonapeptide-1, the decarboxylated carnosine hydrochloride, the 3-o-ethyl ascorbic acid, the tocopheryl acetate, the costustoot extract, the hop extract and the apple extract is as follows: 4-8:0.08-0.12:4-8:4-8:1-5:2-3:1-2:3-5.

Further, the whitening nanoemulsion containing 4-butylresorcinol, wherein the emulsifier further comprises at least one of cetostearyl alcohol polyether-20, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil and acrylic acid (esters) or C10-30 alkanol acrylate crosslinked polymer.

Further, the weight ratio of the poloxamer to the polyvinylpyrrolidone in the whitening nanoemulsion containing 4-butylresorcinol is 1-10:1-10:2-3.

Further, the whitening nanoemulsion containing 4-butylresorcinol is characterized in that the organic phase consists of isopropyl palmitate, 1, 2-hexanediol, 1, 2-pentanediol and ethoxydiglycol according to the mass ratio of 1-10:1-10.

Further, the whitening nanoemulsion containing 4-butylresorcinol is prepared by the following method of the hop extract:

(1) Collecting hops, naturally drying, crushing, and sieving with a 20-40 mesh sieve to obtain beer pollen;

(2) Adding ethanol with the mass concentration of 50-70% into beer pollen according to the volume ratio of 6-8:1, soaking the beer pollen for 30-50 minutes, ultrasonically extracting for 15-30 minutes at the frequency of 40-60 kHz and the power of 150-300W and the temperature of 40-55 ℃, filtering the extracting solution,

(3) Adding ethanol solution with the mass concentration of 50-70% into filter residues again according to the volume ratio of ethanol to beer pollen of 3-5:1, and performing ultrasonic extraction for 10-20 min at the frequency of 40-60 kHz and the power of 150-300W at the temperature of 40-50 ℃; filtering to obtain a second extracting solution, combining the extracting solutions, evaporating and concentrating at the temperature of between 30 and 50kPa and between 35 and 45 ℃ to recover ethanol to obtain an extract primary extract;

(4) Dissolving the extract in an aqueous solution with the mass concentration of 2t% of ethanol, carrying out component enrichment and separation by adopting AB-8 type macroporous adsorption resin, eluting with 20% of ethanol, and then eluting with 70% of ethanol;

(5) Collecting 70% ethanol eluate, and concentrating under reduced pressure of 30kPa to obtain 70% ethanol-eluted flos Lupuli extract;

(6) The hop extract is obtained at the temperature of between 50 ℃ below zero and 30 ℃ below zero for 18 to 20 hours.

Further, the whitening nanoemulsion containing 4-butylresorcinol is prepared from the apple extract by the following method:

1) Selecting fresh apples, cleaning, removing seeds, and crushing the apples to obtain apple paste;

2) Adding 0.1-0.25% of cellulase and 0.2-0.5% of pectase into the apple paste according to the mass of the apple paste, adding 50wt% of ethanol which is 20 times of the mass of the apple paste, adding citric acid to adjust the PH to 5.5-6.0, carrying out enzymolysis for 1-2 h at 50-60 ℃, and then inactivating enzyme in boiling water for 20-30 min;

3) Placing the substances after enzyme deactivation into an ultrasonic extractor, performing ultrasonic treatment at 600W and 50-55 ℃ for 15-20 min, centrifuging at 5000rpm for 12-20 min, centrifuging the extracting solution, and collecting supernatant;

4) Adding 50wt% ethanol with 20 times of the volume of the filter residue into the filter residue, placing into an ultrasonic extractor, performing ultrasonic treatment at 600W and 50-55 ℃ for 15-20 min, centrifuging at 4000rpm for 12-20 min, centrifuging the extracting solution, and collecting supernatant;

5) Combining the supernatant collected in the step 4) and the step 5), concentrating the supernatant in a vacuum rotary evaporator for 1-2 hours until no liquid is evaporated, and freeze-drying the concentrated supernatant at 20Kpa and-25 ℃ for 24-48 hours to obtain an apple crude extract;

6) Dissolving the apple crude extract in distilled water, stirring to dissolve completely, pre-removing impurities with 0.45 μm organic phase microfiltration membrane, collecting filtrate, ultrafiltering the filtrate with ultrafiltration membrane with molecular cut-off of 2kDa at normal temperature and 0.1MPa to obtain ultrafiltrate, and freeze-drying the ultrafiltrate at-25deg.C for 24-48 hr to obtain apple extract.

The second technical scheme provided by the invention is that the preparation method of the whitening nanoemulsion containing 4-butylresorcinol sequentially comprises the following steps:

1) Weighing each substance;

2) Mixing the weighed organic phases in the step 1), heating to 50 ℃, adding 4-butyl resorcinol, tocopheryl acetate and 3-o-ethyl ascorbic acid, and mixing; adding each component of the emulsifier phase, stirring and dissolving;

3) And adding the rest components into the water phase for dissolution, heating the water phase to 50 ℃ after dissolution, slowly adding the organic phase while stirring at the maintained temperature, and stirring until the organic phase is uniform and transparent.

The final technical scheme of the application is the application of the whitening nanoemulsion containing 4-butylresorcinol as an additive for preparing cosmetics.

Compared with the prior art, the technical scheme provided by the invention has the following technical advantages:

1. according to the technical scheme provided by the invention, the 4-butyl resorcinol whitening nanoemulsion is prepared by mutually composing poloxamer, polyvinylpyrrolidone and an organic phase, so that the problem of water solubility of the 4-butyl resorcinol is effectively solved, and the problem of difficult compatibility with other components is solved; the prepared 4-butyl resorcinol whitening nanoemulsion is stable in property, not easy to oxidize, small in skin irritation and easy to absorb.

2. According to the technical scheme provided by the application, poloxamer is adopted as a nonionic surfactant, and the compound polyvinylpyrrolidone improves the solubility and biocompatibility, effectively prolongs the permeation and residence time of the functional components, increases the skin care effect of the functional components, and reduces the irritation of the components such as 4-butylresorcinol and the like to the skin; the stability and the permeability of the cetostearyl alcohol polyether-20, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil and acrylic acid (esters)/C10-30 alkanol acrylate crosslinked polymer provided by the application are further improved by compounding the cetostearyl alcohol polyether-20, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil and acrylic acid (esters) and C10-30 alkanol acrylate crosslinked polymer; the compound isopropyl palmitate, 1, 2-hexanediol, 1, 2-pentanediol and ethoxydiglycol further reduce the oil-water interfacial tension, increase the solubility of the emulsifier, and enable the emulsion to form smaller liquid drops, thereby improving the stability of the nano emulsion system.

3. The whitening active factors provided by the technical scheme comprise 4-butylresorcinol, nonapeptide-1, decarboxylated carnosine hydrochloride, 3-o-ethyl ascorbic acid and tocopheryl acetate, so that the whitening active factors are easy to permeate into the muscle base, inhibit the activity of tyrosinase, prevent melanin from generating and solve color spots at multiple angles; can be used as a contrast agent of melanocyte stimulating hormone, and can be combined with MC1 receptor in a competition way by compounding with nonapeptide-1 to prevent tyrosinase from being further activated to generate melanin; the compound decarboxylated carnosine hydrochloride can effectively capture hydroxyl free radicals, elemental oxygen and hydrogen peroxide free radicals, so that the purpose of scavenging the free radicals is achieved, the oxidation of liposome is inhibited, and cell membranes can be stably protected; inhibit glycosylation of structural proteins, and protect collagen and elastin. The compound 3-o-ethyl ascorbic acid and the tocopheryl acetate lead the active components to be easy to be used by skin, directly participate in the synthesis of collagen after penetrating through the stratum corneum to reach the dermis, restore the activity of skin cells and increase the collagen, thereby leading the skin to become full and elastic and leading the skin to be fine and smooth; has strong antibacterial and antiinflammatory effects, and can further reduce irritation of other components to skin.

4. The whitening activity provided by the invention also comprises costustoot extract, hop extract and apple extract, and the plant extract contains rich flavonoids, vitamins, minerals, polyphenols, humulone, lupulone and the like, and can further effectively remove free radicals in skin such as DPPH free radicals, superoxide anions and the like by mixing with 4-butylresorcinol, nonapeptide-1, decarboxylated carnosine hydrochloride, 3-o-ethyl ascorbic acid and tocopheryl acetate, thereby reducing oxidative stress, protecting cells from oxidative damage, environmental pollution and ultraviolet injury, promoting the renewal of skin cells, improving the elasticity and compactness of skin, reducing the generation of wrinkles and color spots, improving skin color darkness and delaying skin aging; and inhibit the generation of various inflammatory factors, reduce skin inflammatory reaction, relieve skin redness and swelling, pruritus and other symptoms, and improve acne, sensitive skin problems and the like; meanwhile, the moisture content of the skin can be improved, the evaporation of the skin moisture can be inhibited, the moisture retention capacity of the skin can be improved, and the elasticity and luster of the skin can be improved.

5. The hop extract provided by the invention is subjected to ultrasonic extraction and then is subjected to macroporous resin adsorption purification, the apple extract is subjected to ultrasonic extraction and ultrafiltration purification after enzymolysis, the content and purity of active ingredients such as flavonoids, vitamins, minerals and polyphenols are high, and melanin generation is inhibited by a multichannel target spot, so that color spots are solved at multiple angles; and effectively repairing skin barrier, moisturizing, anti-inflammatory, reducing skin stress reaction and relieving skin discomfort.

Drawings

FIG. 1 is a particle size distribution chart of example 1

FIG. 2 is a particle size distribution chart of example 8

FIG. 3 is a TEM image of example 9 after addition of emulsion essence

FIG. 4 is a TEM image of example 10 after addition of emulsion essence

FIG. 5 is a TEM image of example 11 after addition of emulsion essence

FIG. 6 shows the skin condition before the start of the experiment

FIG. 7 shows the skin condition after the end of the experiment (48 h)

FIG. 8 is a statistical histogram of data at 32℃and 37℃for example 1 (4-butylresorcinol nanoemulsion group, NE);

FIG. 9 is a statistical histogram of data at 32℃and 37℃for the 4-butylresorcinol solution group;

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the specific embodiments.

The whitening nanoemulsion containing 4-butylresorcinol provided in the application of example 1 to example 14 comprises the following components:

table 1 examples 1 to 7

Table 2 examples 8 to 14

The costustoot extracts described in examples 1 to 14 were prepared by the following method: pulverizing radix aucklandiae decoction pieces to below 100 mesh, adding 4 equivalent 60% ethanol solution, ultrasonic extracting at room temperature for 1 hr, removing ethanol phase by rotary evaporation, filtering with microporous membrane, and lyophilizing to obtain extract.

The hop extracts described in examples 1 to 14 were prepared by the following method:

The apple extracts described in examples 1 to 14 were prepared by the following method:

2) Adding 0.1% -0.25% (preferably 0.2%) of cellulase and 0.2% -0.5% (preferably 0.3%) of pectase into the apple paste according to the mass of the apple paste, adding 50wt% of ethanol which is 20 times of the mass of the apple paste, adding citric acid to adjust the PH to 5.5-6.0, carrying out enzymolysis at 50-60 ℃ (preferably 55 ℃) for 1-2 hours (preferably 1.5 hours), and then inactivating enzyme in boiling water for 20-30 minutes;

3) Placing the materials after enzyme deactivation into an ultrasonic extractor, performing ultrasonic treatment at 600W and 50-55 ℃ (preferably 55 ℃) for 15-20 min (preferably 20 min), centrifuging at 5000rpm for 12-20 min, centrifuging the extracting solution, and collecting supernatant;

4) Adding 50wt% ethanol which is 20 times the volume of the filter residue into the filter residue, placing into an ultrasonic extractor, performing ultrasonic treatment at 600W and 50-55 ℃ and preferably 55 ℃ for 15-20 min (preferably 20 min), centrifuging at 4000rpm for 12-20 min, centrifuging the extracting solution, and collecting supernatant;

5) Combining the supernatant collected in the step 4) and the step 5), concentrating the supernatant in a vacuum rotary evaporator for 1-2h (preferably 1.5 h) until no liquid is distilled out, and freeze-drying the concentrated supernatant at 20Kpa and-25 ℃ for 24-48 h (preferably 36 h) to obtain an apple crude extract;

The preparation method of the whitening nanoemulsion containing 4-butylresorcinol sequentially comprises the following steps of:

1) Weighing each of the substances according to the components provided in examples 1 to 14;

3) The remaining components are added to the aqueous phase.

Example 15

The product prepared in the example 9 is added into the emulsion according to the mass ratio of 1:9, and the mixture is uniformly mixed.

Example 16

The product prepared in example 10 is added into the emulsion according to the mass ratio of 1:9, and the mixture is uniformly mixed.

Example 17

The product prepared in example 11 is added into the emulsion according to the mass ratio of 1:9, and the mixture is uniformly mixed.

Comparative example 1

The individual substances and their components are the same as those provided in example 8, except that the hop extract is prepared by conventional aqueous extraction.

10G of hops are taken, 80g of 60wt% ethanol solution is added, after soaking for 1 hour, reflux extraction is carried out for 1 hour under the water bath condition of 80 ℃, the extracting solution is separated and repeated twice, and the two extracting solutions are combined and filtered, thus obtaining the clarified hops extracting solution. And (3) placing the clear solution in a vacuum drying oven, continuously drying at 80 ℃ and under the vacuum degree of-0.05 MPa for 48 hours to obtain a dried product, and crushing to obtain the hop extract powder.

Comparative example 2

The individual substances and their components are the same as those provided in example 8, except that the apple extract is obtained by direct purchase.

In order to prove the effect of the technical scheme provided by the application, experimental data of the technical scheme provided by the application are given below:

test example one: nanoemulsion particle size distribution

The particle size distribution of the nanoemulsions in example 1 and example 8 is detected by a Markov laser particle sizer, and the results are shown in fig. 1 (example 1) and fig. 2 (example 8), so that the nanoemulsions prepared by the method are in normal distribution, the median diameter is 10-18nm, the particle size distribution is uniform, and the PDI is less than 0.1.

Test example two: nanoemulsion placement stability study

Examples 1-14, comparative example 1 were placed at room temperature protected from light, taken out at different time points, tested for macroscopic state and particle size distribution, and characterized for nanoemulsion stability, with the following results:

TABLE 3 nanoemulsion shelf stability results

The nanoemulsion prepared by the invention has good placement stability.

Test example three: structural stability study in nanoemulsion

The structural stability of nanoemulsion after being added into essence is observed by a Transmission Electron Microscope (TEM), and the results are shown in fig. 3 (example 15), fig. 4 (example 16) and fig. 5 (example 17), so that the distribution of nanoemulsion drops of 10-20nm is clearly shown around emulsion essence large-particle-size emulsion drops, and a stable nanostructure is maintained and cannot be damaged due to the addition into the emulsion essence.

Test example four: 4-butyl resorcinol nanoemulsion and solution transdermal test

1.32 ℃ Transdermal test

Experimental conditions: 32 ℃ (according to the requirements of Chinese pharmacopoeia, simulate the skin temperature after warm water skin cleaning)

And (3) comparing products: the product provided in example 1 (designated 4-butylresorcinol nanoemulsion) and 4-butylresorcinol solution (designated solution set).

Dissolution medium: physiological saline

Experimental skin: standard skin after dehairing and degreasing treatment of skin of Kunming mouse

Transdermal test results:

TABLE 4 cumulative 48h transmittance of solution set (%)

Table 5 4-butyl resorcinol nanoemulsion 48h cumulative transmittance (%)

Time (h)	Cumulative permeability 1	Cumulative permeability 2	Cumulative permeability 3	Average cumulative transmittance%	SD
						0.5h	0.4669	0.4706	0.4595	0.46567	0.00565
1h	0.8595	0.8646	0.8823	0.8688	0.01197
						2h	1.4488	1.4617	1.4742	1.46157	0.0127
4h	2.4322	2.4388	2.4417	2.43757	0.00487
						6h	3.1343	3.1376	3.141	3.13763	0.00335
8h	3.8971	3.9115	3.9078	3.90547	0.00748
						10h	4.7805	4.7886	4.7883	4.7858	0.00459
12h	7.9016	7.8697	7.9428	7.9047	0.03665
						24h	11.4087	11.3364	11.2699	11.33833	0.06942
48h	17.0738	17.1043	17.0672	17.08177	0.01979

Table 6 solution set to 4-butylresorcinol nanoemulsion cumulative transdermal rate comparison

Statistical analysis was performed on 4-butylresorcinol nanoemulsion group (NE) and solution group (solution) data;

0.5h：P＝0.00000004101＜0.01；

1h：P＝0.0000001899＜0.01；

2h：P＝0.00000082＜0.01；

4h：P＝0.0000002814＜0.01。

After the end of the 48-hour experiment, it was observed that: the skin tissue for the 4-butyl resorcinol nanoemulsion group experiment has no obvious difference from the skin tissue before the experiment in terms of appearance and touch, but after the skin penetration experiment, the central round area contacted by the solution group is obviously yellow, the skin is thinned, the skin is broken when touched lightly, and the skin is seriously damaged, as shown in figure 6.

The transdermal efficiency of the 4-butyl resorcinol nanoemulsion group is obviously higher than that of a solution group within 4 hours, the transdermal efficiency of the solution group is increased from 6 hours later, and the transdermal efficiency is higher than that of the nanoemulsion group, which indicates that the skin is thinned and damaged, and the permeability is obviously enhanced.

And performing t-test analysis of variance on the data of the time point when the accumulated permeability of the nanoemulsion group is higher than that of the solution group, wherein the result shows that the P value is smaller than 0.01 within 4 hours of the test, namely the accumulated permeability of the nanoemulsion group is obviously higher than that of the solution group, and the accumulated permeability of the nanoemulsion group and the solution group have statistical differences.

Transdermal experiments at 2.37 ℃

Experimental conditions: 37 ℃ (simulating skin temperature after hot water cleansing)

Dissolution medium: physiological saline

TABLE 7 cumulative transmittance of 48h for solution set (%)

Table 8 4-butyl resorcinol nanoemulsion 48h cumulative transmittance (%)

Time of	Cumulative permeability 1	Cumulative permeability 2	Cumulative permeability 3	Average cumulative transmittance%	SD
						0.5h	0.3626	0.3549	0.3500	0.3559	0.0064
1h	0.8486	0.8525	0.8436	0.8482	0.0045
						2h	1.1990	1.1931	1.2010	1.1977	0.0041
4h	2.0954	2.0962	2.0911	2.0942	0.0028
						6h	3.0822	3.0588	3.0498	3.0636	0.0167
8h	3.7682	3.7637	3.7681	3.7667	0.0025
						10h	4.5530	4.5485	4.5326	4.5447	0.0107
12h	5.4281	5.4166	5.4202	5.4216	0.0059
						24h	9.9234	9.9227	9.8478	9.8979	0.0435
48h	20.6089	20.7437	20.7341	20.6956	0.0752

Table 9 solution set comparison of cumulative transdermal rate with 4-butylresorcinol nanoemulsion

Time (h)	Cumulative transdermal rate of 4-butylresorcinol solution%	Accumulated transdermal rate of 4-butyl resorcinol nanoemulsion%
			0.5	0.5442±0.0007	0.3559±0.0064
1	1.6124±0.0034	0.8482±0.0045
			2	3.9286±0.0167	1.1977±0.0041
4	7.9511±0.0841	2.0942±0.0028
			6	13.5432±0.0592	3.0636±0.0167
8	18.5827±0.1012	3.7667±0.0025
			10	22.2143±0.0905	4.5447±0.0107
12	25.2030±0.3144	5.4216±0.0059
			24	32.2237±0.3357	98979±0.0435
48	36.8139±0.4703	20.6956±0.0752

As can be seen from the above table, in the transdermal experiments at 37 ℃, the cumulative skin permeation rate of the 4-butylresorcinol solution group from 0.5h is greater than that of the 4-butylresorcinol nanoemulsion group, and the P value is less than 0.05, with significant difference. The difference between the solution group and the nanoemulsion group is not large in the first 2 hours, the difference of the permeability of the 4-butyl resorcinol nanoemulsion and the solution group is gradually increased along with the deepening of the skin damage degree after 2 hours, and the solution group maintains higher skin permeability until the end of the 48-hour experiment. It is presumed that the temperature dependence of 4-butyl resorcinol on skin irritation injury is enhanced with the increase of temperature, and the nanoemulsion can encapsulate 4-butyl resorcinol to form a nanocarrier, and has the functions of slow release and mild delivery into cells. Therefore, after the temperature is increased, the skin of the solution group is obviously damaged within half an hour, the permeability is obviously increased, and the accumulated skin permeability of the solution group is obviously higher than that of the nanoemulsion group.

Comparison of cumulative skin penetration of 4-butylresorcinol solution with nanoemulsions at different temperatures (32 ℃ and s37 ℃) nanoemulsion group 4-butylresorcinol nanoemulsion group (NE) data at 32 ℃ and 37 ℃ were statistically analyzed:

0.5h：P＝0.000023622＜0.01；

1h：P＝0.04934＜0.05；

2h：P＝0.000004341＜0.01；

4h：P＝0.00000004675＜0.01；

6h：P＝0.001675896＜0.01；

8h：P＝0.000006972＜0.01；

10h：P＝0.00000362818＜0.01；

12h：P＝0.0000000333＜0.01；

24h：P＝0.000006917＜0.01；

48h：P＝0.0000001428＜0.01；

Statistical analysis of data at 32℃and 37℃for the 4-butylresorcinol solution group (solution group):

0.5h：P＝0.0000000000095445＜0.01；

1h：P＝0.00000000009＜0.01；

2h：P＝0.000000001433＜0.01；

4h：P＝0.0000000282＜0.01；

6h：P＝0.00000000337＜0.01；

8h：P＝0.00000000665＜0.0 1；

10h：P＝0.0000000012＜0.01；

12h：P＝0.0000000982＜0.0 1；

24h：P＝0.000000165＜0.01；

48h：P＝0.000002941＜0.01。

From fig. 8, fig. 9 and data analysis, it is known that the skin permeability of the nanoemulsion group is not increased along with the temperature rise to 37 ℃, but is slightly lower than 32 ℃ instead, and the damage capability of the 4-butylresorcinol to the skin is obviously increased along with the temperature rise only in 48 hours of 37 ℃ groups, so that the nanoemulsion has a wrapping effect on the nanoemulsion, the skin damage is small, the skin state is good in 24 hours, and the skin permeability is obviously increased due to the fact that the 4-butylresorcinol wrapped in the nanoemulsion has a certain damage to the skin at 48 hours. The phenomenon that the damage capability of 4-butyl resorcinol to skin is obviously increased along with the temperature rise is more obvious, 4-butyl resorcinol directly acts on the skin, the skin permeability is higher than 32 ℃ from 0.5h to 48h at 37 ℃, and the difference between the 4-butyl resorcinol and the skin permeability is increased along with the time, so that the skin damage is more serious and the skin permeability is obviously increased.

In combination with the experimental results and data analysis, it can be seen that the 4-butyl resorcinol nanoemulsion group is compared with the solution group: the skin irritation and injury effects of the 4-butylresorcinol are reduced through the encapsulation of the nano-carrier; the skin permeability of the 4-butyl resorcinol nanoemulsion group is obviously higher than that of the solution group within 2 hours (the acting time of the skin care product is met) under the condition of normal skin temperature (32 ℃); increasing the temperature (e.g., 37 ℃) significantly increases the skin damage of 4-butylresorcinol.

Test example five: freckle removing and whitening efficacy test

1 Principle and method

(1) Principle and purpose of testing

The freckle removing and whitening effects of the test object are evaluated by inducing a melanin model generated by melanoma cells and detecting the relative concentration of melanin in the cells.

And evaluating the freckle removing and whitening effects of the test product by detecting the melanin content of the test sample in the cell model as an index.

(2) Evaluation index and determination criterion

Table 10 evaluation index and judgment Standard

(3) Test subjects

Positive control: 0.05% vitamin C

Sample: examples 1-14, comparative examples 1-2;

(4) Instrument reagent

Enzyme-labeled instrument (BioTek), analytical balance (Shimadzu), pipette (Beijing Dalong)

Cell line: melanoma cells (B16)

Culture solution: DMEM medium (sameifer) experimental kit containing 10% fbs (Shanghai dashi biotechnology): CCK-8 kit (Solarbio), melanin kit (Xinquan technology)

5. Environmental conditions and culture conditions

Environmental conditions: the temperature is 18-27 ℃, and the humidity is 30-70% RH

Culture conditions: culturing at 37deg.C under 5% CO ₂ and saturated humidity

6. Test method

6.1 Cell Activity test

1. Cell suspensions (100. Mu.L/well) were seeded in 96-well plates. The plates were placed in an incubator for preculture.

2. 10 Mu LCCK-8 solutions were added to each well.

3. The plates were incubated in the incubator for 2 hours.

4 Absorbance at 450nm was measured with a microplate reader.

6.2 Melanin content determination

1. Melanoma cells are inoculated into a 96-well plate and cultured for 18-24 hours.

2. The blank groups were replaced with maintenance medium and the remaining groups were incubated with maintenance medium containing sample or positive control for 48h.3. The original medium was discarded and washed 3 times with PBS

4. Adding DMEM medium (except blank group), inducing for 48 hr, and making cells produce melanin

5. Cell supernatants were collected and assayed for melanin content according to kit instructions.

6. Data analysis:

data were analyzed using SPSS and expressed as mean ± standard deviation.

1. Cell activity relative to each group was calculated with the cell activity of the control group being 100%.

Relative cell activity (%) = [ (OD sample-OD blank)/(OD control-OD blank) ] ×100%

2. Melanin downregulation.

Down-regulation (%) = (1-T/C) ×100%

Wherein:

Mean value of melanin content of T-test object;

C-blank/solvent control melanin content average.

2. Test results

1. Cell activity analysis of the product provided in example 8, table 11

TABLE 11 analysis of cell Activity

2. Product fine melanin downregulation assay provided in example 8

TABLE 12 Effect of test substances on cellular melanin levels

Group of	Down regulation (%)	P value
			Blank control group	/	/
Sample (0.05 mg/mL)	31.28±1.24	＜0.001
			Sample (0.025 mg/mL)	20.06±2.23	＜0.001
Sample (0.0125 mg/mL)	14.53±1.54	＜0.001
			Positive control group	35.23±1.68	＜0.001

3. The cell activities of the products provided in comparative examples 1-2, examples 3, examples 6, examples 8, examples 10 and examples 13 were measured using a sample concentration of 0.025mg/mL, and the cell melanin level downregulation (%), table 13.

TABLE 13

	Cell relative Activity (%)	Down regulation (%)	P value
				Example 1	99.00±1.78	16.02±2.47	＜0.001
Example 3	99.00±1.91	15.45±2.06	＜0.001
				Example 6	99.00±1.89	14.27±1.97	＜0.001
Example 8	99.00±1.65	20.06±2.23	＜0.001
				Example 10	99.00±1.78	17.05±2.09	＜0.001
Example 13	99.00±2.05	16.08±1.79	＜0.001
				Comparative example 1	99.00±1.79	17.02±1.75	＜0.001
Comparative example 2	99.00±1.68	16.96±2.12	＜0.001

Remarks: p > 0.05, representing no statistical difference between the two sets of data; 0.01< P < 0.05 means statistically significant; 0.001 < P < 0.01 indicates significant differences; p < 0.001, which indicates a significant difference.

According to the test results, the positive control group showed significantly reduced relative melanin content (P < 0.05) compared with the blank group, and the test results were valid. The concentrations of the samples are respectively 0.05mg/mL, 0.025mg/mL and 0.0125mg/mL, the relative content of melanin can be reduced, and the samples have obvious differences (P is less than 0.05), so that the samples have the effects of removing freckles and whitening; the increase of the content of the 4-butyl resorcinol is beneficial to improving the whitening effect of the product; in addition, the 4-butyl resorcinol compound costustoot extract, the hop extract and the apple extract further improve the whitening effect of the product; as can be seen from comparative examples 1,2 and 8, the hop extract and apple extract prepared by the extraction method provided by the present application have significantly higher whitening effect than the hop extract and apple extract provided in comparative examples 1 and 2 and other components in the formula, probably due to higher activity of the hop extract and apple extract provided by the present application.

Claims

1. The whitening nanoemulsion containing 4-butylresorcinol is characterized by comprising the following components in percentage by mass: 13.08 to 29.12 percent of active factor, 4 to 40.5 percent of emulsifier phase, 2.2 to 41.8 percent of organic phase, 0.1 to 1 percent of preservative and a proper amount of water;

the active factors comprise 4-butylresorcinol, nonapeptide-1, decarboxylated carnosine hydrochloride, 3-o-ethyl ascorbic acid, tocopheryl acetate, radix aucklandiae extract, hops extract, and apple extract;

The mass ratio of the 4-butylresorcinol to the nonapeptide-1 to the decarboxylated carnosine hydrochloride to the 3-o-ethyl ascorbic acid to the tocopheryl acetate to the costustoot extract to the hop extract to the apple extract is 4-8:0.08-0.12:4-8:4-8:1-5:2-3:1-2:3-5.

2. The whitening nanoemulsion comprising 4-butylresorcinol of claim 1, wherein said emulsifier further comprises at least one of cetostearyl alcohol polyether-20, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, acrylates/C10-30 alkanol acrylate cross-linked polymer.

3. The whitening nanoemulsion containing 4-butylresorcinol according to claim 1, wherein the mass ratio of poloxamer, cetostearyl alcohol polyether-20 and polyvinylpyrrolidone is 1-10:1-10:2-3.

4. The whitening nanoemulsion containing 4-butylresorcinol according to claim 1, wherein the organic phase consists of isopropyl palmitate, 1, 2-hexanediol, 1, 2-pentanediol and ethoxydiglycol in a mass ratio of 1-10:1-1.

5. The 4-butylresorcinol-containing whitening nanoemulsion of claim 1, wherein the elecampane extract is prepared by the following method: pulverizing radix aucklandiae decoction pieces to below 100 meshes, adding 3-5 equivalents of 50% -80% ethanol solution, performing ultrasonic extraction at 20-35 ℃ for 0.5-1 h, removing an alcohol phase by rotary evaporation of an extract liquid, filtering by a microporous filter membrane, and freeze-drying to obtain an extract.

6. The 4-butylresorcinol-containing whitening nanoemulsion of claim 1, wherein the hops extract is prepared by the following method:

(2) Adding ethanol with the mass concentration of 50-70% into beer pollen according to the volume ratio of 6-8:1, soaking the beer pollen for 30-50 minutes, performing ultrasonic extraction for 15-30 minutes at the frequency of 40-60 kHz and the power of 150-300W at the temperature of 40-55 ℃, and filtering the extracting solution;

(6) Eluting the hop extract with 70% ethanol at-50deg.C to-30deg.C for 18-20 hr to obtain hop extract.

7. The whitening nanoemulsion comprising 4-butylresorcinol according to claim 1, wherein the apple extract is prepared by the following method:

8. The method for preparing the whitening nanoemulsion containing 4-butylresorcinol according to claim 1, which is characterized by comprising the following steps in sequence:

1) Weighing each substance according to the mass fraction of any one of claims 1 to 4;

2) Mixing the organic phases weighed in the step 1), heating to 50 ℃, and then adding 4-butylresorcinol, tocopheryl acetate and 3-o-ethyl ascorbic acid, and mixing; adding each component of the emulsifier phase, stirring and dissolving;

9. Use of the 4-butylresorcinol-containing whitening nanoemulsion of claim 1 as an additive for cosmetics.