Preparation method, product and application of a Panax japonicus Torr. extract with repair and moisturizing effects

Abstract

The present invention relates to a preparation method, product and application of a Panax japonicus Torr. extract with the functions of repair and moisturization, and comprises the following steps: after the Panax japonicus Torr. medicinal materials are crushed, heating reflux extraction is carried out with a 60-80% ethanol aqueous solution; after the extract is filtered and concentrated, macroporous resin is used for purification and elution; after the eluate is concentrated, flocculation treatment and decolorization treatment are carried out, and then drying is carried out to obtain the product. The extract prepared by this preparation method has the advantages of light color, few impurities and high extraction efficiency of effective active ingredients; it also has the excellent effects of promoting the expression of aquaporin-3 in epidermal cells and promoting epidermal repair. The Panax japonicus Torr. extract prepared by this preparation method can be applied to cosmetics with skin moisturizing or skin repair functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and relates to a preparation method, product and application of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract, specifically to a preparation method, product and application of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract with repair and moisturizing effects. Background Art

[0002] Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng is the dried rhizome of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng, a plant of the genus Panax in the Araliaceae family. Its rhizome is in the shape of Buddhist beads or spherical, and it is a variant of Panax japonicus Torr. It is mainly produced in high-altitude areas such as Yunnan, Guizhou, and Sichuan in China. It was first recorded in "Compendium of Materia Medica in Yunnan", and is one of the seven famous herbs in Taibai Mountain. It has the effects of nourishing the lungs and yin, removing stasis and relieving pain, and stopping bleeding. Modern research shows that the content of saponin components in the rhizome of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng is relatively high. The main chemical components are triterpenoid saponins, which are also the main active components, mainly including oleanane-type saponins such as chikusetsusaponin IVa, chikusetsusaponin IV, and chikusetsusaponin V (ginsenoside R0). Some studies have pointed out that chikusetsusaponin IVa has the effect of promoting autophagy, showing significant anti-aging potential. There are also studies proving that ginsenoside R0 can inhibit the production of MMP-2 in fibroblasts induced by UVB, showing significant anti-skin photoaging effects.

[0003] At present, the extraction and purification processes of the active ingredients in Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng mainly include alcohol extraction method, ultrasonic-assisted extraction method, etc. For example, CN101947249A discloses a method for ultrasonic extraction of total saponins from Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng. The Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng is dried, crushed and sieved through a 40-mesh sieve. The crude powder is weighed and placed in a Soxhlet extractor, petroleum ether is added, and reflux is carried out for 2 h. The filter paper cylinder is taken out, and the medicinal materials are dried and reserved. Ethanol with a concentration of 30%-70% is added, and after shaking well, it is soaked for 1 h, and ultrasonic extraction is carried out at 30-50 °C for 10-30 min. However, the ultrasonic-assisted extraction has high energy consumption, is not suitable for industrial production, and the content of active ingredients in the obtained extract is low. And CN102603851A discloses a method for extracting and purifying chikusetsusaponin IVa from Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng. The Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng is appropriately crushed and extracted with an ethanol aqueous solution or a methanol aqueous solution. The extract is filtered by suction, and concentrated under reduced pressure to obtain a paste. The paste is dispersed in water, extracted with an organic solvent, and concentrated to obtain a concentrated solution. Then, it is dispersed in water again, and the dispersion is separated and prepared into high-concentration chikusetsusaponin IVa by a macroporous resin column and high-speed countercurrent chromatography in sequence. However, the obtained product has a heavy color, many impurities, and it is difficult to ensure stability.

[0004] In addition, there are few reports on applying the active ingredients of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng to skin repair and moisturizing. Aquaporin-3 (AQP3) plays a key role in the process of keratinocytes performing functions, plays an important role in the hydration of the skin, plays an important role in maintaining skin moisture and skin function, and can promote the proliferation and migration of human keratinocytes, and is closely related to the generation and repair of the skin barrier. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method, product and application of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract, specifically to provide a preparation method, product and application of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract with repair and moisturizing effects.

[0006] To achieve the purpose of this invention, the following technical solutions are adopted:

[0007] In the first aspect, the present invention provides a preparation method of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract with repair and moisturizing effects, and the preparation method includes the following steps:

[0008] (1) After crushing the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng medicinal materials, heat reflux extraction is carried out with 60 - 80% ethanol aqueous solution, and after filtering and concentrating the extract, a crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng is obtained;

[0009] (2) Purify and elute the crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng with macroporous resin, and collect the eluate;

[0010] (3) After concentrating the eluate, carry out flocculation treatment and decolorization treatment, and then dry to obtain the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract with repair and moisturizing effects.

[0011] The 60 - 80% ethanol aqueous solution can select ethanol aqueous solutions with concentrations of 60%, 65%, 70%, 75%, 80%, etc. Other specific point values within this numerical range can be selected, and they will not be elaborated one by one here.

[0012] The preparation method of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract involved in the present invention creatively adopts ethanol thermal extraction, macroporous resin purification and enrichment, and combines flocculation treatment and decolorization treatment. On the one hand, the prepared extract has the advantages of light color, few impurities and high extraction efficiency of effective active ingredients (mainly ginsenoside R0, chikusetsusaponin Ⅳa, polysaccharide); on the other hand, the obtained extract also has the excellent effect of promoting the expression of aquaporin - 3 in epidermal cells and the excellent effect of promoting epidermal repair. Therefore, the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by this preparation method can be applied to cosmetics with skin moisturizing or skin repair effects.

[0013] Preferably, the macroporous resin includes any one of SP850, HP2MGL or X - 5.

[0014] The present invention also creatively discovers that compared with other types of macroporous resins, adopting the above - mentioned specific macroporous resin type in this preparation method can significantly improve the content of effective active ingredients (mainly ginsenoside R0, chikusetsusaponin Ⅳa, polysaccharide) in the prepared Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract and the total yield of the extract, and has more excellent skin moisturizing and repair effects.

[0015] Preferably, the process of purification and elution is as follows: first, elute the impurities at the front end with 5-7 BV of 0-10% ethanol aqueous solution or do not elute the impurities at the front end, and then elute with 35-45% ethanol aqueous solution for 5-7 BV, and collect the eluate (0% ethanol aqueous solution is pure water).

[0016] Adopting the above specific elution procedure, that is, the combination method of ethanol aqueous solution with specific concentration, can significantly improve the concentration and total extraction content of the effective active ingredients (mainly ginsenoside R0, panax japonicus Torr. saponin Ⅳa, polysaccharide).

[0017] The 0-10% ethanol aqueous solution can be an ethanol aqueous solution with a concentration of 0% (pure water), 2%, 5%, 8%, 10%, etc. The 35-45% ethanol aqueous solution can be an ethanol aqueous solution with a concentration of 35%, 37%, 40%, 43%, 45%, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0018] The 5-7 BV can be, for example, 5 BV, 6 BV, 7 BV, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0019] Preferably, a flocculant is used for the flocculation treatment, and the preparation raw materials of the flocculant include chitosan and ZTC1+1 clarifying agent.

[0020] The mass ratio of the chitosan to the ZTC1+1 clarifying agent is (3-1):1, such as 3:1, 2:1, 1:1, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0021] It is also unexpectedly found here that preferably, a flocculant composed of chitosan and ZTC1+1 compounded in a mass ratio of (3-1):1 is used, and the relative amount of the saponin and polysaccharide active ingredients in the product after the flocculation treatment is significantly increased, and at the same time, the extract has more excellent moisturizing and repair effects.

[0022] Preferably, the flocculant is prepared by the following method: first, mix the preparation raw materials with 0.5-2% acetic acid aqueous solution and stir until it becomes a paste, and then mix it with water and swell for more than 20 h to obtain it.

[0023] The usage amount of the acetic acid aqueous solution is small relative to the water used subsequently, and its concentration can be 0.5%, 0.7%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, etc. Swelling for more than 20 h can be 22 h, 23 h, 24 h, 25 h, 27 h, 28 h, 30 h, 32 h, etc.

[0024] The dosage of the flocculant is 1-3% of the concentrated eluate, such as 1%, 1.2%, 1.5%, 2%, 3%, etc.

[0025] Preferably, the flocculation treatment is carried out at 40 - 60 °C for 20 - 60 min.

[0026] Preferably, the decolorization treatment uses a decolorizing agent, and the decolorizing agent includes any one or a combination of at least two of diatomaceous earth, silica gel or neutral alumina.

[0027] The dosage of the decolorizing agent is 1 - 3% of the concentrated eluate, such as 1%, 1.2%, 1.5%, 2%, 3%, etc.

[0028] Preferably, the decolorization treatment is carried out at 40 - 60 °C for 20 - 60 min.

[0029] The flocculation treatment and the decolorization treatment are carried out separately or simultaneously.

[0030] After the pulverization in step (1), it further includes a sieving treatment through a 20 - 60 mesh sieve, such as 25 mesh, 30 mesh, 35 mesh, 40 mesh, 45 mesh, 50 mesh, 55 mesh, 60 mesh, etc.

[0031] The drying in step (3) includes any one of spray drying, freeze drying or vacuum drying.

[0032] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0033] In a second aspect, the present invention provides a Panax japonicus Torr. extract prepared by the preparation method of the Panax japonicus Torr. extract with repair and moisturizing effects according to the first aspect.

[0034] In a third aspect, the present invention provides the application of the Panax japonicus Torr. extract according to the second aspect in the preparation of a cosmetic having skin moisturizing and / or skin repair effects.

[0035] Preferably, the content of the Panax japonicus Torr. extract in the cosmetic is 0.01 - 15%, such as 0.01%, 0.05%, 0.08%, 1%, 1.2%, 1.5%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 12%, 13%, 15%, etc.

[0036] In a fourth aspect, the present invention provides the application of the Panax japonicus Torr. extract according to the second aspect in the preparation of an aquaporin - 3 expression promoter.

[0037] According to the research results of the present invention, it can be known that the Panax japonicus Torr. extract prepared by the above - mentioned specific method has the effect of promoting the expression of aquaporin - 3. Therefore, the Panax japonicus Torr. extract prepared by the above - mentioned specific method is used in the preparation of an aquaporin - 3 expression promoter and plays a role in the systematic theoretical research on the physiological action mechanism of aquaporin - 3.

[0038] In a fifth aspect, the present invention provides the application of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract according to the second aspect in the preparation of a product having the effect of promoting the healing of epidermal cell scratches.

[0039] According to the research results of the present invention, it can be seen that the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by the above specific method has the effect of promoting the healing of epidermal cell scratches. Therefore, the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by the above specific method can also be used to promote the healing of epidermal cell scratches in related scientific research.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The preparation method of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract involved in the present invention creatively adopts ethanol thermal extraction, macroporous resin purification and enrichment, and combines flocculation treatment and decolorization treatment. On the one hand, the prepared extract can have the advantages of light color, few impurities and high extraction efficiency of effective active ingredients (mainly ginsenoside R0, chikusetsusaponin IVa, polysaccharide); on the other hand, the obtained extract also has a more excellent effect of promoting the expression of aquaporin-3 in epidermal cells and a more excellent effect of promoting epidermal repair. Therefore, the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by this preparation method can be applied to cosmetics with skin moisturizing or skin repair effects. Description of the Drawings

[0042] Figure 1 is the liquid chromatogram of the ginsenoside R0 reference substance;

[0043] Figure 2 is the liquid chromatogram of the chikusetsusaponin IVa reference substance;

[0044] Figure 3 is the liquid chromatogram of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared in Example 1;

[0045] Figure 4 is the immunofluorescence detection map of the expression level of aquaporin-3 in each group;

[0046] Figure 5 is the microscope field view map of the cell scratch repair test in each group. Detailed Embodiments

[0047] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0048] The Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng medicinal materials involved in the following embodiments were purchased from Lijiang Shengtai Agricultural Development Co., Ltd.

[0049] Chitosan is a product with the model number S11064-500g purchased from Yuanye Biotechnology Co., Ltd.; ZTC1+1 is purchased from Henan Bawei Food Ingredients Business.

[0050] The HaCaT cells involved in the following test examples are from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0051] The yield calculation formula for the bead ginseng extract involved below is: yield = (M1 / M2) × 100%, where M1 is the weight of the bead ginseng extract and M2 is the weight of the bead ginseng medicinal material.

[0052] The detection of the contents of ginsenoside R0 and japonicoside IVa involved below adopts the liquid phase method, specifically as follows:

[0053] The reference standards of ginsenoside R0 and japonicoside IVa are purchased from Shanghai Yuanye Biotechnology Co., Ltd. The detection conditions refer to the "Chinese Pharmacopoeia" 2020 edition. Using octadecylsilyl silica gel as the filler, acetonitrile - 0.2% phosphoric acid water (65:35) as the mobile phase, the flow rate is 1 mL / min, the detection wavelength is 203 nm, the column temperature is 30 °C, and the number of theoretical plates calculated by the japonicoside IVa peak is not less than 3000. The liquid chromatograms of the ginsenoside R0 reference standard and the japonicoside IVa reference standard are respectively as Figure 1 and Figure 2 shown, and the liquid chromatogram of the bead ginseng extract prepared in Example 1 is as Figure 3 shown.

[0054] The method for detecting the polysaccharide content involved below is as follows:

[0055] (1) Preparation of standard solutions: Prepare a 0.1 mg / mL glucose standard solution. Sequentially pipette 0, 0.1, 0.2, 0.4, 0.8, 1.6, 2 mL into test tubes, add ddH2O to each test tube to make up 2 mL, and set aside. Preparation of sample solutions: Accurately weigh the sample powder, add ddH2O to prepare a 0.1 mg / mL sample solution for testing. Preparation of 5% phenol solution: Accurately weigh 2.5 g of phenol solid powder, add 47.5 mL of ddH2O and dissolve at 75 °C, and store in the dark.

[0056] (2) Reaction of standard product solutions and sample solutions: Sequentially pipette 1 mL of the prepared standard product solutions and sample solutions into test tubes, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution for reaction, gently shake, use ddH2O as the blank control, and measure the absorbance with a microplate reader at 490 nm. Plot a standard curve with the concentration as the abscissa and the absorbance as the ordinate, which is y = 0.0049x + 0.0018 (R 2 = 0.9984). Substitute the absorbance of the sample solution into the standard curve for calculation.

[0057] Example 1

[0058] This example provides a method for preparing an extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng:

[0059] (1) After crushing the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng medicinal materials through a 40-mesh sieve, 100 g was taken and extracted by heating under reflux with 10 times the mass of 70% ethanol aqueous solution for two times, 2 h each time. After combining the extraction solutions, they were filtered and concentrated under reduced pressure to obtain the crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng;

[0060] (2) Weigh 300 g of HP2MGL macroporous resin and pack the column with pure water. Dissolve the crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng in 10 times the mass of water and load the sample. Elute with 40% ethanol aqueous solution for 6 BV, and collect the eluate;

[0061] (3) After concentrating the mixed solution until the alcohol smell disappears, add 2.5% of the flocculant and 2.5% of the decolorizing agent diatomaceous earth to the solution, and stir at 50 °C for 30 min. The flocculant is obtained by first mixing chitosan and ZTC1 + 1 with a mass ratio of 3:1 with 5 times the mass of 1% acetic acid aqueous solution and stirring until it becomes pasty, and then mixing with 100 times the mass of water and swelling for 24 h;

[0062] (4) Then let it stand, cool, filter, and freeze-dry to obtain the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng.

[0063] Example 2

[0064] This example provides a method for preparing an extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng:

[0065] (1) After crushing the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng medicinal materials through a 40-mesh sieve, 100 g was taken and extracted by heating under reflux with 10 times the mass of 60% ethanol aqueous solution for two times, 3 h each time. After combining the extraction solutions, they were filtered and concentrated under reduced pressure to obtain the crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng;

[0066] (2) Weigh 300 g of X-5 macroporous resin and pack the column with pure water. Dissolve the crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng in 10 times the mass of water and load the sample. First, elute with 10% ethanol aqueous solution for 7 BV to elute the impurities at the front end, and then elute with 35% ethanol aqueous solution for 7 BV, and collect the eluate;

[0067] (3) After concentrating the eluate until the alcohol smell disappears, add 1% of the flocculant and 1% of the decolorizing agent diatomaceous earth to the solution, and stir at 50 °C for 30 min. The flocculant is obtained by first mixing chitosan and ZTC1 + 1 with a mass ratio of 1:1 with 5 times the mass of 1% acetic acid aqueous solution and stirring until it becomes pasty, and then mixing with 100 times the mass of water and swelling for 24 h;

[0068] (4) Then let it stand, cool, filter, and freeze-dry to obtain the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng.

[0069] Example 3

[0070] This example provides a preparation method of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract:

[0071] (1) After pulverizing the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng medicinal materials and passing them through a 40-mesh sieve, 100 g was taken and extracted by heating under reflux with 10 times the mass of 80% ethanol aqueous solution for two times, 2 h each time. After combining the extraction solutions, filtering and concentrating under reduced pressure until the alcohol smell disappeared, a crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng was obtained;

[0072] (2) Weigh 300 g of SP850 macroporous resin and pack it into a column with pure water. Dissolve the crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng in 10 times the mass of water and load the sample. First, elute with pure water for 5 BV to elute the impurities at the front end, and then elute with 45% ethanol aqueous solution for 5 BV, and collect the eluate;

[0073] (3) After concentrating the eluate until the alcohol smell disappeared, add 1.5% of the flocculant and 1.5% of the decolorizing agent neutral alumina to the solution, stir at 50 °C for 30 min. The flocculant is obtained by first mixing chitosan and ZTC1 + 1 with a mass ratio of 2:1 with 5 times the mass of 1% acetic acid aqueous solution and stirring until it becomes a paste, and then mixing with 100 times the mass of water and swelling for 24 h;

[0074] (4) Then let it stand, cool, filter, and freeze-dry to obtain the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract.

[0075] Example 4

[0076] This example provides a preparation method of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract, and the difference from Example 1 is only that HP2MGL macroporous resin is replaced with D101 macroporous resin in equal amount, and other conditions remain unchanged.

[0077] Example 5

[0078] This example provides a preparation method of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract, and the difference from Example 1 is only that HP2MGL macroporous resin is replaced with AB-8 macroporous resin in equal amount, and other conditions remain unchanged.

[0079] Example 6

[0080] This example provides a preparation method of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract, and the difference from Example 1 is only that the elution process in step (2) is: first elute with 20% ethanol aqueous solution for 6 BV to elute the impurities at the front end, and then elute with 40% ethanol aqueous solution for 6 BV, and collect the eluate, and other conditions remain unchanged.

[0081] Example 7

[0082] This example provides a method for preparing the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng, which is only different from Example 1 in that the elution process in step (2) is as follows: the front-end impurities are not eluted, and then 25% ethanol aqueous solution is used to elute 6 BV, and the eluate is collected, and other conditions remain unchanged.

[0083] Example 8

[0084] This example provides a method for preparing the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng, which is only different from Example 1 in that the flocculant used is a single chitosan, but its dosage is still 2.5%, and its preparation method remains unchanged.

[0085] Example 9

[0086] This example provides a method for preparing the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng, which is only different from Example 1 in that the flocculant used is a single ZTC1+1, but its dosage is still 2.5%, and its preparation method remains unchanged.

[0087] Comparative Example 1

[0088] This comparative example provides a method for preparing the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng, which is only different from Example 1 in that no flocculant is added for treatment, and other conditions remain unchanged.

[0089] Comparative Example 2

[0090] This comparative example provides a method for preparing the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng, which is only different from Example 1 in that no decolorizing agent is added for treatment, and other conditions remain unchanged. The color of the product is significantly darker than that of the product in Example 1.

[0091] Comparative Example 3

[0092] This comparative example provides a method for preparing the extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng, which is only different from Example 1 in that no flocculant and decolorizing agent are added for treatment, and other conditions remain unchanged. The color of the product is significantly darker than that of the product in Example 1.

[0093] Test Example 1

[0094] The contents of ginsenoside R0, chikusetsusaponin Ⅳa, and polysaccharide in the extracts of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng prepared in Examples 1-9 and Comparative Examples 1-3 were determined, and the results are shown in Table 1:

[0095] Table 1

[0096]

[0097] As can be seen from the data in Table 1, the preparation method of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract involved in the present invention can not only make the concentration ratios of ginsenoside R0 and chikusetsusaponin Ⅳa in the obtained extract at a high level, but also, by comparing the results of Example 1 and Examples 6 - 7, make the ratios of the contents of ginsenoside R0, chikusetsusaponin Ⅳa and total polysaccharides in the extract within a reasonable range, so that the extract can play a significant role in both repair and moisturization; while the type of macroporous resin and the elution procedure of the macroporous resin, especially the flocculation treatment and decolorization treatment, affect the concentration ratios of ginsenoside R0, chikusetsusaponin Ⅳa and total polysaccharides in the extract and the balance between them, and also affect the efficacy of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract in terms of moisturization and repair.

[0098] Test Example 2

[0099] The moisturizing and repair activities of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extracts prepared in Example 1, Examples 6 - 7 and Comparative Examples 1 - 3 were determined:

[0100] (1) Determination of moisturizing efficacy activity:

[0101] Aquaporin - 3 plays an important role in maintaining skin moisture and skin function. The immunofluorescence method was used in the experiment to determine the effect of the Bletilla striata extract on the expression level of aquaporin - 3. HaCaT cells were inoculated in 24 - well plates. The experimental groups included a normal control group, a positive control group (CaCl2 3 mM), and the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract administration groups (120 μg / mL) obtained from different groups. They were incubated in an incubator (37 °C, 5% CO2) for 12 h. When the plating rate reached 40% - 60%, grouped administration was carried out. After the administration was completed, they were incubated in the incubator for 24 h. After the incubation ended, the cells were fixed with 4% paraformaldehyde. After 24 h, immunofluorescence detection was carried out, and fluorescence microscopy was used for shooting. The results are as Figure 4 shown, and Image - image processing software was used for analysis. The test results are shown in Table 2:

[0102] Table 2

[0103]

[0104]

[0105] As can be seen from the data in Table 2, the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by the specific extraction process of the present invention has a significantly excellent effect of promoting the expression of aquaporin-3, namely, a moisturizing effect. At the same time, flocculation treatment and decolorization treatment significantly affect the promoting effect on the expression of aquaporin-3. Moreover, by comparing the results of Example 1 and Examples 6-7, it can be seen that when the proportions of ginsenoside R0, chikusetsusaponin IVa, and total polysaccharides in the extract are within a reasonable range, the extract can exert a significant effect in terms of moisturization.

[0106] (2) Determination of scratch repair activity:

[0107] Vertically place a cell scratch insert in the center of each 12-well cell culture plate. Add HaCaT cell suspension to the two small chambers of each insert and culture at 37 °C under 5% CO2 for 24 h. Gently remove the insert with sterilized forceps, gently rinse the cells one to two times with 1 mL of PBS solution and aspirate all the PBS. Set up a normal control group (DMEM medium without serum), a positive control group (epidermal growth factor EGF solution with a concentration of 10,000 IU / mL), and a test substance treatment group (Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract extracted from different groups at 120 μg / mL). Observe the cell growth status at the scratch area at 0 h, 16 h, 24 h, and 48 h under a microscope and take pictures respectively. As Figure 5 shown, and calculate the healing area using ImageJ software. The healing rate calculation formula is as follows: Healing rate = (Initial scratch area - Scratch area at the time to be measured) / Initial scratch area. The test results are shown in Table 3:

[0108] Table 3

[0109]

[0110]

[0111] As can be seen from the data in Table 3, the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by the specific extraction process of the present invention has a significantly excellent effect of promoting the healing and repair of epidermal cells. At the same time, flocculation treatment and decolorization treatment significantly affect the effect of promoting the healing and repair of epidermal cells. Moreover, by comparing the results of Example 1 and Examples 6-7, it can be seen that when the proportions of ginsenoside R0, chikusetsusaponin IVa, and total polysaccharides in the extract are within a reasonable range, the extract can exert a significant effect in terms of repair.

[0112] Test Example 3

[0113] Determine the skin irritation and eye irritation of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extracts prepared in Examples 1-3 according to the "Technical Specifications for Cosmetic Safety" (2015 edition). The results are shown in Table 4.

[0114] Table 4

[0115]

[0116] The results show that the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by the present invention has no skin irritation and eye irritation, and has good safety.

[0117] The applicant declares that the present invention illustrates a preparation method, its products and applications of a Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the products of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

[0118] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0119] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A preparation method of Panax japonicus Torr. extract with the efficacy of repair and moisturization, characterized in that, The preparation method includes the following steps: (1) After pulverizing the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng medicinal material, heat reflux extraction is carried out with 60 - 80% ethanol aqueous solution. After filtering and concentrating the extract, the crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng is obtained; (2) The crude extract of Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng is purified and eluted with macroporous resin, and the eluate is collected; The macroporous resin is any one of SP850, HP2MGL or X - 5; The process of purification and elution is as follows: First, elute the front - end impurities with 5 - 7BV of 0 - 10% ethanol aqueous solution or do not elute the front - end impurities, and then elute with 35 - 45% ethanol aqueous solution for 5 - 7BV, and collect the eluate; (3) After concentrating the eluate, flocculation treatment and decolorization treatment are carried out, and then it is dried to obtain the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract with the efficacy of repair and moisturization; The flocculation treatment uses a flocculant, and the preparation raw materials of the flocculant are a combination of chitosan and ZTC1 + 1 clarifying agent with a mass ratio of (3 - 1):

1.

2. The preparation method of the Panax japonicus Torr. extract with repair and moisturizing effects according to claim 1, characterized in that, The flocculant is prepared by the following method: First, mix the preparation raw materials with 0.5 - 2% acetic acid aqueous solution and stir until it becomes a paste, and then mix with water and swell for more than 20h to obtain it; The dosage of the flocculant is 1 - 3% of the concentrated eluate.

3. The preparation method of the Panax japonicus Torr. extract with repair and moisturizing effects according to claim 1, characterized in that, The decolorization treatment uses a decolorizing agent, and the decolorizing agent includes any one or a combination of at least two of diatomaceous earth, silica gel or neutral alumina; The dosage of the decolorizing agent is 1 - 3% of the concentrated eluate; The flocculation treatment and the decolorization treatment are carried out separately or simultaneously; After pulverization in step (1), it also includes sieving through a 20 - 60 - mesh sieve; The drying in step (3) includes any one of spray drying, freeze drying or vacuum drying.

4. The Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract prepared by the preparation method of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract with the efficacy of repair and moisturization according to any one of claims 1 - 3.

5. The application of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract according to claim 4 in the preparation of cosmetics with skin moisturizing and / or skin repair effects.

6. The application of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract according to claim 4 in the preparation of an aquaporin - 3 expression promoter.

7. The application of the Panax japonicus Torr. var. wangianus (Sun) Tsai & Tseng extract according to claim 4 in the preparation of a product with the effect of promoting the scratch healing of epidermal cells.

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