CN112386622B - Preparation method and application of freckle-removing active substance - Google Patents
Preparation method and application of freckle-removing active substance Download PDFInfo
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Abstract
The invention relates to a preparation method and application of an efficient freckle-removing active substance, which is characterized by comprising the following steps: A. drying fresh folium Artemisiae Argyi, adding into round-bottom flask together with deionized water, adjusting pH to 2.0-3.0 with concentrated hydrochloric acid, heating in water bath for extraction, and receiving folium Artemisiae Argyi essential oil with conical flask at liquid outlet of condenser tube; carrying out water bath extraction on the first filter residue under the environment of pH6.5-7.0; extracting the second filter residue in water bath under the environment of pH8.2-8.6; B. mixing the first filtrate, the second filtrate and the third filtrate, and filtering to obtain folium Artemisiae Argyi extract; C. distilling folium Artemisiae Argyi extract under reduced pressure, concentrating to obtain folium Artemisiae Argyi extract, adding appropriate amount of extract into ethanol/ammonium sulfate aqueous two-phase system, adjusting pH to 3-4 with concentrated hydrochloric acid, shaking, and standing; collecting the ethanol phase at the upper layer, and concentrating under reduced pressure to obtain folium Artemisiae Argyi flavonoids; D. mixing the folium artemisiae argyi essential oil and the folium artemisiae argyi flavonoid to obtain the high-efficiency freckle removing active substance. The high-efficiency freckle-removing active substance has a good improvement effect on chloasma.
Description
Technical Field
Chloasma is a pigmentation disorder frequently occurring on women's face. Its clinical reaction is mainly light brown to dark brown pigment spots, and some pigment spots have skin lesions of a certain degree and are mostly distributed on the face, cheeks, nose and other parts. The pathogenesis of chloasma is complex, and at present, endocrine, heredity, sunlight irradiation, free radicals and the like are considered as main factors causing the chloasma. Therefore, the desire to improve chloasma requires attention to mood regulation and reduction of sunlight irradiation, and control of free radicals and inflammation is also important.
Referring to domestic and foreign documents, the main intervention treatment of chloasma comprises topical application of drugs, chemical skin change, laser treatment, oral administration and the like. In the intervention means of various chloasma in cosmetics, the most commonly used whitening agents comprise fruit acid and derivatives thereof, vitamin C, arbutin, tranexamic acid and the like, but in the practical application process, the fruit acid and the derivatives thereof have strict pH dependence, the vitamin C also has the characteristics of stronger thermal instability and extremely easy color change, the arbutin is extremely easy to decompose in an acidic environment, and the tranexamic acid has a certain degree of sensitization. Meanwhile, the traditional Chinese medicine external application method based on the theory of traditional Chinese medicine has better universality due to the mild property and lower sensitization, some pure natural Chinese herbal medicine plants can also show better tyrosinase inhibitory activity and B-16 melanocyte inhibitory activity, and the active ingredient extract has higher research value for improving chloasma.
As a traditional Chinese herbal medicine used as both medicine and food in China, folium artemisiae argyi is often found in an anti-inflammatory compatible formula and is used less singly. Such as lignum sappan and flos Carthami external lotion prepared from folium Artemisiae Argyi, lignum sappan, flos Carthami, pericarpium Granati, cortex Phellodendri, Kochiae fructus Atriplicis Sibiricae, and Natrii sulfas at certain ratio. The main active ingredients of folium Artemisiae Argyi include volatile oil, flavonoids, eucalyptol, triterpenes, etc. The flavone in folium Artemisiae Argyi is mainly free flavone, and has good free radical scavenging activity. The argy wormwood leaves are reported to have better inhibition on early, middle and late stages of inflammation. The folium Artemisiae Argyi volatile oil mainly contains monoterpene, terpineol, sesquiterpene, aromatic compound, etc., wherein the substance providing antiinflammatory activity is mainly concentrated in sesquiterpene and monoterpene. The research proves that the blumea oil can inhibit the activation of NF-kB; down-regulating JNK, P38, MAPK phosphorylation levels; blocks a plurality of inflammatory pathways such as JAK2/STAT3, reduces the release of inflammatory factors such as NO, PGE2, TNF-1 and the like, and reduces the expression of TNF-alpha, iNOS and COX-2, thereby inhibiting the generation of inflammation at multiple levels.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method and application of a high-efficiency freckle-removing active substance, wherein the high-efficiency freckle-removing active substance simultaneously contains folium artemisiae argyi volatile oil and folium artemisiae argyi flavonoid substances and has a good improvement effect on chloasma; the high-efficiency freckle-removing active substance prepared by the method can be directly applied to freckle-removing and whitening cosmetics.
In order to solve the technical problems, the invention adopts the following technical scheme: the preparation method of the high-efficiency freckle-removing active substance is characterized by comprising the following steps of:
A. picking fresh folium Artemisiae Argyi, spreading on a drying sieve, drying in a dryer at 60 deg.C for 6-8 hr until the water content of folium Artemisiae Argyi is lower than 2% to obtain dried folium Artemisiae Argyi; weighing a proper amount of dried folium artemisiae argyi, adding the dried folium artemisiae argyi into a round-bottom flask, and then adding deionized water, wherein the mass of the deionized water is 5-10 times of that of the dried folium artemisiae argyi; adjusting pH of the liquid in the flask to 2.0-3.0 with chemical pure concentrated hydrochloric acid, heating and extracting the round-bottom flask in 75-95 deg.C water bath, and collecting folium Artemisiae Argyi essential oil in a conical flask filled with deionized water at the liquid outlet of the condenser tube; stopping heating and extracting when the yellow oily liquid is not generated any more; filtering the mixture in the round-bottom flask, collecting first filtrate, reloading first filter residue into the round-bottom flask, adding deionized water with the mass of 5-10 times that of the first filter residue, adding sodium hydroxide solution with appropriate concentration to adjust the pH value of liquid in the flask to 6.5-7.0, heating and extracting at the water bath temperature of 75-95 ℃ until no yellow oily liquid is generated, and stopping heating and extracting; filtering the mixture in the round-bottom flask, collecting a second filtrate, reloading a second filter residue into the round-bottom flask, adding deionized water with the mass 5-10 times that of the second filter residue, adding a sodium hydroxide solution with a proper concentration to adjust the pH value of the liquid in the flask to 8.2-8.6, heating and extracting at the water bath temperature of 75-95 ℃ until no yellow oily liquid is generated, and stopping heating and extracting; filtering the mixture in the round-bottom flask, collecting a third filtrate, and discarding filter residues; taking the upper layer yellow oily liquid in the conical flask to obtain folium Artemisiae Argyi essential oil;
B. mixing the first filtrate, the second filtrate and the third filtrate obtained in the step A, and filtering the mixture by a filter bag of 200 meshes to obtain a coarse extract of the folium artemisiae argyi; adding active carbon which accounts for 2% of the mass of the folium artemisiae argyi crude extract into the folium artemisiae argyi crude extract, wherein the particle size of the active carbon is 200 meshes, and adsorbing for 20min under the condition of 80 ℃ water bath; paving diatomite with the mass being 8% of that of the folium artemisiae argyi crude extract in a Buchner funnel, wherein the particle size of the diatomite is 150 meshes, and performing suction filtration on the folium artemisiae argyi crude extract through the Buchner funnel to obtain an folium artemisiae argyi extract;
C. distilling the folium artemisiae argyi extract obtained in the step B at 70 ℃ under reduced pressure and concentrating to obtain folium artemisiae argyi extract, weighing a proper amount of folium artemisiae argyi extract and putting the extract into an ethanol/ammonium sulfate double-aqueous phase system to obtain a premixed solution; the mass concentration of absolute ethyl alcohol in the ethanol/ammonium sulfate double-aqueous phase system is 27%, the mass concentration of ammonium sulfate is 17% -22%, and the balance is deionized water; the folium artemisiae argyi extract accounts for 5% of the total mass of the ethanol/ammonium sulfate aqueous two-phase system; adjusting the pH of the premix solution to 3-4 with chemically pure concentrated hydrochloric acid, stirring for 10min, transferring to a separating funnel, fully shaking for 10 times, and standing to obtain a clear layered solution, wherein the upper layer is an ethanol phase and the lower layer is a salt phase; collecting upper ethanol phase, and concentrating under reduced pressure at 70-80 deg.C to obtain extract, to obtain folium Artemisiae Argyi flavonoids;
D. and D, mixing the folium artemisiae argyi essential oil obtained in the step A with the folium artemisiae argyi flavonoid substances obtained in the step C to obtain the efficient freckle removing active substance.
The high-efficiency freckle-removing active substance is added into whitening cream and freckle-removing cream products as an active substance, and the adding amount is 0.05-0.2% by mass ratio.
In the prior art, the folium artemisiae argyi is used for fumigation and foot bath independently, the external application is usually compatible with other traditional Chinese medicine components, and the application for removing chloasma is less when the external application is used for removing chloasma independently. The high-efficiency freckle-removing active substance prepared by adopting the folium artemisiae argyi as the raw material has a better in-vitro test effect, and has a better freckle removing effect when being used alone. In addition, the method improves the comprehensive utilization rate of the folium artemisiae argyi plants.
The process of the invention has the following advantages: the effective components in the folium artemisiae argyi are efficiently extracted by adopting a semi-bionic extraction method, and the folium artemisiae argyi is separated and purified by adopting a double-aqueous-phase extraction method, so that the folium artemisiae argyi efficient freckle removing active substance is finally obtained.
1. Adopts a semi-bionic extraction method to extract the essential oil of the folium artemisiae argyi and simultaneously extract flavonoids of the folium artemisiae argyi with high efficiency. According to the bionics principle, the digestion and absorption process of the traditional Chinese medicine under the pH value of body fluid of stomach, small intestine and large intestine of a human body is simulated by controlling the change of the pH value in the extraction process, so that the active ingredients in the traditional Chinese medicine are extracted efficiently. The semi-bionic extraction method can effectively destroy plant cells and swell cell tissues, so that the dissolving capacity and the penetrating capacity of the solution are enhanced, the high-efficiency extraction of the volatile oil of the folium artemisiae argyi and the flavonoids of the folium artemisiae argyi is facilitated, and the process can realize the simultaneous extraction of the essential oil of the folium artemisiae argyi and the flavonoids of the folium artemisiae argyi.
2. Adopts a novel liquid-liquid extraction purification technology, namely a two-aqueous phase purification method. The method can realize the high-efficiency separation and enrichment of the flavonoid substances, has the advantages of simple and convenient operation compared with the purification by macroporous adsorption resin, reduces the dosage of the organic solvent, and is beneficial to the application of the flavonoid substances in skin care. According to the invention, the essential oil and flavonoids of the folium artemisiae argyi with anti-inflammatory and anti-free radical activities in the folium artemisiae argyi are extracted at the same time, so that the comprehensive utilization rate of folium artemisiae argyi plants is improved.
In order to verify the chloasma-improving effect of the high-efficiency freckle-removing active substance extracted by the method, the following in vitro tests were performed by comparing the high-efficiency freckle-removing active substance obtained in example 1 with a conventional folium artemisiae argyi extract obtained by a common extraction method of traditional Chinese medicine active ingredients. The preparation method of the folium artemisiae argyi conventional extracting solution comprises the following steps: extracting with 70% ethanol under reflux, adsorbing the extract with active carbon, filtering, distilling at 70 deg.C under reduced pressure to obtain extract, and mixing the extract with distilled water and propylene glycol at a ratio of 1:20 to obtain folium Artemisiae Argyi conventional extract.
In vitro test of whitening Activity
1. Tyrosinase inhibition assay
Taking the first 3 substances by a pipette according to the table 1, adding the substances into a centrifuge tube, uniformly mixing, putting the centrifuge tube into a water bath at 37 ℃ for warm bath for 10min, adding 200U/ml tyrosinase solution, putting the centrifuge tube into a water bath at 37 ℃ for warm bath for 40min, stopping the reaction at 90 ℃, adding 200ul of the tyrosinase solution into a 96-well plate, and measuring the absorbance under the wavelength of 475nm of an enzyme labeling instrument.
TABLE 1 tyrosinase inhibition test reagent addition Table
Tyrosinase inhibition% = (blank absorbance-extract absorbance)/blank absorbance × 100%;
the experimental results are shown in table 2, compared with the 70% ethanol reflux extraction method, the high-efficiency freckle removing active substance obtained by the invention with the same concentration has higher inhibitory activity on tyrosinase, and the whitening activity of the high-efficiency freckle removing active substance is dose-dependent. Indicated as very significant difference compared to the arbutin positive control group (p < 0.01) and significant difference compared to the arbutin positive control group (p < 0.05).
TABLE 2 high efficacy spot removal active tyrosinase inhibition assay
Note: table indicates significant difference between data in the same columnp<0.05, in the table, indicates that the data in the same column are extremely significant in differencep<0.01。
2. B-16 melanocyte inhibition assay
B16 cell source: a sanggo organism; the goods number is: ATCC CRL-6475;
generation number: generation 16; counting: the number of cells is 2 multiplied by 104 per well, 24-well plate;
the concentration of the high-efficiency freckle-removing active substance is diluted to 1mg/mL, 0.5mg/mL and 0.25 mg/mL. Based on the melanin content in the blank control as 100%, IBMX (as positive stimulation, the final concentration is 22.2 mug/ml) is used for stimulating the cells to generate melanin, so that the cells with the melanin content of 261.71% are obtained. The spot samples are added with high-efficiency spot-removing active substances with different concentrations in the cell sap respectively. Arbutin (0.3 mg/ml) is set as a control group, and the change of the melanin content of the cells is determined after incubating samples for 48 hours.
Melanin inhibition/% = (IBMX melanin content-melanin content after incubation)/IBMX melanin content × 100%.
TABLE 3 high-potency Spot-removing active B16 cell melanin activity assay
Note: table indicates significant difference between data in the same columnp<0.05, in the table, indicates that the data in the same column are extremely significant in differencep<0.01。.
The experimental results are shown in table 3, and compared with the 70% ethanol reflux extraction method, the high-efficiency freckle removing active substance with the same concentration has obviously improved melanin inhibiting activity on B16 cells. Table 3 shows that the difference was very significant compared to the arbutin positive control group (p < 0.01) and significant compared to the arbutin positive control group (p < 0.05).
3. Anti-inflammatory activity in vitro test, i.e. RAW264.7 cell NO content inhibition test:
marking the NO content generated by rat macrophage RAW264.7 after being acted by 1 mu g/mL LPS as 100% (positive control), setting a group without adding LPS as negative control, carrying out sample application on folium artemisiae argyi extract after gradient dilution, and recording the change condition of NO release amount. The reduction of the release amount of NO represents the weakening of the activity of inflammatory cells, and the anti-inflammatory effect is better.
TABLE 4 variation of NO release amount of highly effective spot-removing active substance
Note: table indicates significant difference between data in the same columnp<0.05, in the table, indicates that the data in the same column are extremely significant in differencep<0.01
As shown in Table 4, the highly effective speckle-removing active substance obtained by the present invention has very good anti-inflammatory activity and shows a dose-dependent trend. In table 4, the difference was very significant compared to the LPS positive control group (p < 0.01), and the difference was significant compared to the LPS positive control group (p < 0.05).
4. Cell activity assay (CCK-8 kit)
The high-efficiency freckle-removing active substance is subjected to sample application after being diluted in a gradient manner, and the cell activity condition under the folium artemisiae argyi extract with each concentration is recorded.
TABLE 5 cytotoxicity test results of highly effective spot-removing active substances
Note: table indicates significant difference between data in the same columnp<0.05, in the table, indicates that the data in the same column are extremely significant in differencep<0.01
As shown in Table 5, the cell viability exceeding 80% was considered to be low in cytotoxicity and was used. CCK-8 cell survival rate tests prove that the high-efficiency freckle-removing active substance has stronger cytotoxicity at the concentration of more than 25 mu g/mL after being purified by AB-8 resin. Therefore, the concentration of the effective freckle removing active substance can be selected to be lower. In table 5, the difference was very significant compared to the blank positive control group (p < 0.01), and the difference was significant compared to the blank positive control group (p < 0.05).
5. Evaluation test of human body efficacy
Subject: the total 54 people are female patients with chloasma, and the age distribution is 30 plus or minus 3 years old. (wherein 1. people allergic to azelaic acid; 2. pregnant or lactating women; 3. patients receiving other treatment) the test subjects were divided into 6 groups of 9 subjects, blank group, positive control group and groups of high-efficacy spot-removing active substances 1, 2, 3 and 4.
Sample preparation: a stable oil-in-water sample a containing azelaic acid at a concentration of 0.5% and a stable oil-in-water sample B, C, D, E containing high spot removing actives 0.5%, 1%, 1.5%, 2%, respectively, were prepared, all with identical sample matrix components. In the test process, the sample painter and the data collector do not know the concentration of the sample, and the concentration of each sample is revealed after the test is finished, and statistical analysis is carried out.
The efficacy evaluation method comprises the following steps: the product is used for chloasma in the morning and evening, and is continuously used for 8 weeks, and no product is used in the blank control group.
The freckle removing effect evaluation method comprises the following steps: the depigmenting efficacy of the samples was evaluated by measuring the melanin value and the colour intensity L x value of the skin before, 2 weeks, 4 weeks, 6 weeks, 8 weeks of use of the product, respectively using a spectrophotometer (MX 18 type) and a colorimeter (CD-2600), by comparing the change in the melanin value and L x value before and after self-application of the samples. The indoor light, temperature (20 +/-2) DEG C and humidity (40 +/-5)% are ensured to be uniform during each measurement, a test is carried out after a test subject takes a rest for 15min without using any product, and the measurement is carried out 3 times on the same part. The average value was taken.
Colorimetric value variation amount: Δ L value = nth day L value-base L value;
melanin value change amount: Δ melanin value = day n melanin value-basal melanin value.
TABLE 6 high-efficacy depigmenting active substance human body test delta L value variation
As shown in table 6, the sample B, C, D, E with the high spot removing active added thereto showed no significant improvement in skin color L over the first 4 weeks compared to the blank group (p>0.05), but the skin color was significantly improved after 30 days of continuous use (p<0.05) and, at the same time, the degree of improvement was slightly lower compared to the positive control azelaic acid.
TABLE 7 variation of melanin values in human body test of highly effective spot-removing active substance
As shown in Table 7, the A, B, C, D melanin values for the sample using the high efficacy depigmenting active were significantly improved over the initial levels (p<0.05), and the improvement ability is dose-dependent. The change situation of the melanin value and the L-value proves that the high-efficiency freckle removing active substance has better activity of improving chloasma.
In conclusion, the high-efficiency freckle-removing active substance prepared by the invention has a remarkable tyrosinase inhibition effect, has a remarkable melanin inhibition activity on B16 cells, has a very good whitening effect, and also has a better NO inhibition activity of rat macrophage RAW 264.7. The combination with the confirmation of human body experiment results can be directly used as an active substance to be added into spot-removing and whitening cosmetics, thereby achieving the effect of removing chloasma.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.
Example 1: the preparation method of the high-efficiency freckle-removing active substance is characterized by comprising the following steps of: the method comprises the following steps:
A. picking fresh folium Artemisiae Argyi, spreading on a drying sieve, drying in a dryer at 60 deg.C for 6 hr until the water content of folium Artemisiae Argyi is lower than 2% to obtain dried folium Artemisiae Argyi; weighing a proper amount of dried folium artemisiae argyi, adding the weighed dried folium artemisiae argyi into a round-bottom flask, and then adding deionized water, wherein the mass of the deionized water is 5 times of that of the dried folium artemisiae argyi; adjusting the pH of the liquid in the flask to 2.0 by using chemical pure concentrated hydrochloric acid, placing the round-bottom flask in a water bath at 75 ℃ for heating and extracting, and receiving folium artemisiae argyi essential oil by adopting a conical flask filled with deionized water at a liquid outlet of a condensation pipe; stopping heating and extracting when the yellow oily liquid is not generated any more; filtering the mixture in the round-bottom flask, collecting first filtrate, reloading first filter residue into the round-bottom flask, adding deionized water with the mass of 5 times that of the first filter residue, adding sodium hydroxide solution with proper concentration to adjust the pH value of liquid in the flask to 6.5, heating and extracting at the water bath temperature of 75 ℃ until no yellow oily liquid is generated, and stopping heating and extracting; and filtering the mixture in the round-bottom flask, collecting a second filtrate, reloading the second filter residue into the round-bottom flask, adding deionized water with the mass of 5 times that of the second filter residue, adding a sodium hydroxide solution with an appropriate concentration to adjust the pH value of the liquid in the flask to 8.2, heating and extracting the solution at the water bath temperature of 75 ℃ until no yellow oily liquid is generated, and stopping heating and extracting. Filtering the mixture in the round-bottom flask, collecting a third filtrate, and discarding filter residues; taking the upper layer yellow oily liquid in the conical flask to obtain folium Artemisiae Argyi essential oil;
B. mixing the first filtrate, the second filtrate and the third filtrate obtained in the step A, and filtering the mixture by a filter bag of 200 meshes to obtain a coarse extract of the folium artemisiae argyi; adding active carbon which accounts for 2% of the mass of the folium artemisiae argyi crude extract into the folium artemisiae argyi crude extract, wherein the particle size of the active carbon is 200 meshes, and adsorbing for 20min under the condition of 80 ℃ water bath; paving diatomite with the mass being 8% of that of the folium artemisiae argyi crude extract in a Buchner funnel, wherein the particle size of the diatomite is 150 meshes, and performing suction filtration on the folium artemisiae argyi crude extract through the Buchner funnel to obtain an folium artemisiae argyi extract;
C. distilling the folium artemisiae argyi extract obtained in the step B at 70 ℃ under reduced pressure and concentrating to obtain folium artemisiae argyi extract, weighing a proper amount of folium artemisiae argyi extract and putting the extract into an ethanol/ammonium sulfate double-aqueous phase system to obtain a premixed solution; the mass concentration of absolute ethyl alcohol in the ethanol/ammonium sulfate double-aqueous phase system is 27%, the mass concentration of ammonium sulfate is 17%, and the balance is deionized water; the folium artemisiae argyi extract accounts for 5% of the total mass of the ethanol/ammonium sulfate aqueous two-phase system; adjusting the pH value of the premix to 3 by using chemically pure concentrated hydrochloric acid, stirring for 10min, transferring to a separating funnel, fully shaking for 10 times, and standing to obtain a clear layered solution, wherein the upper layer is an ethanol phase and the lower layer is a salt phase; collecting the upper ethanol phase, and concentrating under reduced pressure at 70 deg.C to obtain extract, to obtain folium Artemisiae Argyi flavonoids;
D. and D, mixing the folium artemisiae argyi essential oil obtained in the step A with the folium artemisiae argyi flavonoid substances obtained in the step C to obtain the efficient freckle removing active substance.
Example 2:
the preparation method of the high-efficiency freckle-removing active substance is characterized by comprising the following steps of: the method comprises the following steps:
A. picking fresh folium Artemisiae Argyi, spreading on a drying sieve, drying in a dryer at 60 deg.C for 7 hr until the water content of folium Artemisiae Argyi is lower than 2% to obtain dried folium Artemisiae Argyi; weighing a proper amount of dried folium artemisiae argyi, adding the weighed dried folium artemisiae argyi into a round-bottom flask, and then adding deionized water, wherein the mass of the deionized water is 8 times of that of the dried folium artemisiae argyi; adjusting the pH of the liquid in the flask to 2.5 by using chemical pure concentrated hydrochloric acid, placing the round-bottom flask in a water bath at 85 ℃ for heating and extracting, and receiving folium artemisiae argyi essential oil by adopting a conical flask filled with deionized water at a liquid outlet of a condensation pipe; stopping heating and extracting when the yellow oily liquid is not generated any more; filtering the mixture in the round-bottom flask, collecting first filtrate, reloading first filter residue into the round-bottom flask, adding deionized water with the mass of 8 times that of the first filter residue, adding sodium hydroxide solution with proper concentration to adjust the pH value of liquid in the flask to 6.8, heating and extracting at 85 ℃ in a water bath until no yellow oily liquid is generated, and stopping heating and extracting; and filtering the mixture in the round-bottom flask, collecting a second filtrate, reloading the second filter residue into the round-bottom flask, adding deionized water with the mass of 8 times that of the second filter residue, adding a sodium hydroxide solution with an appropriate concentration to adjust the pH value of the liquid in the flask to 8.4, heating and extracting the solution at the water bath temperature of 85 ℃ until no yellow oily liquid is generated, and stopping heating and extracting. Filtering the mixture in the round-bottom flask, collecting a third filtrate, and discarding filter residues; taking the upper layer yellow oily liquid in the conical flask to obtain folium Artemisiae Argyi essential oil;
B. mixing the first filtrate, the second filtrate and the third filtrate obtained in the step A, and filtering the mixture by a filter bag of 200 meshes to obtain a coarse extract of the folium artemisiae argyi; adding active carbon which accounts for 2% of the mass of the folium artemisiae argyi crude extract into the folium artemisiae argyi crude extract, wherein the particle size of the active carbon is 200 meshes, and adsorbing for 20min under the condition of 80 ℃ water bath; paving diatomite with the mass being 8% of that of the folium artemisiae argyi crude extract in a Buchner funnel, wherein the particle size of the diatomite is 150 meshes, and performing suction filtration on the folium artemisiae argyi crude extract through the Buchner funnel to obtain an folium artemisiae argyi extract;
C. distilling the folium artemisiae argyi extract obtained in the step B at 70 ℃ under reduced pressure and concentrating to obtain folium artemisiae argyi extract, weighing a proper amount of folium artemisiae argyi extract and putting the extract into an ethanol/ammonium sulfate double-aqueous phase system to obtain a premixed solution; the mass concentration of absolute ethyl alcohol in the ethanol/ammonium sulfate double-aqueous phase system is 27%, the mass concentration of ammonium sulfate is 20%, and the balance is deionized water; the folium artemisiae argyi extract accounts for 5% of the total mass of the ethanol/ammonium sulfate aqueous two-phase system; adjusting the pH of the premix solution to 3.5 by using chemically pure concentrated hydrochloric acid, stirring for 10min, transferring to a separating funnel, fully shaking for 10 times, and standing to obtain a clear layered solution, wherein the upper layer is an ethanol phase and the lower layer is a salt phase; collecting the upper ethanol phase, and concentrating under reduced pressure at 75 deg.C to obtain extract, to obtain folium Artemisiae Argyi flavonoids;
D. and D, mixing the folium artemisiae argyi essential oil obtained in the step A with the folium artemisiae argyi flavonoid substances obtained in the step C to obtain the efficient freckle removing active substance.
Example 3:
the preparation method of the high-efficiency freckle-removing active substance is characterized by comprising the following steps of: the method comprises the following steps:
A. picking fresh folium Artemisiae Argyi, spreading on a drying sieve, drying in a dryer for 8h at 60 deg.C until the water content of folium Artemisiae Argyi is lower than 2%, to obtain dried folium Artemisiae Argyi; weighing a proper amount of dried folium artemisiae argyi, adding the weighed dried folium artemisiae argyi into a round-bottom flask, and then adding deionized water, wherein the mass of the deionized water is 10 times of that of the dried folium artemisiae argyi; adjusting the pH of the liquid in the flask to 3.0 by using chemical pure concentrated hydrochloric acid, placing the round-bottom flask in a water bath at 95 ℃, heating and extracting, and receiving folium artemisiae argyi essential oil by adopting a conical flask filled with deionized water at a liquid outlet of a condensation pipe; stopping heating and extracting when the yellow oily liquid is not generated any more; filtering the mixture in the round-bottom flask, collecting first filtrate, reloading first filter residue into the round-bottom flask, adding deionized water with the mass of 10 times that of the first filter residue, adding sodium hydroxide solution with proper concentration to adjust the pH value of liquid in the flask to 7.0, heating and extracting at the water bath temperature of 95 ℃ until no yellow oily liquid is generated, and stopping heating and extracting; and filtering the mixture in the round-bottom flask, collecting a second filtrate, reloading a second filter residue into the round-bottom flask, adding deionized water which is 10 times of the mass of the second filter residue, adding a sodium hydroxide solution with a proper concentration to adjust the pH value of the liquid in the flask to 8.6, heating and extracting the solution at the water bath temperature of 95 ℃ until no yellow oily liquid is generated, and stopping heating and extracting. Filtering the mixture in the round-bottom flask, collecting a third filtrate, and discarding filter residues; taking the upper layer yellow oily liquid in the conical flask to obtain folium Artemisiae Argyi essential oil;
B. mixing the first filtrate, the second filtrate and the third filtrate obtained in the step A, and filtering the mixture by a filter bag of 200 meshes to obtain a coarse extract of the folium artemisiae argyi; adding active carbon which accounts for 2% of the mass of the folium artemisiae argyi crude extract into the folium artemisiae argyi crude extract, wherein the particle size of the active carbon is 200 meshes, and adsorbing for 20min under the condition of 80 ℃ water bath; paving diatomite with the mass being 8% of that of the folium artemisiae argyi crude extract in a Buchner funnel, wherein the particle size of the diatomite is 150 meshes, and performing suction filtration on the folium artemisiae argyi crude extract through the Buchner funnel to obtain an folium artemisiae argyi extract;
C. distilling the folium artemisiae argyi extract obtained in the step B at 70 ℃ under reduced pressure and concentrating to obtain folium artemisiae argyi extract, weighing a proper amount of folium artemisiae argyi extract and putting the extract into an ethanol/ammonium sulfate double-aqueous phase system to obtain a premixed solution; the mass concentration of absolute ethyl alcohol in the ethanol/ammonium sulfate double-aqueous phase system is 27%, the mass concentration of ammonium sulfate is 22%, and the balance is deionized water; the folium artemisiae argyi extract accounts for 5% of the total mass of the ethanol/ammonium sulfate aqueous two-phase system; adjusting the pH value of the premix solution to 4 by using chemically pure concentrated hydrochloric acid, stirring for 10min, transferring to a separating funnel, fully shaking for 10 times, and standing to obtain a clear layered solution, wherein the upper layer is an ethanol phase and the lower layer is a salt phase; collecting the upper ethanol phase, and concentrating under reduced pressure at 80 deg.C to obtain extract, to obtain folium Artemisiae Argyi flavonoids;
D. and D, mixing the folium artemisiae argyi essential oil obtained in the step A with the folium artemisiae argyi flavonoid substances obtained in the step C to obtain the efficient freckle removing active substance.
Claims (2)
1. The preparation method of the freckle-removing active matter is characterized by comprising the following steps:
A. picking fresh folium Artemisiae Argyi, spreading on a drying sieve, drying in a dryer at 60 deg.C for 6-8 hr until the water content of folium Artemisiae Argyi is lower than 2% to obtain dried folium Artemisiae Argyi; weighing a proper amount of dried folium artemisiae argyi, adding the dried folium artemisiae argyi into a round-bottom flask, and then adding deionized water, wherein the mass of the deionized water is 5-10 times of that of the dried folium artemisiae argyi; adjusting pH of the liquid in the flask to 2.0-3.0 with chemical pure concentrated hydrochloric acid, heating and extracting the round-bottom flask in 75-95 deg.C water bath, and collecting folium Artemisiae Argyi essential oil in a conical flask filled with deionized water at the liquid outlet of the condenser tube; stopping heating and extracting when the yellow oily liquid is not generated any more; filtering the mixture in the round-bottom flask, collecting first filtrate, reloading first filter residue into the round-bottom flask, adding deionized water with the mass of 5-10 times that of the first filter residue, adding sodium hydroxide solution with appropriate concentration to adjust the pH value of liquid in the flask to 6.5-7.0, heating and extracting at the water bath temperature of 75-95 ℃ until no yellow oily liquid is generated, and stopping heating and extracting; filtering the mixture in the round-bottom flask, collecting a second filtrate, reloading a second filter residue into the round-bottom flask, adding deionized water with the mass 5-10 times that of the second filter residue, adding a sodium hydroxide solution with a proper concentration to adjust the pH value of the liquid in the flask to 8.2-8.6, heating and extracting at the water bath temperature of 75-95 ℃ until no yellow oily liquid is generated, and stopping heating and extracting; filtering the mixture in the round-bottom flask, collecting a third filtrate, and discarding filter residues; taking the upper layer yellow oily liquid in the conical flask to obtain folium Artemisiae Argyi essential oil;
B. mixing the first filtrate, the second filtrate and the third filtrate obtained in the step A, and filtering the mixture by a filter bag of 200 meshes to obtain a coarse extract of the folium artemisiae argyi; adding active carbon which accounts for 2% of the mass of the folium artemisiae argyi crude extract into the folium artemisiae argyi crude extract, wherein the particle size of the active carbon is 200 meshes, and adsorbing for 20min under the condition of 80 ℃ water bath; paving diatomite with the mass being 8% of that of the folium artemisiae argyi crude extract in a Buchner funnel, wherein the particle size of the diatomite is 150 meshes, and performing suction filtration on the folium artemisiae argyi crude extract through the Buchner funnel to obtain an folium artemisiae argyi extract;
C. distilling the folium artemisiae argyi extract obtained in the step B at 70 ℃ under reduced pressure and concentrating to obtain folium artemisiae argyi extract, weighing a proper amount of folium artemisiae argyi extract and putting the extract into an ethanol/ammonium sulfate double-aqueous phase system to obtain a premixed solution; the mass concentration of absolute ethyl alcohol in the ethanol/ammonium sulfate double-aqueous phase system is 27%, the mass concentration of ammonium sulfate is 17% -22%, and the balance is deionized water; the folium artemisiae argyi extract accounts for 5% of the total mass of the ethanol/ammonium sulfate aqueous two-phase system; adjusting the pH of the premix solution to 3-4 with chemically pure concentrated hydrochloric acid, stirring for 10min, transferring to a separating funnel, fully shaking for 10 times, and standing to obtain a clear layered solution, wherein the upper layer is an ethanol phase and the lower layer is a salt phase; collecting upper ethanol phase, and concentrating under reduced pressure at 70-80 deg.C to obtain extract, to obtain folium Artemisiae Argyi flavonoids;
D. and D, mixing the folium artemisiae argyi essential oil obtained in the step A with the folium artemisiae argyi flavonoid substances obtained in the step C to obtain the freckle removing active substance.
2. The use of the depigmenting active agent according to claim 1, wherein: the freckle-removing active substance is applied to preparing whitening cream or freckle-removing cream products, and the adding amount is 0.05-0.2% by mass.
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| KR20180000513A (en) * | 2016-06-23 | 2018-01-03 | 랜기어 테크놀로지 컴퍼니 리미티드 | Cosmetics Composition |
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