CN114306163A - Preparation method and application of bidens pilosa extract with whitening effect - Google Patents
Preparation method and application of bidens pilosa extract with whitening effect Download PDFInfo
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Abstract
The invention provides a preparation method of a bidens pilosa extract with a whitening effect, which comprises the following steps: step one, adding bidens pilosa into a reaction container of a flash extractor, and adding an extraction solvent; supplementing inactive gas into the reaction container to remove air, and reducing the pressure to 0.5-0.8 bar; performing flash extraction by adopting ultrasonic assistance, wherein the power of the ultrasonic assistance is 400-700W, the mechanical rotation speed of the flash extraction is 2000-4000 r/min, the flash extraction is repeated for 2-4 times, and each time lasts for 6-9 min, and filtering is performed to obtain a bidens pilosa extracting solution; and step four, carrying out rotary evaporation and concentration on the bidens pilosa extracting solution, centrifuging to obtain supernatant, and sealing and filling the supernatant to obtain the bidens pilosa extract. The preparation method of the bidens pilosa extract provided by the invention can be stably stored for a long time without adding artificial additives, and can show prominent whitening effect.
Description
Technical Field
The invention relates to the technical field of cosmetics, and particularly relates to a preparation method of a bidens pilosa extract with a whitening effect and application of the bidens pilosa extract in cosmetics.
Background
With the improvement of living standard, the requirements of people on whitening cosmetics are higher and higher. In addition to skin whitening, there are many aspects to be enhanced, such as delaying skin aging, reducing wrinkles, smoothing skin, which can be achieved by increasing the superoxide dismutase activity of the body and eliminating excessive superoxide radicals; melanocyte-stimulating hormone (alpha-MSH) is produced by melanocyte, and combined with melanocortin-1 (MCR-1) receptor, the melanocyte-stimulating hormone can activate synthesis expression of tyrosinase through a series of reactions, thereby promoting melanin synthesis to increase, and if the melanocyte-stimulating hormone can inhibit alpha-MSH production, melanogenesis can be prevented from the source of tyrosinase synthesis, and finally whitening effect is achieved. In addition, the whitening product with a single mechanism has a poor overall effect, and therefore, in order to develop a whitening cosmetic with more outstanding efficacy, the comprehensive application of multiple mechanisms is needed to better achieve the purpose of whitening skin.
Herba Bidentis Bipinnatae contains polyphenols, coumarin, amino acids, vitamins, etc., and can be used for treating furuncle, toxic swelling, common cold, and sore throat, wherein the herba Bidentis Bipinnatae contains total alkaloids and steroid substances, and has antiinflammatory effect. The extract has antibacterial effect and can inhibit Staphylococcus aureus activity. The total flavonoids in herba Bidentis Bipinnatae can inhibit tyrosinase activity in melanocyte, inhibit melanin transfer from melanocyte to stratum corneum, and remove advanced glycosylation product to achieve antioxidant effect and promote skin whitening.
However, since the chemical components of plant herbs are very complex, and usually contain alkaloids, amino acids, organic acids, phenols, saponins, steroids, terpenoids, proteins, mucilages, tannins, saccharides, starches, celluloses, inorganic salts, etc., the stability of natural plant extracts is difficult to control, and phenomena such as aggregation, precipitation, oxidative discoloration, etc. often occur. Similarly, when the Bidens pilosa is extracted by using a traditional extraction method, active substances with strong reducibility, such as vitamin C and the like, are difficult to stably exist in the extract, so that the Bidens pilosa extract is easy to oxidize and discolor, and the activity loss is caused. The conventional antioxidant means is adding a chemical antioxidant, but on one hand, the stability of the extract is influenced by adding the antioxidant, and on the other hand, the addition of the chemical additive into the cosmetic not only reduces the comfort of the cosmetic to the skin, but also runs counter to the concept of the existing 'natural and green' cosmetic, and reduces the purchasing desire of consumers.
Therefore, the prior art fails to provide an extraction method for bidens pilosa which can effectively maintain the stability and improve the activity of bidens pilosa.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a preparation method of bidens pilosa extract with good stability and outstanding whitening efficacy, comprising the following steps:
step one, adding bidens pilosa into a reaction container of a flash extractor, and adding an extraction solvent;
supplementing inactive gas into the reaction container to remove air, and reducing the pressure to 0.5-0.8 bar;
performing flash extraction by adopting ultrasonic assistance, wherein the power of the ultrasonic assistance is 400-700W, the mechanical rotation speed of the flash extraction is 2000-4000 r/min, the flash extraction is repeated for 2-4 times, and each time lasts for 6-9 min, and filtering is performed to obtain a bidens pilosa extracting solution;
and step four, carrying out rotary evaporation on the bidens pilosa extracting solution to remove an extracting solvent, carrying out centrifugation to obtain a supernatant, and sealing and filling the supernatant to obtain the bidens pilosa extract.
Further, in the first step, the extraction solvent is an ethanol solution with a mass concentration of 65-95%.
Further, in the second step, the inert gas includes carbon dioxide.
Further, in the second step, carbon dioxide was supplied and the pressure was reduced to 0.7 bar.
Further, in the third step, the power of ultrasonic assistance is 600W, and the mechanical rotating speed of flash extraction is 3500 r/min.
Further, in the third step, the filtering is performed by vacuum filtration of diatomite cakes, and the diatomite cakes are prepared from the following raw materials in a mass ratio of (3-9): (0.5-3) mixing diatomite and EDTA-2 Na.
Further, in the fourth step, the conditions of rotary evaporation concentration are as follows: the vacuum degree is 150-250 mbar, and the water bath temperature is 60-70 ℃.
Further, in the fourth step, the centrifugation conditions are as follows: centrifuging at 2000-4000 r/min for 2-8 min.
In another aspect, the application also provides the bidens pilosa extract prepared by the method.
In another aspect, the application also provides the application of the bidens pilosa extract in the preparation of cosmetics, and the dosage forms of the cosmetics comprise creams, lotions, gels, masks, embrocations and lotions.
The beneficial effect of this application lies in:
1. according to the preparation method provided by the application, the bidens pilosa is extracted by adopting a flash extraction method capable of extracting at normal temperature, wherein the flash extraction method is particularly suitable for directly extracting plant leaves with larger fibers, and effective components in the bidens pilosa can be more effectively reserved by combining a normal-temperature and ultrasonic-assisted extraction mode;
2. according to the preparation method provided by the application, the inactive gas is added to exhaust air, particularly oxygen in the air, during flash extraction, the flash extraction is used for highly crushing plant materials, so that a large amount of plant contents are exposed, and a strong vortex is formed in the center of an inner blade of a flash extractor and drives the crushed materials to turn over inside and outside, so that a violent stirring effect is generated, the plant extracts are greatly contacted with the external oxygen to oxidize and discolor or even inactivate, and the inactive gas is introduced to exhaust the oxygen, so that the contact between active substances with strong reducibility in the bidens pilosa and the air can be effectively reduced, and the oxidative discoloration is avoided;
3. according to the preparation method provided by the application, the inactive gas adopts carbon dioxide, the carbon dioxide can be combined with water in the extraction solvent to form an acidic environment, and the acidic environment is particularly suitable for maintaining the stability of active substances such as flavone, vitamin C and the like in bidens pilosa, so that the stability of the bidens pilosa is improved, and the whitening effect of the bidens pilosa is favorably improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a bar graph showing the melanogenesis inhibitory effect of Bidens pilosa extract prepared in example 1 of the present application;
FIGS. 2 to 6 are graphs showing the results of the inhibition of melanosome production by Bidens pilosa extract obtained in example 1 of the present application, in which FIG. 2 is a graph showing the results of a blank control, FIG. 3 is a graph showing the results of a Bidens pilosa extract 1 at a concentration of 0.625%, FIG. 4 is a graph showing the results of a Bidens pilosa extract 2 at a concentration of 0.3125%, FIG. 5 is a graph showing the results of a Bidens pilosa extract 3 at a concentration of 0.156%, and FIG. 6 is a graph showing the results of a Bidens pilosa extract 4 at a concentration of 0.078%.
The specific implementation mode is as follows:
in the following description, numerous specific details are set forth by way of examples in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, well-known features have not been described in order to avoid obscuring the invention.
The carbon dioxide gas is purchased from the popular chemical products company Limited in Henan; the vacuumizing machine is a product of Shanghai Axin electromechanical equipment Limited company, the model is BDR16, and the vacuum degree is less than or equal to 0.05 Pa; the flash extractor is a Shanghai vanadium flag precision equipment company Limited product, the model is SC 200, the motor speed: 0-10000 r/min; the ultrasonic vibrating spear is a product of Shenzhen Shandong ultrasonic cleaning equipment Limited, the model is LDX1000A-20, and the power: 0-1000W; the rotary evaporation evaporator is a product of Kexing instruments company, the model is R1020, the temperature is controlled to be between room temperature and 99 ℃, and the vacuum degree is less than or equal to 399.9 Pa; the centrifuge is a product of Kate laboratory instruments Limited in salt cities, and has a model number of TD6M, and the rotating speed is less than or equal to 6000 r/min.
The materials and equipment used in the following examples are commercially available, and if not specifically mentioned, the raw material grades in the following examples are all cosmetic grades and are all commercially available.
In addition, the "water" in the present invention includes any available water that can be used in the cosmetic field such as deionized water, distilled water, ion-exchanged water, double distilled water, high purity water, purified water, and the like.
Example 1
The embodiment provides a preparation method of a bidens pilosa extract, which comprises the following specific steps:
the first step is as follows: shearing the bidens pilosa to obtain bidens pilosa fragments with the diameter of 1.7cm, adding the bidens pilosa fragments into a reaction container of a flash extractor, and adding ethanol with the mass concentration of 85% as an extraction solvent;
the second step is that: supplementing carbon dioxide into the reaction container to remove air, and controlling the pressure of the supplemented carbon dioxide in the reaction container to be 0.7bar after the reaction container is sealed;
the third step: in a reaction container, partially immersing an ultrasonic vibration rod into a solvent, carrying out ultrasonic treatment at 600W power, simultaneously extracting the bidens pilosa fragments for 3 times at 3500r/min by using a flash extractor, wherein each time is 8min, obtaining an alcohol extraction mixed solution, and then carrying out the mass ratio of the alcohol extraction mixed solution to the dosage of diatomite and EDTA-2Na to be 90: 7: 2, carrying out vacuum filtration on the diatomite cake to obtain a bidens pilosa extract;
the fourth step: concentrating herba Bidentis Bipinnatae extractive solution by rotary evaporation at water bath temperature of 60 deg.C under vacuum degree of 190mbar to remove extraction solvent to obtain herba Bidentis Bipinnatae concentrated solution, centrifuging at 3500r/min for 6min to obtain clear and transparent supernatant, sealing and packaging the supernatant to obtain herba Bidentis Bipinnatae extract.
Examples 2 to 7
Examples 2 to 7 were substantially the same as the preparation method of example 1, except that the pressure after carbon dioxide was supplied in the second step was different, the power of ultrasonic vibration was different in the third step, and the rotation speed of the flash extractor was different, and the parameters of each example are shown in table 1.
Comparative example 1
This comparative example is made in substantially the same manner as example 1 except that the second step is not employed.
Comparative example 2
This comparative example is substantially the same as the preparation of example 1, except that no ultrasonic assistance is employed in the third step.
Comparative example 3
This comparative example is about the same as the preparation method of example 1 except that the third extraction method is 90 c ethanol reflux extraction.
The Bidens pilosa extract obtained in each of the above examples and comparative examples was stored in a dark and ventilated environment with an opening, and the presence or absence of precipitation of the precipitated substance and the stability of the solution were observed, and the number of days for the precipitation of the precipitated substance to start and the coagulation of the solution to start was recorded for 15 consecutive days. The results obtained are shown in Table 1:
TABLE 1
As can be seen from the data in table 1, the bidens pilosa extract prepared by the method provided in the present application has significantly improved storage stability compared to the comparative example, especially in example 1, the bidens pilosa extract under the parameters can be clear and transparent after 15 days, and shows the optimal stability improvement effect. In the following examples, efficacy tests were conducted by taking the Bidens pilosa extract obtained in example 1, which is most effective, as an example.
Efficacy evaluation experiments:
firstly, a melanin content detection experiment:
an experimental instrument: lifting oil bath pan (ZKYY-5L) and CO2Incubator (Thermo, 150i), clean bench (Sujing Antai, SW-CJ-1F), inverted microscope (Olympus, CKX41), microplate reader (BioTek, Epoch), and fluorescence quantitative PCR instrument (BioRad, CFX-96). A rotary evaporator (B-300 Base).
The experimental method comprises the following steps:
1) inoculation: cells were seeded at a seeding density of 3E5 cells/well in 6-well plates, incubators (37 ℃, 5% CO)2) And incubated overnight.
2) Preparing liquid: and preparing the working solution of the tested object according to a specific test scheme.
3) Administration: bidens pilosa extract provided in example 1 was formulated to concentrations of 0.625%, 0.3125%, 0.156%, and 0.078%, respectively, as Bidens pilosa extract 1, Bidens pilosa extract 2, Bidens pilosa extract 3, and Bidens pilosa extract 4. When the cell plating rate in the 6-hole plate reaches 40% -60%, the medicine is administered in groups, the dosage of each hole is 2mL, each group is provided with 3 multiple holes, and the incubator (37 ℃ and 5% CO)2) The medium was incubated for 72h, and after 72h, the culture medium was discarded and washed 1 time with PBS.
4) Cell digestion: melanocyte (700 μ L/hole) is digested with 0.25% pancreatin, the temperature is 37 ℃, 1-2min, when the observation is carried out under a microscope, when 80% of cells retract to be spherical and are not separated from an attached surface, 700 μ L/hole DMEM containing 10% FBS is immediately added to stop the reaction, the cells are collected into a 1.5mLEP tube after being blown uniformly, 10000r/min is centrifuged for 10min, and the supernatant is discarded.
5) Mixing distilled water, anhydrous ethanol and diethyl ether at a ratio of 2:5:5 in a fume hood, adding into an EP tube at a volume of 1.2 mL/tube after mixing, shaking, standing at room temperature for 30min, and centrifuging at 10000r/min for 10 min.
6) The supernatant was discarded, and 1mL of a 1mol/L NaOH aqueous solution containing 10% (volume fraction) DMSO was added thereto, followed by sealing with a sealing film.
7) The sample and the melanin standard solution with different concentrations are respectively placed in a water bath at 80 ℃ to be heated for 30 min.
8) After heating, after the temperature is balanced to room temperature, sequentially adding 200 mu L/hole of the solution into a 96-well plate, arranging 2 multiple holes in each EP tube, and selecting a wavelength of 405nm to measure the absorbance value on a microplate reader.
9) And (3) determining the OD values of the standard solutions with different concentrations as horizontal coordinates and the melanin content as vertical coordinates, making a standard curve, obtaining a regression equation, substituting the OD values of the samples into a formula, and calculating the melanin content of the sample group.
The results of the experiment are shown in FIG. 1. As shown in fig. 1, the melanin content in the positive control (glabridin) group was significantly reduced compared to the blank control group, indicating that this experiment was effective. The melanin content of bidens pilosa extracts 1, 2, 3, and 4 was significantly reduced compared to the blank control group. It is demonstrated that Bidens pilosa prepared in example 1 of the present application has a very significant inhibitory effect on melanin production.
Secondly, melanosome synthesis inhibition detection experiment:
an experimental instrument: lifting oil bath pan (ZKYY-5L) and CO2Incubator (Thermo, 150i), clean bench (Sujing Antai, SW-CJ-1F), inverted microscope (Olympus, CKX41), microplate reader (BioTek, Epoch), and fluorescence quantitative PCR instrument (BioRad, CFX-96). A rotary evaporator (B-300 Base).
The experimental process comprises the following steps:
1) and preparing an experimental solution.
2) Inoculation: according to 7X 104Cells were seeded at density per well in 6-well plates, 3mL melanin medium was added, 37 ℃, 5% CO2The incubator continues to culture for 24 h.
3) And (3) drug treatment: preparing and replacing 3mL epidermal culture medium containing working concentration of active substance to be detected, and performing reaction at 37 ℃ and 5% CO2The incubator continues to culture for 24 h.
4) Melanosome treatment: the cell well plate was washed and fresh keratinocyte culture medium was added. Melanosomes collected at the pre-resuspension stage were placed in 100uL keratinocyte medium, and 15uL of the resuspension solution was added to 2mL of the mediumIn the nutrient hole. 37 ℃ and 5% CO2The incubator continues to culture for 72 h.
5) Sampling, immunofluorescence: carrying out immunohistochemical detection on the melanosome, carrying out photographing observation by using laser confocal technology, and collecting pictures; data analysis was performed using Image-ProPlus Image processing software, plotted using GraphPadPrismProgram software, and statistically analyzed between groups using t-test, with P < 0.05 indicating significant differences and P < 0.01 indicating differential polarity.
The results obtained are shown in FIGS. 2 to 6. As shown in fig. 2-6, the bidens pilosa extract 1, 2, 3, 4 has a significant inhibitory effect on melanosome production compared to the BC group, which indicates that the bidens pilosa extract prepared in example 1 of the present application has a good skin whitening effect and can be used as an additive of whitening cosmetics.
In summary, the bidens pilosa extract with significantly improved stability obtained in example 1 of the present application contains a plurality of active ingredients, and the active ingredients can generate a synergistic effect, so as to reduce skin melanin from various ways. Therefore, the bidens pilosa extract can be applied to skin products to play a whitening effect. Wherein the total flavonoids can inhibit the activity of tyrosinase in melanocytes, inhibit the transfer of melanin from melanocytes to stratum corneum, and remove late glycosylation products to achieve antioxidant effect, thereby promoting skin whitening. The total saponin can effectively inhibit tyrosinase activity increase and melanin synthesis increase caused by alpha-MSH, inhibit tyrosinase protein expression caused by alpha-MSH, and remove glycosylation products so as to achieve antioxidant effect. The vitamins can assist in reducing oxygen free radicals to realize antioxidation, and further assist in further improving the inhibition effect of the flavones and the saponins on melanocytes or melanosomes, so that the skin whitening can be effectively promoted.
As demonstrated by the above experiments, Bidens pilosa extract reduces the production of melanin in the skin by inhibiting the synthesis of melanosomes, it being understood that the exemplary embodiments described herein are illustrative and not limiting. Although one or more embodiments of the present invention have been described in connection with the accompanying drawings, it will be understood by those of ordinary skill in the art that various forms and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims (10)
1. A preparation method of bidens pilosa extract with whitening effect is characterized by comprising the following steps: the method comprises the following steps:
step one, adding bidens pilosa into a reaction container of a flash extractor, and adding an extraction solvent;
supplementing inactive gas into the reaction container to remove air, and reducing the pressure to 0.5-0.8 bar;
performing flash extraction by adopting ultrasonic assistance, wherein the power of the ultrasonic assistance is 400-700W, the mechanical rotation speed of the flash extraction is 2000-4000 r/min, the flash extraction is repeated for 2-4 times, and each time lasts for 6-9 min, and filtering is performed to obtain a bidens pilosa extracting solution;
and step four, carrying out rotary evaporation on the bidens pilosa extracting solution to remove an extracting solvent, carrying out centrifugation to obtain a supernatant, and sealing and filling the supernatant to obtain the bidens pilosa extract.
2. The preparation method of a bidens pilosa extract with a whitening effect according to claim 1, wherein in the first step, the extraction solvent is an ethanol solution with a mass concentration of 65-95%.
3. The method for preparing a bidens pilosa extract with a whitening effect according to claim 1, wherein the inactive gas comprises carbon dioxide in the second step.
4. The method for preparing Bidens pilosa extract with whitening efficacy according to claim 3, wherein in the second step, carbon dioxide is supplemented and the pressure is reduced to 0.7 bar.
5. The method for preparing a bidens pilosa extract with a whitening effect according to claim 1, wherein in the third step, the power of ultrasonic assistance is 600W, and the mechanical rotation speed of flash extraction is 3500 r/min.
6. The preparation method of a bidens pilosa extract with a whitening effect according to claim 1, wherein in the third step, the filtering is performed by vacuum filtration with a diatomite cake, and the diatomite cake is prepared from the following raw materials in a mass ratio of (3-9): (0.5-3) mixing diatomite and EDTA-2 Na.
7. The method for preparing bidens pilosa extract with whitening efficacy according to claim 1, wherein in the fourth step, the conditions of the rotary evaporation concentration are as follows: the vacuum degree is 150-250 mbar, and the water bath temperature is 60-70 ℃.
8. The method for preparing bidens pilosa extract with whitening efficacy according to claim 1, wherein in the fourth step, the centrifugation conditions are as follows: centrifuging at 2000-4000 r/min for 2-8 min.
9. A Bidens pilosa extract prepared by the process of any one of claims 1 to 8.
10. Use of the Bidens pilosa extract according to claim 9 for the preparation of a cosmetic in the form of a cream, lotion, gel, mask, embrocation and lotion.
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