Preparation method of Selaginella tamariscina extract and skin care products
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-15
- Publication Date
- 2026-08-14
AI Technical Summary
[0004]穗花杉双黄酮的抗氧化活性较差，因此，富集黄酮类化合物或穗花杉双黄酮的卷柏提取物在抗氧化方面差强人意
[0019]本公开的上述各个实施例具有如下有益效果：通过本公开的一些实施例卷柏提取物制备方法，提高了卷柏提取物的抗氧化活性。具体的，首先，针对卷柏的醇提取物，通过分别测定乙酸乙酯萃取部位、正丁醇萃取部位、水部位的抗氧化活性，发现正丁醇部位的抗氧化活性最高。进一步的，本公开尝试采用体积分数为30％的乙醇水溶液进行洗脱以进一步提高抗氧化活性，进而得到洗脱液I(样品IV)的抗氧化活性最高。然而，在实际生产过程中，乙酸乙酯和正丁醇存在应用限制较大、对生产环境要求较高、回收难度较大等问题，具体的，如乙酸乙酯、正丁醇均易燃，需要匹配特殊的安全防护措施；提取后难以完全去除乙酸乙酯、正丁醇，进而导致卷柏提取物中残留有害物质；乙酸乙酯、正丁醇在生物降解和非生物降解过程中，需要一定降解周期，处理不当时可能会对环境产生不当影响，需要妥善处理废物；乙酸乙酯、正丁醇作为溶剂成本较高，可能增加生产成本。基于此，本公开尝试在不使用乙酸乙酯和正丁醇萃取的情况下制备高抗氧化活性的卷柏提取物。具体的，本公开尝试采用体积分数为30％的乙醇水溶液直接进行吸附-洗脱，得到洗脱液III(样品VI)的抗氧化活性仅为洗脱液I的50％左右。通过观察洗脱液I和洗脱液III的HPLC图谱发现，洗脱液III的高极性部位的含量偏高于洗脱液I，因此，本公开采用通过大孔树脂对上述卷柏粗提物进行分离，得到分离后卷柏提取物，其中，包括：通过第一洗脱液，对上述悬浮于水中的上述卷柏粗提物进行一次洗脱，得到第一洗脱后卷柏粗提物，其中，上述第一洗脱液是体积分数为5％～15％的乙醇水溶液；通过第二洗脱液，对上述第一洗脱后卷柏粗提物进行二次洗脱，得到分离后卷柏提取物，其中，上述第二洗脱液是体积分数为25％～35％的乙醇水溶液的方式制备洗脱液IV(卷柏提取物)，测定发现，洗脱液IV的抗氧化活性与洗脱液I相当，由此实现了不使用乙酸乙酯和正丁醇萃取的情况下，制备高抗氧化活性的卷柏提取物的目的。
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Figure CN118845556B_ABST
Abstract
Description
Technical Field
[0001] The embodiments disclosed herein relate to the field of cosmetic technology, specifically to a method for preparing Selaginella tamariscina extract and skin care products. Background Technology
[0002] Selaginella tamariscina is a terrestrial or lithophytic, cushion-forming resurrection herb. It possesses a complex chemical structure and strong biological activity. Among its components, flavonoids, phenolic compounds, and glycosides exhibit good pharmacological activity, making it widely used in the treatment of diseases such as tumors, hyperglycemia, and thrombocytopenic purpura. Specifically, flavonoids are an important chemical component of Selaginella tamariscina, particularly paclitaxel flavonoids, which constitute a high proportion of the flavonoids. Pyracalitaxel flavonoids have positive effects in antiviral, antitumor, and anti-inflammatory aspects. Therefore, a major research direction in the extraction process of Selaginella tamariscina is how to improve the extraction rate of paclitaxel flavonoids.
[0003] However, the following technical problems exist in the preparation of Selaginella extracts enriched with flavonoids, especially those enriched with Taxodium spp. flavonoids:
[0004] Taxodium biflavonoids have poor antioxidant activity; therefore, extracts of Selaginella tamariscina enriched with flavonoids or taxodium biflavonoids are unsatisfactory in terms of antioxidant properties.
[0005] The information disclosed in this background section is only intended to enhance the understanding of the background of the inventive concept, and therefore may contain information that does not form prior art known to those skilled in the art. Summary of the Invention
[0006] The summary portion of this disclosure is intended to provide a brief overview of the concepts, which will be described in detail in the detailed description portion. This summary portion is not intended to identify key or essential features of the claimed technical solutions, nor is it intended to limit the scope of the claimed technical solutions.
[0007] Some embodiments of this disclosure provide compositions comprising retinol, methods for their preparation, and skin care products to address one or more of the technical problems mentioned in the background section above.
[0008] In a first aspect, some embodiments of this disclosure provide a method for preparing Selaginella tamariscina extract, comprising: soaking and refluxing pulverized Selaginella tamariscina medicinal material in a soaking solution to obtain Selaginella tamariscina extract; concentrating the above Selaginella tamariscina extract under reduced pressure to obtain crude Selaginella tamariscina extract; separating the above crude Selaginella tamariscina extract using a macroporous resin to obtain separated Selaginella tamariscina extract, wherein the method comprises: eluting the above crude Selaginella tamariscina extract suspended in water once with a first eluent to obtain a first eluted crude Selaginella tamariscina extract, wherein the first eluent is an ethanol aqueous solution with a volume fraction of 5% to 15%; eluting the above crude Selaginella tamariscina extract a second time with a second eluent to obtain separated Selaginella tamariscina extract, wherein the second eluent is an ethanol aqueous solution with a volume fraction of 25% to 35%; and drying the separated Selaginella tamariscina extract to obtain Selaginella tamariscina extract.
[0009] Optionally, the above-mentioned method of soaking and refluxing the pulverized Selaginella tamariscina medicinal material in the soaking solution to obtain Selaginella tamariscina extract includes: soaking the pulverized Selaginella tamariscina medicinal material in the soaking solution and refluxing for 1 to 3 hours, and repeating the process a preset number of times to obtain the above-mentioned Selaginella tamariscina extract, wherein the preset number of times is ≥1.
[0010] Optionally, the above 1 ≤ preset number of times ≤ 3.
[0011] Optionally, the above-mentioned Selaginella extract is concentrated under reduced pressure to obtain a crude Selaginella extract, including: rotary evaporation of the above-mentioned Selaginella extract to obtain the above-mentioned crude Selaginella extract.
[0012] Optionally, the weight ratio of the pulverized Selaginella tamariscina to the soaking solution is 1:10 to 30.
[0013] Optionally, the soaking solution is an aqueous ethanol solution or water. When the soaking solution is an aqueous ethanol solution, the volume fraction of the soaking solution is 70% to 95%. When the soaking solution is water, the soaking solution is one or more of pure water, deionized water, and ultrapure water.
[0014] Optionally, when the soaking solution is pure water, the soaking solution may be one or more of distilled water and reverse osmosis water.
[0015] Optionally, the ratio of the volume of the first eluent to the volume of the macroporous resin column is ≥5:1, and the ratio of the volume of the second eluent to the volume of the macroporous resin column is ≥5:1.
[0016] Optionally, drying the separated Selaginella extract to obtain the Selaginella extract includes drying the separated Selaginella extract by one or more of the following drying methods: evaporative drying, hot air drying, vacuum drying, freeze drying, spray drying, and microwave drying.
[0017] Secondly, some embodiments of this disclosure provide a skin care product comprising Selaginella extract as described in any of the first aspects.
[0018] Optionally, the aforementioned skin care products include one or more of the following: serums, lotions, emulsions, creams, ointments, and masks.
[0019] The above-described embodiments of this disclosure have the following beneficial effects: the preparation methods of Selaginella tamariscina extract according to some embodiments of this disclosure improve the antioxidant activity of Selaginella tamariscina extract. Specifically, firstly, for the alcoholic extract of Selaginella tamariscina, the antioxidant activity of the ethyl acetate extract, n-butanol extract, and water extract were measured respectively, and it was found that the n-butanol extract had the highest antioxidant activity. Furthermore, this disclosure attempts to use a 30% (v / v) ethanol aqueous solution for elution to further improve the antioxidant activity, thus obtaining eluent I (sample IV) with the highest antioxidant activity. However, in actual production, ethyl acetate and n-butanol have problems such as significant application limitations, high requirements for the production environment, and difficulty in recycling. Specifically, ethyl acetate and n-butanol are both flammable, requiring special safety protection measures; it is difficult to completely remove ethyl acetate and n-butanol after extraction, resulting in residual harmful substances in the Selaginella tamariscina extract; ethyl acetate and n-butanol require a certain degradation period in the biodegradation and non-biodegradation processes, and improper handling may have adverse environmental impacts, requiring proper waste disposal; ethyl acetate and n-butanol are expensive solvents, which may increase production costs. Based on this, this disclosure attempts to prepare a Selaginella tamariscina extract with high antioxidant activity without using ethyl acetate and n-butanol extraction. Specifically, this disclosure attempts to use a 30% (v / v) aqueous ethanol solution for direct adsorption-elution, and the antioxidant activity of eluent III (sample VI) is only about 50% of that of eluent I. By observing the HPLC chromatograms of eluent I and eluent III, it was found that the content of the highly polar fraction in eluent III was higher than that in eluent I. Therefore, this disclosure uses macroporous resin to separate the above-mentioned crude Selaginella tamariscina extract to obtain a separated Selaginella tamariscina extract. The process includes: eluting the above-mentioned crude Selaginella tamariscina extract suspended in water once with a first eluent to obtain a first eluted crude Selaginella tamariscina extract, wherein the first eluent is an aqueous ethanol solution with a volume fraction of 5% to 15%; and eluting the first eluted crude Selaginella tamariscina extract a second time with a second eluent to obtain a separated Selaginella tamariscina extract, wherein the second eluent is an aqueous ethanol solution with a volume fraction of 25% to 35% to prepare eluent IV (Selaginella tamariscina extract). It was found that the antioxidant activity of eluent IV was comparable to that of eluent I, thus achieving the goal of preparing a Selaginella tamariscina extract with high antioxidant activity without using ethyl acetate and n-butanol extraction. Attached Figure Description
[0020] The above and other features, advantages, and aspects of the embodiments of this disclosure will become more apparent from the accompanying drawings and the following detailed description. Throughout the drawings, the same or similar reference numerals denote the same or similar elements. It should be understood that the drawings are schematic and the elements are not necessarily drawn to scale.
[0021] Figure 1 This is a comparison chart of the ABTS free radical scavenging rates of samples I, II, III, IV, V, VI, and VII in some embodiments of this disclosure;
[0022] Figure 2 This is a comparison chart of the ABTS free radical scavenging rates of samples VIII, IX, X, XI and XII in some embodiments of this disclosure;
[0023] Figure 3 This is a comparison chart of the ABTS free radical scavenging rates of samples XIII, XIV, XV, XVI, and XVII in some embodiments of this disclosure;
[0024] Figure 4 These are HPLC chromatograms of sample IV from some of the disclosed embodiments;
[0025] Figure 5 These are HPLC chromatograms of sample V from some of the disclosed embodiments;
[0026] Figure 6 These are the HPLC chromatograms of sample VI from some of the disclosed embodiments;
[0027] Figure 7 These are the HPLC chromatograms of sample VIII from some of the disclosed embodiments;
[0028] Figure 8 These are the HPLC chromatograms of sample XIII from some of the disclosed embodiments;
[0029] Figure 9 These are HPLC chromatograms of sample VII from some of the disclosed embodiments. Detailed Implementation
[0030] Embodiments of this disclosure will now be described in more detail with reference to the accompanying drawings. While some embodiments of this disclosure are shown in the drawings, it should be understood that this disclosure can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of this disclosure. It should be understood that the accompanying drawings and embodiments of this disclosure are for illustrative purposes only and are not intended to limit the scope of protection of this disclosure.
[0031] Unless otherwise stated, all technical and scientific terms used herein have the meanings as commonly understood by one of ordinary skill in the art. Generally, the terminology used herein is well-known and conventionally used in the art. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or a range defined by a series of upper and lower preferred values, it should be understood as specifically disclosing all ranges formed by any pairing of any upper limit or upper preferred value with any lower limit or right-hand suspending of the lower limit. Whether or not a range is disclosed individually, when a numerical range is described herein, unless otherwise stated, the range should include its endpoints and all integers and fractions within that range.
[0032] It should also be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings. Unless otherwise specified, the embodiments and features described in this disclosure can be combined with each other.
[0033] It should be noted that the terms "a" and "a plurality of" used in this disclosure are illustrative rather than restrictive, and those skilled in the art should understand that, unless otherwise expressly indicated in the context, they should be understood as "one or more".
[0034] The main terms used in this disclosure are defined as follows:
[0035] The term "n-butanol" refers to a colorless liquid with an alcoholic odor and the molecular formula C4H. 10 O.
[0036] The term "ethyl acetate" refers to a colorless, flammable, and volatile liquid with a distinctive odor. It is slightly soluble in water and readily soluble in organic solvents, with the molecular formula C4H8O2.
[0037] The term "macroporous resin," also known as macroporous adsorption resin, refers to a class of polymeric adsorption resins that do not contain exchange groups and have a macroporous structure.
[0038] The term "pure water" refers to water that is free of impurities and has extremely high chemical purity.
[0039] The term "deionized water" refers to pure water after removing impurities in ionic form.
[0040] The term "ultrapure water" refers to water that is close to high purity, meaning it contains almost no electrolytes other than hydrogen ions and hydroxide ions.
[0041] The term "distilled water" is a type of pure water that is obtained through distillation and condensation. Depending on the number of distillations, water that has been distilled twice is called double-distilled water, and water that has been distilled three times is called triple-distilled water.
[0042] The term "reverse osmosis water" refers to water obtained by filtration using a membrane separation technology that employs a semi-permeable membrane and pressure as the driving force.
[0043] The term "methanol," also known as hydroxymethane, is a highly volatile, colorless, flammable, toxic organic compound with an odor similar to ethanol.
[0044] The term "Carbomer U20" is a hydrophobically modified, cross-linked acrylate copolymer.
[0045] The term "panthenol" is the reduced form of pantothenic acid (vitamin B5), which belongs to the provitamin family.
[0046] The term "acetophenone," also known as p-hydroxyacetophenone, possesses antioxidant, anti-irritant, emulsion-stabilizing, and broad-spectrum preservative properties.
[0047] The term "Carbopol 940" is a white, powdery, cross-linked polyacrylic acid polymer with extremely short rheological properties, high viscosity, moderate clarity, low ionic resistance, and high shear resistance. The method for preparing the Selaginella tamariscina extract disclosed herein may include the following steps:
[0048] The first step is to soak and reflux the pulverized Selaginella tamariscina medicinal material in a soaking solution to obtain Selaginella tamariscina extract.
[0049] The second step is to concentrate the above-mentioned Selaginella extract under reduced pressure to obtain concentrated crude Selaginella extract.
[0050] The fourth step involves separating the crude extract of Selaginella tamariscina using macroporous resin to obtain the separated Selaginella tamariscina extract.
[0051] Optionally, the macroporous resin used is AB-8 type macroporous resin.
[0052] The crude extract of Selaginella tamariscina was separated using macroporous resin to obtain a separated Selaginella tamariscina extract, comprising:
[0053] The first sub-step involves eluting the crude Selaginella extract suspended in water with a first eluent to obtain a first eluted Selaginella extract, wherein the first eluent is an aqueous ethanol solution with a volume fraction of 5% to 15%.
[0054] The second sub-step involves eluting the crude Selaginella extract obtained after the first elution with a second eluent to obtain a separated Selaginella extract. The second eluent is an aqueous ethanol solution with a volume fraction of 25% to 35%.
[0055] Step 5: Dry the separated Selaginella extract to obtain Selaginella extract.
[0056] Optionally, the separated Selaginella extract is dried by rotary evaporation to obtain Selaginella extract.
[0057] Optionally, the above-mentioned method of soaking and refluxing the pulverized Selaginella tamariscina medicinal material in the soaking solution to obtain Selaginella tamariscina extract includes: soaking the pulverized Selaginella tamariscina medicinal material in the soaking solution and refluxing for 1 to 3 hours, and repeating the process a preset number of times to obtain the above-mentioned Selaginella tamariscina extract, wherein the preset number of times is ≥1.
[0058] Optionally, 1 ≤ the above-preset number of times ≤ 3.
[0059] Optionally, the above-mentioned concentration of the Selaginella extract under reduced pressure to obtain a crude Selaginella extract includes: rotary evaporation of the Selaginella extract to obtain the crude Selaginella extract. In practice, the crude Selaginella extract can be obtained by rotary evaporation using a rotary evaporator.
[0060] Optionally, the weight ratio of the pulverized Selaginella tamariscina to the soaking solution is 1:10 to 30.
[0061] Optionally, the soaking solution is an aqueous ethanol solution or water. When the soaking solution is an aqueous ethanol solution, the volume fraction of the soaking solution is 70% to 95%. When the soaking solution is water, the soaking solution is one or more of pure water, deionized water, and ultrapure water.
[0062] Optionally, when the soaking solution is pure water, the soaking solution can be one or more of distilled water and reverse osmosis water.
[0063] Optionally, the ratio of the volume of the first eluent to the volume of the macroporous resin column is ≥5:1, and the ratio of the volume of the second eluent to the volume of the macroporous resin column is ≥5:1.
[0064] Optionally, drying the separated Selaginella extract as described above to obtain the Selaginella extract includes: drying the separated Selaginella extract using one or more of the following drying methods: evaporative drying, hot air drying, vacuum drying, freeze drying, spray drying, and microwave drying. Evaporative drying can refer to a method of vaporizing and escaping moisture (generally water or other volatile liquids) by heating. Hot air drying can refer to a method of accelerating airflow with hot air to promote drying. Vacuum drying refers to a drying method of reaching the boiling point by appropriate heating or reaching the melting point by appropriate cooling under negative pressure. Freeze drying can refer to a method of first freezing and then converting ice into vapor under vacuum to achieve the drying purpose. Spray drying can refer to a method of achieving water vaporization by fully contacting hot air through a spray. Microwave drying can refer to a method of converting microwave energy into thermal energy of a medium to achieve water vaporization.
[0065] The Selaginella extract disclosed herein can be used to prepare skin care products.
[0066] Optionally, the aforementioned skin care products include one or more of the following: serums, lotions, emulsions, creams, ointments, and masks.
[0067] As an example, when the skin care product is a face mask, the skin care product may use a substrate commonly used in the art and the Selaginella extract of this disclosure applied or impregnated onto the substrate. The substrate includes, but is not limited to, silk mask sheets, cotton mask sheets, polyester mask sheets, or blended mask sheets. The choice of substrate can be made by those skilled in the art according to actual needs. The form of the face mask may include, but is not limited to, gel-type masks, cream-type masks, and essential oil masks.
[0068] The materials and instruments involved are described, including:
[0069] (1) Selaginella: The place of origin is Linyi, Shandong Province. The source is the dried whole herb of Selaginella or Selaginella materia var. ...
[0070] (2) Macroporous resin: Model AB-8, selected from Xi'an Lanxiao Technology New Material Co., Ltd., batch number 20220318G2034.
[0071] (3) 95% volume fraction of ethanol: selected from Shandong Xiekang Medical Technology Co., Ltd.
[0072] (4) Rotary evaporator: Selected from Shanghai Ailang Instrument Co., Ltd., model N-1300.
[0073] (5) Cooling circulator, selected from Shanghai Airong Instrument Co., Ltd., model CA-1330.
[0074] (6) High-performance liquid chromatograph, selected from Waters TM .
[0075] This disclosure will now be described in detail with reference to the accompanying drawings and embodiments.
[0076] Comparative Example 1:
[0077] Comparative Example 1 was used to prepare Sample I, Sample II, and Sample III.
[0078] Preparation method:
[0079] The first step is to soak 100 grams of Selaginella tamariscina in an ethanol aqueous solution (90% by volume) at a weight ratio of 1:20, and then reflux for 2 hours. This process is repeated 3 times to obtain the Selaginella tamariscina extract.
[0080] In practice, a 90% ethanol aqueous solution can be obtained by diluting it with 95% ethanol.
[0081] The second step is to concentrate the above-mentioned Selaginella extract under reduced pressure to obtain concentrated Selaginella extract.
[0082] The third step is to dilute the concentrated Selaginella extract with an appropriate amount of water to obtain crude Selaginella extract.
[0083] The fourth step involves sequentially extracting the crude extract of Selaginella tamariscina using ethyl acetate and n-butanol to obtain the ethyl acetate fraction (named Sample I), the n-butanol fraction (named Sample II), and the aqueous fraction (named Sample III).
[0084] Comparative Example 2
[0085] Comparative Example 2 was used to prepare Sample IV and Sample V.
[0086] Preparation method:
[0087] The first step was to elute the n-butanol fraction of Selaginella tamariscina (sample II) with a 30% (v / v) aqueous ethanol solution to obtain an eluent containing 30% ethanol (named sample IV).
[0088] In practice, a 30% ethanol aqueous solution can be obtained by diluting it with 95% ethanol.
[0089] The second step involves eluting the n-butanol fraction of Selaginella tamariscina (sample II) with a 50% (v / v) ethanol aqueous solution to obtain an eluent (named sample V) containing a 50% (v / v) ethanol aqueous solution.
[0090] In practice, a 50% ethanol aqueous solution can be obtained by diluting it with 95% ethanol.
[0091] Comparative Example 3
[0092] Comparative Example 3 was used to prepare Sample VI.
[0093] Preparation method:
[0094] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0095] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 between the pulverized Selaginella tamariscina and an aqueous ethanol solution (90% by volume). This extraction is repeated 3 times to obtain the Selaginella tamariscina extract.
[0096] The third step is to concentrate the Selaginella extract under reduced pressure to obtain a crude Selaginella extract.
[0097] The fourth step involves column chromatography separation of the crude extract of Selaginella tamariscina using AB-8 macroporous resin, including eluting the crude extract with a 30% (v / v) ethanol aqueous solution to obtain an eluent containing a 30% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named Sample VI).
[0098] Comparative Example 4
[0099] Comparative Example 4 was used to prepare Sample VII.
[0100] Preparation method:
[0101] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0102] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 between the pulverized Selaginella tamariscina and an aqueous ethanol solution (90% by volume). This extraction is repeated 3 times to obtain the Selaginella tamariscina extract.
[0103] The third step is to concentrate the Selaginella extract under reduced pressure to obtain a crude Selaginella extract.
[0104] The fifth step involves column chromatography separation of the crude extract of Selaginella tamariscina using AB-8 macroporous resin, including: the first sub-step, eluting the crude extract of Selaginella tamariscina once with a 30% (v / v) ethanol aqueous solution to obtain the first eluted crude extract of Selaginella tamariscina.
[0105] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 50% (v / v) ethanol aqueous solution to obtain an eluent containing a 50% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named Sample VII).
[0106] Example 1
[0107] Example 1 was used to prepare sample VIII.
[0108] Preparation method:
[0109] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0110] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 between the pulverized Selaginella tamariscina and an aqueous ethanol solution (90% by volume). This extraction is repeated 3 times to obtain the Selaginella tamariscina extract.
[0111] The third step involves concentrating the Selaginella extract under reduced pressure to obtain a crude extract. The fourth step involves column chromatography using AB-8 macroporous resin to separate the crude extract, yielding the separated Selaginella extract, which includes:
[0112] The first sub-step involves eluting the crude extract of Selaginella tamariscina using a 10% (v / v) ethanol aqueous solution to obtain the first eluted crude extract of Selaginella tamariscina.
[0113] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 30% (v / v) ethanol aqueous solution to obtain an eluent containing a 30% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named Sample VIII).
[0114] Example 2
[0115] In Example 2, sample IX was prepared.
[0116] Preparation method:
[0117] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0118] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 between the pulverized Selaginella tamariscina and an aqueous ethanol solution (90% by volume). This extraction is repeated 3 times to obtain the Selaginella tamariscina extract.
[0119] The third step is to concentrate the Selaginella extract under reduced pressure to obtain a crude Selaginella extract.
[0120] Step 5: The crude extract of Selaginella tamariscina was separated by column chromatography using AB-8 macroporous resin to obtain the separated Selaginella tamariscina extract, which includes:
[0121] The first sub-step involves eluting the crude extract of Selaginella tamariscina using a 5% (v / v) ethanol aqueous solution to obtain the first eluted crude extract of Selaginella tamariscina.
[0122] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 30% (v / v) ethanol aqueous solution to obtain an eluent containing a 30% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named Sample IX).
[0123] Example 3
[0124] Example 3 was used to prepare sample X.
[0125] Preparation method:
[0126] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0127] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 between the pulverized Selaginella tamariscina and an aqueous ethanol solution (90% by volume). This extraction is repeated 3 times to obtain the Selaginella tamariscina extract.
[0128] The third step involves concentrating the Selaginella extract under reduced pressure to obtain a crude extract. The fifth step involves column chromatography using AB-8 macroporous resin to separate the crude extract, yielding the separated Selaginella extract, which includes:
[0129] The first sub-step involves eluting the crude extract of Selaginella tamariscina using a 20% (v / v) ethanol aqueous solution to obtain the first eluted crude extract of Selaginella tamariscina.
[0130] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 30% (v / v) ethanol aqueous solution to obtain an eluent containing a 30% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named Sample X).
[0131] Example 4
[0132] Example 4 was used to prepare sample XI.
[0133] Preparation method:
[0134] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0135] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 between the pulverized Selaginella tamariscina and an aqueous ethanol solution (90% by volume). This extraction is repeated 3 times to obtain the Selaginella tamariscina extract.
[0136] The third step involves concentrating the Selaginella extract under reduced pressure to obtain a crude extract. The fifth step involves column chromatography using AB-8 macroporous resin to separate the crude extract, yielding a water-based Selaginella extract, comprising:
[0137] The first sub-step involves eluting the Selaginella extract once with a 10% (v / v) ethanol aqueous solution to obtain the first eluted crude Selaginella extract.
[0138] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 20% (v / v) ethanol aqueous solution to obtain an eluent containing a 20% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named sample XI).
[0139] Example 5
[0140] Example 5 was used to prepare sample XII.
[0141] Preparation method:
[0142] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0143] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 between the pulverized Selaginella tamariscina and an aqueous ethanol solution (90% by volume). This extraction is repeated 3 times to obtain the Selaginella tamariscina extract.
[0144] The third step is to concentrate the Selaginella extract under reduced pressure to obtain a crude Selaginella extract.
[0145] The fourth step involves column chromatography separation of the crude Selaginella tamariscina extract using AB-8 macroporous resin to obtain the separated Selaginella tamariscina extract, which includes:
[0146] The first sub-step involves eluting the crude extract of Selaginella tamariscina using a 10% (v / v) ethanol aqueous solution to obtain the first eluted crude extract of Selaginella tamariscina.
[0147] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 40% (v / v) ethanol aqueous solution to obtain an eluent containing a 40% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named sample XII).
[0148] Example 6
[0149] Example 6 was used to prepare sample XIII.
[0150] Preparation method:
[0151] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0152] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 for the crushed Selaginella tamariscina medicinal material to water, and repeating the extraction 3 times to obtain Selaginella tamariscina extract.
[0153] The third step is to concentrate the Selaginella extract under reduced pressure to obtain crude Selaginella extract (Selaginella aqueous extract).
[0154] Step 5: The Selaginella tamariscina aqueous extract was separated by column chromatography using AB-8 macroporous resin. The separated Selaginella tamariscina extract contained the following components:
[0155] The first sub-step involves eluting the crude extract of Selaginella tamariscina with a 10% (v / v) ethanol aqueous solution to obtain the first eluted crude extract. The second sub-step involves eluting the first eluted crude extract of Selaginella tamariscina with a 30% (v / v) ethanol aqueous solution to obtain an eluent containing a 30% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named Sample XIII).
[0156] Example 7
[0157] Example 7 was used to prepare sample XIV.
[0158] Preparation method:
[0159] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0160] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 for the crushed Selaginella tamariscina medicinal material to water, and repeating the extraction 3 times to obtain Selaginella tamariscina extract.
[0161] The third step is to concentrate the Selaginella extract under reduced pressure to obtain crude Selaginella extract (Selaginella aqueous extract).
[0162] The fourth step involves column chromatography separation of the crude Selaginella tamariscina extract using AB-8 macroporous resin to obtain the separated Selaginella tamariscina extract, which includes:
[0163] The first sub-step involves eluting the crude extract of Selaginella tamariscina using a 5% (v / v) ethanol aqueous solution to obtain the first eluted crude extract. The second sub-step involves eluting the first eluted crude extract of Selaginella tamariscina using a 30% (v / v) ethanol aqueous solution to obtain an eluent containing a 30% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named sample XIV).
[0164] Example 8
[0165] Example 8 was used to prepare sample XV.
[0166] Preparation method:
[0167] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0168] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 for the crushed Selaginella tamariscina medicinal material to water, and repeating the extraction 3 times to obtain Selaginella tamariscina extract.
[0169] The third step is to concentrate the Selaginella extract under reduced pressure to obtain crude Selaginella extract (Selaginella aqueous extract).
[0170] The fourth step involves column chromatography separation of the Selaginella tamariscina aqueous extract using AB-8 macroporous resin to obtain the separated Selaginella tamariscina extract, which includes:
[0171] The first sub-step involves eluting the crude extract of Selaginella tamariscina using a 20% (v / v) ethanol aqueous solution to obtain the first eluted crude extract. The second sub-step involves eluting the first eluted crude extract of Selaginella tamariscina using a 30% (v / v) ethanol aqueous solution to obtain an eluent containing a 30% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named sample XV).
[0172] Example 9
[0173] Example 9 was used to prepare sample XVI.
[0174] Preparation method:
[0175] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0176] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 for the crushed Selaginella tamariscina medicinal material to water, and repeating the extraction 3 times to obtain Selaginella tamariscina extract.
[0177] The third step is to concentrate the Selaginella extract under reduced pressure to obtain crude Selaginella extract (Selaginella aqueous extract).
[0178] The fourth step involves column chromatography separation of the Selaginella extract using AB-8 macroporous resin to obtain the separated Selaginella extract, which includes:
[0179] The first sub-step involves eluting the Selaginella extract once with a 10% (v / v) ethanol aqueous solution to obtain the first eluted crude Selaginella extract.
[0180] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 20% (v / v) ethanol aqueous solution to obtain an eluent containing a 20% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named sample XVI).
[0181] Example 10
[0182] Example 10 was used to prepare sample XVII.
[0183] Preparation method:
[0184] The first step is to crush 100 grams of Selaginella tamariscina into powder to obtain the crushed Selaginella tamariscina.
[0185] The second step involves reflux extraction for 2 hours at a weight ratio of 1:20 for the crushed Selaginella tamariscina medicinal material to water, and repeating the extraction 3 times to obtain Selaginella tamariscina extract.
[0186] The third step is to concentrate the Selaginella extract under reduced pressure to obtain crude Selaginella extract (Selaginella aqueous extract).
[0187] The fourth step involves column chromatography separation of the crude Selaginella tamariscina extract using AB-8 macroporous resin to obtain the separated Selaginella tamariscina extract, which includes:
[0188] The first sub-step involves eluting the crude extract of Selaginella tamariscina using a 10% (v / v) ethanol aqueous solution to obtain the first eluted crude extract of Selaginella tamariscina.
[0189] The second sub-step involves eluting the crude extract of Selaginella tamariscina after the first elution with a 40% (v / v) ethanol aqueous solution to obtain an eluent containing a 40% (v / v) ethanol aqueous solution, thus obtaining the active fraction of Selaginella tamariscina (named sample XVII).
[0190] Sample testing
[0191] The sample testing employed two methods: high-performance liquid chromatography (HPLC) and antioxidant activity detection.
[0192] High performance liquid chromatography detection
[0193] Sample preparation method: Weigh the sample into a 15 mL centrifuge tube, add an appropriate amount of methanol or water according to the solubility of the sample, and use sonication to fully dissolve the sample. After dissolution, filter through a 0.45 μm filter membrane for detection by high performance liquid chromatography.
[0194] Chromatographic conditions:
[0195] Chromatographic column: C18 column, column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm.
[0196] Mobile phase: Gradient elution method is selected, as follows:
[0197] (1) Acetonitrile / water: 0 to 20 minutes, the flow strength is: 10% / 90% → 95% / 5%; 20 to 25 minutes, the flow strength remains at 95% / 5%.
[0198] (2) Acetonitrile / water: 0 to 30 minutes, the flow strength is 5% / 95% → 50% / 50%; 30 to 36 minutes, the flow strength is maintained at 95% / 5%; 37 to 41 minutes, the flow strength is maintained at 5% / 95%.
[0199] Flow rate: 1.0 ml / min.
[0200] Antioxidant activity detection
[0201] Sample processing methods:
[0202] The first step is to prepare an ABTS (7mM) deionized water solution.
[0203] The second step is to prepare an aqueous solution of K2S2O8 (potassium persulfate, 2.45 mM).
[0204] The third step involves mixing ABTS deionized aqueous solution and K2S2O8 aqueous solution in equal proportions and reacting at room temperature in the dark for 12 to 16 hours to form an ABTS+ free radical stock solution.
[0205] The ABTS+ free radical stock solution needs to be diluted with distilled water or ethanol before use until the absorbance value (OD value) at 734 nm is 0.7 ± 0.02.
[0206] The fourth step is to prepare the sample solution.
[0207] The sample solutions for samples I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, and XVII were prepared respectively, as detailed in Table 1 below.
[0208] Table 1
[0209]
[0210]
[0211] The fifth step involves reacting the sample solution with the ABTS working solution in the dark for 6 minutes, then measuring the absorbance (OD value) at 734 nm at 20 degrees Celsius and calculating the ABTS free radical scavenging rate of the sample.
[0212] Wherein, the ABTS free radical scavenging rate = [1 - (A1 - A2) / A0] × 100%.
[0213] Wherein, A1 represents the absorbance value at 734 nm at 20°C after 20 μL of sample solution reacts with 180 μL of ABTS working solution in the dark for 6 minutes. A2 represents the absorbance value at 734 nm at 20°C after 20 μL of sample solution reacts with 180 μL of blank solvent (ethanol / water) in the dark for 6 minutes. A0 represents the absorbance value at 734 nm at 20°C after 20 μL of blank solvent (ethanol / water) reacts with 180 μL of ABTS working solution in the dark for 6 minutes.
[0214] The ABTS radical scavenging rates for samples I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, and XVII are shown in Table 2 below.
[0215] Table 2
[0216]
[0217]
[0218] For details, see Figure 1 The graph shows a comparison of ABTS radical scavenging rates for samples I, II, III, IV, V, VI, and VII. Figure 2 The graph shows a comparison of ABTS radical scavenging rates for samples VIII, IX, X, XI, and XII. Figure 3 The chart shows a comparison of ABTS radical scavenging rates for samples XIII, XIV, XV, XVI, and XVII. For specific ABTS radical scavenging rates for each sample, please refer to Table 2. The comparison revealed that sample VIII (using an ethanol-water solution as the soaking solution) and sample XIII (using water as the soaking solution) exhibited significantly higher ABTS radical scavenging rates. Furthermore, comparing the ABTS radical scavenging rates of samples VII and VIII, it was found that the antioxidant activity of the Selaginella tamariscina extract enriched with Taxodium biflavonoids was significantly lower than that of the other component extracts.
[0219] In addition, see Figure 4 The HPLC chromatogram of sample IV is shown below. Figure 5 The HPLC chromatogram of sample V shown is as follows: Figure 6 The HPLC chromatogram of sample VI shown is as follows: Figure 7 The HPLC chromatogram of sample VIII shown is as follows: Figure 8 The HPLC chromatogram of sample XIII is shown below. Figure 9 The HPLC chromatogram of sample VII is shown. Comparison revealed that the content of the highly polar component in sample VI (eluent III) was higher than that in sample IV (eluent I), and the antioxidant activity of sample VI was lower than that of sample IV (eluent I). It was speculated that the highly polar component in sample VI affected the activity. Therefore, sample VIII was prepared using the same method as sample VIII, and the corresponding HPLC chromatogram was observed. It was found to be similar to that of sample IV (eluent I). Comparing the corresponding ABTS radical scavenging rates, sample VIII exhibited higher antioxidant activity. Furthermore, Figure 9 The peak corresponds to paclitaxel flavonoids, indicating that the preparation method of sample VII, namely, 50% ethanol aqueous solution can elute paclitaxel flavonoids, thus achieving the purpose of enriching paclitaxel flavonoids.
[0220] Application Example 1
[0221] Application Example 1 is used to prepare an aqueous skin care product containing Selaginella tamariscina extract. The detailed formulation is shown in Table 3 below:
[0222] Table 3
[0223] 1 water margin 2 Capcom U20 0.1 3 glycerin 6.0 4 Panthenol 1.0 5 EDTA-2NA (Disodium ethylenediaminetetraacetate) 0.02 6 Xinxiantong 0.3 7 Hexanediol 0.5 8 Pentylene glycol 0.8 9 Arginine 0.08 10 water 2 11 Methyl glucetol polyether-20 0.5 12 Selaginella extract 0.11
[0224] Preparation method:
[0225] First, weigh out ingredients 1 and 2 according to the required amounts and add them to the emulsifying pot. Stir until the mixture is fully mixed and free of particles and clumps. Then, heat the mixture to 80-90℃.
[0226] The second step is to add raw materials No. 3, 4, and 5 in sequence, stir at a constant speed until a transparent solution is obtained, and then cool it down to 50-60℃.
[0227] Third, in a separate container, heat raw materials 6, 7, and 8 to 70-85℃ until dissolved into a clear, particle-free solution, then cool to 50℃ and add to the emulsification pot.
[0228] Fourth step: In a separate container, stir ingredients 9 and 10 until they are transparent and free of granules, then add them to the emulsifying pot.
[0229] Fifth step: After the emulsification pot cools down to 40-45℃, add ingredients No. 11 and No. 12 in sequence and stir evenly.
[0230] The sixth step is to take samples for testing. Once the samples pass the test, the product is discharged, yielding a water-based skin care product containing Selaginella extract.
[0231] Application Example 2
[0232] Application Example 3 was used to prepare a water-in-oil type skin care product containing Selaginella tamariscina extract. The detailed formulation is shown in Table 4 below:
[0233] Table 4
[0234]
[0235]
[0236] Preparation method:
[0237] First, weigh out raw materials 1 and 2 according to the required amount, and then add them into the emulsifying pot in sequence. Stir them thoroughly until there are no particles or clumps. Then heat the pot to 80-90℃.
[0238] The second step is to add ingredients No. 3 and No. 4 in sequence, stir at a constant speed until a transparent solution is obtained, and keep it warm while stirring.
[0239] The third step is to add ingredients 5, 6, 7, 8, and 9 to the oil pan, heat it to 80-90℃, and stir well.
[0240] The fourth step is to add the processed oil phase into the emulsifying pot, homogenize for 3-5 minutes, and then stir and cool to 45-50℃.
[0241] Fifth step: Take another container and stir ingredients 10 and 11 until they are transparent and free of particles, then add them to the emulsification pot.
[0242] Step 6: In another container, heat raw materials No. 12, 13, and 14 to 70-85℃ to dissolve them into a clear, particle-free solution, then cool the solution to 40-50℃ and add it to the emulsification pot.
[0243] Step 7: Once the emulsification pot has cooled to 40-45℃, add ingredients 15 and 16 in sequence and stir well.
[0244] Step 8: Sampling and testing. Once qualified, the product is discharged to obtain a water-in-oil type skin care product containing Selaginella extract.
[0245] The above description is merely a selection of preferred embodiments of this disclosure and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in the embodiments of this disclosure is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in the embodiments of this disclosure.
Claims
1. A method for preparing Selaginella tamariscina extract, comprising: Selaginella extract is obtained by soaking and refluxing the pulverized Selaginella herb in a soaking solution. The Selaginella herb is the dried whole herb of Selaginella tamariscina or Selaginella materia var. pachycarpa, a plant of the Selaginellaceae family. The soaking solution is an aqueous solution of ethanol or water with a volume fraction of 90% to 95%. The Selaginella extract was concentrated under reduced pressure to obtain a crude Selaginella extract. The crude extract of Selaginella tamariscina was separated using AB-8 type macroporous resin to obtain a separated Selaginella tamariscina extract, which includes: The crude extract of Selaginella tamariscina was eluted for the first time by an aqueous ethanol solution with a volume fraction of 5% to 15% to obtain the crude extract of Selaginella tamariscina after the first elution. The crude extract of Selaginella tamariscina after the first elution was eluted a second time by an aqueous ethanol solution with a volume fraction of 25%~35% to obtain the corresponding eluent, which is denoted as the separated Selaginella tamariscina extract. The separated Selaginella extract was dried to obtain Selaginella extract.
2. The method according to claim 1, wherein, The process of soaking and refluxing the pulverized Selaginella tamariscina medicinal material in the soaking solution to obtain Selaginella tamariscina extract includes: The pulverized Selaginella tamariscina herb is soaked in a soaking solution and then refluxed for 1 to 3 hours, and the process is repeated a preset number of times to obtain the Selaginella tamariscina extract, wherein the preset number of times is ≥1.
3. The method according to claim 1, wherein, The Selaginella extract is concentrated under reduced pressure to obtain a crude Selaginella extract, comprising: The Selaginella extract was rotary evaporated to obtain the crude Selaginella extract.
4. The method according to claim 1, wherein, The weight ratio of the pulverized Selaginella tamariscina to the soaking solution is 1:10~30.
5. The method according to claim 1, wherein, When the soaking solution is water, the soaking solution is one or more of pure water, deionized water, and ultrapure water.
6. The method according to claim 1, wherein, The ratio of the volume of the 5%~15% ethanol aqueous solution used to the volume of the macroporous resin column is ≥5:1, and the ratio of the volume of the 25%~35% ethanol aqueous solution used to the volume of the macroporous resin column is ≥5:
1.
7. The method according to claim 1, wherein, The dried and separated Selaginella extract yields Selaginella extract, comprising: The separated Selaginella extract was obtained by evaporation and drying.
8. The method according to claim 1, wherein, The dried and separated Selaginella extract yields Selaginella extract, comprising: The separated Selaginella extract is dried by one or more of the following drying methods: hot air drying, vacuum drying, freeze drying, spray drying, and microwave drying, to obtain the Selaginella extract.
9. A skin care product comprising the Selaginella extract as described in any one of claims 1-8.
10. The skin care product according to claim 9, wherein, The skin care products include one or more of the following: serums, lotions, emulsions, creams, ointments, and masks.