Preparation method of taxus chinensis extract, product and application thereof

Abstract

This invention belongs to the field of material extraction technology, and relates to a method for preparing an extract of *Taxus wallichiana*, its product, and its application. The preparation method includes: mixing *Taxus wallichiana* with an aqueous two-phase system, extracting, allowing to stand and separate phases, collecting phase A and filtering to obtain a filtrate; treating the filtrate with an adsorbent and filtering to obtain the final product; the aqueous two-phase system includes phase A and phase B; phase A includes ethanol and behenamidopropyl dimethylamine; phase B includes inorganic salts and water. The *Taxus wallichiana* extract prepared by this invention does not contain four substances: taxine B, paclitaxel, 10-deacetylated paclitaxel, and cephalotaxine, and the extract has excellent antioxidant, soothing, moisturizing, and anti-aging effects.

Description

Technical Field

[0001] This invention belongs to the field of material extraction technology, and relates to a method for preparing extracts of Taxus wallichiana, as well as its products and applications. Background Technology

[0002] Southern yew is a plant of the Taxaceae family, also known as yew. It is an evergreen tree or shrub that is extremely rare due to its slow growth and difficult development.

[0003] The main potential risk substances in *Taxus wallichiana* include taxine, paclitaxel, 10-deacetylated paclitaxel, and cephalotaxine. Taxine is the main potential risk substance in *Taxus wallichiana*, primarily found in its leaves, branches, and bark. Taxine is a diterpenoid alkaloid whose main toxic effect is the inhibition of calcium and sodium ion channels in cardiomyocytes, leading to cardiotoxicity. Taxine B is one of the main toxic components of taxine. Although paclitaxel is the most well-known anticancer component in *Taxus wallichiana*, it also has certain toxicity. Paclitaxel's toxicity mainly manifests as bone marrow suppression, neurotoxicity, and cardiotoxicity. Paclitaxel's anticancer mechanism involves binding to microtubules, promoting their polymerization, and inhibiting the mitosis of cancer cells. However, this effect can also affect the division of normal cells, leading to certain toxic side effects. 10-Deacetylated paclitaxel is a derivative similar to paclitaxel, exhibiting similar toxicity, but with slightly weaker toxicity. The main toxicities include bone marrow suppression (neutropenia), neurotoxicity, and allergic reactions. Homoharringtonine is an active ingredient with potent anti-inflammatory and immunosuppressive effects, but it does exhibit certain toxicities, primarily hepatotoxicity, nephrotoxicity, and reproductive toxicity. This is a major challenge it faces in clinical application.

[0004] There is no report on a method that can simultaneously remove the above four substances from Taxus wallichiana extract. Therefore, providing a method that removes the above four substances while retaining the effective components of Taxus wallichiana has become an urgent problem to be solved in this field. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing extract of Taxus wallichiana, as well as its products and applications.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a *Taxus wallichiana* extract, the method comprising:

[0008] (1) Mix the southern yew with the aqueous two-phase system, extract, let stand to separate the phases, collect phase A and filter to obtain the filtrate;

[0009] (2) The filtrate is treated with an adsorbent and then filtered to obtain the final product;

[0010] The aqueous two-phase system includes phase A and phase B;

[0011] Phase A comprises ethanol and behenamidopropyl dimethylamine;

[0012] Phase B comprises inorganic salts and water.

[0013] This invention uses a combination of aqueous two-phase extraction and adsorbent treatment to prepare Taxus wallichiana extract. While ensuring that the extract has excellent antioxidant, soothing, moisturizing and anti-aging effects, it removes four substances including taxine B, paclitaxel, 10-deacetylpaclitaxel and cephalotaxine, making it safe and non-irritating.

[0014] The selection of components in phases A and B of an aqueous two-phase system is crucial. The ratio of phases A to B is particularly critical; only under specific conditions can an aqueous two-phase extraction system be formed. Phase A is primarily used to extract the desired main target components. Furthermore, by creatively introducing a surfactant such as behenamidopropyl dimethylamine, phase A possesses a certain degree of weak alkalinity, which facilitates the subsequent removal of four substances, including taxine B, paclitaxel, 10-deacetylpaclitaxel, and holotaxine. Replacing behenamidopropyl dimethylamine in phase A with other surfactants significantly reduces its effectiveness. Phase B is mainly used to dissolve and remove non-target components such as water-soluble proteins, tannins, and water-soluble pigments. The selection of inorganic salts for phase B not only needs to ensure the formation of an aqueous two-phase extraction system, but specific types of inorganic salts can further amplify the "salting-out effect," making taxine B, paclitaxel, 10-deacetylpaclitaxel, and holotaxine more soluble in phase A, thus increasing the subsequent removal efficiency.

[0015] Preferably, the southern yew includes branches and leaves of the southern yew.

[0016] Specifically, these are the tender shoots and leaves that grow in the first 1-2 years on trees that are 10-20 years old, such as Southern Yew.

[0017] Preferably, the aqueous two-phase system comprises 10-20 parts of phase A and 80-100 parts of phase B by mass.

[0018] The mass fractions of phase A can be selected from 10, 12, 14, 16, 18, 20, etc., and the mass fractions of phase B can be selected from 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0019] Preferably, the mass ratio of inorganic salt to water in phase B is (20-30):(50-70).

[0020] The specific point values ​​in (20-30) can be selected from 20, 22, 24, 26, 28, 30, etc., and the specific point values ​​in (50-70) can be selected from 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0021] Preferably, the mass ratio of ethanol to behenamidopropyl dimethylamine in phase A is (500-1000):1.

[0022] The specific point values ​​in (500-1000) can be selected from 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0023] Preferably, the inorganic salt includes any one or a combination of at least two of sodium carbonate, sodium bicarbonate, and ammonium sulfate.

[0024] Preferably, the ratio of the Southern Yew to the aqueous two-phase system is 1 g:(10-40) mL.

[0025] The specific point values ​​in (10-40) can be selected from 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0026] Preferably, the extraction temperature is 60-80℃, the extraction time for a single extraction is 1-3 hours, and the number of extractions is 1-4 times.

[0027] Temperatures can be selected from 60℃, 62℃, 65℃, 68℃, 70℃, 72℃, 75℃, 78℃, 80℃, etc.; time can be selected from 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, etc.; number of times can be selected from 1 time, 2 times, 3 times, 4 times, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0028] Preferably, the adsorbent comprises behenamidopropyl dimethylamine and behenyltrimethylammonium chloride.

[0029] Treatment of the filtrate after aqueous two-phase extraction with an adsorbent can remove hazardous substances present in the filtrate, reduce the irritation of the product, and behenamidopropyl dimethylamine and behentrimethylammonium chloride have a certain synergistic effect.

[0030] Preferably, the mass ratio of behenamidopropyl dimethylamine to behenyltrimethylammonium chloride is (1-5):(1-5).

[0031] The specific point values ​​in (1-5) can all be selected from 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0032] Preferably, the temperature for the adsorbent treatment is 85-95℃, the time is 3-4 h, and the stirring speed is 100-500 rpm.

[0033] Temperatures can be selected from 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, etc.; time can be selected from 3 h, 3.2 h, 3.4 h, 3.6 h, 3.8 h, 4 h, etc.; stirring speed can be selected from 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0034] Preferably, before using the adsorbent, the pH of the filtrate is adjusted to 9-10 using a pH adjuster, such as 9, 9.2, 9.4, 9.6, 9.8, 10, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0035] The pH value of the adsorbent treatment also affects the adsorption effect.

[0036] Preferably, the mass of the adsorbent is 0.01-0.05% of the mass of the filtrate, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0037] Preferably, the pH adjuster includes any one or a combination of at least two of sodium hydroxide, arginine, and aminomethylpropanol.

[0038] In a second aspect, the present invention provides a *Taxus wallichiana* extract prepared according to the method for preparing *Taxus wallichiana* extract described in the first aspect.

[0039] Thirdly, the present invention provides the use of the southern yew extract according to the second aspect in the preparation of products with antioxidant, soothing, moisturizing or anti-aging effects.

[0040] Preferably, the product includes cosmetics or skincare products.

[0041] Fourthly, the present invention provides the use of the Taxus wallichiana extract according to the second aspect in the preparation of inflammatory factor inhibitors, AQP3 gene activators or MMP-1 gene inhibitors.

[0042] According to the research results of this invention, the Taxus wallichiana extract prepared by this invention has a significant inhibitory effect on the secretion of inflammatory factors, an activating effect on the AQP3 gene, and an inhibitory effect on the MMP-1 gene. Therefore, the Taxus wallichiana extract prepared by this invention can be used as an in vitro preparation for basic scientific research, such as exploring the mechanism of action of physiological activities related to inflammatory response, or for studying the mechanism of gene expression activation.

[0043] Preferably, the inflammatory factor includes NO.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention employs a specific preparation method that can effectively remove four substances: taxine B, paclitaxel, 10-deacetylated paclitaxel, and cephalotaxine. The resulting southern yew extract has excellent antioxidant, soothing, moisturizing, and anti-aging effects, and is safe and non-irritating. Attached Figure Description

[0046] Figure 1 This is a flowchart of the preparation process of Taxus chinensis extract.

[0047] Figure 2 It is the aqueous two-phase extraction solvent used in Example 1.

[0048] Figure 3 This is the result of phase separation after two-phase extraction of Taxus wallichiana and standing to remove the residue in Example 1.

[0049] Figure 4 This is a liquid phase diagram of a mixture of four standards: taxine B, paclitaxel, 10-deacetylated paclitaxel, and cephalotaxine.

[0050] Figure 5 This is a liquid phase diagram of the Taxus wallichiana extract obtained in Example 1. Detailed Implementation

[0051] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0052] The tender shoots of Southern Yew used in the following content are the tender shoots and leaves that grow in the first 1-2 years from Southern Yew trees that are 10-20 years old.

[0053] Example 1

[0054] This embodiment provides a method for preparing extracts of Taxus chinensis var. mongolica, the preparation method comprising:

[0055] (1) The tender branches of Southern Yew were crushed and passed through a 60-mesh sieve, and mixed with a two-phase system at a material-liquid ratio of 1 g:20 mL (the two-phase system includes 20 parts of phase A and 80 parts of phase B by mass. Phase A is anhydrous ethanol and behenamidopropyl dimethylamine with a mass ratio of 800:1, and phase B is sodium carbonate and water with a mass ratio of 25:60). The mixture was extracted at 70℃ for 2 hours each time, and extracted twice. After standing and phase separation, phase A was collected and filtered to obtain the filtrate.

[0056] (2) The pH of the filtrate was adjusted to 9.5 with sodium hydroxide, and the filtrate was treated with an adsorbent at 90°C under stirring for 3.5 h with a stirring speed of 200 rpm. The adsorbent was behenamidopropyl dimethylamine and behentrimethylammonium chloride in a mass ratio of 1:1. The amount of adsorbent added was 0.05% of the mass of the filtrate. After filtration, concentration and drying, the extract of Taxus wallichiana was obtained.

[0057] Preparation flow chart as follows Figure 1 As shown, the aqueous two-phase extraction solvent used is as follows: Figure 2 As shown, the phase separation results after settling to remove the residue are as follows: Figure 3 As shown.

[0058] Example 2

[0059] This embodiment provides a method for preparing extracts of Taxus chinensis var. mongolica, the preparation method comprising:

[0060] (1) The tender branches of southern yew were crushed and passed through a 60-mesh sieve, and mixed with a two-phase system at a material-liquid ratio of 1 g:10 mL (the two-phase system includes 15 parts of phase A and 85 parts of phase B by mass, phase A is anhydrous ethanol and behenamidopropyl dimethylamine with a mass ratio of 500:1, and phase B is sodium bicarbonate and water with a mass ratio of 30:50). The mixture was extracted at 60℃ for 3 hours each time, and extracted twice. After standing and phase separation, phase A was collected and filtered to obtain the filtrate.

[0061] (2) The pH of the filtrate was adjusted to 9 with sodium hydroxide, and the filtrate was treated with an adsorbent at 85°C under stirring for 3 h at a stirring speed of 100 rpm. The adsorbent was behenamidopropyl dimethylamine and behentrimethylammonium chloride in a mass ratio of 5:1. The amount of adsorbent added was 0.01% of the mass of the filtrate. After filtration, concentration and drying, the extract of Taxus wallichiana was obtained.

[0062] Example 3

[0063] This embodiment provides a method for preparing extracts of Taxus chinensis var. mongolica, the preparation method comprising:

[0064] (1) The tender branches of Southern Yew were crushed and passed through a 60-mesh sieve, and mixed with a two-phase system at a material-liquid ratio of 1 g:40 mL (the two-phase system includes 10 parts of phase A and 90 parts of phase B by mass. Phase A is anhydrous ethanol and behenamidopropyl dimethylamine with a mass ratio of 1000:1, and phase B is ammonium sulfate and water with a mass ratio of 20:70). The mixture was extracted at 80℃ for 1 hour each time, and extracted 4 times. After standing and phase separation, phase A was collected and filtered to obtain the filtrate.

[0065] (2) The pH of the filtrate was adjusted to 10 with sodium hydroxide, and the filtrate was treated with an adsorbent at 95°C for 4 hours under stirring. The stirring speed was 500 rpm. The adsorbent was behenamidopropyl dimethylamine and behentrimethylammonium chloride in a mass ratio of 1:5. The amount of adsorbent added was 0.03% of the mass of the filtrate. After filtration, concentration and drying, the extract of Taxus wallichiana was obtained.

[0066] Example 4

[0067] This embodiment provides a method for preparing Southern Yew extract, which differs from Example 1 only in that step (2) is: "The pH of the filtrate is adjusted to 12 using sodium hydroxide, and the filtrate is treated at 90°C under stirring conditions for 3.5 h with an adsorbent at a stirring speed of 200 rpm. The adsorbent is behenamidopropyl dimethylamine and behenyltrimethylammonium chloride in a mass ratio of 1:1. The amount of adsorbent added is 0.05% of the mass of the filtrate. After filtration, the Southern Yew extract is obtained after concentration and drying." Other operations remain unchanged.

[0068] Example 5

[0069] This embodiment provides a method for preparing Southern Yew Extract, which differs from Example 1 only in that step (2) is "adjusting the pH of the filtrate to 7, treating it with an adsorbent at 90°C under stirring conditions for 3.5 h, with a stirring speed of 200 rpm, the adsorbent being behenamidopropyl dimethylamine and behenyltrimethylammonium chloride in a mass ratio of 1:1, the amount of adsorbent added being 0.05% of the mass of the filtrate, filtering, and obtaining Southern Yew Extract after concentration and drying", with other operations remaining unchanged.

[0070] Example 6

[0071] This embodiment provides a method for preparing Southern Yew Extract, which differs from Example 1 only in that step (2) is: "adjust the pH of the filtrate to 3, treat it with an adsorbent at 90°C under stirring conditions for 3.5 h, the stirring speed is 200 rpm, the adsorbent is behenamidopropyl dimethylamine and behentrimethylammonium chloride in a mass ratio of 1:1, the amount of adsorbent added is 0.05% of the mass of the filtrate, filter, concentrate and dry to obtain Southern Yew Extract", and other operations remain unchanged.

[0072] Example 7

[0073] This embodiment provides a method for preparing Southern Yew Extract, which differs from Example 1 only in that step (2) is "adjusting the pH of the filtrate to 9.5 with sodium hydroxide, treating it with an adsorbent at 90°C under stirring conditions for 3.5 hours, stirring at 200 rpm, the adsorbent being behenamidopropyl dimethylamine, the amount of adsorbent added being 0.05% of the mass of the filtrate, filtering, and obtaining Southern Yew Extract after concentration and drying", with other operations remaining unchanged.

[0074] Example 8

[0075] This embodiment provides a method for preparing Southern Yew Extract, which differs from Example 1 only in that step (2) is "adjusting the pH of the filtrate to 9.5 with sodium hydroxide, treating it with an adsorbent at 90°C under stirring conditions for 3.5 hours, stirring at 200 rpm, the adsorbent being behenyltrimethylammonium chloride, the amount of adsorbent added being 0.05% of the mass of the filtrate, filtering, and obtaining Southern Yew Extract after concentration and drying", with other operations remaining unchanged.

[0076] Example 9

[0077] This embodiment provides a method for preparing Southern Yew Extract, which differs from Example 1 only in that step (2) is "adjusting the pH of the filtrate to 9.5 with sodium hydroxide, treating it with activated carbon at 90°C under stirring conditions for 3.5 hours, stirring at 200 rpm, adding activated carbon at 0.05% of the filtrate mass, filtering, and obtaining Southern Yew Extract after concentration and drying", while other operations remain unchanged.

[0078] Comparative Example 1

[0079] This comparative example provides a method for preparing extract of Taxus wallichiana, which differs from Example 1 only in that step (1) is to "crush the tender branches of Taxus wallichiana through a 60-mesh sieve, mix with an ethanol aqueous solution with a volume percentage of 80%, the material-to-liquid ratio is 1 g: 20 mL, reflux for extraction, the extraction time for each extraction is 2 h, extract twice, filter, and collect the filtrate", while other operations remain unchanged.

[0080] Comparative Example 2

[0081] The method was completely based on the article "Differences in the main components and in vitro antioxidant activity of Taxus wallichiana from different producing areas" by Xie Tao et al., published in the journal Chinese Medicinal Herbs in 2025.

[0082] The specific method includes: taking 1.0 g of Taxus wallichiana powder (Taxus wallichiana branches and leaves), accurately weighing it, placing it in a stoppered conical flask, adding 30 mL of 80% ethanol, weighing it (i.e., the material-to-liquid ratio is 1 g: 30 mL), sonicating (280 W, 40 Hz) for 30 min, adding 80% ethanol to make up the weight, filtering, and evaporating the supernatant to dryness in a water bath to obtain Taxus wallichiana extract.

[0083] Comparative Example 3

[0084] This comparative example provides a method for preparing extract of Taxus wallichiana, which differs from Example 1 only in that step (1) is to "crush the tender branches of Taxus wallichiana through a 60-mesh sieve and mix them with a two-phase system at a material-liquid ratio of 1 g:20 mL (the two-phase system includes 20 parts of phase A and 80 parts of phase B by mass, phase A is anhydrous ethanol, and phase B is sodium carbonate and water with a mass ratio of 25:60), extract at 70°C, the extraction time for each extraction is 2 h, extract twice, allow to stand and separate phases, collect phase A and filter to obtain filtrate", and other operations remain unchanged.

[0085] Comparative Example 4

[0086] This comparative example provides a method for preparing extract of Taxus wallichiana, which differs from Example 1 only in that step (1) is to "crush the tender branches of Taxus wallichiana through a 60-mesh sieve and mix them with a two-phase system at a material-liquid ratio of 1 g:20 mL (the two-phase system includes 20 parts of phase A and 80 parts of phase B by mass, phase A is polyethylene glycol with a molecular weight of 400, and phase B is sodium carbonate and water with a mass ratio of 25:60), extract at 70°C, the extraction time is 2 h per extraction, extract twice, allow to stand and separate phases, collect phase A and filter to obtain filtrate", and other operations remain unchanged.

[0087] Comparative Example 5

[0088] This comparative example provides a method for preparing extract of Taxus wallichiana, which differs from Example 1 only in that step (1) is to "crush the tender branches of Taxus wallichiana and pass them through a 60-mesh sieve, mix them with a two-phase system at a material-liquid ratio of 1 g:20 mL (the two-phase system includes 20 parts of phase A and 80 parts of phase B by mass, phase A is anhydrous ethanol and behenamidopropyl dimethylamine with a mass ratio of 800:1, phase B is dextran (molecular weight: 10000) and water with a mass ratio of 25:60), extract at 70°C, the extraction time is 2 h per extraction, extract twice, allow to stand and separate phases, collect phase A and filter to obtain filtrate", and other operations remain unchanged.

[0089] Comparative Example 6

[0090] This comparative example provides a method for preparing extract of Taxus wallichiana, which differs from Example 1 only in that step (1) is to "crush the tender branches of Taxus wallichiana through a 60-mesh sieve and mix them with a two-phase system at a material-liquid ratio of 1 g:20 mL (the two-phase system includes 20 parts of phase A and 80 parts of phase B by mass, phase A is anhydrous ethanol and behenamidopropyl dimethylamine with a mass ratio of 800:1, and phase B is dipotassium hydrogen phosphate and water with a mass ratio of 25:60), extract at 70°C, the extraction time is 2 h per extraction, extract twice, allow to stand and separate phases, collect phase A and filter to obtain filtrate", and other operations remain unchanged.

[0091] Comparative Example 7

[0092] This comparative example provides a method for preparing extract of Taxus wallichiana, which differs from Example 1 only in that step (1) is to "crush young branches of Taxus wallichiana through a 60-mesh sieve and mix them with a two-phase system at a material-to-liquid ratio of 1 g:20 mL (the two-phase system includes 20 parts of phase A and 80 parts of phase B by mass. Phase A is anhydrous ethanol and polysorbate-60 (Croy Chemicals (Shanghai) Co., Ltd., trade name: SP TWEEN 60 MBAL-SS-(SG)) at a mass ratio of 800:1, and phase B is sodium carbonate and water at a mass ratio of 25:60), extract at 70°C, with a single extraction time of 2 h, extract twice, allow to stand and separate phases, collect phase A and filter to obtain filtrate", and other operations remain unchanged.

[0093] Test Example 1

[0094] Content detection of 4 substances

[0095] Liquid chromatography detection methods:

[0096] Testing instrument: Agilent 1290 Infinity II high-performance liquid chromatograph;

[0097] Chromatographic column: Aglient ZORBAX SB-AQ 100*3 mm, 3.5 μm;

[0098] Mobile phase: Mobile phase A is water, mobile phase B is acetonitrile; gradient elution program: 0 min: 60% mobile phase A; 20 min: 20% mobile phase A; 25 min: 60% mobile phase A;

[0099] Flow rate: 0.4 mL / min;

[0100] Column temperature: 30 ℃;

[0101] Detection wavelength: 227 nm;

[0102] Injection volume: 4 μL.

[0103] The contents of the four substances in the extracts of Taxus wallichiana obtained in Examples 1-9 and Comparative Examples 1-7 are shown in Table 1 below.

[0104] Table 1

[0105]

[0106] As shown in Table 1, the southern yew extract prepared by the method of the present invention does not contain taxine B, paclitaxel, 10-deacetylpaclitaxel and cephalotaxine, and has a good removal effect on these four substances. The selection of adsorbent, pH of adsorption treatment and extraction method will affect the removal effect of the four substances.

[0107] The liquid chromatography chromatograms of four standards—taxine B, paclitaxel, 10-deacetylated paclitaxel, and cephalotaxine—are shown below. Figure 4 As shown (numbers 1, 2, 3, and 4 in the figure correspond to 10-deacetylated paclitaxel, taxaneine, paclitaxel, and taxine B, respectively). The liquid chromatography diagram of the Taxus wallichiana extract obtained in Example 1 is shown below. Figure 5 As shown.

[0108] Test Example 2

[0109] Antioxidant efficacy test

[0110] Test Method: Vitamin C was used as a positive control. Both the *Taxus wallichiana* extract and the positive control solution were prepared at an initial concentration of 0.5 mg / mL (dissolved in 50% ethanol) and diluted 1 / 2 times. The 96-well plates were divided into sample and control groups, with three replicates in each group. A blank control was included in both groups. An equal volume of DPPH solution was added to each well. The 96-well plates were incubated at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm. The DPPH free radical scavenging effect was measured using the half-inhibitory concentration (IC50). 50 The value is expressed in μg / mL.

[0111] The calculation formula is as follows:

[0112] Free radical scavenging rate (%) = (1 - (A1 - A2) / A0) × 100%

[0113] Where: A1 - OD value of the sample group; A2 - OD value of the sample control group; A0 - OD value of the blank group

[0114] OD value of sample group: OD value of the sample after the colorimetric reaction.

[0115] The OD value of the sample control group is the initial OD value of the sample, that is, the OD value after dissolution before the colorimetric reaction.

[0116] The OD value of the blank group was the OD value of 50% ethanol.

[0117] The DPPH free radical scavenging effects (IC50) of the extracts of Taxus wallichiana obtained in Examples 1-9 and Comparative Examples 1-7 50 (See Table 2 below).

[0118] Table 2

[0119]

[0120] The above results indicate that the Taxus wallichiana extract prepared by this invention has good antioxidant effects, and the choice of adsorbent, pH of adsorption treatment, extraction method, and the choice of phase A and phase B in aqueous two-phase extraction all affect the antioxidant effects.

[0121] Test Example 3

[0122] Soothing efficacy test

[0123] Test Method: RAW264.7 cells in logarithmic growth phase with good morphology were seeded into 24-well plates and incubated for 24 h. A model group (LPS (lipopolysaccharide), 0.1 μg / mL) and a sample group were set up. LPS was added to each well, and the plates were incubated at 37 ℃ in a 5% CO2 incubator for 2 h. After drug administration, the plates were incubated for another 24 h. The cell supernatant was analyzed according to the NO detection kit instructions. The NO content in the collected cell supernatant was measured, and the experimental results were analyzed using data processing software.

[0124] The effects of the southern yew extract (1 μg / mL) obtained in Examples 1-9 and Comparative Examples 1-7 on the relative expression level of NO (the relative expression level was lower than that of the model group, and the lower the relative expression level, the stronger the soothing effect; while the relative expression level was higher than that of the model group, it had a certain pro-inflammatory effect), are shown in Table 3 below.

[0125] Table 3

[0126]

[0127] The above results indicate that the Taxus wallichiana extract prepared by this invention has good soothing effects, and the choice of adsorbent, pH of adsorption treatment, extraction method, and the choice of phase A and phase B in aqueous two-phase extraction all affect the soothing effects.

[0128] Test Example 4

[0129] Moisturizing efficacy test

[0130] Test method: 4.4 × 10 4 Cells were seeded at a density of cells / well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2). A control group and a sample group were set up. When the cell deposition rate in the 24-well plates reached approximately 40%–60%, the cells were administered drug to each group at a dose of 1 mL per well, with three replicates per group. The cells were cultured for another 24 hours. The control group received no treatment but was fixed with 4% paraformaldehyde for 30 minutes. Immunofluorescence detection of aquaporin 3 (AQP3) was performed, and images were taken and analyzed under a microscope.

[0131] The effects of the Taxus wallichiana extract (1 μg / mL) obtained in Examples 1-9 and Comparative Examples 1-7 on the relative expression level of AQP3 (the relative expression level was higher than that of the blank group, and the higher the relative expression level, the stronger the moisturizing effect; while the relative expression level was lower than that of the blank group, it had no moisturizing effect), are shown in Table 4 below.

[0132] Table 4

[0133]

[0134] The above results indicate that the Taxus wallichiana extract prepared by this invention has good moisturizing effects, and the selection of adsorbent, pH of adsorption treatment, extraction method, and the selection of phase A and phase B in aqueous two-phase extraction all affect the moisturizing effect.

[0135] Test Example 5

[0136] Anti-aging efficacy test

[0137] Test method: Human fibroblasts in logarithmic growth phase with good morphology were seeded in 6-well plates at a density of 2.5 × 10⁻⁶. 5Cells / wells were incubated in an incubator for 24 h. A model group (H2O2 group) and a sample group were set up, with three replicates for each concentration. A suitable concentration of H2O2 (1.4 mM) was added for induction treatment for 2 h, followed by drug administration and incubation at 37 ℃ in a 5% CO2 incubator for 24 h. Cell supernatants were analyzed according to the instructions of the MMP-1 ELISA kit, and the MMP-1 content in the collected cell supernatants was measured. The experimental results were analyzed using data processing software.

[0138] The effects of Taxus wallichiana extract (50 μg / mL) obtained in Examples 1-9 and Comparative Examples 1-7 on the relative expression level of MMP1 (the relative expression level was lower than that in the model group, and the lower the relative expression level, the stronger the anti-aging effect) are shown in Table 5 below.

[0139] Table 5

[0140]

[0141] The above results indicate that the southern yew extract prepared by this invention has good anti-aging effects, and the choice of adsorbent, pH of adsorption treatment, extraction method, and the choice of phase A and phase B in aqueous two-phase extraction all affect the anti-aging effects.

[0142] Test Example 6

[0143] Security test

[0144] This test case refers to the "Cosmetic Safety Technical Specifications" (2015 edition) and conducts human skin patch experiments on the Taxus wallichiana extract (mass concentration of 0.3%) obtained in Examples 1-9 and Comparative Examples 1-7.

[0145] The human skin patch test was conducted using the following method:

[0146] Thirty volunteers were randomly recruited (male to female ratio 1:1), with no history of allergies, and aged 20-50 years. Sixteen samples (0.01 g each) were weighed from each volunteer, and 0.01 g of pure water was added and mixed thoroughly. The mixture was then placed in a patch and pressed onto the volunteer's designated upper back, gently pressing to remove air bubbles. During the experiment, volunteers were instructed to avoid strenuous exercise, scratching, and bathing. Skin reactions were observed at 0.5 h, 24 h, and 48 h.

[0147] The criteria for evaluating adverse skin reactions in patch testing are shown in Table 6.

[0148] Table 6

[0149]

[0150] The results of the patch test are summarized in Table 7.

[0151] Table 7

[0152]

[0153] The patch test results above indicate that, according to the relevant provisions of the "Cosmetic Safety Technical Specifications," the samples of *Taxus chinensis* extract obtained in Examples 1-9 and Comparative Examples 1-7 will not cause adverse reactions to human skin. However, in the comparative examples, Comparative Examples 2 and 6 showed 5 and 4 cases of Grade 2 reactions, respectively, approaching the safety threshold. This suggests that using conventional methods to prepare *Taxus chinensis* extract carries a certain potential for irritation, and arbitrarily changing the types of inorganic salts in the aqueous two-phase extraction technique may also pose a potential for irritation.

[0154] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing an extract of Taxus chinensis, characterized in that, The preparation method includes: (1) Mix the southern yew with the aqueous two-phase system, extract, let stand to separate the phases, collect phase A and filter to obtain the filtrate; (2) The filtrate is treated with an adsorbent and then filtered to obtain the final product; The aqueous two-phase system includes phase A and phase B; Phase A comprises ethanol and behenamidopropyl dimethylamine; Phase B comprises inorganic salts and water.

2. The method for preparing the Taxus wallichiana extract according to claim 1, characterized in that, The aqueous two-phase system comprises, by mass, 10-20 parts of phase A and 80-100 parts of phase B; Preferably, the mass ratio of inorganic salt to water in phase B is (20-30):(50-70); Preferably, the mass ratio of ethanol to behenamidopropyl dimethylamine in phase A is (500-1000):1; Preferably, the inorganic salt includes any one or a combination of at least two of sodium carbonate, sodium bicarbonate, and ammonium sulfate.

3. The method for preparing the extract of Taxus wallichiana according to claim 1 or 2, characterized in that, The ratio of the Southern Yew to the aqueous two-phase system is 1 g:(10-40) mL.

4. The method for preparing the extract of Taxus wallichiana according to any one of claims 1-3, characterized in that, The extraction temperature is 60-80℃, the extraction time for a single extraction is 1-3 hours, and the number of extractions is 1-4 times.

5. The method for preparing the extract of Taxus wallichiana according to any one of claims 1-4, characterized in that, The adsorbent includes behenamidopropyl dimethylamine and behenyltrimethylammonium chloride; Preferably, the mass ratio of behenamidopropyl dimethylamine to behenyltrimethylammonium chloride is (1-5):(1-5).

6. The method for preparing the extract of Taxus wallichiana according to any one of claims 1-5, characterized in that, The adsorbent treatment is carried out at a temperature of 85-95℃ for 3-4 hours, with a stirring speed of 100-500 rpm.

7. The method for preparing the extract of Taxus wallichiana according to any one of claims 1-6, characterized in that, Before using the adsorbent, the pH of the filtrate is adjusted to 9-10 using a pH adjuster. Preferably, the pH adjuster comprises any one or a combination of at least two of sodium hydroxide, arginine, and aminomethylpropanol; Preferably, the mass of the adsorbent is 0.01-0.05% of the mass of the filtrate.

8. The Taxus chinensis extract prepared by the method according to any one of claims 1-7.

9. The use of the Taxus wallichiana extract according to claim 8 in the preparation of products with antioxidant, soothing, moisturizing or anti-aging effects.

10. The use of the Taxus wallichiana extract according to claim 8 in the preparation of inflammatory factor inhibitors, AQP3 gene activators, or MMP-1 gene inhibitors.

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