Fermented folium artemisiae argyi extract as well as preparation method and application thereof

Abstract

The invention relates to the technical field of natural products, and particularly discloses a fermented folium artemisiae argyi extract as well as a preparation method and application thereof. The preparation method of the fermented folium artemisiae argyi extract comprises the following steps: (1) fermenting folium artemisiae argyi to obtain fermented folium artemisiae argyi; (2) extracting the fermented folium artemisiae argyi with a solvent; after the extraction is finished, taking an extracting solution, and concentrating and drying the extracting solution to obtain a fermented folium artemisiae argyi solvent extract; and taking the fermented folium artemisiae argyi solvent extract to obtain the fermented folium artemisiae argyi extract. Research shows that compared with an unfermented folium artemisiae argyi extract, the fermented folium artemisiae argyi extract prepared by the method disclosed by the invention has a more excellent melanocyte generation inhibition effect and has an important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural products, in particular to a fermented Artemisia argyi extract and a preparation method and application thereof. BACKGROUND

[0002] Artemisia argyi, belonging to the family Asteraceae, is widely used in traditional Chinese medicine for treating various diseases such as gynecological diseases and rheumatic pain. Artemisia argyi is rich in flavonoids, volatile oils, triterpenoids and other active ingredients, which endow Artemisia argyi with antioxidant, anti-inflammatory and potential skin whitening effects.

[0003] With the increasing demand for natural skincare products, especially the growing interest in products that can provide efficient and safe whitening effects, the cosmetic industry has been seeking new natural ingredients as active substances for their products. Artemisia argyi, as a traditional Chinese medicinal material, has become one of the research hotspots due to its containing various bioactive ingredients. Recent studies have shown that Artemisia argyi extract has the ability to inhibit melanocyte production, which provides a theoretical basis for its application in whitening products. However, due to the poor effect of single Artemisia argyi extract in inhibiting melanocyte production, it often needs to be compounded with other active ingredients to achieve better inhibition of melanocyte production. Therefore, how to improve the melanocyte production inhibition effect of single Artemisia argyi extract and improve the whitening effect of single Artemisia argyi extract is a subject worthy of in-depth study for those skilled in the art. SUMMARY

[0004] In order to overcome at least one of the technical problems in the prior art, the present application provides The present application first provides a preparation method of a fermented Artemisia argyi extract, comprising the following steps: (1) fermenting Artemisia argyi to obtain fermented Artemisia argyi; (2) taking the fermented Artemisia argyi and extracting it with a solvent; after the extraction is completed, taking the extract liquid and concentrating the extract liquid drying to obtain a fermented Artemisia argyi solvent extract; taking the fermented Artemisia argyi solvent extract to obtain the fermented Artemisia argyi extract.

[0005] The present application provides a novel preparation method of a fermented Artemisia argyi extract; studies have shown that the fermented Artemisia argyi extract obtained by fermenting Artemisia argyi and then extracting it with ethanol has a significantly higher effect on inhibiting melanocyte production than the Artemisia argyi extract obtained by extracting Artemisia argyi without fermentation with ethanol.

[0006] Preferably, the fermentation in step (1) is specifically fermentation with Eurotium cristatum.

[0007] Preferably, the specific method of fermenting the Artemisia argyi in step (1) is as follows: Artemisia argyi is mixed with water, and then is piled up at 30-50℃ for 1-2h, and then is sterilized; after sterilization, Eurotium cristatum bacterial suspension is added and mixed, and then is fermented at 25-35℃ and 40-60% RH under non-sealed conditions for 10-20d; the fermented Artemisia argyi is dried to obtain the fermented Artemisia argyi.

[0008] Most preferably, the specific method of fermenting the Artemisia argyi in step (1) is as follows: Artemisia argyi is mixed with water, and then is piled up at 40℃ for 1.5h, and then is sterilized; after sterilization, Eurotium cristatum bacterial suspension is added and mixed, and then is fermented at 30℃ and 50% RH under non-sealed conditions for 15d; the fermented Artemisia argyi is dried to obtain the fermented Artemisia argyi.

[0009] Preferably, the weight ratio of Artemisia argyi to water is 1:0.5-1.

[0010] Preferably, the weight ratio of Artemisia argyi to Eurotium cristatum bacterial suspension is 1:0.5-1.

[0011] Preferably, the content of Eurotium cristatum in the Eurotium cristatum bacterial suspension is 1-2×10 8 CFU / ml.

[0012] Most preferably, the content of Eurotium cristatum in the Eurotium cristatum bacterial suspension is 1.5×10 8 CFU / ml.

[0013] Preferably, the Eurotium cristatum is from China General Microbiological Culture Collection Center, and the strain preservation number is CICC 2099.

[0014] Preferably, the solvent in step (2) is ethanol.

[0015] Preferably, the ethanol used is an ethanol aqueous solution with a volume fraction of 70%-95%.

[0016] Most preferably, the ethanol used is an ethanol aqueous solution with a volume fraction of 80%.

[0017] Preferably, the use amount ratio of the fermented Artemisia argyi to the solvent in step (2) is 1kg:5-10L.

[0018] Most preferably, the use amount ratio of the fermented Artemisia argyi to the solvent in step (2) is 1kg:8L.

[0019] Preferably, the preparation method further comprises the following steps: The fermented Artemisia argyi solvent extract was loaded onto a macroporous resin column and eluted with 3-6 BV of 31-34% ethanol aqueous solution at a flow rate of 1-3 BV / h to obtain macroporous resin eluent A, which was then discarded. Next, it was eluted with 3-6 BV of 56-58% ethanol aqueous solution at a flow rate of 1-3 BV / h to obtain macroporous resin eluent B. Macroporous resin eluent B was concentrated and dried to obtain the fermented Artemisia argyi macroporous resin eluent fraction. The fermented mugwort extract is obtained by eluting the macroporous resin fraction of fermented mugwort leaves.

[0020] Preferably, the preparation method further includes the following steps: The fermented Artemisia argyi solvent extract was loaded onto a macroporous resin column and eluted with 5 BV of 33% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A, which was then discarded. Next, it was eluted with 5 BV of 57% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent B. Macroporous resin eluent B was concentrated and dried to obtain the fermented Artemisia argyi macroporous resin eluent fraction. The fermented mugwort extract is obtained by eluting the macroporous resin fraction of fermented mugwort leaves.

[0021] Preferably, the macroporous resin column is a D101 type macroporous resin column.

[0022] Further research by the inventors revealed that the macroporous resin elution fraction of fermented Artemisia argyi obtained by further eluting the fermented Artemisia argyi solvent extract through the macroporous resin column of this invention exhibits a significantly higher inhibitory effect on melanocyte production than the fermented Artemisia argyi solvent extract.

[0023] The inventors should note that the elution conditions of the macroporous resin column in this invention are crucial. Only the fermented Artemisia argyi macroporous resin elution fraction prepared under the macroporous resin column elution conditions of this invention exhibits a significantly higher inhibitory effect on melanocyte production than the fermented Artemisia argyi solvent extract. However, the fermented Artemisia argyi macroporous resin elution fraction prepared under other macroporous resin column elution conditions does not exhibit a significantly higher inhibitory effect on melanocyte production than the fermented Artemisia argyi solvent extract.

[0024] Preferably, the preparation method further includes the following steps: eluting the macroporous resin eluent from fermented Artemisia argyi onto a silica gel column, first eluting with 2-4 BV of mixed solvent A at a flow rate of 1-3 BV / h to obtain silica gel column eluent A, which is then discarded; next, eluting with 3-5 BV of mixed solvent B at a flow rate of 1-3 BV / h to obtain silica gel column eluent B; concentrating and drying silica gel column eluent B to obtain the fermented Artemisia argyi silica gel column eluent; and finally, obtaining the fermented Artemisia argyi extract from the fermented Artemisia argyi silica gel column eluent. The mixed solvent A is composed of methanol and chloroform in a volume ratio of 100:16~18; the mixed solvent B is composed of methanol and chloroform in a volume ratio of 100:31~33.

[0025] Most preferably, the preparation method further includes the following steps: eluting the macroporous resin eluent from fermented Artemisia argyi onto a silica gel column, first eluting with 3 BV of mixed solvent A at a flow rate of 2 BV / h to obtain silica gel column eluent A, which is then discarded; next, eluting with 4 BV of mixed solvent B at a flow rate of 2 BV / h to obtain silica gel column eluent B; concentrating and drying silica gel column eluent B to obtain the fermented Artemisia argyi silica gel column eluent; and finally, obtaining the fermented Artemisia argyi extract from the fermented Artemisia argyi silica gel column eluent. The mixed solvent A is composed of methanol and chloroform in a volume ratio of 100:17; the mixed solvent B is composed of methanol and chloroform in a volume ratio of 100:32.

[0026] Further research by the inventors revealed that the fermented Artemisia argyi extract obtained by further eluting the macroporous resin-eluted portion of fermented Artemisia argyi through the silica column of this invention exhibits a significantly higher inhibitory effect on melanocyte production than the macroporous resin-eluted portion of fermented Artemisia argyi; it possesses an excellent inhibitory effect on melanocyte production.

[0027] The inventors should note that the silica gel column elution conditions of this invention are crucial; only fermented Artemisia argyi extract prepared under the silica gel column elution conditions of this invention can significantly inhibit melanocyte production more effectively than the macroporous resin elution fraction of fermented Artemisia argyi. However, fermented Artemisia argyi extract prepared under other silica gel column elution conditions cannot significantly inhibit melanocyte production more effectively than the macroporous resin elution fraction of fermented Artemisia argyi.

[0028] The present invention also provides a fermented Artemisia argyi extract prepared by the above preparation method.

[0029] The present invention also provides an application of the above-mentioned fermented Artemisia argyi extract in the preparation of a product with the effect of inhibiting melanocyte production.

[0030] The present invention also provides an application of the above-mentioned fermented Artemisia argyi extract in the preparation of products with whitening effects.

[0031] Preferably, the product is a drug or cosmetic.

[0032] Beneficial Effects: This invention provides a novel method for preparing fermented Artemisia argyi extract. Studies have shown that the fermented Artemisia argyi extract prepared by the method of this invention exhibits superior melanin production inhibition compared to unfermented Artemisia argyi extract. Because the fermented Artemisia argyi extract of this invention has a superior inhibitory effect on melanin production, it has significant application value as an active ingredient in the preparation of whitening drugs or cosmetics. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] The *Aurotriarcha* strain described in the following examples was obtained from the China Industrial Microbial Culture Collection Center, strain preservation number: CICC 2099.

[0035] Example 1: Preparation of fermented Artemisia argyi extract (1) Take Artemisia argyi leaves and mix with water, pile them at 40℃ for 1.5h, and sterilize them after the pile is finished; after sterilization, add a suspension of *Aspergillus cristatus* and mix well, then ferment for 15 days under non-sealed conditions at 30℃ and 50%RH; take the fermented Artemisia argyi leaves and dry them to obtain the fermented Artemisia argyi leaves; wherein, the weight ratio of Artemisia argyi leaves to water is 1:0.5; the weight ratio of Artemisia argyi leaves to *Aspergillus cristatus* suspension is 1:0.5; the content of *Aspergillus cristatus* in the *Aspergillus cristatus* suspension is 1.5×10 8 CFU / ml; (2) Take fermented Artemisia argyi leaves, heat to boiling with solvent and then reflux for 1.5 h; after extraction, strain... The extract was filtered, concentrated and dried to obtain fermented Artemisia argyi solvent extract; the fermented Artemisia argyi solvent extract was obtained; wherein, the ratio of fermented Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% ethanol aqueous solution.

[0036] Example 2 Preparation of fermented Artemisia argyi extract (1) Take Artemisia argyi leaves and mix with water, pile them at 40℃ for 1.5h, and sterilize them after the pile is finished; after sterilization, add a suspension of *Aspergillus cristatus* and mix well, then ferment for 15 days under non-sealed conditions at 30℃ and 50%RH; take the fermented Artemisia argyi leaves and dry them to obtain the fermented Artemisia argyi leaves; wherein, the weight ratio of Artemisia argyi leaves to water is 1:0.5; the weight ratio of Artemisia argyi leaves to *Aspergillus cristatus* suspension is 1:0.5; the content of *Aspergillus cristatus* in the *Aspergillus cristatus* suspension is 1.5×10 8 CFU / ml; (2) Take fermented Artemisia argyi leaves, heat to boiling with solvent and then reflux for 1.5 h; after extraction, strain... The extract was filtered, concentrated, and dried to obtain fermented Artemisia argyi solvent extract; wherein the ratio of fermented Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% (v / v) aqueous ethanol solution. (3) Take the fermented Artemisia argyi solvent extract and place it on a D101 type macroporous resin column. First, elute with 5 BV of 33% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A. Discard macroporous resin column eluent A. Then, elute with 5 BV of 57% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin column eluent B. Take macroporous resin column eluent B, concentrate and dry it to obtain the fermented Artemisia argyi macroporous resin eluted fraction. The fermented mugwort extract is obtained by eluting the macroporous resin fraction of fermented mugwort leaves.

[0037] Example 3 Preparation of fermented Artemisia argyi extract (1) Take Artemisia argyi leaves and mix with water, pile them at 40℃ for 1.5h, and sterilize them after the pile is finished; after sterilization, add a suspension of *Aspergillus cristatus* and mix well, then ferment for 15 days under non-sealed conditions at 30℃ and 50%RH; take the fermented Artemisia argyi leaves and dry them to obtain the fermented Artemisia argyi leaves; wherein, the weight ratio of Artemisia argyi leaves to water is 1:0.5; the weight ratio of Artemisia argyi leaves to *Aspergillus cristatus* suspension is 1:0.5; the content of *Aspergillus cristatus* in the *Aspergillus cristatus* suspension is 1.5×10 8 CFU / ml; (2) Take fermented Artemisia argyi leaves, heat to boiling with solvent and then reflux for 1.5 h; after extraction, strain... The extract was filtered, concentrated, and dried to obtain fermented Artemisia argyi solvent extract; wherein the ratio of fermented Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% (v / v) aqueous ethanol solution. (3) Take the fermented Artemisia argyi solvent extract and place it on a D101 type macroporous resin column. First, elute with 5 BV of 33% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A. Discard macroporous resin column eluent A. Then, elute with 5 BV of 57% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin column eluent B. Take macroporous resin column eluent B, concentrate and dry it to obtain the fermented Artemisia argyi macroporous resin eluted fraction. (4) Take the macroporous resin elution fraction of fermented Artemisia argyi and place it on a silica gel column. First, elute with 3 BV of mixed solvent A at a flow rate of 2 BV / h to obtain silica gel column eluent A, and discard silica gel column eluent A. Then, elute with 4 BV of mixed solvent B at a flow rate of 2 BV / h to obtain silica gel column eluent B. Take silica gel column eluent B, concentrate and dry it to obtain the silica gel column elution fraction of fermented Artemisia argyi. The mixed solvent A is composed of methanol and chloroform in a volume ratio of 100:17, and the mixed solvent B is composed of methanol and chloroform in a volume ratio of 100:32.

[0038] The fermented mugwort extract was obtained by eluting the fermented mugwort leaves using a silica gel column.

[0039] Comparative Example 1: Preparation of Fermented Artemisia argyi Extract (1) Take Artemisia argyi leaves and mix with water, pile them at 40℃ for 1.5h, and sterilize them after the pile is finished; after sterilization, add a suspension of *Aspergillus cristatus* and mix well, then ferment for 15 days under non-sealed conditions at 30℃ and 50%RH; take the fermented Artemisia argyi leaves and dry them to obtain the fermented Artemisia argyi leaves; wherein, the weight ratio of Artemisia argyi leaves to water is 1:0.5; the weight ratio of Artemisia argyi leaves to *Aspergillus cristatus* suspension is 1:0.5; the content of *Aspergillus cristatus* in the *Aspergillus cristatus* suspension is 1.5×10 8 CFU / ml; (2) Take fermented Artemisia argyi leaves, heat to boiling with solvent and then reflux for 1.5 h; after extraction, strain... The extract was filtered, concentrated, and dried to obtain fermented Artemisia argyi solvent extract; wherein the ratio of fermented Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% (v / v) aqueous ethanol solution. (3) Take the fermented Artemisia argyi solvent extract and place it on a D101 type macroporous resin column. First, elute with 5 BV of 15% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A. Discard macroporous resin column eluent A. Then, elute with 5 BV of 30% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin column eluent B. Take macroporous resin column eluent B, concentrate and dry it to obtain the fermented Artemisia argyi macroporous resin eluted fraction. The fermented mugwort extract is obtained by eluting the macroporous resin fraction of fermented mugwort leaves.

[0040] Comparative Example 2: Preparation of Fermented Artemisia argyi Extract (1) Take Artemisia argyi leaves and mix with water, pile them at 40℃ for 1.5h, and sterilize them after the pile is finished; after sterilization, add a suspension of *Aspergillus cristatus* and mix well, then ferment for 15 days under non-sealed conditions at 30℃ and 50%RH; take the fermented Artemisia argyi leaves and dry them to obtain the fermented Artemisia argyi leaves; wherein, the weight ratio of Artemisia argyi leaves to water is 1:0.5; the weight ratio of Artemisia argyi leaves to *Aspergillus cristatus* suspension is 1:0.5; the content of *Aspergillus cristatus* in the *Aspergillus cristatus* suspension is 1.5×10 8 CFU / ml; (2) Take fermented Artemisia argyi leaves, heat to boiling with solvent and then reflux for 1.5 h; after extraction, strain... The extract was filtered, concentrated, and dried to obtain fermented Artemisia argyi solvent extract; wherein the ratio of fermented Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% (v / v) aqueous ethanol solution. (3) Take the fermented Artemisia argyi solvent extract and place it on a D101 type macroporous resin column. First, elute with 5 BV of 60% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A. Discard macroporous resin column eluent A. Then, elute with 5 BV of 80% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin column eluent B. Take macroporous resin column eluent B, concentrate and dry it to obtain the fermented Artemisia argyi macroporous resin eluted fraction. The fermented mugwort extract is obtained by eluting the macroporous resin fraction of fermented mugwort leaves.

[0041] Comparative Example 3: Preparation of Fermented Artemisia argyi Extract (1) Take Artemisia argyi leaves and mix with water, pile them at 40℃ for 1.5h, and sterilize them after the pile is finished; after sterilization, add a suspension of *Aspergillus cristatus* and mix well, then ferment for 15 days under non-sealed conditions at 30℃ and 50%RH; take the fermented Artemisia argyi leaves and dry them to obtain the fermented Artemisia argyi leaves; wherein, the weight ratio of Artemisia argyi leaves to water is 1:0.5; the weight ratio of Artemisia argyi leaves to *Aspergillus cristatus* suspension is 1:0.5; the content of *Aspergillus cristatus* in the *Aspergillus cristatus* suspension is 1.5×10 8 CFU / ml; (2) Take fermented Artemisia argyi leaves, heat to boiling with solvent and then reflux for 1.5 h; after extraction, strain... The extract was filtered, concentrated, and dried to obtain fermented Artemisia argyi solvent extract; wherein the ratio of fermented Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% (v / v) aqueous ethanol solution. (3) Take the fermented Artemisia argyi solvent extract and place it on a D101 type macroporous resin column. First, elute with 5 BV of 33% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A. Discard macroporous resin column eluent A. Then, elute with 5 BV of 57% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin column eluent B. Take macroporous resin column eluent B, concentrate and dry it to obtain the fermented Artemisia argyi macroporous resin eluted fraction. (4) Take the macroporous resin elution fraction of fermented Artemisia argyi and place it on a silica gel column. First, elute with 3 BV of mixed solvent A at a flow rate of 2 BV / h to obtain silica gel column eluent A, and discard silica gel column eluent A. Then, elute with 4 BV of mixed solvent B at a flow rate of 2 BV / h to obtain silica gel column eluent B. Take silica gel column eluent B, concentrate and dry it to obtain the silica gel column elution fraction of fermented Artemisia argyi. The mixed solvent A is composed of methanol and chloroform in a volume ratio of 100:1. The mixed solvent B is composed of methanol and chloroform in a volume ratio of 100:15.

[0042] The fermented mugwort extract was obtained by eluting the fermented mugwort leaves using a silica gel column.

[0043] Comparative Example 4: Preparation of Fermented Artemisia argyi Extract (1) Take Artemisia argyi leaves and mix with water, pile them at 40℃ for 1.5h, and sterilize them after the pile is finished; after sterilization, add a suspension of *Aspergillus cristatus* and mix well, then ferment for 15 days under non-sealed conditions at 30℃ and 50%RH; take the fermented Artemisia argyi leaves and dry them to obtain the fermented Artemisia argyi leaves; wherein, the weight ratio of Artemisia argyi leaves to water is 1:0.5; the weight ratio of Artemisia argyi leaves to *Aspergillus cristatus* suspension is 1:0.5; the content of *Aspergillus cristatus* in the *Aspergillus cristatus* suspension is 1.5×10 8 CFU / ml; (2) Take fermented Artemisia argyi leaves, heat to boiling with solvent and then reflux for 1.5 h; after extraction, strain... The extract was filtered, concentrated, and dried to obtain fermented Artemisia argyi solvent extract; wherein the ratio of fermented Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% (v / v) aqueous ethanol solution. (3) Take the fermented Artemisia argyi solvent extract and place it on a D101 type macroporous resin column. First, elute with 5 BV of 33% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A. Discard macroporous resin column eluent A. Then, elute with 5 BV of 57% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin column eluent B. Take macroporous resin column eluent B, concentrate and dry it to obtain the fermented Artemisia argyi macroporous resin eluted fraction. (4) Take the macroporous resin elution fraction of fermented Artemisia argyi and place it on a silica gel column. First, elute with 3 BV of mixed solvent A at a flow rate of 2 BV / h to obtain silica gel column eluent A, and discard silica gel column eluent A. Then, elute with 4 BV of mixed solvent B at a flow rate of 2 BV / h to obtain silica gel column eluent B. Take silica gel column eluent B, concentrate and dry it to obtain the silica gel column elution fraction of fermented Artemisia argyi. The mixed solvent A is composed of methanol and chloroform in a volume ratio of 100:30. The mixed solvent B is composed of methanol and chloroform in a volume ratio of 100:50.

[0044] The fermented mugwort extract was obtained by eluting the fermented mugwort leaves using a silica gel column.

[0045] Comparative Example 5: Preparation of Artemisia argyi extract Take Artemisia argyi leaves, heat to boiling with solvent, and then reflux for 1.5 hours; after extraction, filter and collect the extract. The extract was concentrated and dried to obtain Artemisia argyi solvent extract; the Artemisia argyi solvent extract was then taken to obtain the Artemisia argyi extract; wherein the ratio of Artemisia argyi to solvent was 1 kg: 8 L; the solvent used was an 80% ethanol aqueous solution by volume.

[0046] Experiment Example 1: Melanocyte Production Inhibition Experiment 50 μL of human epidermal melanocytes containing HEM-L (concentration 2 × 10⁻⁶) were added to a 96-well plate. 5 Cells were cultured in a medium containing (number of cells / mL) for 24 hours. Then, different concentrations of the test sample were added to each well in the experimental groups, and the cells were cultured for another 48 hours. The supernatant was discarded, and the cells were washed with PBS. Each experimental group had six replicates. Three replicates were used to count cell numbers, and 50 μL of 3 mol / L NaOH solution was added to the other three replicates, which were then incubated at 90°C for 60 min for lysis. The OD value (490 nm) of each well was measured, and the melanocyte production inhibition rate was calculated based on the OD value. A blank control group without the test sample was set up using the same method. The IC50 was further calculated based on the melanocyte production inhibition rate of each group. 50 Value; melanocyte production inhibition rate (%) = [1 - (OD value of experimental group / number of cells in experimental group) / (OD value of blank control group / number of cells in blank control group)] × 100%; test results are shown in Table 1.

[0047] The samples to be tested are fermented Artemisia argyi extracts prepared in Examples 1-3, Comparative Examples 1-4, and Artemisia argyi extract prepared in Comparative Example 5.

[0048] Table 1. Results of the experiment on melanocyte production inhibition

[0049] As can be seen from the experimental results in Table 1, the fermented Artemisia argyi extract prepared in Example 1 has an IC50 value that inhibits melanocyte production. 50 The value was significantly lower than that of the Artemisia argyi extract prepared in Comparative Example 5. This indicates that the fermented Artemisia argyi extract obtained by fermenting Artemisia argyi and then extracting it with ethanol has a significantly higher inhibitory effect on melanocyte production than the Artemisia argyi extract obtained by extracting unfermented Artemisia argyi with ethanol.

[0050] As can be seen from the experimental results in Table 1, the fermented Artemisia argyi extract prepared in Example 2 has an IC50 value that inhibits melanocyte production. 50 The value was significantly lower than that of the fermented Artemisia argyi extract prepared in Example 1. This indicates that the macroporous resin elution fraction of fermented Artemisia argyi obtained by further eluting the fermented Artemisia argyi solvent extract through the macroporous resin column of the present invention has a significantly higher inhibitory effect on melanocyte production than the fermented Artemisia argyi solvent extract.

[0051] As can be seen from the experimental results in Table 1, the fermented Artemisia argyi extracts prepared in Comparative Examples 1 and 2 have an IC50 value for inhibiting melanin production. 50 The value was not significantly lower than that of the fermented Artemisia argyi extract prepared in Example 1. This indicates that the macroporous resin column elution conditions of the present invention are crucial; only the macroporous resin elution fraction of fermented Artemisia argyi prepared under the macroporous resin column elution conditions of the present invention can significantly inhibit melanocyte production more effectively than the solvent extract of fermented Artemisia argyi; however, the macroporous resin elution fraction of fermented Artemisia argyi prepared under other macroporous resin column elution conditions cannot significantly inhibit melanocyte production more effectively than the solvent extract of fermented Artemisia argyi.

[0052] As can be seen from the experimental results in Table 1, the fermented Artemisia argyi extract prepared in Example 3 has an IC50 value that inhibits melanocyte production. 50 The value was significantly lower than that of the fermented Artemisia argyi extract prepared in Example 2. This indicates that the fermented Artemisia argyi extract obtained by further eluting the macroporous resin-eluted portion of fermented Artemisia argyi through the silica column of this invention has a significantly higher inhibitory effect on melanocyte production than the macroporous resin-eluted portion of fermented Artemisia argyi.

[0053] As can be seen from the experimental results in Table 1, the fermented Artemisia argyi extracts prepared in Comparative Examples 3 and 4 have an IC50 value for inhibiting melanocyte production. 50The value was not significantly lower than that of the fermented Artemisia argyi extract prepared in Example 2. This indicates that the silica gel column elution conditions of the present invention are crucial; only the fermented Artemisia argyi extract prepared under the silica gel column elution conditions of the present invention can have a significantly higher inhibitory effect on melanocyte production than the macroporous resin elution fraction of fermented Artemisia argyi. However, the fermented Artemisia argyi extract prepared under other silica gel column elution conditions does not have a significantly higher inhibitory effect on melanocyte production than the macroporous resin elution fraction of fermented Artemisia argyi.

Claims

1. A method for preparing fermented Artemisia argyi extract, characterized in that, It includes the following steps: (1) Take mugwort leaves and ferment them to obtain fermented mugwort leaves; (2) Take fermented mugwort leaves and extract them with a solvent; after the extraction is completed, take the extract, concentrate and dry the extract to obtain fermented mugwort leaf solvent extract; take the fermented mugwort leaf solvent extract to obtain the fermented mugwort leaf extract.

2. The method for preparing fermented Artemisia argyi extract according to claim 1, characterized in that, The fermentation mentioned in step (1) specifically refers to fermentation using *Aspergillus cristatus*.

3. The method for preparing fermented Artemisia argyi extract according to claim 1, characterized in that, The solvent mentioned in step (2) is ethanol.

4. The method for preparing fermented Artemisia argyi extract according to claim 3, characterized in that, The ethanol used is an aqueous solution of ethanol with a volume fraction of 70% to 95%.

5. The method for preparing fermented Artemisia argyi extract according to claim 3, characterized in that, The preparation method further includes the following steps: The fermented Artemisia argyi solvent extract was loaded onto a macroporous resin column and eluted with 3-6 BV of 31-34% ethanol aqueous solution at a flow rate of 1-3 BV / h to obtain macroporous resin eluent A, which was then discarded. Next, it was eluted with 3-6 BV of 56-58% ethanol aqueous solution at a flow rate of 1-3 BV / h to obtain macroporous resin eluent B. Macroporous resin eluent B was concentrated and dried to obtain the fermented Artemisia argyi macroporous resin eluent fraction. The fermented mugwort extract is obtained by eluting the macroporous resin fraction of fermented mugwort leaves.

6. The method for preparing fermented Artemisia argyi extract according to claim 5, characterized in that, The preparation method further includes the following steps: The fermented Artemisia argyi solvent extract was loaded onto a macroporous resin column and eluted with 5 BV of 33% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent A, which was then discarded. Next, it was eluted with 5 BV of 57% ethanol aqueous solution at a flow rate of 2 BV / h to obtain macroporous resin eluent B. Macroporous resin eluent B was concentrated and dried to obtain the fermented Artemisia argyi macroporous resin eluent fraction. The fermented mugwort extract is obtained by eluting the macroporous resin fraction of fermented mugwort leaves.

7. The method for preparing fermented Artemisia argyi extract according to claim 5, characterized in that, The macroporous resin column is a D101 type macroporous resin column.

8. The fermented Artemisia argyi extract prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the fermented Artemisia argyi extract according to claim 8 in the preparation of a product having the effect of inhibiting melanocyte production.

10. The use of the fermented Artemisia argyi extract according to claim 8 in the preparation of products with whitening effects.

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