Method for purifying selaginella tamariscina extract, skin care composition and use of purified product

Abstract

The embodiment of the invention discloses a selaginella tamariscina extract purification method, a skin care composition and application of a purified product. A specific embodiment of the method comprises the following steps: mixing a selaginella tamariscina extract with a first hydrochloric acid solution to obtain a first mixture; after standing the first mixture for a first time, separating the first mixture to obtain a first precipitate and a first filtrate; mixing the first filtrate with a second hydrochloric acid solution to obtain a second mixture; after standing the second mixture for a second time, separating the second mixture to obtain a second precipitate and a second filtrate; and drying and grinding the second precipitate to obtain a selaginella tamariscina purified product. According to the embodiment, the content of flavonoid chemical components in the selaginella tamariscina extract, especially the content of amentotaxus biflavone, can be further increased, and compared with the prior art, the method has higher amentotaxus biflavone process transfer rate.

Description

Technical Field

[0001] The embodiments of the present disclosure relate to the field of biotechnology, and in particular to a method for purifying a Selaginella extract, a skin care composition, and uses of the purified product. Background Art

[0002] Selaginella (scientific name: Selaginella tamariscina) is a native or rocky fern of the Selaginella genus of the Selaginellaceae family. It is widely distributed throughout the world, especially in tropical and subtropical regions. Specifically, some species of Selaginella are extremely drought-resistant. During droughts, the roots can separate from the soil by themselves and curl up into a ball. After encountering water, the roots will drill back into the soil to find water and then recover vitality, so it is called "resurrection grass". In addition, Selaginella has a long history of application in traditional Chinese medicine. It is recorded in ancient books such as "Shennong's Herbal Classic". It is believed to have multiple medicinal effects, including but not limited to: clearing away heat and detoxification, stopping bleeding, anti-inflammatory and anti-tumor.

[0003] Specifically, Selaginella is rich in a variety of flavonoids, acetylenic phenols, and phenylpropanoid chemical components. Among them, selaginella bisflavonoids, as one of the chemical components in Selaginella, has multiple biological activities. Therefore, how to develop Selaginella extracts with higher content of selaginella bisflavonoids has become a research and development focus.

[0004] The above information disclosed in this Background section is only for enhancement of understanding of the background of the inventive concept and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art. Summary of the invention

[0005] The content of this disclosure is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this disclosure is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[0006] Some embodiments of the present disclosure provide a method for purifying a Selaginella extract, a skin care composition, and uses of the purified product to solve one or more of the technical problems mentioned in the above background technology section.

[0007] In the first aspect, some embodiments of the present disclosure provide a method for purifying a Selaginella extract, the method comprising: mixing a Selaginella extract with a first hydrochloric acid solution to obtain a first mixture, wherein the Selaginella extract is a flavonoid extract extracted from Selaginella, and the volume fraction of hydrochloric acid in the first hydrochloric acid solution is 0.1% to 1%; after the first mixture is allowed to stand for a first period of time, separating the first mixture to obtain a first precipitate and a first filtrate; mixing the first filtrate with a second hydrochloric acid solution to obtain a second mixture, wherein the volume fraction of hydrochloric acid in the second hydrochloric acid solution is 0.1% to 1%; after the second mixture is allowed to stand for a second period of time, separating the second mixture to obtain a second precipitate and a second filtrate; drying and grinding the second precipitate to obtain a purified Selaginella.

[0008] Optionally, the Selaginella extract is a Selaginella ethanol extract, or a Selaginella methanol extract, or a mixture of the Selaginella ethanol extract and the Selaginella methanol extract.

[0009] Optionally, the volume ratio of the Selaginella extract and the first hydrochloric acid solution in the first mixed solution is 1:1-3.

[0010] Optionally, the volume ratio of the Selaginella extract and the first hydrochloric acid solution in the first mixed solution may be any one of 1:1, 1:1.5, 1:2, 1:2.5, and 1:3.

[0011] Optionally, the ambient temperature of the first mixture when it is at rest is 2°C to 10°C.

[0012] Optionally, the first duration is 15 minutes to 60 minutes.

[0013] Optionally, the first duration may be any one of 15 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0014] Optionally, the first mixture may be separated by any one of methods including but not limited to gravity filtration, pressure filtration, vacuum filtration, and centrifugal filtration to obtain a first precipitate and a first filtrate.

[0015] Optionally, the volume ratio of the first filtrate to the second hydrochloric acid solution in the second mixed liquid is 1:1-3.

[0016] Optionally, the ambient temperature of the second mixed liquid when it is at rest is 2°C to 10°C.

[0017] Optionally, the volume fraction of hydrochloric acid in the first hydrochloric acid solution may be any one of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%.

[0018] Optionally, the volume fraction of hydrochloric acid in the second hydrochloric acid solution may be any one of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%.

[0019] Optionally, the second duration is 120 minutes to 600 minutes.

[0020] Optionally, the second mixture may be separated by any one of methods including but not limited to gravity filtration, pressure filtration, vacuum filtration, and centrifugal filtration to obtain a second precipitate and a second filtrate.

[0021] In a second aspect, some embodiments of the present disclosure provide a skin care composition, wherein the skin care composition comprises the purified Selaginella as described above in the first aspect.

[0022] Optionally, the skin care composition can be in any form of an aqueous solution, an oily solution, an aqueous cream, an aqueous gel, or an oily gel.

[0023] Optionally, the skin care composition can be any one of a lotion, a microemulsion, a nanoemulsion, a bath gel, a facial mask, an essence, a lotion, a liquid soap, a skin cake, an ointment, a mousse, a spray, a conditioner, and a shampoo.

[0024] Optionally, the skin care composition may further comprise at least one cosmetically acceptable excipient.

[0025] Optionally, the excipients may include, but are not limited to, surfactants, emulsifiers, preservatives, buffers, chelating agents, denaturants, opacifiers, pH adjusters, reducing agents, stabilizers, thickeners, gelling agents, film-forming polymers, matting agents, glossing agents, pigments, fragrances, and essential oils.

[0026] Optionally, the weight of the purified Selaginella can be 0.0001% to 10% of the total weight of the skin care composition.

[0027] Optionally, the weight of the purified Selaginella can be 0.001% to 5% of the total weight of the skin care composition.

[0028] In a third aspect, some embodiments of the present disclosure provide a use of a purified product, which includes use of the Selaginella purified product as described above in the first aspect in the preparation of a product for inhibiting photodamage of skin cells.

[0029] Optionally, the above-mentioned product for inhibiting photodamage of skin cells has any of the following effects: protecting against cell damage and apoptosis caused by UVB irradiation; preventing thickening of epidermal tissue, loss of collagen fibers, and loss of water in epidermal tissue caused by UVB irradiation; improving the oxidative stress response of epidermal tissue caused by UVB irradiation; reducing the production of matrix metalloproteinases to alleviate the degradation of collagen and elastin in the skin.

[0030] The above-mentioned embodiments of the present disclosure have the following beneficial effects: the purification method of the Selaginella extract of some embodiments of the present disclosure can achieve the purification of the Selaginella extract. Specifically, the test found that by adding an appropriate amount of hydrochloric acid solution, the Selaginella extract can be placed in a weak acid environment, under which the flavonoid chemical components including but not limited to selaginella biflavonoids can still remain dissolved rather than forming a precipitate. Therefore, after adding the first hydrochloric acid solution, the flavonoid chemical components including but not limited to selaginella biflavonoids are present in the supernatant (first filtrate). In addition, further tests found that the pH value of the first filtrate can be changed by adding the hydrochloric acid solution (the second hydrochloric acid solution) again, and as the pH value decreases, the flavonoid chemical components including but not limited to selaginella biflavonoids dissolved in the first filtrate can be formed into a precipitate. Compared with the conventional "acid extraction and alkali precipitation" or "alkali extraction and acid precipitation" method, the content of flavonoid chemical components in the Selaginella extract, especially the content of selaginella biflavonoids, can be further increased, and the selaginella biflavonoids process transfer rate is higher than that of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the accompanying drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and the elements are not necessarily drawn to scale.

[0032] Figure 1 is a chromatogram of the purified Selaginella and aconitum biflavonoids corresponding to sample 1-1 of some embodiments of the present disclosure;

[0033] Figure 2 is a comparison chart of the effects of sample 1-1 on HaCat cell activity in some embodiments of the present disclosure;

[0034] Figure 3 This is a comparison chart of the effects of sample 1-1 of some embodiments of the present disclosure on the proliferation activity of HaCat cells after UV (UVB) irradiation;

[0035] Figure 4 is a comparison chart of the effects of sample 1-1 of some embodiments of the present disclosure on the apoptosis rate of HaCat cells after UV (UVB) irradiation;

[0036] Figure 5 Comparison of the effects of sample 1-1 of some embodiments of the present disclosure on the moisture content of mouse epidermis after UV (UVB) irradiation;

[0037] Figure 6 Comparison of the effects of sample 1-1 of some embodiments of the present disclosure on the epidermal thickness of mice after UV (UVB) irradiation;

[0038] Figure 7 Comparison of mouse epidermal thickness in stained sections of some embodiments of the present disclosure;

[0039] Figure 8 Comparison of the changes in collagen fibers in mouse epidermal tissue after UV (UVB) irradiation of sample 1-1 in some embodiments of the present disclosure;

[0040] Fig. 9 Comparison of the effects of sample 1-1 of some embodiments of the present disclosure on the malondialdehyde content in mouse epidermal tissue after UV (UVB) irradiation;

[0041] Fig.10 Comparison of the effects of sample 1-1 of some embodiments of the present disclosure on the superoxide dismutase activity of mouse epidermal tissue after UV (UVB) irradiation;

[0042] Fig.11 A comparison chart of the effects of sample 1-1 of some embodiments of the present disclosure on the content of matrix metalloproteinase-3 in mouse epidermal tissue after UV (UVB) irradiation. DETAILED DESCRIPTION

[0043] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0044] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0045] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0046] It should be noted that the term “about” mentioned in the present disclosure, when used to define a numerical value, means ±5% of the specified numerical value.

[0047] It should be noted that the "normal temperature" mentioned in the present disclosure refers to the ambient temperature, that is, no special heating or cooling treatment has been performed.

[0048] Terminology Explanation: The term "Aconitum sutchuenensis biflavonoids" is a natural biflavonoid compound with a molecular formula of C 30 H 18 O 10 , which can be extracted from plants such as Cupressaceae and Taxaceae.

[0049] The term "acid extraction and alkaline precipitation" is a commonly used extraction and purification technique. Specifically, compounds with good solubility under acidic conditions are first extracted. Among them, impurities that are insoluble in acidic solutions or have low solubility will not be extracted. Then the acidic extract is adjusted to alkaline. Due to the change in pH, the compounds that are soluble under acidic conditions will become difficult to dissolve or insoluble, and precipitate from the acidic extract in the form of precipitation.

[0050] The term "alkali extraction and acid precipitation" is a commonly used extraction and purification technique. Specifically, compounds with good solubility under alkaline conditions are first extracted. Among them, impurities that are insoluble in alkaline solutions or have low solubility will not be extracted. Then the alkaline extract is adjusted to acidic. Due to the change in pH, the compounds that are soluble under alkaline conditions will become difficult to dissolve or insoluble, and precipitate from the alkaline extract in the form of precipitation.

[0051] The term "ethanol solution" refers to a mixture of ethanol and water.

[0052] The term "volume-to-mass ratio" refers to the ratio of the volume of liquid A to the mass of solid B.

[0053] The term "MTT solution" refers to a solution containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is a yellow soluble dye. MTT is commonly used in cell proliferation and cytotoxicity assays.

[0054] The term "DMSO solution" is a versatile organic solvent with extremely strong dissolving power and permeability.

[0055] The term "ICR mouse" is an experimental animal named by the Institute of Cancer Research (ICR) of the United States. It is small in size, has strong adaptability, strong disease resistance, high reproductive capacity and fast growth rate.

[0056] Hereinafter, the present disclosure will be described in detail with reference to the accompanying drawings.

[0057] Purification method description

[0058] The method for purifying the Selaginella extract disclosed in the present invention may include the following steps:

[0059] In the first step, the Selaginella extract is mixed with a first hydrochloric acid solution to obtain a first mixture.

[0060] Wherein, the above-mentioned Selaginella extract is a flavonoid extract extracted from Selaginella, and the volume fraction of hydrochloric acid in the above-mentioned first hydrochloric acid solution is 0.1% to 1%;

[0061] In the second step, after the first mixture is allowed to stand for a first period of time, the first mixture is separated to obtain a first precipitate and a first filtrate.

[0062] The third step is to mix the first filtrate with the second hydrochloric acid solution to obtain a second mixture.

[0063] Wherein, the volume fraction of hydrochloric acid in the second hydrochloric acid solution is 0.1% to 1%;

[0064] The fourth step is to separate the second mixture after the second mixture is allowed to stand for a second period of time to obtain a second precipitate and a second filtrate.

[0065] The fifth step is to dry and grind the second precipitate to obtain a purified Selaginella product.

[0066] Alternatively, as a non-limiting illustration, when the Selaginella extract is a Selaginella ethanol extract, the Selaginella extract can be obtained by the following steps:

[0067] In the first step, the Selaginella powder and the ethanol solution I are mixed and extracted and filtered to obtain an extract solution I.

[0068] In practice, the Selaginella powder can be obtained by grinding Selaginella. Specifically, the mesh size of the Selaginella powder is not limited as long as it does not affect the dissolution of the Selaginella powder in the ethanol solution I.

[0069] In practice, the volume fraction of ethanol in the ethanol solution I may be 60% to 95%. Specifically, the volume fraction of ethanol in the ethanol solution I may be any volume fraction of 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0070] In practice, the mixing volume ratio of Selaginella powder and ethanol solution I can be 1:5 to 10. Specifically, the mixing volume ratio of Selaginella powder and ethanol solution I can be any mixing volume ratio of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0071] In practice, in order to ensure the extraction rate, at least one re-extraction and filtration step can be performed after obtaining the extract I. The re-extraction and filtration step includes: mixing the drug residue filtered in the first step with the ethanol solution I for extraction and filtering, and combining the extract with the extract I in the first step as the extract I.

[0072] In practice, in the re-extraction and filtration steps, the mixing volume ratio of the drug residue to the ethanol solution I can be 1:5 to 10. Specifically, the mixing volume ratio of the drug residue to the ethanol solution I can be any mixing volume ratio of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0073] In practice, the mixed extraction of Selaginella powder and ethanol solution I can be carried out by any one of the extraction methods including but not limited to: heating reflux extraction, ultrasonic assisted extraction, microwave assisted extraction, immersion extraction, and osmosis extraction. Specifically, when the heating reflux extraction method is adopted, the temperature of the heating reflux extraction can be 50°C to 90°C. For example, the temperature of the heating reflux extraction can be any one of 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, and 90°C. Specifically, the duration of the heating reflux extraction can be 1 hour to 3 hours. For example, the duration of the heating reflux extraction can be any one of 1 hour, 1.5 hours, 2 hours, 2.5 hours, and 3 hours.

[0074] In practice, the extract I can also be concentrated to obtain concentrated solution I as the extract I.

[0075] In practice, the volume ratio of the concentrated solution I to the Selaginella powder can be 1:1 to 3. Specifically, the volume ratio of the concentrated solution I to the Selaginella powder can be any one of 1:1, 1:2, and 1:3.

[0076] In practice, the concentration temperature when the extract I is concentrated can be 60°C.

[0077] In practice, the concentration method for concentrating the extract I may include, but is not limited to, any one of the following concentration methods: atmospheric pressure evaporation concentration, reduced pressure evaporation concentration, vacuum evaporation concentration, and nitrogen blowing concentration.

[0078] In the second step, the extract I and the ethanol solution II are mixed and allowed to stand, and extract II is obtained after separation.

[0079] Among them, the extract II is the ethanol extract of Selaginella.

[0080] In practice, the volume fraction of ethanol in the ethanol solution II may be 90% to 98%. Specifically, the volume fraction of ethanol in the ethanol solution II may be any volume fraction of 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.

[0081] In practice, the volume ratio of the extract I to the ethanol solution II can be 1:5 to 10. Specifically, the volume ratio of the extract I to the ethanol solution II can be any one of 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0082] In practice, the temperature at which the extract I and the ethanol solution II are left to stand after being mixed is 4°C.

[0083] In practice, the extract I and the ethanol solution II are mixed and allowed to stand at a temperature of 48 hours.

[0084] In practice, when the extract I and the ethanol solution II are mixed and separated, any filtration method including but not limited to gravity filtration, pressure filtration, and vacuum filtration can be used.

[0085] In practice, the extract II may be concentrated to obtain a concentrated solution II as the extract II.

[0086] In practice, the volume mass ratio of the concentrated solution II to the Selaginella powder can be 1 to 3: 1. Specifically, the volume mass ratio of the concentrated solution II to the Selaginella powder can be any one of 1: 1, 1: 2, and 1: 3.

[0087] Experiment A

[0088] Among them, the samples participating in Experiment A include: Sample 1-1, Sample 1-2, Sample 1-3, Sample 1-4, Sample 1-5, Sample 2-1, Sample 2-2, Sample 2-3, Sample 2-4 and Sample 2-5.

[0089] The preparation method of sample 1-1 includes:

[0090] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume ethanol solution was added to obtain a mixed solution A.

[0091] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0092] The third step is to mix the medicinal residue with 80 mL of 80% by volume ethanol solution to obtain a mixed solution B.

[0093] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0094] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0095] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain concentrated solution A.

[0096] The concentration temperature is 60°C.

[0097] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0098] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0099] In the ninth step, 15 mL of a 0.5% volume fraction hydrochloric acid solution is added to the concentrated solution B, and the solution is allowed to stand at 4° C. for 30 minutes, and after the standing period is completed, the solution is filtered to obtain a filtrate B.

[0100] In the tenth step, the filtrate B is mixed with 20 mL of a hydrochloric acid solution having a volume fraction of 0.5%, and the mixture is allowed to stand at 4° C. for 12 hours, and after the standing period is completed, the mixture is filtered to obtain a precipitate.

[0101] In the eleventh step, the precipitate is dried in a vacuum drying oven, and then ground to obtain a purified Selaginella in a powdery state, which is used as sample 1-1.

[0102] The preparation method of sample 1-2 includes:

[0103] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume ethanol solution was added to obtain a mixed solution A.

[0104] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0105] The third step is to mix the medicinal residue with 80 mL of 80% by volume ethanol solution to obtain a mixed solution B.

[0106] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0107] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0108] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain concentrated solution A.

[0109] The concentration temperature is 60°C.

[0110] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0111] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0112] In the ninth step, 15 mL of a 0.5% volume fraction sodium hydroxide solution was added to the concentrated solution B, and the solution was allowed to stand at 4° C. for 30 minutes, and then filtered to obtain a filtrate B.

[0113] In the tenth step, the filtrate B is mixed with 20 mL of a hydrochloric acid solution having a volume fraction of 0.5%, and the mixture is allowed to stand at 4° C. for 12 hours, and after the standing period is completed, the mixture is filtered to obtain a precipitate.

[0114] In the eleventh step, the precipitate is dried in a vacuum drying oven, and then ground to obtain a purified Selaginella in a powder state, which is used as sample 1-2.

[0115] The preparation method of samples 1-3 includes:

[0116] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume ethanol solution was added to obtain a mixed solution A.

[0117] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0118] The third step is to mix the medicinal residue with 80 mL of 80% by volume ethanol solution to obtain a mixed solution B.

[0119] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0120] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0121] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain concentrated solution A.

[0122] The concentration temperature is 60°C.

[0123] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0124] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0125] In the ninth step, 15 mL of a 0.5% volume fraction hydrochloric acid solution is added to the concentrated solution B, and the solution is allowed to stand at 4° C. for 60 minutes, and after the standing period is completed, the solution is filtered to obtain a filtrate B.

[0126] In the tenth step, the filtrate B is mixed with 15 mL of a sodium hydroxide solution having a volume fraction of 0.5%, and the mixture is allowed to stand at 4° C. for 12 hours, and after the standing period is completed, the mixture is filtered to obtain a precipitate.

[0127] In the eleventh step, the precipitate is dried in a vacuum drying oven, and then ground to obtain a purified Selaginella in a powder state, which is used as sample 1-3.

[0128] The preparation method of samples 1-4 includes:

[0129] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume ethanol solution was added to obtain a mixed solution A.

[0130] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0131] The third step is to mix the medicinal residue with 80 mL of 80% by volume ethanol solution to obtain a mixed solution B.

[0132] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0133] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0134] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain concentrated solution A.

[0135] The concentration temperature is 60°C.

[0136] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0137] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0138] In the ninth step, 200 mL (600 mL in total) of ethyl acetate was added to the concentrated solution B in three times for extraction and separation, and the ethyl acetate layers were combined and concentrated to obtain a concentrated solution C.

[0139] The tenth step is to use a vacuum drying oven to dry the above concentrate, and grind it after drying to obtain a powdered purified Selaginella thunbergii product as sample 1-4.

[0140] The preparation method of samples 1-5 includes:

[0141] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume ethanol solution was added to obtain a mixed solution A.

[0142] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0143] The third step is to mix the medicinal residue with 80 mL of 80% by volume ethanol solution to obtain a mixed solution B.

[0144] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0145] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0146] In the sixth step, the extract C is purified by an ultrafiltration membrane, the permeate is collected, and the permeate is concentrated to 5 mL by a rotary evaporator to obtain a concentrated solution A.

[0147] The cut-off molecular weight of the ultrafiltration membrane is 1000D. The temperature during the purification process is 35°C. The pressure during the purification process is 0.5Mpa. The membrane permeability of the ultrafiltration membrane is 5L / h. The concentration temperature is 60°C.

[0148] In the eleventh step, the concentrated solution A is dried in a vacuum drying oven, and then ground to obtain a purified Selaginella in a powder state, which is used as sample 1-5.

[0149] The preparation method of sample 2-1 includes:

[0150] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume methanol solution was added to obtain a mixed solution A.

[0151] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0152] The third step is to mix the medicinal residue with 80 mL of 80% by volume methanol solution to obtain a mixed solution B.

[0153] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0154] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0155] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain a concentrated solution A.

[0156] The concentration temperature is 60°C.

[0157] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0158] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0159] In the ninth step, 15 mL of a 0.5% volume fraction hydrochloric acid solution is added to the concentrated solution B, and the solution is allowed to stand at 4° C. for 30 minutes, and after the standing period is completed, the solution is filtered to obtain a filtrate B.

[0160] In the tenth step, the filtrate B is mixed with 20 mL of a hydrochloric acid solution having a volume fraction of 0.5%, and the mixture is allowed to stand at 4° C. for 12 hours, and after the standing period is completed, the mixture is filtered to obtain a precipitate.

[0161] In the eleventh step, the precipitate is dried in a vacuum drying oven, and then ground to obtain a purified Selaginella in a powder state, which is used as sample 2-1.

[0162] The preparation method of sample 2-2 includes:

[0163] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume methanol solution was added to obtain a mixed solution A.

[0164] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0165] The third step is to mix the medicinal residue with 80 mL of 80% by volume methanol solution to obtain a mixed solution B.

[0166] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0167] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0168] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain a concentrated solution A.

[0169] The concentration temperature is 60°C.

[0170] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0171] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0172] In the ninth step, 15 mL of a 0.5% volume fraction sodium hydroxide solution was added to the concentrated solution B, and the solution was allowed to stand at 4° C. for 30 minutes, and then filtered to obtain a filtrate B.

[0173] In the tenth step, the filtrate B is mixed with 20 mL of a hydrochloric acid solution having a volume fraction of 0.5%, and the mixture is allowed to stand at 4° C. for 12 hours, and after the standing period is completed, the mixture is filtered to obtain a precipitate.

[0174] In the eleventh step, the precipitate is dried in a vacuum drying oven, and then ground to obtain a purified Selaginella in a powder state, which is used as sample 2-2.

[0175] The preparation method of sample 2-3 includes:

[0176] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume methanol solution was added to obtain a mixed solution A.

[0177] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0178] The third step is to mix the medicinal residue with 80 mL of 80% by volume methanol solution to obtain a mixed solution B.

[0179] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0180] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0181] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain concentrated solution A.

[0182] The concentration temperature is 60°C.

[0183] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0184] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0185] In the ninth step, 15 mL of a 0.5% volume fraction hydrochloric acid solution is added to the concentrated solution B, and the solution is allowed to stand at 4° C. for 30 minutes, and after the standing period is completed, the solution is filtered to obtain a filtrate B.

[0186] In the tenth step, the filtrate B is mixed with 20 mL of a sodium hydroxide solution having a volume fraction of 0.5%, and the mixture is allowed to stand at 4° C. for 12 hours, and after the standing period is completed, the mixture is filtered to obtain a precipitate.

[0187] In the eleventh step, the precipitate is dried in a vacuum drying oven, and then ground to obtain a purified Selaginella in a powder state as sample 2-3.

[0188] The preparation method of sample 2-4 includes:

[0189] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume methanol solution was added to obtain a mixed solution A.

[0190] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0191] The third step is to mix the medicinal residue with 80 mL of 80% by volume methanol solution to obtain a mixed solution B.

[0192] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0193] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0194] Step 6: Use a rotary evaporator to concentrate the extract C to 5 mL to obtain a concentrated solution A.

[0195] The concentration temperature is 60°C.

[0196] Step 7: Add 45 mL of 95% by volume ethanol solution to the concentrated solution A, and perform alcohol precipitation at 4° C. for 48 hours, and perform suction filtration after the alcohol precipitation to obtain filtrate A.

[0197] Step 8: Use a rotary evaporator to concentrate the filtrate A to 20 mL to obtain a concentrated solution B.

[0198] In the ninth step, 200 mL (600 mL in total) of ethyl acetate was added to the concentrated solution B in three times for extraction and separation, and the ethyl acetate layers were combined and concentrated to obtain a concentrated solution C.

[0199] The tenth step is to use a vacuum drying oven to dry the above concentrate, and grind it after drying to obtain a powdered purified Selaginella thunbergii product as sample 2-4.

[0200] The preparation method of samples 2-5 includes:

[0201] In the first step, 10 g of Selaginella powder was weighed and 100 mL of 80% by volume methanol solution was added to obtain a mixed solution A.

[0202] The second step is to heat the mixed solution A at 90°C for reflux extraction for 2 hours, and filter to obtain the extract A and the medicinal residue.

[0203] The third step is to mix the medicinal residue with 80 mL of 80% by volume ethanol solution to obtain a mixed solution B.

[0204] The fourth step is to heat the mixed solution B at 90°C for 2 hours to obtain an extract solution B.

[0205] The fifth step is to combine the extract A and the extract B to obtain the extract C.

[0206] In the sixth step, the extract C is purified by an ultrafiltration membrane, the permeate is collected, and the permeate is concentrated to 5 mL by a rotary evaporator to obtain a concentrated solution A.

[0207] The cut-off molecular weight of the ultrafiltration membrane is 1000D. The temperature during the purification process is 35°C. The pressure during the purification process is 0.5Mpa. The membrane permeability of the ultrafiltration membrane is 5L / h. The concentration temperature is 60°C.

[0208] In the eleventh step, the concentrated solution A is dried in a vacuum drying oven, and then ground to obtain a powdered purified Selaginella thunbergii product as sample 2-5.

[0209] Description of the assay method for Experiment A

[0210] Among them, the determination of Experiment A is to determine the content of flavonoids in Metasequoia glyptostroboides.

[0211] In practice, the preparation of the test samples includes sample 1-1, sample 1-2, sample 1-3, sample 1-4, sample 1-5, sample 2-1, sample 2-2, sample 2-3, sample 2-4, and sample 2-5. Specifically, 5 mg of the test sample is accurately weighed in a 50 mL volumetric flask, dissolved in methanol to a fixed volume, and filtered through a 0.22 μm filter membrane.

[0212] In practice, the control sample is prepared by accurately weighing 5 mg of the reference substance for the determination of the content of the flavonoids of Achyranthes bidentata in a 50 mL volumetric flask, and then dissolving it in methanol, making up the volume, shaking well and filtering it.

[0213] In practice, the content of aconitum biflavonoids in the test sample and the control sample was determined according to the external standard one-point method. 18(250L*4.6mm, 5μm); detection wavelength: 330nm; flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 10μ; mobile phase: phase A is 0.1% phosphoric acid aqueous solution; phase B is acetonitrile. Wherein, phase A: phase B = 65:35.

[0214] As an example, Figure 1 Shown are sample 1 - corresponding chromatograms of (purified) Selaginella extract and aconitum biflavonoids.

[0215] Calculation method of process transfer rate of flavonoids from Metasequoia gracile

[0216] The process transfer rate of aconitum flavonoids=100%×the mass of the purified Selaginella×the content of aconitum flavonoids in the purified Selaginella / (the mass of Selaginella powder×the content of aconitum flavonoids in the Selaginella powder).

[0217] Here, the yield of the purified Selaginella product = 100% x (mass of the purified Selaginella product / mass of Selaginella powder).

[0218] For each test product, three replicates were selected for repeated determination, and the RSD (Relative Standard Deviation) was calculated.

[0219] The experimental results corresponding to Experiment A are shown in Table 1 below:

[0220]

[0221] Analysis of the above Table 1 shows that, firstly, in terms of the content of sedum biflavonoids, sample 1-1 is increased by 97%, 440%, 63% and 124% compared with samples 1-2 to 1-5, respectively; sample 2-1 is increased by 124%, 610%, 45% and 164% compared with samples 2-2 to 2-5, respectively. This shows that the method for purifying the Selaginella extract disclosed in the present invention can effectively achieve the purpose of purifying the Selaginella extract, that is, to increase the content of sedum biflavonoids. Secondly, in terms of the process transfer rate of sedum biflavonoids, the process transfer rate of sedum biflavonoids of sample 1-1 is much higher than that of samples 1-2, sample 1-3 and sample 1-5. Although the process transfer rates of sedum biflavonoids of samples 1-4 and sample 1-1 are comparable, since the content of sedum biflavonoids of sample 1-4 is lower than that of sample 1-1, the advantage of sample 1-1 is still very obvious. Similarly, the process transfer rate of sugihara-flavonoids in sample 2-1 is much higher than that in sample 2-2, sample 2-3 and sample 2-5. Although the process transfer rate of sugihara-flavonoids in sample 2-4 is higher than that in sample 2-1, the content of sugihara-flavonoids in sample 2-4 is still significantly lower than that in sample 2-1.

[0222] In summary, it is proved that the purified Selaginella obtained by the method for purifying the Selaginella extract disclosed in the present invention has the obvious advantages of higher content of aconitum flavonoids and higher process transfer rate of aconitum flavonoids.

[0223] Experiment B

[0224] In Experiment B, the sample 1-1 (purified Selaginella) in Experiment A was used as the experimental sample. Therefore, the preparation method of Sample 1-1 is not described in Experiment B.

[0225] Among them, Experiment B includes:

[0226] (1) Toxicity detection experiment of HaCat cells;

[0227] (2) HaCat cell proliferation activity detection experiment after UV (UVB) irradiation;

[0228] (3) HaCat cell apoptosis rate detection experiment after UV (UVB) irradiation;

[0229] (4) Experiment on the detection of water content in mouse epidermal tissue after UV (UVB) irradiation;

[0230] (5) Experiment on the thickness of mouse epidermis after UV (UVB) irradiation;

[0231] (6) Detection of collagen fibers in mouse epidermis after UV (UVB) irradiation

[0232] (7) Detection of malondialdehyde content and superoxide dismutase activity in mouse epidermal tissue after UV (UVB) irradiation;

[0233] (8) Experiment on detection of matrix metalloproteinase-3 (MMP-3) in mouse epidermal tissue after UV (UVB) irradiation.

[0234] Next, the experimental method and experimental results of each of the above experiments are described.

[0235] (1) Toxicity detection experiment of HaCat cells

[0236] Experimental method: First, HaCat cells were inoculated in a 96-well cell culture plate at a density of 1×104 wells, and the cells were attached overnight. Secondly, 1-1 0.1mL of samples of different concentrations were added, wherein the concentrations were: 0, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 200μg / m. Then, 6 replicate wells were set for each concentration, and after incubation in a cell culture incubator for 24 hours, 20μL of MTT solution was added to each well. Further, after incubation in a cell culture incubator for 4 hours, the cell culture medium was aspirated and DMSO solution was added. In addition, the plate was shaken at a low speed for 10 minutes on a shaker to completely dissolve the crystals. Finally, after complete dissolution, the absorbance of each well in the 96-well plate was detected at a wavelength of 490nm by an ELISA reader.

[0237] Experimental results: Figure 2 The comparison chart of the effect of sample 1-1 on HaCat cell activity shows that when the concentration of Selaginella extract was between 3.125 and 50 μg / mL, there was no significant difference in cell survival rate between the Selaginella extract and the blank group, that is, there was no cytotoxicity to HaCat cells.

[0238] (2) Detection of proliferation activity of HaCat cells after UV (UVB) irradiation

[0239] Experimental method: First, HaCat cells were cultured at 1×10 4 / well were inoculated into 96-well cell culture plates, and the cells adhered overnight. Secondly, the cells were divided into a blank group (Control), a model group (UV), a low-dose Selaginella extract group (STE-L, 5ug / mL), and a high-dose Selaginella extract group (STE-H, 10ug / mL), wherein each group was added with a drug-containing culture medium and a blank culture medium, and each group was set up with 6 parallel wells. Then, the edge wells were filled with sterile PBS buffer to avoid the interference of edge effects on the test results. Further, after incubation in a cell culture incubator for 4 hours, the supernatant of each well was aspirated, and the cells in the 96-well plate were exposed to ultraviolet rays, and the supernatant of each well was added back after 12 minutes of irradiation. In addition, after incubation in a cell culture incubator for 24 hours, 20μL of MTT solution was added. Then, after incubation in a cell culture incubator for 4 hours, the cell culture medium was aspirated and DMSO solution was added. Then, the crystals were completely dissolved by low-speed shaking on a shaker for 10 minutes. Finally, after complete dissolution, the absorbance of each well in the 96-well plate was detected by an enzyme reader at a wavelength of 490 nm.

[0240] Experimental results: Figure 3 The results of the sample 1-1 on the proliferation activity of HaCat cells after UV (UVB) irradiation are compared. Figure 3The cell survival rate in the model group was compared with that in the blank group. The analysis found that, first, a large number of cells in the model group died after ultraviolet irradiation, and the cell proliferation activity was significantly lower than that in the blank group. The cell survival rates corresponding to the low-dose group and the high-dose group of Selaginella extract were analyzed, and different doses of Selaginella extract can effectively reduce cell death. It can be seen that Selaginella extract can significantly improve the phenomenon of the effect of cell proliferation activity after ultraviolet irradiation (P<0.05), and it is dose-dependent, indicating that Selaginella extract has a good protective effect on cell damage caused by UV (UVB) irradiation.

[0241] (3) HaCat cell apoptosis rate detection experiment after UV (UVB) irradiation

[0242] Experimental method: First, equal amounts of HaCat cells were inoculated into 60mm dishes, and the cells adhered to the wall overnight. Then, the cells were divided into four groups: blank group (Control), model group (UV), low-dose Selaginella extract group (STE-L, 5ug / mL), and high-dose Selaginella extract group (STE-H, 10ug / mL). Each group was added with drug-containing culture medium and blank culture medium, and 3 parallel groups were set up in each group. Secondly, after incubation in a cell culture incubator for 4 hours, the supernatant of each dish was aspirated, and the cells in the dish were exposed to ultraviolet rays. After irradiation for 12 minutes, the supernatant of each dish was added back. Further, after incubation in a cell culture incubator for 24 hours, the culture medium was aspirated, the cells were digested, centrifuged at 1000r / min for 5 minutes, and the supernatant was discarded, and PBS was used to wash twice. After centrifugation, the cells were collected in a 1.5mL EP tube. Finally, the cells were treated according to the cell apoptosis reagent detection kit, and the cell apoptosis rate was detected by flow cytometry after treatment.

[0243] Experimental results: see Figure 4 The results of the sample 1-1 on the apoptosis rate of HaCat cells after UV (UVB) irradiation are shown in the comparison chart. The analysis found that, first, the apoptosis rate of HaCat cells after UV (UVB) irradiation significantly increased when the blank group and the model group were compared. Secondly, when the model group, the low-dose Selaginella extract group and the high-dose Selaginella extract group were compared, the addition of Selaginella extract can effectively alleviate the apoptosis caused by UV (UVB) irradiation.

[0244] (4) Experiment on the detection of water content in mouse epidermal tissue after UV (UVB) irradiation;

[0245] Experimental method: First, ICR mice were allowed to drink water and eat freely, and the bedding was changed regularly. Among them, ICR mice were raised in a quiet environment, with a temperature of 23-25°C, a relative humidity of 40%-60%, and a 12-hour day and night alternation. Secondly, after 3 days of adaptive feeding, ICR mice were divided into 4 groups using a random number method, a blank group (Control), a model group (UV), a low-dose group of Selaginella extract (STE-L, 5ug / mL), and a high-dose group of Selaginella extract (STE-H, 10ug / mL). Before the experiment, mice in each group were anesthetized with isoflurane gas to facilitate shaving. Specifically, the hair on the back of the mouse was shaved with a shaver one day before irradiation, and the hair on the back was removed with a depilatory cream, and the depilatory cream remaining on the back was washed off with warm water, and wiped dry with a cotton ball, so that the exposed area of ​​the back was about 3cm×3cm. (Among them, mice with skin abnormalities such as erosion and scars on the back were eliminated and did not participate in the experiment.) In addition, the exposed skin of the backs of mice in other groups except the blank group was placed 2 cm below the UV (UVB) light source, exposing the exposed skin of the backs to a power of 2 mW / cm 2 The skin was exposed to ultraviolet light for six weeks. In the first three weeks, the exposure time was 50 seconds each time and the radiation dose was 100mJ / cm 2 ; Each irradiation time for the next three weeks was 100 seconds, and the radiation dose was 200mJ / cm 2 Each irradiation area was 2 cm × 2 cm. All groups except the blank group were irradiated once every other day, with a total radiation dose of 3.1 J / cm 2 . 30 minutes after each irradiation, the drug was applied to the exposed part of the mouse. After application, it was given enough space to move alone for 1 hour to allow the drug to fully penetrate and absorb, once a day for a total of six weeks. 24 hours after the last irradiation and administration, the mice were depilated, anesthetized and killed by cervical vertebrae removal, and the skin tissue of the depilated area on the back was collected, the connective tissue and subcutaneous fat were peeled off, and fixed with 4% paraformaldehyde solution or stored in an ice box, and some were stored at -80°C. Finally, the moisture content of mouse skin was measured by direct drying method. About 0.1g of mouse epidermal tissue was quickly cut off, accurately weighed, placed in an oven at 105°C to dry to constant weight, and accurately weighed after being placed at room temperature, and the skin moisture content was calculated (wherein, skin moisture content (%) = (wet weight-dry weight) / wet weight*100%).

[0246] Experimental results: see Figure 5 The results of the sample 1-1 on the effect of UV (UVB) irradiation on the epidermal water content of mice are shown in the comparative diagram, wherein the analysis found that, first, the epidermal water content of mice after UV irradiation was significantly lower than that of the blank group. In addition, the low-dose group of Selaginella extract and the high-dose group of Selaginella extract were analyzed, and the epidermal water content of mice increased significantly compared with the model group, indicating that the Selaginella extract has a good protective effect on the loss of epidermal water flow in UV-irradiated mice.

[0247] (5) Experiment on the thickness of mouse epidermis after UV (UVB) irradiation;

[0248] Experimental method: First, take the mouse epidermal tissue obtained in the experiment of detecting the moisture content of mouse epidermal tissue after (4) UV (UVB) irradiation and put it into 4% paraformaldehyde. Then, the mouse epidermal tissue is made into slices after dehydration, transparency and embedding. Secondly, after dewaxing, the slices are stained with H&E. Finally, after staining, the thickness of the mouse epidermis is measured by ImageJ software to observe the thickness change of the mouse epidermal tissue.

[0249] Experimental results: see Figure 6 The results of the sample 1-1 on the effect of UV (UVB) irradiation on the epidermal thickness of mice are shown in the figure, and see Figure 7 The comparison of mouse epidermal thickness in the stained sections shown in the figure shows that, first, the skin structure of the mice in the blank group is intact, the epidermis is thin, and the dermis is tightly connected to the epidermis. Secondly, the epidermis of the skin of the mice in the model group was significantly thickened, and local epidermal keratinization was incomplete. Next, the low-dose group and the high-dose group of Selaginella extract were analyzed, and the epidermal thickness of the mice was significantly reduced compared with the model group, that is, different doses of Selaginella extract can effectively improve the increase in epidermal thickness caused by UV (UVB) radiation (P<0.01), indicating that Selaginella extract has a good restorative effect on the increase in mouse epidermal thickness caused by UV (UVB) irradiation.

[0250] (6) Detection of collagen fibers in mouse epidermis after UV (UVB) irradiation

[0251] Experimental method: First, the mouse epidermal tissue obtained in the experiment of detecting the water content of mouse epidermal tissue after (4) UV (UVB) irradiation was placed in 4% paraformaldehyde. Then, the mouse epidermal tissue was made into slices after dehydration, transparency, and embedding. Secondly, the slices were Masson stained after dewaxing, and the changes in collagen fibers in the mouse epidermal tissue were observed.

[0252] Experimental results: see Figure 8 The sample 1-1 shown is a comparison chart of the changes in collagen fibers in the mouse epidermal tissue after UV (UVB) irradiation. The analysis found that, first, the skin structure of the mice in the blank group was intact, and the wavy collagen fiber bundles were arranged in an orderly and evenly distributed manner. Secondly, the number of collagen fiber bundles in the epidermal tissue of the mice in the model group was significantly reduced and sparsely distributed. Then, the number of collagen fiber bundles in the epidermal tissue of mice with different doses of Selaginella extract (Selaginella extract low-dose group and Selaginella extract high-dose group) was significantly restored, indicating that Selaginella extract has a good restorative effect on the loss of mouse epidermal collagen fibers caused by UV (UVB) irradiation.

[0253] (7) Detection of malondialdehyde content and superoxide dismutase activity in mouse epidermal tissue after UV (UVB) irradiation;

[0254] Experimental method: First, the mouse epidermal tissue obtained in the experiment of detecting the water content of mouse epidermal tissue after (4) UV (UVB) irradiation was taken, and the protein was extracted by chemical method. After quantification, the malondialdehyde content and superoxide dismutase activity of the mouse epidermal tissue were determined according to the requirements of the lipid oxidation detection kit and the total SOD activity detection kit.

[0255] Experimental results: see Fig. 9 The comparison chart of the effect of sample 1-1 on the malondialdehyde content in mouse epidermal tissue after UV (UVB) irradiation is shown, and Fig.10 The results of the sample 1-1 on the superoxide dismutase activity of mouse epidermal tissue after UV (UVB) irradiation are shown in the comparative diagram. The analysis shows that after ultraviolet irradiation, the malondialdehyde content of mouse epidermal tissue increases significantly, and the superoxide dismutase activity decreases significantly. Different doses of Selaginella extract (low-dose group of Selaginella extract and high-dose group of Selaginella extract) significantly reduce the malondialdehyde content of mouse epidermal tissue and increase the superoxide dismutase activity compared with the model group. This shows that sample 1-1 can effectively improve the oxidative stress reaction caused by ultraviolet rays, that is, it has a good antioxidant effect.

[0256] (8) Detection of matrix metalloproteinase-3 (MMP-3) in mouse epidermal tissue after UV (UVB) irradiation

[0257] Experimental method: First, the mouse epidermal tissue obtained in the experiment of detecting the water content of mouse epidermal tissue after (4) UV (UVB) irradiation was placed in 4% paraformaldehyde. Then, the mouse epidermal tissue was made into slices after dehydration, transparency, and embedding. Secondly, after dewaxing, antigen retrieval buffer was used to repair the antigen, and the slices were incubated with MMP-3 antibody. After washing with PBS, the slices were incubated with goat anti-rabbit secondary antibody solution and then stained. After the slices were sealed, they were observed under a microscope and photographed.

[0258] Experimental results: see Fig.11 The results of the sample 1-1 on the content of matrix metalloproteinase-3 in mouse epidermal tissue after UV (UVB) irradiation are shown in the comparative diagram. The analysis found that, first, the expression of MMP-3 protein in the tissue after ultraviolet irradiation was significantly increased compared with the blank group. Secondly, different doses of Selaginella extract (low-dose group of Selaginella extract and high-dose group of Selaginella extract) can reduce the expression of MMP-3 protein in mouse epidermal tissue in a dose-dependent manner. This shows that sample 1-1 can alleviate skin damage caused by ultraviolet irradiation by reducing the degradation of collagen and elastin.

[0259] Combined with Experiment A and Experiment B, firstly, it is proved that the Selaginella extract purification method disclosed in the present invention is stable and reliable. At the same time, it is proved that the Selaginella purified product prepared by the Selaginella extract purification method disclosed in the present invention can protect against cell loss and apoptosis caused by UVB irradiation; prevent the thickening of epidermal tissue, loss of collagen fibers and water loss of epidermal tissue caused by UVB irradiation; can effectively improve the oxidative stress response of epidermal tissue caused by UV irradiation; and can also alleviate the degradation of collagenase and elastase to alleviate the problem of skin damage caused by ultraviolet irradiation.

[0260] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

Claims

1. A method for purifying a Selaginella extract, comprising: Mixing the Selaginella extract with a first hydrochloric acid solution to obtain a first mixture, wherein the Selaginella extract is a flavonoid extract extracted from Selaginella, and the volume fraction of hydrochloric acid in the first hydrochloric acid solution is 0.1% to 1%; After the first mixture is allowed to stand for a first period of time, the first mixture is subjected to separation treatment to obtain a first precipitate and a first filtrate; Mixing the first filtrate with a second hydrochloric acid solution to obtain a second mixture, wherein the volume fraction of hydrochloric acid in the second hydrochloric acid solution is 0.1% to 1%; After the second mixture is allowed to stand for a second period of time, the second mixture is subjected to separation treatment to obtain a second precipitate and a second filtrate; The second precipitate is dried and ground to obtain a purified Selaginella thunbergii product.

2. The method according to claim 1, wherein: The Selaginella extract is a Selaginella ethanol extract, a Selaginella methanol extract, or a mixture of the Selaginella ethanol extract and the Selaginella methanol extract.

3. The method according to claim 1, wherein: The volume ratio of the Selaginella extract to the first hydrochloric acid solution in the first mixed solution is 1:1-3.

4. The method according to claim 1, wherein: The ambient temperature of the first mixture when it is at rest is 2°C to 10°C.

5. The method according to claim 1, wherein: The volume ratio of the first filtrate to the second hydrochloric acid solution in the second mixed solution is 1:1-3.

6. The method according to claim 1, wherein: The ambient temperature of the second mixed solution when it is at rest is 2°C to 10°C.

7. A skin care composition, wherein: The skin care composition comprises the purified Selaginella according to any one of claims 1 to 6.

8. Use of a purified product, comprising use of the purified Selaginella according to any one of claims 1 to 6 in the preparation of a product for inhibiting photodamage of skin cells.

9. The use of the purified product according to claim 8, wherein The product for inhibiting photodamage of skin cells has any of the following effects: Protect against cell damage and apoptosis caused by UVB irradiation; Prevent the thickening of epidermal tissue, loss of collagen fibers, and loss of water in epidermal tissue caused by UVB irradiation; Improve the oxidative stress response of epidermal tissue caused by UVB irradiation; Reduces the production of matrix metalloproteinases to alleviate the degradation of collagen and elastin in the skin.

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