An extract of taxodium distichum and its preparation method and application

Abstract

This invention provides an extract of *Lycopodium clavatum*, its preparation method, and its application. The preparation method includes the following steps: *Lycopodium clavatum* powder is extracted with alcohol; the resulting extract is concentrated under reduced pressure to obtain a crude extract; the crude extract is centrifuged, and supernatant I is collected; the precipitate is reconstituted and centrifuged again to collect supernatant II; supernatant I and supernatant II are combined to obtain a purified solution; the purified solution is eluted in stages using a macroporous resin, and the last eluent is collected, concentrated, and dried to obtain the *Lycopodium clavatum* extract. The preparation method provided by this invention can effectively enrich saponins, flavonoids, polyphenols, and lycoclavanin in *Lycopodium clavatum*, allowing for precise control and stable content of active ingredients; the prepared *Lycopodium clavatum* extract has good antioxidant and soothing effects and can be used in cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to an extract of Lycopodium clavatum, its preparation method, and its application. Background Technology

[0002] *Palhinhaea cernua* (L.) Vasc. et Franco is a medium to large-sized terrestrial plant belonging to the genus *Palhinhaea* (Lycopodiaceae P. Beauv. ex Mirb.). It is mainly distributed in South China and eastern Southwest China, and is native to China. In China, it primarily grows in shady places under forests and shrubs, or on rocks at altitudes of 100-1800 meters, and is found in Yunnan, Guizhou, Jiangxi, Hunan, and other regions.

[0003] Lycopodium is a traditional Chinese medicine and one of the source plants of the Chinese herbal medicine *Shenjincao*. The whole plant and its spores can be used medicinally. According to *Lu Chuan Ben Cao* (a traditional Chinese medicine text), Lycopodium has effects such as "anti-inflammatory and hemostatic, clearing the liver and improving eyesight." Modern pharmacological studies have shown that Lycopodium can reduce fever, promote blood circulation, soothe the liver and detoxify, promote intestinal peristalsis, and aid sleep. It can be used to treat numbness in the limbs, hematochezia, infantile convulsions, burns, traumatic injuries, hepatitis, and urticaria. Clinically, it is also commonly used to treat acute gastroenteritis and rheumatoid arthritis. However, research on *Lycopodium clavatum* is relatively limited.

[0004] Current research on Lycopodium clavatum mainly focuses on its chemical composition. The active ingredients found in Lycopodium clavatum are mainly alkaloids such as Lycopodium clavatum alkaloid, hydroxy Lycopodium clavatum alkaloid, and nicotine, as well as flavonoids and triterpenoids. A total of 34 chemical components have been identified.

[0005] The compound lycoclavanin has not been previously reported in the literature for plants of the genus Lycopodium.

[0006] There is relatively little research on Lycopodium clavatum. The separation and purification of its chemical components, such as CN108721343A, often uses reagents such as ethyl acetate and petroleum ether, which are harmful to the environment and health. This is not conducive to environmentally friendly and sustainable research. Furthermore, no research has yet been published on the application of Lycopodium clavatum extract in the field of skin care products.

[0007] Therefore, developing a simple process, a clearly defined active ingredient, and a well-defined quality standard for the extract of Lycopodium clavatum is a key research focus in this field. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a *Lycopodium clavatum* extract, its preparation method, and its application. The preparation method is simple, the effective components in the obtained *Lycopodium clavatum* extract are clearly defined, and it can be applied to cosmetics to exert antioxidant, anti-inflammatory, and soothing effects.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing an extract of Lycopodium clavatum, characterized in that the preparation method includes the following steps:

[0011] (1) Extract the powder of Lycopodium clavatum with alcohol, and concentrate the resulting extract under reduced pressure to obtain a crude extract.

[0012] (2) Centrifuge the crude extract, collect supernatant I, redissolve the precipitate and centrifuge again, collect supernatant II, combine supernatant I and supernatant II to obtain the purified solution;

[0013] (3) The solution to be purified is eluted in stages using macroporous resin. The last eluent is collected, concentrated and dried to obtain the extract of Lycopodium clavatum.

[0014] Preferably, the particle size of the *Lycopodium clavatum* powder is 70-100 mesh, for example, 70 mesh, 80 mesh, 90 mesh, 100 mesh, etc.

[0015] Preferably, the *Lycopodium yunnanense* is the dried whole herb of *Lycopodium yunnanense*, a plant belonging to the genus *Lycopodium* of the family Lycopodiaceae.

[0016] Preferably, the extraction agent used is an aqueous ethanol solution, wherein the mass percentage of ethanol in the aqueous ethanol solution is 60-80%, for example, it can be 65%, 70%, 75%, 80%, etc., preferably 70%.

[0017] Preferably, the material-to-liquid ratio during extraction is 1:(5-10), for example, it can be 1:6, 1:7, 1:8, 1:9, 1:10, etc., and preferably 1:10.

[0018] Preferably, the extraction is performed independently 1-3 times, for example, once, twice or three times, preferably twice.

[0019] Preferably, the extraction time is 1-3 hours, for example, 1.5 hours, 2 hours, 2.5 hours, etc.

[0020] Preferably, the temperature for vacuum concentration is 45-65℃, for example, it can be 46℃, 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, etc.

[0021] Preferably, the mass ratio of the crude extract to the powdered Lycopodium clavatum is (0.5-1.5):1, for example, it can be 0.6:1, 0.8:1, 1:1, 1.2:1, 1.4:1, etc.

[0022] Preferably, in step (2), the centrifugation speed is independently 3000-16000 rpm, for example, it can be 4000 rpm, 6000 rpm, 8000 rpm, 10000 rpm, 12000 rpm, 14000 rpm, etc.

[0023] Preferably, the resolution is performed using a solvent to crude extract volume ratio of (0.8-1.2):1, for example, 0.9:1, 1:1, 1.1:1, etc.

[0024] Preferably, the solvent used for the resolution is water.

[0025] Preferably, the mass ratio of the macroporous resin to the *Lycopodium clavatum* powder is (3-5):1, for example, it can be 3.5:1, 4:1, 4.5:1, etc.

[0026] Preferably, the macroporous resin is a D-101 type macroporous resin.

[0027] Preferably, the loading rate during segmented elution is 0.4-0.6 column volumes / h, for example, 0.45 column volumes / h, 0.5 column volumes / h, 0.55 column volumes / h, etc.

[0028] Preferably, the segmented elution is performed sequentially using solvent I, solvent II, and solvent III.

[0029] Preferably, the solvent I is water, and the elution is carried out for 2-5 column volumes, for example, 2 column volumes, 3 column volumes, 4 column volumes or 5 column volumes.

[0030] Preferably, solvent II is an aqueous ethanol solution, wherein the mass percentage of ethanol in the aqueous ethanol solution is 15-25% (e.g., 16%, 18%, 20%, 22%, 24%, etc.), and the elution is performed for 3-8 column volumes, such as 4, 5, 6, 7 column volumes, etc.

[0031] Preferably, solvent III is an aqueous ethanol solution, wherein the mass percentage of ethanol in the aqueous ethanol solution is 80-90% (e.g., 82%, 84%, 86%, 88%, etc.), and the elution is performed for 5-10 column volumes, such as 6, 7, 8, 9 column volumes, etc.

[0032] Preferably, the preparation method includes the following steps:

[0033] (1) Extract 70-100 mesh Lycopodium clavatum powder with alcohol at a material-to-liquid ratio of 1:(5-10). The extractant is an aqueous ethanol solution with a mass percentage of 60-80%. The extraction is carried out independently 1-3 times, with each extraction lasting 1-3 hours. The resulting extract is concentrated under reduced pressure at 45-65℃ to obtain a crude extract. The mass ratio of the crude extract to the Lycopodium clavatum powder is (0.5-1.5):1.

[0034] (2) Centrifuge the crude extract at 3000-16000 rpm and collect supernatant I. Redissolve the precipitate with 0.8-1.2 times the amount of solvent and centrifuge again at 3000-16000 rpm to collect supernatant II. Combine supernatant I and supernatant II to obtain the solution to be purified.

[0035] (3) The solution to be purified is eluted in stages using D-101 macroporous resin. The mass ratio of macroporous resin to Lycopodium clavatum powder is (3-5):1. The loading rate is 0.4-0.6 column volumes / h. During the staged elution, water is used to elute for 2-5 column volumes, followed by elution with 15-25% ethanol aqueous solution for 3-8 column volumes, and finally elution with 80-90% ethanol aqueous solution for 5-10 column volumes. The last eluent is collected, concentrated and dried to obtain the Lycopodium clavatum extract.

[0036] In a second aspect, the present invention provides a *Lycopodium clavatum* extract, which is prepared by the preparation method described in the first aspect.

[0037] Preferably, the saponin content in the *Lycopodium clavatum* extract is 30.74-42.96% by mass, for example, it can be 31%, 34%, 36%, 38%, 40%, 42%, etc.

[0038] Preferably, the polyphenol content in the *Lycopodium clavatum* extract is 8.96-12.67% by mass, for example, it can be 9%, 10%, 11%, 12%, etc.

[0039] Preferably, the flavonoid content in the *Lycopodium clavatum* extract is 7.91-10.48% by mass, for example, it can be 8%, 8.5%, 9%, 9.5%, 10%, etc.

[0040] Preferably, the mass percentage of lycoclavanin in the *Lycoclavaninus sylvestris* extract is 11.32-14.76%, for example, it can be 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, etc.

[0041] Thirdly, the present invention provides an application of the *Lycopodium clavatum* extract described in the second aspect in cosmetics.

[0042] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The preparation method provided by the present invention is simple and can effectively enrich saponins, flavonoids, polyphenols and lycoclavanin in Lycopodium clavatum. The effective components in the obtained Lycopodium clavatum extract can be precisely controlled and the content is stable.

[0045] 2. The *Lycopodium clavatum* extract provided by this invention can effectively remove DPPH and inhibit NO and TRPV1, exhibiting good antioxidant and soothing effects, and can be applied in cosmetics. Attached Figure Description

[0046] Figure 1 The HPLC chromatogram of the *Lycopodium clavatum* extract prepared in Example 1 is shown below.

[0047] Figure 2 The HPLC chromatogram of lycoclavanin isolated from the lycoclavanin extract prepared in Example 1 is shown below.

[0048] Figure 3 The DPPH free radical scavenging rate of the *Lycopodium clavatum* extract prepared in Example 1 at different concentrations. Detailed Implementation

[0049] 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.

[0050] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not limited to those elements and may also include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0051] "Optional" or "any one" means that the matter or event described thereafter may or may not occur, and the description includes both the possibility that the event will occur and the possibility that the event will not occur.

[0052] The indefinite articles “a” and “an” preceding an element or component of this invention do not impose any limitation on the quantity (i.e., number of times) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0053] The terms "one embodiment," "some embodiments," "exemplary," "specific example," or "some examples," etc., used in this invention refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this document, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example.

[0054] In this invention, unless otherwise explicitly stated, percentages and contents are all by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available.

[0055] Example 1

[0056] This embodiment provides an extract of Lycopodium clavatum, the preparation method of which is as follows:

[0057] (1) Take 50g of dried whole herb of Yunnan Lycopodium clavatum, grind it into 80-mesh fine powder, put it into an extraction tank, and extract it twice with 500g of 70% ethanol for 2 hours each time. Combine the extracts and concentrate them under reduced pressure at 60℃ to 25g to obtain crude extract.

[0058] (2) Centrifuge the crude extract at 3000 rpm, keep the supernatant for later use, redissolve the precipitate in 30g of water, centrifuge again, combine the supernatant obtained from centrifugation with the supernatant from the first centrifugation to obtain the solution to be purified;

[0059] (3) Take 220g of D-101 macroporous resin and pack it into the column. Wash it with water until there is no alcohol smell. Then, take the purification solution from step (2) and load it onto the column for adsorption. The adsorption rate is 0.5 column volumes per hour. After loading, let it stand for 2 hours.

[0060] (4) Perform segmented elution on the adsorption column from step (3):

[0061] ① Wash with water for 2 column volumes;

[0062] ② Elute with 20% ethanol for 4 column volumes;

[0063] ③ Elute with 85% ethanol for 8 column volumes;

[0064] The eluent obtained from the third elution was collected, concentrated, and dried to obtain the *Lycopodium clavatum* extract.

[0065] Example 2

[0066] This embodiment provides an extract of Lycopodium clavatum, the preparation method of which is as follows:

[0067] (1) Take 50g of dried whole herb of Yunnan Lycopodium clavatum, grind it into 80-mesh fine powder, put it into an extraction tank, and extract it three times with 250g of 80% ethanol for 1 hour each time. Combine the extracts and concentrate them under reduced pressure at 55℃ to 30g to obtain crude extract.

[0068] (2) Centrifuge the crude extract at 5000 rpm, keep the supernatant for later use, redissolve the precipitate in 30g of water, centrifuge again, combine the supernatant obtained from centrifugation with the supernatant from the first centrifugation to obtain the solution to be purified;

[0069] (3) Take 150g of D-101 macroporous resin and pack it into the column. Wash it with water until there is no alcohol smell. Then, take the purification solution from step (2) and load it onto the column for adsorption. The adsorption rate is 0.5 column volumes per hour. After loading, let it stand for 2 hours.

[0070] (4) Perform segmented elution on the adsorption column from step (3):

[0071] ① Wash with water for 4 column volumes;

[0072] ② Elute with 25% ethanol for 3 column volumes;

[0073] ③ Elute with 90% ethanol for 5 column volumes;

[0074] The eluent obtained from the third elution was collected, concentrated, and dried to obtain the *Lycopodium clavatum* extract.

[0075] Example 3

[0076] This embodiment provides an extract of Lycopodium clavatum, the preparation method of which is as follows:

[0077] (1) Take 50g of dried whole herb of Lycopodium yunnanense, grind it into 80-mesh fine powder, put it into an extraction tank, and extract it once by reflux with 450g of 60% ethanol for 3 hours. Combine the extracts and concentrate them under reduced pressure at 45℃ to 40g to obtain crude extract.

[0078] (2) Centrifuge the crude extract at 5000 rpm, keep the supernatant for later use, redissolve the precipitate in 40g of water, centrifuge again, combine the supernatant obtained from centrifugation with the supernatant from the first centrifugation to obtain the solution to be purified;

[0079] (3) Take 250g of D-101 macroporous resin and pack it into the column. Wash it with water until there is no alcohol smell. Then, take the purification solution from step (2) and load it onto the column for adsorption. The adsorption rate is 0.5 column volumes per hour. After loading, let it stand for 2 hours.

[0080] (4) Perform segmented elution on the adsorption column from step (3):

[0081] ① Wash with water for 5 column volumes;

[0082] ② Elute with 15% ethanol for 8 column volumes;

[0083] ③ Elute with 80% ethanol for 10 column volumes;

[0084] The eluent obtained from the third elution was collected, concentrated, and dried to obtain the *Lycopodium clavatum* extract.

[0085] Example 4

[0086] This embodiment provides a *Lycopodium clavatum* extract, which differs from Example 1 only in that the D-101 macroporous resin in step (3) is replaced with an equal amount of AB-8 macroporous resin; other raw materials, dosages and preparation methods are the same as in Example 1.

[0087] Example 5

[0088] This embodiment provides a *Lycopodium clavatum* extract, which differs from Example 1 only in that the 20% ethanol in step (4) is replaced with an equal amount of 55% ethanol; other raw materials, dosages and preparation methods are the same as in Example 1.

[0089] Example 6

[0090] This embodiment provides a *Lycopodium clavatum* extract, which differs from Example 1 only in that 85% ethanol in step (4) is replaced with an equal amount of 70% ethanol; other raw materials, dosages and preparation methods are the same as in Example 1.

[0091] Example 7

[0092] This embodiment provides a Lycopodium clavatum extract, which differs from Example 1 only in that the 70% ethanol in step (1) is replaced with an equal amount of 30% ethanol; other raw materials, dosages and preparation methods are the same as in Example 1.

[0093] Example 8

[0094] This embodiment provides a Lycopodium clavatum extract, which differs from Example 1 only in that the 70% ethanol in step (1) is replaced with an equal amount of 95% ethanol; other raw materials, dosages and preparation methods are the same as in Example 1.

[0095] Comparative Example 1

[0096] This comparative example provides a *Lycopodium clavatum* extract, which differs from Example 1 only in that 70% ethanol in step (1) is replaced with an equal amount of water; other raw materials, dosages and preparation methods are the same as in Example 1.

[0097] Test Example 1

[0098] Lycoclavanin content test

[0099] The monomeric compound lycoclavanin in the extract of Lycopodium clavatum from Example 1 was separated and purified by high performance liquid chromatography.

[0100] The preparative liquid chromatography column used was an XBridge Prep; the inner diameter of the column was 19 mm, the length of the column was 150 mm, the packing material was OBD C18, and the particle size of the packing material was 5 micrometers.

[0101] The conditions used for preparative liquid chromatography were as follows: mobile phase A was water; mobile phase B was acetonitrile, with gradient elution. The elution flow rate was 10 mL / min; the detection wavelength was 256 nm; and the preparative column temperature was 25 °C.

[0102] The obtained monomer compound lycoclavanin had a purity greater than 99.0%. NMR analysis confirmed that its structure was consistent with that of the reported triterpenoid compound lycoclavanin from *Lycopodium clavatum* and *Lycopodium clavatum* from Yunnan. Therefore, the monomer compound in the *Lycopodium clavatum* extract of Example 1 was identified as lycoclavanin. The test results are compared with those in the references in Table 1.

[0103] Using the lycoclavanin obtained from the aforementioned isolation and purification as a reference standard, the lycoclavanin content in the *Lycopodium clavatum* extracts prepared in each example and comparative example was determined: lycoclavanin was prepared into solutions with concentrations of 0.1250 mg / mL, 0.1500 mg / mL, 0.2500 mg / mL, 0.3750 mg / mL, 0.6250 mg / mL, and 0.8750 mg / mL; the *Lycopodium clavatum* extracts prepared in each example and comparative example were prepared into solutions with a concentration of 2 mg / mL. The standard and the analyte were analyzed using LC-MS, and lycoclavanin was determined using MS and retention time. A standard curve was plotted using the integrated area and lycoclavanin concentration.

[0104] Y = 2513.6X + 3.9328, R 2 =1 (Y: concentration, X: integral area). The test results of the *Lycopodium clavatum* extract prepared in each example and comparative example are shown in Table 2.

[0105] For example, the HPLC chromatogram of the Lycopodium clavatum extract prepared in Example 1 is as follows: Figure 1 As shown; the HPLC chromatogram of the isolated lycoclavanin is as follows. Figure 2 As shown.

[0106] Table 1

[0107]

[0108]

[0109] Test Example 2

[0110] Using rutin as a reference standard, the total flavonoid content in the extracts of *Lycopodium clavatum* prepared in each example and comparative example was determined by the aluminum nitrate colorimetric method.

[0111] Prepare solutions of rutin standard with concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0.03125 mg / mL; prepare solutions of each lycopodium extract to be tested with a concentration of 1 mg / mL. Perform colorimetric reactions of total flavonoids using the aluminum nitrate method on both the standard and the test samples, and measure the absorbance. Plot a standard curve for the rutin standard using absorbance and flavonoid concentration as follows:

[0112] Y = 0.7654X - 0.0044, R 2 =0.9992 (Y: flavonoid concentration, X: absorbance);

[0113] The test results are shown in Table 2.

[0114] Test Example 3

[0115] Using ursolic acid as a reference standard, the total saponin content in the extracts of *Lycopodium clavatum* prepared in each example and comparative example was determined by the perchloric acid method.

[0116] Prepare a 1 mg / mL solution of ursolic acid standard. Take 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of each solution and place them in test tubes, then dry them. Prepare a 2 mg / mL solution of each of the tested *Lycopodium clavatum* extracts. Take 100 μL of each solution and place it in a test tube, then dry it. Add 400 μL of 5% vanillin-acetic acid solution and 1.6 mL of perchloric acid sequentially. After reacting for 20 min, add 5 mL of acetic acid and measure the absorbance. Plot a standard curve using absorbance and standard concentration as shown below:

[0117] Y = 4.0592X + 0.0293, R 2 =0.9903 (Y: absorbance, X: total saponin concentration);

[0118] The test results are shown in Table 2.

[0119] Test Example 4

[0120] The total polyphenol content in the extracts of *Lycopodium clavatum* prepared in each example and comparative example was determined using gallic acid as a reference standard.

[0121] Gallic acid was prepared into standard solutions at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0.03125 mg / mL. The *Lycopodium clavatum* extract from Example 1 was prepared into a 0.03 mg / mL sample solution. 100 μL of each of the standard solutions and sample solutions were added to folin-Ciocalteu and sodium carbonate solutions, and the mixtures were reacted in the dark for 1 hour. The absorbance was measured at 760 nm, and the standard curves were plotted as follows:

[0122] Y = 37.933X + 0.0787, R 2 =0.9984 (Y: absorbance, X: total polyphenol concentration);

[0123] The test results are shown in Table 2.

[0124] Table 2

[0125]

[0126] According to the table data, the extract of *Lycopodium clavatum* prepared using the method provided by this invention has a high content of total flavonoids, total saponins, total polyphenols, and lycoclavanin. Examples 1-3 and 4 show that replacing the D-101 macroporous resin with other types of resin reduces the adsorption and enrichment of effective components such as total saponins, total flavonoids, and total polyphenols. Examples 1 and 5-6 show that when eluting the adsorption column, a segmented process of water-low concentration ethanol-high concentration ethanol should be used. If the concentration of low concentration ethanol is slightly higher, or the concentration of high concentration ethanol is lower... Slightly lower concentrations will reduce the enrichment of effective components such as total saponins, total flavonoids, and total polyphenols in the product. As shown in Examples 1 and 7-8, when extracting Lycopodium clavatum with alcohol, if the concentration of the ethanol solution is too low, the effective components such as total saponins, total flavonoids, and total polyphenols cannot be fully extracted. If the concentration is too high, although the content of effective components such as total saponins, total flavonoids, and total polyphenols is considerable, there are more impurities. As shown in Examples 1 and Comparative Example 1, when extracting Lycopodium clavatum with water, the total saponins cannot be fully extracted. The content of total saponins and Lycoclavanin is significantly reduced compared to alcohol extraction.

[0127] Test Example 5

[0128] Using vitamin C as a control, the DPPH free radical scavenging activity of the *Lycopodium clavatum* extract in Example 1 was determined.

[0129] Prepare solutions of vitamin C standard with concentrations of 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.0312 mg / mL, and 0.0156 mg / mL, and take 1 mL of each solution for later use. Prepare solutions of *Lycopodium clavatum* extract prepared in each example and comparative example with a concentration of 1 mg / mL, and take 1 mL of each solution for later use. Prepare a solution of DPPH with a concentration of 0.51 mg / mL, and add 1 mL of each solution to the test sample and the control sample. After reacting for 30 minutes, measure the absorbance and plot a curve using absorbance and sample concentration.

[0130] The results showed that under these conditions, vitamin C had an IC50 value for scavenging DPPH free radicals. 50 The IC50 concentration of the *Lycopodium clavatum* extract prepared in each example and comparative example was 0.024 mg / mL, indicating its effectiveness against DPPH free radical scavenging. 50 The values ​​are shown in Table 3. For example, the DPPH free radical scavenging rate of the *Lycopodium clavatum* extract prepared in Example 1 at different concentrations is shown in Table 3. Figure 3 As shown.

[0131] Test Example 6

[0132] The NO-inhibiting effect of the extracts of *Lycopodium clavatum* prepared in each example and comparative example was determined using the mouse macrophage RAW264.7 model.

[0133] Mouse macrophages RAW264.7 were selected and seeded in 96-well plates with an initial cell number of 10. 4 -10 5 After the cell number increased to a certain extent, the cells were treated with 60 μg / mL of the tested *Lycopodium clavatum* extract and 10 μg / mL of the positive control drug dexamethasone sodium phosphate for 1 h. After 1 h of drug intervention, LPS lipopolysaccharide was added, and the cells were cultured for another 24 h. The supernatant was collected, and the NO production capacity of the samples was detected using a NO kit. The absorbance was measured at 540 nm to test the anti-inflammatory efficacy of the *Lycopodium clavatum* extract. The results showed that the relative expression level of NO secretion by LPS lipopolysaccharide was set at 1.0, and the relative expression level of NO secretion by the positive control drug dexamethasone sodium phosphate (DEX) was 0.8. The test results of the *Lycopodium clavatum* extract prepared in the examples and comparative examples are shown in Table 3.

[0134] Test Example 7

[0135] A keratinocyte model was selected to determine the inhibitory effect of capsaicin receptor (TRPV1) on the extracts of *Lycopodium clavatum* prepared in each example and comparative example.

[0136] Keratinocytes were selected and revived. Once the cell deposition rate reached approximately 60%, cells were seeded into 24-well plates and incubated overnight in CO2. When the cell deposition rate in the 24-well plates reached 40-60%, 0.0031 mg / mL of *Lycopodium clavatum* extract and 15.6 μg / mL of the positive control drug trans-4-tert-butylcyclohexanol were administered for stimulation. After administration, the 24-well plates were cultured for 24 h. The plates were then fixed with 4% paraformaldehyde for 30 min, and immunofluorescence assays of the capsaicin receptor (TRPV1) were performed. Microscopic observation and calculation of the capsaicin receptor (TRPV1) inhibition of the *Lycopodium clavatum* extracts prepared in each example and comparative example were performed. The results are shown in Table 3.

[0137] Table 3

[0138]

[0139]

[0140] According to the table data, the *Lycopodium clavatum* extract prepared using the method provided in this invention exhibits the highest DPPH free radical scavenging rate and the strongest inhibitory effect on NO generation and capsaicin receptor (TRPV1), demonstrating excellent antioxidant and anti-inflammatory soothing effects. Examples 1 and 4 show that replacing the D-101 macroporous resin with other types of resin reduces the extract's DDPH free radical scavenging ability and decreases its anti-inflammatory and soothing effects. Examples 1 and 5-6 indicate that when eluting the adsorption column, a water-low concentration ethanol-high concentration ethanol separation process should be used. The extraction process was carried out in stages, and the effect was best when the ethanol concentration was within a specific range. Otherwise, the extract's ability to scavenge DPPH free radicals and its inhibitory effect on NO and TRPV1 activity decreased. As shown in Examples 1 and 7-8, when extracting Lycopodium clavatum with ethanol, if the concentration of the ethanol solution was too low, the scavenging ability of DPPH free radicals and the inhibitory effect on NO and TRPV1 activity were not good. If the concentration was too high, better antioxidant, anti-inflammatory and soothing effects could not be achieved. As shown in Examples 1 and Comparative Example 1, when extracting Lycopodium clavatum with water, the antioxidant, anti-inflammatory and soothing effects of the extract were not as good as those of the ethanol extract.

[0141] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0142] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0143] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing an extract of Lycopodium clavatum, characterized in that, The preparation method includes the following steps: (1) Extract the powder of Lycopodium clavatum with alcohol, and concentrate the resulting extract under reduced pressure to obtain a crude extract; (2) Centrifuge the crude extract, collect supernatant I, redissolve the precipitate and centrifuge again, collect supernatant II, combine supernatant I and supernatant II to obtain the solution to be purified; (3) The solution to be purified is eluted in stages using macroporous resin. The last eluent is collected, concentrated and dried to obtain the extract of Lycopodium clavatum. The extraction agent used is an aqueous ethanol solution, and the material-to-liquid ratio during extraction is 1:(5-10). The ethanol solution contains 60-80% ethanol by mass. The extraction is carried out independently 1-3 times, and the extraction time for each extraction is 1-3 hours. The macroporous resin is type D-101 macroporous resin; The segmented elution process uses solvent I, solvent II, and solvent III sequentially. Solvent I is water, and elution is performed for 2-5 column volumes. Solvent II is an aqueous ethanol solution, wherein the ethanol aqueous solution contains 15-25% ethanol by mass, and the elution is performed for 3-8 column volumes. Solvent III is an aqueous ethanol solution with a mass percentage of 80-90% ethanol, and the elution time is 5-10 column volumes.

2. The preparation method according to claim 1, characterized in that, The particle size of the *Lycopodium clavatum* powder is 70-100 mesh.

3. The preparation method according to claim 1, characterized in that, The *Lycopodium yunnanense* is the dried whole herb of *Lycopodium yunnanense*, a plant belonging to the genus *Lycopodium* in the family Lycopodiaceae.

4. The preparation method according to claim 1, characterized in that, The temperature for vacuum concentration is 45-65℃.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the crude extract to the powdered Lycopodium clavatum is (0.5-1.5):

1.

6. The preparation method according to claim 1, characterized in that, In step (2), the centrifugation speed is independently 3000-16000 rpm.

7. The preparation method according to claim 1, characterized in that, The resolution is performed using a solvent to crude extract volume ratio of (0.8-1.2):

1.

8. The preparation method according to claim 1, characterized in that, The mass ratio of the macroporous resin to the *Lycopodium clavatum* powder is (3-5):

1.

9. The preparation method according to claim 1, characterized in that, The loading rate for the segmented elution is 0.4-0.6 column volumes / h.

10. The preparation method according to any one of claims 1-9, characterized in that, The preparation method includes the following steps: (1) Extract 70-100 mesh Lycopodium clavatum powder with alcohol at a material-to-liquid ratio of 1:(5-10). The extractant is an aqueous ethanol solution with a mass percentage of 60-80%. The extraction is carried out independently 1-3 times, with each extraction lasting 1-3 hours. The resulting extract is concentrated under reduced pressure at 45-65℃ to obtain a crude extract. The mass ratio of the crude extract to the Lycopodium clavatum powder is (0.5-1.5):

1. (2) Centrifuge the crude extract at 3000-16000 rpm, collect supernatant I, redissolve the precipitate with 0.8-1.2 times the amount of solvent, centrifuge again at 3000-16000 rpm, collect supernatant II, combine supernatant I and supernatant II to obtain the purified solution; (3) The solution to be purified is eluted in stages using D-101 macroporous resin. The mass ratio of macroporous resin to Lycopodium clavatum powder is (3-5):

1. The loading rate is 0.4-0.6 column volumes / h. During the staged elution, water is used to elute for 2-5 column volumes, followed by elution with 15-25% ethanol aqueous solution for 3-8 column volumes, and finally elution with 80-90% ethanol aqueous solution for 5-10 column volumes. The last eluent is collected, concentrated and dried to obtain the Lycopodium clavatum extract.

11. An extract of *Lycopodium clavatum*, characterized in that, The *Lycopodium clavatum* extract was prepared using the preparation method described in any one of claims 1-10.

12. The *Lycopodium clavatum* extract according to claim 11, characterized in that, The saponin content in the *Lycopodium clavatum* extract reached a maximum of 42.96% by mass.

13. The Lycopodium extract according to claim 11, characterized in that, The polyphenol content in the *Lycopodium clavatum* extract reached a maximum of 12.67% by mass.

14. The Lycopodium extract according to claim 11, characterized in that, The flavonoid content in the *Lycopodium clavatum* extract reached a maximum of 10.48% by mass.

15. The Lycopodium extract according to claim 11, characterized in that, The lycoclavanin content in the *Lycopodium clavatum* extract reached a maximum of 14.76% by mass.

16. The use of the *Lycopodium clavatum* extract as described in any one of claims 11-15 in the preparation of cosmetics having antioxidant or soothing effects.

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