A selenium-rich Fucha tea extract, its preparation method and application in preparing a whitening and freckle-removing agent

Abstract

The present invention provides a selenium-rich Fucha tea extract, a preparation method thereof, and an application thereof in the preparation of a whitening and freckle-removing agent, relating to the technical field of tea extracts. The present invention uses selenium-rich Fucha tea as a raw material and performs vacuum extraction using water, methanol, and ethanol as extraction agents, which improves the extraction rate of whitening active ingredients and also significantly reduces the extraction temperature, avoiding the loss of whitening active ingredients in the selenium-rich Fucha tea; impurities are removed through purification (one or several of petroleum ether extraction, macroporous resin purification, and reverse column chromatography purification), improving the concentration of whitening active ingredients; the raw material selenium-rich Fucha tea used is rich in organic selenium compounds, enhancing the whitening effect of the selenium-rich Fucha tea extract. The prepared selenium-rich Fucha tea extract has excellent effects of inhibiting tyrosinase from synthesizing melanin and inhibiting the synthesis of melanin in cells, has excellent whitening effects, and has good application prospects in cosmetics, especially in whitening and freckle-removing products.

Description

Technical Field

[0001] The present invention relates to the technical field of tea extracts, and particularly relates to a selenium-rich Fucha extract, a preparation method thereof, and an application thereof in the preparation of a whitening and freckle-removing agent. Background Art

[0002] During the processing and deep processing of tea leaves, a series of complex biochemical changes occur in the active ingredients, resulting in the generation of new compounds. Especially during the processing of Fucha tea, due to the fermentation effects of piling, fermentation, and the unique Eurotium cristatum in Fucha tea, a series of Fucha tea element compounds will be produced in Fucha tea, and these substances are considered to have the effect of regulating the skin. For example, Chinese Patent CN111053716A discloses the use of a Fucha tea extract in the preparation of a skin-regulating product, and specifically discloses the effects of Fucha tea on anti-acne, antioxidant, anti-inflammatory, anti-irritant, anti-aging, or enhancing skin elasticity. Among them, the preparation method of the aqueous extract of Fucha tea includes the following steps: grinding Fucha tea into powder, adding water according to a solid-liquid ratio of 1:10, stirring evenly, ultrasonic cell disruption until there is no visible tea powder and bacteria, standing at room temperature for 2 h, boiling in a boiling water bath for 2 h, cooling, ultracentrifuging, filtering and sterilizing the supernatant with a 0.22 μm filter membrane, concentrating and evaporating to dryness, and freeze-drying at low temperature to obtain the aqueous extract of Fucha tea; the preparation method of the organic solvent extract includes the following steps: adding 85-95% ethanol to the Fucha tea powder according to a solid-liquid ratio of 1:20, ultrasonic cell disruption until there is no visible tea powder and bacteria, then cold soaking for 18-28 h, collecting the filtrate, repeating 2-5 times, distilling the filtrate under reduced pressure, concentrating the filtrate, drying at low temperature to obtain a crude extract, adsorbing and decolorizing the crude extract with macroporous resin, eluting with 85-95% ethanol, concentrating and evaporating to dryness, and freeze-drying at low temperature to obtain the organic solvent extract of Fucha tea. However, the above Fucha tea extract does not have the effect of whitening and freckle-removing. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a selenium-rich Fucha extract, a preparation method thereof, and an application thereof in the preparation of a whitening and freckle-removing agent. The selenium-rich Fucha extract prepared by the present invention has excellent whitening effect.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a preparation method of a selenium-rich Fucha extract, including the following steps:

[0006] Mixing selenium-rich Fucha tea powder with an extractant, and performing vacuum extraction to obtain a selenium-rich Fucha crude extract; the extractant includes one or several of water, methanol, and ethanol;

[0007] Refining the selenium-rich Fucha crude extract to obtain a selenium-rich Fucha extract; the refining includes one or several of petroleum ether extraction, macroporous resin purification, and reverse column chromatography purification.

[0008] Preferably, when the extractant contains methanol and / or ethanol, the volume fraction of methanol and / or ethanol in the extractant is 60-100%;

[0009] The mass ratio of the selenium-enriched Fucha powder to the extractant is 1:5-20;

[0010] The temperature of the reduced-pressure extraction is ≤40°C, the time is 30-120 min, and the absolute pressure is 2.5-10.5 kPa.

[0011] Preferably, the petroleum ether extraction includes: dispersing the selenium-enriched Fucha crude extract in water to obtain a turbid solution; mixing the turbid solution with petroleum ether for extraction, and concentrating the extracted aqueous phase under reduced pressure to 20-50% of the volume of the aqueous phase and then filtering to obtain an aqueous solution containing the selenium-enriched Fucha extract.

[0012] Preferably, the volume ratio of the turbid solution to the petroleum ether used for single extraction is 1:0.3-1, and the number of times of the petroleum ether extraction is 2-5 times.

[0013] Preferably, the macroporous resin used for the macroporous resin purification includes LX-8 resin and / or DIAION HP20 resin.

[0014] Preferably, the eluent used for the macroporous resin purification includes methanol and / or ethanol.

[0015] Preferably, the eluent used for the reverse column chromatography purification includes 10-30 v / v% aqueous alcohol solution, the alcohol in the 10-30 v / v% aqueous alcohol solution includes methanol or ethanol, and the chromatographic column is a reverse C18 column.

[0016] The present invention provides a selenium-enriched Fucha extract prepared by the preparation method described in the above technical solution.

[0017] The present invention provides the application of the selenium-enriched Fucha extract described in the above technical solution in the preparation of a whitening and freckle-removing agent.

[0018] Preferably, the whitening and freckle-removing agent includes essence, lotion or cream.

[0019] The present invention provides a method for preparing a selenium-rich Fucha tea extract, comprising the following steps: mixing selenium-rich Fucha tea powder with an extractant, and performing extraction under reduced pressure to obtain a selenium-rich Fucha tea crude extract; the extractant includes one or more of water, methanol and ethanol; refining the selenium-rich Fucha tea crude extract to obtain a selenium-rich Fucha tea extract; the refining includes one or more of petroleum ether extraction, macroporous resin purification and reverse column chromatography purification. The present invention uses selenium-rich Fucha tea as a raw material, and uses one or more of water, methanol and ethanol for extraction under reduced pressure, which improves the extraction rate of the whitening active ingredients in selenium-rich Fucha tea, and at the same time significantly reduces the extraction temperature, avoiding the degradation of the whitening active ingredients in selenium-rich Fucha tea; through refining, impurities such as sugar components and caffeine are removed, improving the concentration of the whitening active ingredients in selenium-rich Fucha tea; at the same time, the raw material selenium-rich Fucha tea used is rich in organic selenium compounds that are indispensable for human health, further improving the whitening effect of the selenium-rich Fucha tea extract. The prepared selenium-rich Fucha tea extract has excellent effects of inhibiting the synthesis of melanin by tyrosinase and inhibiting the synthesis of melanin in cells, has excellent whitening effects, and the whitening effect is close to or even exceeds that of arbutin, and has good application prospects in cosmetics, especially in whitening and freckle-removing agents. As shown by the test results of the examples, the inhibition rates of the selenium-rich Fucha tea extracts prepared in Examples 1 to 3 on tyrosinase were 85.31% ± 2.23, 76.23 ± 3.56 and 75.32 ± 4.67% respectively, and the inhibition rates on the formation of melanin in cells were 21.22% ± 2.34, 26.57 ± 4.41 and 20.18 ± 2.07% respectively. It can also significantly reduce the expression of four key genes (MITF, TYR, TRP-1 and TRP-2) for melanin synthesis, and has excellent whitening effects. Description of the Drawings

[0020] Figure 1 It is a comparison result chart of the antioxidant activities of the tea extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5, the selenium-rich Fucha tea crude extract prepared in Example 3, and vitamin C;

[0021] Figure 2 It is a comparison result chart of the selenium element contents of the tea extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5 and the selenium-rich Fucha tea crude extract prepared in Example 3;

[0022] Figure 3 It is a comparison result chart of the in vitro tyrosinase inhibitory activities of the tea extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5, the selenium-rich Fucha tea crude extract prepared in Example 3, and arbutin;

[0023] Figure 4 It is a comparison result chart of the activities of inhibiting the synthesis of melanin in cells of the tea extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5, the selenium-rich Fucha tea crude extract prepared in Example 3, and arbutin;

[0024] Figure 5 Graph showing the comparison results of the activities of inhibiting intracellular tyrosinase of the tea extracts prepared in Examples 1-3 and Comparative Examples 1-5, the crude selenium-enriched Fucha extract prepared in Example 3, and arbutin;

[0025] Figure 6 Graph showing the comparison results of the quantitative determination of genes related to melanin synthesis inhibition of the tea extracts prepared in Examples 1-3 and Comparative Examples 1-5, the crude selenium-enriched Fucha extract prepared in Example 3, and arbutin. Detailed implementation manners

[0026] The present invention provides a preparation method of a selenium-enriched Fucha extract, comprising the following steps:

[0027] Mix the selenium-enriched Fucha powder with an extractant, and perform reduced-pressure extraction to obtain a crude selenium-enriched Fucha extract; the extractant includes one or more of water, methanol, and ethanol;

[0028] Refine the crude selenium-enriched Fucha extract to obtain a selenium-enriched Fucha extract; the refining includes one or more of petroleum ether extraction, macroporous resin purification, and reverse column chromatography purification.

[0029] Unless otherwise specified, the raw materials used in the present invention are all commercially available products.

[0030] The present invention mixes the selenium-enriched Fucha powder with an extractant, and performs reduced-pressure extraction to obtain a crude selenium-enriched Fucha extract; the extractant includes one or more of water, methanol, and ethanol.

[0031] In the present invention, when the extractant contains methanol and / or ethanol, the volume fraction of methanol and / or ethanol in the extractant is preferably 60-100%, more preferably 65-100%, further preferably 70-95%, and most preferably 80-95%.

[0032] In the present invention, the particle size of the selenium-enriched Fucha powder is preferably 5-8 mesh; the selenium-enriched Fucha powder is obtained by crushing selenium-enriched Fucha; the present invention has no special limitation on the crushing method, and a crushing method well-known to those skilled in the art can be used to obtain a selenium-enriched Fucha powder with a particle size of 5-8 mesh. In the present invention, the selenium-enriched Fucha is preferably selenium-enriched Fucha brick tea, which meets the selenium-enriched tea standard of NY / T 600-2002. In the present invention, the mass ratio of the selenium-enriched Fucha powder to the extractant is preferably 1:5-20, more preferably 1:8-18, and further preferably 1:9-15.

[0033] In the present invention, the preferred temperature for reduced-pressure extraction (reduced-pressure boiling) is ≤40°C. At this temperature, reduced-pressure extraction is carried out, and the extractant approaches boiling during the extraction process; the preferred time for reduced-pressure extraction is 80 - 120 min, more preferably 90 - 120 min, and further preferably 100 - 120 min; the absolute pressure of the system during the reduced-pressure extraction process is preferably 2.5 - 10.5 kPa; when the extractant is water, the absolute pressure of the system during the reduced-pressure extraction process is more preferably 2.5 - 3.5 kPa, and more preferably 3 ± 0.2 kPa. During the water extraction process, vacuum boiling can improve the extraction rate of the skin-whitening and freckle-removing components in the selenium-enriched Fucha extract while protecting the skin-whitening and freckle-removing active substances; when the extractant is methanol and / or ethanol, the absolute pressure of the extraction and reduced-pressure process system is preferably 9.5 - 10.5 kPa, and more preferably 10 ± 0.2 kPa. When the extractant is an aqueous solution of alcohol (methanol and / or ethanol), the absolute pressure of the reduced-pressure extraction is more preferably 8.5 - 9.5 kPa, and more preferably 9 ± 0.2 kPa; the reduced-pressure extraction is preferably carried out under stirring conditions; the extractor used for the reduced-pressure extraction is preferably provided with a condensation reflux device. In the present invention, the number of times of reduced-pressure extraction is preferably 1 - 3 times. When the number of extraction times is 2 - 3 times, it is preferred to repeat the above-mentioned reduced-pressure extraction 1 - 2 times for the residue obtained from the reduced-pressure extraction, and the clear liquid obtained from the reduced-pressure extraction is combined.

[0034] A selenium-enriched Fucha crude extract is obtained. In the present invention, the selenium-enriched Fucha crude extract is refined to obtain a selenium-enriched Fucha extract; the refining includes one or more of petroleum ether extraction, macroporous resin purification, and reverse column chromatography purification.

[0035] In the present invention, it is preferred to dry the selenium-enriched Fucha crude extract before refining; the drying preferably includes freeze-drying or vacuum reduced-pressure drying. The present invention has no special limitation on the conditions of the drying, as long as the solvent in the crude extract can be removed.

[0036] In the present invention, the petroleum ether extraction preferably includes: dispersing the crude selenium-enriched Fucha extract in water to obtain a turbid solution; mixing the turbid solution with petroleum ether for extraction, and concentrating the aqueous phase after extraction under reduced pressure to 20-50% of the volume of the aqueous phase and then filtering to obtain an aqueous solution containing the selenium-enriched Fucha extract. In the present invention, the mass ratio of the crude selenium-enriched Fucha extract to water is preferably 1:5-20, more preferably 1:8-18, and further preferably 1:10-15. In the present invention, the number of times of petroleum ether extraction is preferably 2-5 times, more preferably 3-4 times; the volume ratio of the turbid solution to the petroleum ether used for single extraction is preferably 1:0.3-1, more preferably 1:0.5; during the extraction process, the petroleum ether can extract liposoluble components from the system, and the target component (selenium-enriched Fucha extract) is dissolved in water. In the present invention, the temperature of the reduced pressure concentration is preferably ≤40°C, more preferably 35-40°C. In the present invention, when the purification is petroleum ether extraction, the petroleum ether purification further includes drying the obtained aqueous solution containing the selenium-enriched Fucha extract to a constant weight to obtain the selenium-enriched Fucha extract. In the present invention, when the purification is sequentially performing petroleum ether extraction and macroporous resin purification, the aqueous solution containing the selenium-enriched Fucha extract is directly subjected to subsequent macroporous resin purification. In the present invention, when the purification is sequentially performing petroleum ether extraction and reverse column chromatography purification, the petroleum ether purification further includes concentrating the aqueous solution containing the selenium-enriched Fucha extract to a constant weight to obtain a defatted extract. In the present invention, the temperature of the drying is preferably ≤40°C, more preferably 35-40°C; the drying preferably includes reduced pressure concentration drying and / or vacuum freeze drying.

[0037] In the present invention, the macroporous resin preferably used for purification includes LX-8 resin and / or DIAION HP20 resin; the eluent preferably used for the macroporous resin purification includes methanol and / or ethanol. In the present invention, the macroporous resin purification is preferably as follows: the crude selenium-enriched Fucha extract is redissolved in water, macroporous resin is added to the obtained aqueous solution of the crude extract (or directly the aqueous solution containing the selenium-enriched Fucha extract obtained by petroleum ether extraction) for adsorption, solid-liquid separation is carried out, and the obtained solid component (the macroporous resin adsorbed with the target substance) is placed in a glass chromatography column sleeve, and elution is carried out using the eluent. The first 0.1 to 0.3 BV of the eluate is discarded, and the remaining eluate is dried. In the present invention, the redissolution is preferably carried out under ultrasonic conditions. The present invention has no special limitation on the conditions of the ultrasonic treatment, as long as the crude selenium-enriched Fucha extract can be dissolved in water. In the present invention, the concentration of the aqueous solution of the crude extract is preferably 1 to 80 g / L, more preferably 10 to 60 g / L, and further preferably 40 g / L. In the present invention, the volume of the macroporous resin preferably accounts for 20 to 70% of the volume of the aqueous solution of the crude extract (or the aqueous solution containing the selenium-enriched Fucha extract obtained by petroleum ether extraction), more preferably 30 to 60%, and further preferably 40 to 50%. In the present invention, the adsorption is preferably carried out under stirring conditions, and the adsorption time is preferably 0.1 to 5 h, more preferably 0.5 to 1 h. The present invention has no special limitation on the solid-liquid separation, and the solid-liquid separation well-known to those skilled in the art can be used, such as filtration, suction filtration or centrifugal separation. In the present invention, the volume ratio of the eluent to the macroporous resin is preferably 10 to 20:1, more preferably 15 to 20:1. The present invention has no special limitation on the drying, as long as the solvent can be completely removed, such as drying under reduced pressure and / or vacuum drying. In the present invention, the macroporous resin purification can also use the method of column chromatography, that is, the aqueous solution of the crude extract is passed through a chromatography column filled with macroporous resin, and the flow rate is preferably 1 to 10 mL / min, more preferably 2 to 6 mL / min. Elution is carried out using the eluent. The first 0.1 to 0.3 BV of the eluate is discarded, and the remaining eluate is dried to a constant weight; the drying is preferably vacuum drying under reduced pressure or vacuum freeze-drying; the drying temperature is preferably ≤40 °C, more preferably 35 to 40 °C. In the present invention, the macroporous resin purification can remove caffeine and sugar components.

[0038] In the present invention, the conditions for reverse column chromatography purification include: the eluent used includes 10 - 30 v / v% aqueous alcohol solution, more preferably 15 - 25 v / v% aqueous alcohol solution, and further preferably 20 v / v% aqueous alcohol solution. The alcohol in the eluent includes methanol or ethanol; the chromatographic column is preferably a reverse C18 column, more preferably a SiliaSphere PC18 reverse-phase chromatographic column; the detection wavelength is preferably 227 nm. In the present invention, the reverse C18 column chromatography purification preferably includes: dissolving the selenium-enriched Fucha crude extract (or the defatted extract obtained by petroleum ether extraction) in an aqueous alcohol solution, loading the obtained extract alcohol solution onto a C18 reverse chromatographic column, eluting with the eluent, and after the components with an obvious absorption at a wavelength of 227 nm (the components containing caffeine) flow out completely, collecting 20 - 70 BV (more preferably 30 - 50 BV) of the eluate, concentrating and then drying. In the present invention, the volume fraction of alcohol in the aqueous alcohol solution for dissolution is preferably 10 - 30%, more preferably 13 - 25%, and further preferably 20% (methanol aqueous solution) or 14% (ethanol aqueous solution); the alcohol preferably includes methanol and / or ethanol. In the present invention, the temperature for concentration is preferably ≤40°C, more preferably 35 - 40°C; the concentration is preferably vacuum concentration. The present invention has no special limitation on the solid-liquid separation, and the solid-liquid separation well-known to those skilled in the art can be adopted, such as filtration, suction filtration or centrifugal separation. In the present invention, the drying is preferably vacuum freeze-drying, and the present invention has no special limitation on the conditions for the vacuum freeze-drying, as long as the solvent can be completely removed.

[0039] In the present invention, the refining preferably includes purifying with macroporous resin 1 - 3 times (more preferably 2 times), or successively performing petroleum ether extraction, macroporous resin purification and reverse C18 column chromatography purification, or successively performing petroleum ether extraction and purifying with macroporous resin 1 - 3 times (more preferably 2 times).

[0040] The present invention provides a selenium-enriched Fucha extract prepared by the preparation method described in the above technical solution.

[0041] The present invention provides the application of the selenium-enriched Fucha extract described in the above technical solution in the preparation of a whitening and freckle-removing agent. In the present invention, the whitening and freckle-removing agent preferably includes essence, lotion or cream. The present invention first uses the selenium-enriched Fucha extract in a whitening and freckle-removing agent, and the whitening effect is close to or even exceeds that of arbutin, with excellent whitening effect, and is suitable for industrial promotion.

[0042] The following examples are used to illustrate in detail the selenium-enriched Fucha extract provided by the present invention, its preparation method and its application in the preparation of a whitening and freckle-removing agent, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] 1) Mechanically crush the selenium-rich Fuzhuan tea, add deionized water with a mass 10 times that of the tea leaves to obtain a tea-water mixture;

[0045] 2) Load the tea-water mixture into a sealed extraction tank with a condensing reflux device, then evacuate to reduce the absolute pressure of the system to 3 ± 0.2 kPa. Stir and gently heat to maintain the water in the system near the boiling state while ensuring that the temperature of the extract does not exceed 40 °C. After continuous extraction for 30 min, pour out the mixture from the extraction tank and filter press to obtain a clear liquid and residue respectively;

[0046] 3) Vacuum freeze-dry the clear liquid to obtain a crude selenium-rich Fuzhuan tea extract;

[0047] 4) Under ultrasonic conditions, redissolve the crude selenium-rich Fuzhuan tea extract with pure water to prepare a saturated solution, then add LX-8 resin and stir for 30 min. Filter out the resin particles and place them in a glass chromatography column sleeve, and elute with ethanol. Discard the first 0.3 column volumes of the eluate, collect the remaining ethanol eluate, and then vacuum decompression evaporate and dry to constant weight at 40 °C to obtain a resin-purified extract; wherein, the volume ratio of the saturated solution to the LX-8 resin is 1:0.5, and the volume ratio of the ethanol to the LX-8 resin is 20:1;

[0048] 5) Repeat step 4) once for the resin-purified extract to obtain a selenium-rich Fuzhuan tea extract (denoted as refined extract 1).

[0049] Example 2

[0050] 1) Mechanically crush the selenium-rich Fuzhuan tea into particles with a particle size of 8 mesh, add absolute ethanol with a mass 10 times that of the tea leaves to obtain a tea-ethanol mixture;

[0051] 2) Load the tea-ethanol mixture into a sealed extraction tank with a condensing reflux device, then evacuate to reduce the absolute pressure of the system to 10 ± 0.2 kPa. Stir and gently heat to maintain the ethanol in the system near the boiling state while ensuring that the extraction temperature does not exceed 30 °C. After continuous extraction for 30 min, pour out the mixture from the extraction tank and filter to obtain a clear liquid and residue respectively;

[0052] 3) Continuously extract the residue 3 times according to step 2), combine all the clear liquids, and vacuum dry to constant weight to obtain a crude selenium-rich Fuzhuan tea extract;

[0053] 4) Disperse the crude selenium-rich Fuzhuan tea extract in ultrapure water to obtain a turbid liquid, then add petroleum ether for extraction 3 times. Concentrate the obtained aqueous phase in vacuo to 1 / 3 of the original volume of the aqueous phase and filter to obtain a liquid component; wherein, the volume ratio of the turbid liquid to the petroleum ether used for single extraction is 1:0.5;

[0054] 5) Add LX-8 resin to the liquid component and stir for 30 min. Filter out the resin particles and place them in a glass chromatography column sleeve. Elute with ethanol, discard the first 0.3 column volumes of the eluate, collect the remaining ethanol eluate, and then vacuum evaporate and dry to constant weight at 35 °C to obtain a resin-purified extract. Among them, the volume ratio of the liquid component to LX-8 resin is 1:0.5, and the volume ratio of ethanol to LX-8 resin is 20:1.

[0055] 6) Prepare a saturated solution of the resin-purified extract with a 20% v / v methanol solution and load it onto a SiliaSphere PC18 reversed-phase chromatography column (50 μm, ). Elute with a 20 v / v% methanol aqueous solution, with a detection wavelength of 272 nm. Discard the components containing caffeine, collect the other effluent, vacuum concentrate it at 35 °C, and freeze-dry to obtain a selenium-rich Fucha extract (denoted as refined extract 2).

[0056] Example 3

[0057] 1) Mechanically crush the selenium-rich Fucha brick tea into 5-mesh particles, and add an 80 v / v% ethanol solution with a mass 10 times that of the tea leaves to obtain a tea-ethanol-water mixture.

[0058] 2) Load the tea-ethanol-water mixture into a sealed extraction tank with a condensing reflux device, then evacuate to reduce the absolute pressure of the system to 10 ± 0.2 kPa. Stir and slightly heat to maintain the ethanol in the system at a near-boiling state while ensuring that the extraction temperature does not exceed 35 °C. After continuous extraction for 30 min, pour out the mixture from the extraction tank and filter to obtain a clear liquid and a residue respectively.

[0059] 3) Continuously extract the residue 3 times according to step 2), combine all the clear liquids, and vacuum dry to constant weight at 40 °C to obtain a selenium-rich Fucha crude extract.

[0060] 4) Disperse the selenium-rich Fucha crude extract in ultrapure water to obtain a turbid liquid, add petroleum ether for extraction 3 times, vacuum concentrate the aqueous phase to 1 / 3 of the original volume of the aqueous phase, and filter to obtain a liquid component. Among them, the volume ratio of the turbid liquid to the petroleum ether used for single extraction is 1:0.5.

[0061] 5) Under ultrasonic conditions, add the liquid component obtained in step 4) to LX-8 resin and stir for 30 min. Filter out the resin particles and place them in a glass chromatography column sleeve. Elute with ethanol, discard the first 0.3 column volumes of the eluate, collect the remaining ethanol eluate, stop collecting until the eluate is colorless, and then vacuum evaporate and dry to constant weight at 35 °C to obtain a resin-purified extract; wherein, the volume ratio of the liquid component to LX-8 resin is 1:0.5, and the volume ratio of ethanol to LX-8 resin is 20:1;

[0062] 6) Redissolve the resin-purified extract in pure water, and pass the obtained resin-purified extract solution through a chromatography column filled with LX-8 resin at a flow rate of 4 mL / min, then elute with ethanol, discard the first 0.3 column volumes of the eluate, collect the remaining ethanol eluate, and then vacuum evaporate and dry to constant weight at 35 °C to obtain a selenium-rich Fucha extract (denoted as refined extract 3); wherein, the volume ratio of the LX-8 resin to the resin-purified extract solution is 1:3, and the volume ratio of ethanol to LX-8 resin is 10:1.

[0063] Example 4

[0064] 1) Mechanically crush the selenium-rich Fucha brick tea into particles with a particle size of 8 mesh, add methanol with a mass 10 times that of the tea leaves, then put the mixture into a sealed extraction tank with a condensing reflux device, reduce the absolute pressure of the system to 10 ± 0.2 kPa, stir and heat to 30 °C, maintain the liquid in the system at a near-boiling state, while ensuring that the extraction temperature does not exceed 30 °C. After continuously extracting for 30 min, pour out the mixture from the extraction tank and filter it to obtain a clear liquid and a residue respectively;

[0065] 3) Continuously extract the residue 3 times according to step 1), combine all the clear liquids, and vacuum dry to constant weight to obtain a selenium-rich Fucha crude extract;

[0066] 4) Disperse the selenium-rich Fucha crude extract in ultrapure water to obtain a turbid liquid, then add petroleum ether for extraction 3 times. Concentrate the obtained aqueous phase in vacuo to 1 / 3 of the original volume of the aqueous phase and filter it to obtain a liquid component; wherein, the volume ratio of the turbid liquid to the petroleum ether used for single extraction is 1:0.5;

[0067] 5) Add LX-8 resin to the liquid component and stir for 30 min. Filter out the resin particles and place them in a glass chromatography column sleeve. Elute with methanol, discard the first 0.3 column volumes of the eluate, collect the remaining eluate, and vacuum evaporate and dry the collected eluate to constant weight to obtain a resin-purified extract; wherein, the volume ratio of the liquid component to LX-8 resin is 1:0.5, and the volume ratio of methanol to LX-8 resin is 20:1;

[0068] 6) The purified extract of the resin was formulated into a saturated solution with a 20% v / v methanol solution, loaded onto a SiliaSphere PC18 reverse-phase chromatography column (50 μm, 4 × 60 cm), eluted with a 20 v / v% methanol aqueous solution, the detection wavelength was 272 nm, the components containing caffeine were discarded, and other effluents were collected, concentrated under reduced pressure in vacuo, and freeze-dried to obtain a selenium-rich Fucha extract.

[0069] Example 5

[0070] 1) The selenium-rich Fucha brick tea was mechanically crushed into particles with a particle size of 8 mesh, 60% ethanol with a mass 10 times that of the tea leaves was added, and then the mixture was loaded into a sealed extraction tank with a condensing reflux device. After that, the absolute pressure of the system was reduced to 7 ± 0.2 kPa, and the liquid in the system was maintained in a nearly boiling state while ensuring that the extraction temperature did not exceed 30 °C. After continuous extraction for 30 min, the mixture was poured out from the extraction tank and filtered by suction to obtain a clear liquid and a residue respectively.

[0071] 3) The residue was continuously extracted 3 times according to step 1), and all the clear liquids were combined and dried in vacuo to a constant weight to obtain a crude extract of selenium-rich Fucha.

[0072] 4) The crude extract of selenium-rich Fucha was dispersed in ultrapure water to obtain a turbid solution, and then petroleum ether was added for extraction 3 times. The aqueous phase obtained was concentrated in vacuo to 1 / 3 of the original volume of the aqueous phase and filtered to obtain a liquid component; wherein, the volume ratio of the turbid solution to the petroleum ether used for single extraction was 1:0.5.

[0073] 5) LX-8 resin was added to the liquid component and stirred for 30 min, the resin particles were filtered out and placed in a glass chromatography column sleeve, and eluted with methanol. The first 0.3 column volumes of the eluate were discarded, and the remaining eluate was collected. The collected eluate was evaporated to dryness in vacuo to a constant weight to obtain a purified extract of the resin; wherein, the volume ratio of the liquid component to LX-8 resin was 1:0.5, and the volume ratio of the methanol to LX-8 resin was 20:1.

[0074] 6) The purified extract of the resin was formulated into a saturated solution with a 14% v / v ethanol solution, loaded onto a SiliaSphere PC18 reverse-phase chromatography column (50 μm, ) and eluted with a 14 v / v% ethanol aqueous solution, the detection wavelength was 272 nm, the components containing caffeine were discarded, and other effluents were collected. After concentration under reduced pressure in vacuo, it was freeze-dried to obtain a selenium-rich Fucha extract.

[0075] Comparative Example 1

[0076] Using selenium-rich Fuzhuan tea as raw material, prepared according to the method of Example 18 of CN111053716A. Among them, 90 v / v% ethanol aqueous solution was used for extraction and elution, the cold soaking time was 20 h, and the cold soaking was repeated 3 times to obtain the selenium-rich Fuzhuan tea ethanol extract.

[0077] Comparative Example 2

[0078] Using selenium-rich Fuzhuan tea as raw material, prepared according to the method of Example 19 of CN111053716A. After concentration under reduced pressure and freeze-drying, the aqueous extract of Fuzhuan tea was obtained.

[0079] The aqueous extract of Fuzhuan tea was redissolved with water and slowly flowed through a chromatographic column packed with macroporous resin AB-8 for adsorption, and then eluted with 95 v / v% ethanol aqueous solution. The eluate was collected and concentrated to dryness to constant weight to obtain the selenium-rich aqueous extract of Fuzhuan tea.

[0080] Comparative Example 3

[0081] Using commercially available non-selenium-rich Jingyang Fuzhuan tea as raw material, prepared according to the method of Example 3 to obtain the Fuzhuan tea extract.

[0082] Comparative Example 4

[0083] Using dark tea as raw material, prepared according to the method of Example 3 to obtain the dark tea extract.

[0084] Comparative Example 5

[0085] Using the tea green raw material for processing dark tea that has been killed green and dried, prepared according to the method of Example 3 to obtain the tea green extract.

[0086] Test Example

[0087] Performance test of the tea extracts prepared in Examples 1-3 and Comparative Examples 1-5 and the selenium-rich Fuzhuan tea crude extract obtained in step 3) of Example 3

[0088] 1. Determination of antioxidant capacity

[0089] Using ethanol as a solvent, the tea extracts prepared in Examples 1-3 and Comparative Examples 1-5 were respectively formulated into solutions with a concentration of 10 μg / L. Using vitamin C as a positive control, the scavenging ability of these tea extract solutions for DPPH radicals and ABTS radicals was respectively tested, and the results are as Figure 1 shown. It can be Figure 1 seen that the selenium-rich Fuzhuan tea extracts 1-3 all have strong free radical scavenging activity.

[0090] 2. Determination of selenium element content

[0091] Weigh the tea extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5, and the crude selenium-enriched Fucha extract obtained in step 3) of Example 3, each with the same mass. Use the first method, hydride generation atomic fluorescence spectrometry, in the national standard "GB5009.93-2017 National Food Safety Standard Determination of Selenium in Foods" to determine the selenium element in various extracts. The results are as Figure 2 shown. As Figure 2 can be seen, the selenium element contents of the selenium-enriched Fucha extracts prepared in Examples 1 to 3 are 0.89±0.013mg / kg, 0.43±0.025mg / kg, and 0.66±0.009mg / kg respectively. This shows that the refinement of the crude selenium-enriched Fucha extract will cause the loss of a part of selenium element, but the selenium element content in the final extract still reaches the selenium-enriched standard, and the selenium element in it is significantly higher than that of the extracts of other several kinds of tea.

[0092] 3. In vitro tyrosinase inhibitory activity

[0093] Prepare the tea extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5, the crude selenium-enriched Fucha extract obtained in step 3) of Example 3, and arbutin standard product into 1mg / mL solutions with PBS respectively. Mix them with an equal volume of tyrosinase PBS solution respectively, and the enzyme concentration is 200U / mL. Incubate with shaking at 37°C for 10min, and then add the PBS solution of L-tyrosine with 8 times the volume of the enzyme solution respectively, where the concentration of L-tyrosine is 20mg / mL. React at 37°C for 10min, and then measure the absorbance of the whole reaction system at 475nm. The inhibitory activity of tyrosinase is calculated according to formula 1, and the results are as Figure 3 shown.

[0094] Tyrosinase inhibition rate = 100%×(A0 - A) / A0 Formula 1;

[0095] where A0 is the absorbance of the reaction after mixing tyrosinase and tyrosine without adding any compounds and extracts, A is the absorbance of the test group adding extracts and arbutin, and arbutin is used as a positive control compound. All absorbance values are subtracted from the system blank of the reaction system.

[0096] As Figure 3It can be seen that the inhibition rates of the selenium-enriched Fuping tea extracts prepared in Examples 1-3 on tyrosinase were 85.31% ± 2.23, 76.23 ± 3.56, and 75.32 ± 4.67% respectively, and the inhibition rates on tyrosinase were significantly higher than those of the selenium-enriched Fuping tea crude extract and the tea extracts prepared in Comparative Examples 1-5. Among them, the selenium-enriched Fuping tea extracts prepared in Examples 1-2 had significantly stronger inhibitory activity on tyrosinase than the model compound arbutin. Although the alcohol extract of selenium-enriched Fuping tea also had relatively high inhibitory activity on tyrosinase, it was weaker than arbutin, while the inhibitory activity of the water extract of selenium-enriched Fuping tea was lower. It shows that the selenium-enriched Fuping tea extract prepared by the present invention has better whitening efficacy.

[0097] 4. Inhibiting the total amount of melanin in B16 cells

[0098] Using B16-F10 (CRL-6475) melanoma cells (from ATCC), the cells were inoculated in DMEM medium containing 10% fetal bovine serum, and the cell culture conditions were 37 °C, 5% carbon dioxide content, and 98% relative humidity. The cells in the logarithmic growth phase were digested with trypsin and inoculated in a 6-well culture plate at a concentration of 1×10 6 cells / mL, 2 mL per well. After culturing in a CO2 incubator for 8 h, the supernatant was discarded, and the cells were rinsed once with PBS. Then, 2 mL of DEME medium containing the selenium-enriched Fuping tea extracts 1-3, selenium-enriched Fuping tea crude extract, Fuping tea extract, dark tea extract, tea green extract, and arbutin standard were added to each well. The concentration of all test compounds was 50 μg / mL, and the blank group was DEME medium without adding any extract or compound. After culturing for 48 h, the supernatant of the cultured cells was discarded, the cells were collected after digestion, centrifuged at 1500 r / min for 10 min, the supernatant was aspirated, and 1 mL of 1 mol / L sodium hydroxide solution containing 10% DMSO was added. After incubating in a water bath at 80 °C for 1 h, the absorbance was measured at 405 nm with an enzyme-labeling instrument. The inhibition rate of the extract on melanin formation was calculated according to Equation 2, and the results are as Figure 4 shown.

[0099] Melanin formation inhibition rate = 100% × (A1 - A) / A1 Equation 2;

[0100] where A1 is the absorbance of the melanin in the cells without adding any compound and extract, A is the absorbance of the test group adding the extract and arbutin, and arbutin is used as a positive control compound. All absorbance values are subtracted from the system blank of the reaction system.

[0101] From Figure 4It can be seen that the inhibition rates of the selenium-enriched Fucha extracts prepared in Examples 1 to 3 on the formation of melanin in cells were 21.22% ± 2.34, 26.57 ± 4.41, and 20.18 ± 2.07% respectively, which were significantly higher than those of the positive control compound arbutin, the selenium-enriched Fucha crude extract, and the tea extracts prepared in Comparative Examples 1 to 5. It shows that the selenium-enriched Fucha extract prepared by the present invention has good ability to inhibit melanin formation and excellent whitening effect.

[0102] 5. Inhibiting the activity of tyrosinase in B16 cells

[0103] The cell culture conditions and cell treatments were the same as those in "4. Inhibiting the total amount of melanin in B16 cells". The DMEM medium without adding any extracts and compounds was used as the blank group, and arbutin was used as the positive control. The experimental groups were the selenium-enriched Fucha extracts prepared in Examples 1 to 3, the selenium-enriched Fucha crude extract prepared in Example 3, the Fucha extract, the dark tea extract, and the tea shoot extract respectively. The experimental treatment method was the same as that in "4. Inhibiting the total amount of melanin in B16 cells". After centrifugation of the treated cells, they were rinsed with PBS, then 40 μL of 1% Triton-X100 was added, and then transferred to -80 °C for freezing for 30 min, and then placed at 37 °C until completely melted. Subsequently, 10 μL of L-levodopa solution (1 w / w%) was added thereto. The reaction was carried out at 37 °C for 30 min, and then the absorbance of the system was measured at a wavelength of 450 nm. The inhibition rate of the tea extract on the formation of melanin in cells was calculated according to Equation 3, and the results are as Figure 5 shown.

[0104] Inhibition rate of intracellular melanin formation = 100% × (A3 - A) / A3 Equation 3;

[0105] where A3 is the absorbance after the reaction of tyrosinase extracted from cells without adding any compounds and extracts, and A is the absorbance of the test group adding the extract and arbutin. Arbutin is used as the positive control compound, and all absorbance values are subtracted from the system blank of the reaction system.

[0106] It can be Figure 5 seen that the selenium-enriched Fucha extracts prepared in Examples 1 to 3 all have good ability to inhibit the activity of tyrosinase in cells, and the inhibitory effect is significantly stronger than that of the selenium-enriched Fucha crude extract and the tea extracts prepared in Comparative Examples 1 to 5. Moreover, the inhibitory effect of the selenium-enriched Fucha extract prepared in Example 2 is significantly stronger than that of the control compound arbutin. It shows that the selenium-enriched Fucha extract prepared by the present invention has excellent whitening effect.

[0107] 6. Inhibiting the expression of melanin-related genes in B16 cells

[0108] In Application Example 4, the cells treated with each extract and compound and the control group cells were digested and collected respectively. Then, 1 mL of pre-cooled Trizol reagent was added to each sample and lysed for 5 min. Subsequently, after adding 200 μL of chloroform, centrifugation was performed at 4°C and 12,000 r / min for 15 min. Then, 500 μL of the supernatant was mixed with 500 μL of isopropanol and left at room temperature for 10 min. Subsequently, centrifugation was carried out at 4°C and 12,000 r / min for 10 min, and the supernatant was discarded. The precipitate was washed with 1 mL of 75 v / v% ethanol aqueous solution, shaken, and centrifuged at 4°C and 7,500 r / min for 5 min, and the above steps were repeated once. The ethanol aqueous solution was discarded, and it was air-dried until the extracted RNA was colorless and transparent. After dissolving with 20 μL of DEPC water, total cellular RNA was obtained. After detecting the total RNA concentration and determining the RNA integrity, it was used as a transcription template.

[0109] According to the kit Prepare cDNA by reverse transcription according to the instructions of the RT reagent Kit With g DNA Eraser(Perfect RealTime). First, react according to the system in Table 1 at 42°C for 2 min. Then, react according to the system in Table 2 at 37°C for 15 min, and then react at 85°C for 5 s to prepare cDNA.

[0110] Using the cDNA as a template, quantitatively analyze the melanin-related genes with SYBRPremix Ex Taq TM reagent. The reaction system is shown in Table 3, the reaction conditions are shown in Table 4, the primers used are shown in Table 5, and the internal reference gene is β-actin. The results are as Figure 6 shown.

[0111] Table 1 Reagents and Dosages

[0112] Reagent Dosage 5×gDNA Eraser Buffer 2 μL g DNA Eraser 1 μL Total RNA 2 μL <![CDATA[RNase Free dH2O]]> 5 μL

[0113] Table 2 Reagents and Dosages

[0114]

[0115]

[0116] Table 3 Reagents and Dosages

[0117] Reagent Dosage cDNA template 2 μL Forward primer 0.4 μL Reverse primer 0.4 μL ROX 0.4 μL 2×SYBR Green Premix EX Taq 6.8 μL <![CDATA[ddH2O]]> 20 μL

[0118] Table 4 Reaction Conditions

[0119] Temperature Time Pre-denaturation at 95°C 10 min Denaturation at 95°C 10s 57℃ 20s 72℃ 30s

[0120] Table 5 Primers Used

[0121]

[0122]

[0123] It can be seen from Figure 6 that the selenium-enriched Fucha extract prepared by the present invention can significantly reduce the expression of four key genes (MITF, TYR, TRP-1, and TRP-2) for melanin synthesis, and the inhibitory effects of the three selenium-enriched Fucha extracts on the genes related to melanin synthesis are close to those of the control arbutin, indicating that the selenium-enriched Fucha extract prepared by the present invention can jointly promote the whitening effect at the gene level and enzyme activity level.

[0124] Application Example 1

[0125] Selenium-enriched Fucha extract compound essence

[0126] Composition: Calculated by mass / volume percentage, 2% of selenium-enriched Fucha extract, 1% of hyaluronic acid, 5% of L-ascorbic acid, 0.5% of ferulic acid, 0.5% of hydroxyethyl urea, and 0.1% of ethylene glycol phenyl ether are dissolved in ultrapure water; among them, the selenium-enriched Fucha extract is one or several of the selenium-enriched Fucha extracts prepared in Examples 1 to 3.

[0127] Dissolve the selenium-enriched Fucha extract, hyaluronic acid, L-ascorbic acid, ferulic acid, hydroxyethyl urea, and ethylene glycol phenyl ether in ultrapure water to obtain a uniform and transparent selenium-enriched Fucha extract compound essence.

[0128] Determine the selenium element in the selenium-enriched Fucha extract compound essence by hydride generation atomic fluorescence spectrometry, and the selenium element content > 10.0 μg / L.

[0129] Application Example 2

[0130] Selenium-enriched Fucha extract compound emulsion

[0131] Composition: Calculated by mass / volume percentage, 0.3% of bacterial cellulose nanofibers, 2.0% of selenium-enriched Fucha extract, 5.0% of L-ascorbic acid, 0.1% of ethylene glycol phenyl ether, and 1.0% of gelatin are mixed in water to form an emulsion; among them, the selenium-enriched Fucha extract is one or several of the selenium-enriched Fucha extracts prepared in Examples 1 to 3.

[0132] Slowly add bacterial cellulose nanofibers to a 1.0 w / v% aqueous gelatin solution, stir well, add the selenium-enriched Fucha extract, L-ascorbic acid, and ethylene glycol phenyl ether, and use ultrasonic emulsification at a power of 70 W for 2 min for every 100 mL of emulsion to obtain a uniform and stable selenium-enriched Fucha extract compound emulsion.

[0133] Determine the selenium element in the selenium-enriched Fucha extract compound emulsion by hydride generation atomic fluorescence spectrometry, and the selenium element content > 5.0 μg / L.

[0134] Application Example 3

[0135] Compound Cream with Se-enriched Fucha Tea Extract

[0136] Composition: 20 g of shea butter, 5 g of EG emulsifier, 5 g of amino acid moisturizer, 2 g of vitamin E, 2 g of Se-enriched Fucha tea extract, 1 g of vitamin C, 0.1 g of black tea essence, 0.5 mL of phenoxyethanol, 65 g of ultrapure water; among them, the Se-enriched Fucha tea extract is one or several of the Se-enriched Fucha tea extracts prepared in Examples 1 to 3.

[0137] Heat the ultrapure water to 60 °C, then add shea butter and EG emulsifier and stir until completely melted. Cool to room temperature under continuous stirring, then add amino acid moisturizer, vitamin E, Se-enriched Fucha tea extract, vitamin C, black tea essence and phenoxyethanol, and continue to cool to solidify under stirring to obtain the compound cream with Se-enriched Fucha tea extract.

[0138] Determine the selenium element in the compound cream with Se-enriched Fucha tea extract by hydride generation atomic fluorescence spectrometry, and the selenium element content > 10.0 μg / kg.

[0139] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a selenium-rich Fucha tea extract, characterized in that, It includes the following steps: Mix the selenium-rich Fucha powder with an extractant, and perform vacuum extraction to obtain a selenium-rich Fucha crude extract; the extractant is water; the temperature of the vacuum extraction is ≤ 40°C, the time is 30 - 120 min, and the absolute pressure is 2.5 - 10.5 kPa; Refine the selenium-rich Fucha crude extract to obtain a selenium-rich Fucha extract; the refining is macroporous resin purification; the macroporous resin used for the macroporous resin purification is LX-8 resin; the eluent used for the macroporous resin purification is methanol and / or ethanol.

2. A method for preparing a selenium-enriched Fucha extract, characterized in that: It includes the following steps: Mix the selenium-rich Fucha powder with an extractant, and perform vacuum extraction to obtain a selenium-rich Fucha crude extract; the extractant is methanol and / or ethanol with a volume fraction of 60 - 100%; the temperature of the vacuum extraction is ≤ 40°C, the time is 30 - 120 min, and the absolute pressure is 2.5 - 10.5 kPa; Refine the selenium-rich Fucha crude extract to obtain a selenium-rich Fucha extract; the refining is successively carried out with petroleum ether extraction and macroporous resin purification; the petroleum ether extraction is as follows: disperse the selenium-rich Fucha crude extract in water to obtain a turbid solution; mix the turbid solution with petroleum ether for extraction, and after extraction, concentrate the aqueous phase under reduced pressure to 20 - 50% of the volume of the aqueous phase and then filter to obtain an aqueous solution containing the selenium-rich Fucha extract; the macroporous resin used for the macroporous resin purification is LX-8 resin; the eluent used for the macroporous resin purification is methanol and / or ethanol.

3. The preparation method according to claim 2, wherein The volume ratio of the turbid solution to the petroleum ether used for single extraction is 1:0.3 - 1, and the number of times of the petroleum ether extraction is 2 - 5 times.

4. The preparation method according to claim 2, wherein After the macroporous resin purification, the refining also includes reverse column chromatography purification; the eluent used for the reverse column chromatography purification is 10 - 30 v / v% aqueous alcohol solution, the alcohol in the 10 - 30 v / v% aqueous alcohol solution is methanol or ethanol, and the chromatographic column is a reverse C18 column.

5. The preparation method according to claim 1 or 2, characterized in that, The mass ratio of the selenium-rich Fucha powder to the extractant is 1:5 - 20.

6. The selenium-rich Fucha extract prepared by the preparation method according to any one of claims 1 - 5.

7. Use of the selenium-rich Fucha extract according to claim 6 in the preparation of a whitening and freckle-removing agent.

8. The application according to claim 7, wherein The whitening and freckle-removing agent includes essence, lotion or cream.

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