CN116898780A - Anoectochilus roxburghii extract and preparation process and application thereof - Google Patents

Abstract

This application proposes a Anoectochilus extract and its preparation process and application. The preparation process of the Anomatis extract includes: pulverizing the dried whole Alematophora plant with a grinder and sieving to obtain dry powder; adding the dry powder to an ethanol solvent and performing ultrasonic extraction at room temperature to obtain a crude extract of Anomatis; Filter the crude extract of Anomatis to remove the residue and obtain a filtrate; conduct membrane separation and/or concentrate the obtained filtrate under reduced pressure to remove ethanol, and dilute to volume to obtain a concentrated liquid; add cosolvents and stabilizers to the obtained concentrated liquid, and then proceed Decolorize to obtain a Anomatis decolorizing solution; filter and sterilize the Anoectochus depigmenting solution; add a preservative to the sterilized solution to obtain a Anomatis extract. Anomatis extract is prepared by a Anoectochilus extract preparation process. The application of anomatis extract is the application of anomatis extract in skin care products that are soothing, anti-inflammatory, anti-wrinkle, or repairing.

Description

A kind of Anomatis extract and its preparation process and application

Technical field

The present application relates to the technical field of skin care, and in particular to a Anoectochilus extract and its preparation process and application.

Background technique

Anoectochilus roxburghii (Wall.) Lindl., also known as Anoectochilus roxburghii (Wall.) Lindl., also known as Anoectochilus roxburghii (Wall.) Lindl., Anoectochilus roxburghii (Wall.) Lindl., also known as Anoectochilus roxburghii (Wall.) Lindl., Anoectochilus roxburghii (Wall.) Lindl. It is mainly distributed in southern provinces such as Fujian, Guangdong, and Zhejiang in my country. Fujian is the authentic production area of Anomatis. The whole plant of Anomatis can be used as medicine. "Fujian Provincial Traditional Chinese Medicinal Materials Standards" (2006 Edition) records Anoectochilus: "This strain is a rare folk herbal medicine in Fujian and Taiwan and other provinces. Its nature, flavor and functionality are flat and sweet; it clears away heat, cools blood, Dispelling wind and dampness; used for nephritis, bronchitis, cystitis, diabetes, rheumatoid arthritis, convulsions in children, snake bites and other diseases. Anoectochilus is rich in flavonoids, polysaccharides and glycosides, amino acids, triterpenes , esters, sterols and trace elements and other substances, which have rich biological activities and can effectively resist inflammation, scavenge free radicals, anti-aging, etc. At present, the development of Anomatis products is mainly focused on fresh products, dry tea, health tea and other products On the other hand, the structure is single, the innovation ability is weak, and there are few applications and researches in skin care, which still does not meet the market demand.

The currently disclosed preparation process of Anomatis is relatively complex and is not suitable for large-scale production. In addition, the active ingredient content or biological activity obtained by some preparation processes is low, and the active ingredients are unstable, etc., which will cause a waste of Anomatis resources to a certain extent. Anomatis is known as the "King of Medicines" and "Golden Grass" and is one of the unique Chinese herbal medicines in my country. Therefore, optimizing the preparation process of Anomatis extract and improving its application value are important for fully developing Anomatis resources. significance.

Application content

The embodiments of this application provide a Anomatis extract and its preparation process and application to solve the problems existing in related technologies. The technical solutions are as follows:

In the first aspect, the embodiments of the present application provide a preparation process for Anomatis extract, including:

Crush the dried Anoectochilus whole plant with a grinder and then sieve to obtain dry powder;

Add the dry powder to the ethanol solvent and carry out ultrasonic extraction at room temperature to obtain a crude extract of Anomatis;

Filter the crude extract of Anomatis to remove residues and obtain a filtrate;

The obtained filtrate is subjected to membrane separation and/or concentration under reduced pressure to remove ethanol, and the volume is adjusted to obtain a concentrated solution;

Add a co-solvent and a stabilizer to the obtained concentrated liquid, and then perform decolorization to obtain a Anomatis decolorizing liquid;

Filter and sterilize the Anomatis decolorizing solution;

Add preservatives to the sterilized solution to obtain anomatis extract.

In one embodiment, the dry powder is added to the ethanol solvent according to the material-to-liquid ratio of 1:4-1:40, and ultrasonic extraction is performed at room temperature; wherein, the volume fraction of the ethanol solvent is 5%-70%, and the ultrasonic frequency is 480W. The ultrasonic extraction time is 10-60 minutes, and the number of extractions is 1-2 times.

In one embodiment, the volume is adjusted to 10-20 times the mass of Anomatis dry powder to obtain a concentrated solution.

In one embodiment, 20%-50% w/w co-solvent and 0.1%-5% w/w stabilizer are added to the obtained concentrated liquid.

In one embodiment, the co-solvent is one or more of glycerol, 1,3-propanediol, and 1,3-butanediol;

The stabilizer is one or more of 1,2-hexanediol, 1,2-pentanediol and p-hydroxyacetophenone.

In one embodiment, macroporous adsorption resin is used for adsorption and decolorization, and the amount of resin used is 4%-40% w/v.

In one embodiment, the macroporous adsorption resin includes one or more of D101, AB-8, XAD-2, HDP-100, HP-10, and LSD-20;

The adsorption method is static adsorption, and the adsorption time is 0.5-8h.

In one embodiment, 0.1%-0.5% w/w preservative is added to the sterilized solution; the preservative is sodium metabisulfite, phenoxyethanol, benzyl alcohol, potassium sorbate, and sodium sorbate. One or several.

In a second aspect, embodiments of the present application provide a Anomatis extract, which is prepared by any one of the above-described preparation processes for Anomatis extract.

In a third aspect, the embodiments of the present application provide an application of a Anomatis extract, which is an application of the Anomatis extract in skin care products for soothing, anti-inflammation, anti-wrinkle, or repair.

The advantages or beneficial effects of the above technical solutions include at least:

(1) The optimized extraction process of this application, including extraction material-liquid ratio, extraction solvent, extraction time, decolorization process, adding stabilizer, etc., is used to extract Anomatis chinensis to obtain Anomatis chinensis extract. The extraction process is simple and efficient. Suitable for large-scale production.

(2) The ultrasonic extraction method is used to extract the Anomatis plant, so that the rupture of the plant cell wall and the entire organism is completed in an instant, shortening the crushing time. At the same time, the vibration generated by the ultrasonic waves strengthens the release, diffusion and release of intracellular substances of Anomatis. Dissolve, thereby significantly improving the extraction efficiency and obtaining a Anomatis extract with high content of active ingredients and stable properties.

(3) The Anomatis extract obtained by this application is simultaneously enriched with a variety of active ingredients such as Anomatis glycosides, Anoectochus polysaccharides, etc. The nature of the extract is stable, the active ingredient polysaccharide content is high, the extract activity is good, and it has significant soothing, anti-inflammatory, anti-wrinkle, and repair effects. It can be used as a skin protectant in skin care products, thereby realizing the high value of Anoectochus use.

The above summary is for illustration purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the drawings and the following detailed description.

Description of the drawings

In the drawings, unless otherwise specified, the same reference numbers refer to the same or similar parts or elements throughout the several figures. The drawings are not necessarily to scale. It should be understood that these drawings depict only some embodiments disclosed in accordance with the present application and should not be considered as limiting the scope of the present application.

Figure 1 shows the result of the Anoectochilus extract of Example 1 reducing the expression level of the inflammatory factor TNF-α in RAW264.7 cells.

Figure 2 shows the result of the Anoectochilus extract of Example 1 reducing the expression level of the inflammatory factor IL-1α in RAW264.7 cells.

Figure 3 shows the results of the anoectochilus extract in Example 1 promoting the synthesis of type I collagen.

Figure 4 shows a graph showing the results of the HaCaT cell proliferation experiment using the Anomatis extract in Example 1.

Figure 5 shows the imageJ quantitative analysis diagram of the HaCaT cell migration experiment of the Anomatis extract in Example 1.

Figure 6 shows a histogram of the migration mobility of HaCaT cells derived from Anomatis chinensis extract in Example 1.

Detailed ways

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art would realize, the described embodiments may be modified in various different ways without departing from the spirit or scope of the application. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive. Unless otherwise stated, the test methods used in the following examples are all conventional methods. The raw materials, reagents, etc. used, unless otherwise stated, can be obtained from conventional commercial sources such as commercial sources.

In the first aspect, the embodiments of the present application provide a preparation process for Anomatis extract, including:

Step 101, crush.

The dried whole Anomatis plant is crushed with a grinder and then sieved to obtain dry powder.

Step 102, extraction.

The dry powder is added to the ethanol solvent, and ultrasonic extraction is performed at room temperature to obtain a crude extract of Anoectochilus.

In one embodiment, the dry powder is added to the ethanol solvent according to the material-to-liquid ratio of 1:4-1:40, and ultrasonic extraction is performed at room temperature; wherein, the volume fraction of the ethanol solvent is 5%-70%, and the ultrasonic frequency is 480W. The ultrasonic extraction time is 10-60 minutes, and the number of extractions is 1-2 times.

The main driving force of ultrasound to enhance extraction comes from the ultrasonic cavitation effect. The ultrasonic cavitation process is a process that concentrates the sound field energy and releases it quickly. It usually refers to the oscillation caused by the tiny bubbles in the liquid being activated under the action of ultrasonic waves. , growth, shrinkage, collapse and a series of dynamic processes. Ultrasonic cavitation causes turbulence effects, perturbation effects, interface effects and energy concentration effects, etc. The turbulence effect thins the boundary layer and increases the mass transfer rate; the perturbation effect strengthens the micropore diffusion; the interface effect increases the mass transfer surface area; the concentrated energy effect activates the separated material molecules; thereby strengthening the separation and purification process as a whole mass transfer rate and effect. Ultrasonic extraction of Chinese medicinal materials is not limited by the polarity and molecular weight of the ingredients, and is suitable for the extraction of most types of Chinese medicinal materials and various components. When extracting medicinal liquids, the active ingredients are easy to separate and purify; there is no chemical reaction during the entire extraction process. Little effect on the activity of the active ingredients. Ultrasonic extraction can shorten the extraction time and improve the extraction rate. Compared with conventional methods, ultrasonic extraction has the advantages of simple operation, short extraction time, high extraction efficiency and no need for heating.

The ultrasonic extraction method is used to extract the Anomatis plant, so that the rupture of the plant cell wall and the entire organism is completed in an instant, shortening the crushing time. At the same time, the vibration generated by the ultrasonic waves strengthens the release, diffusion and dissolution of the intracellular substances of Anomatis, thereby Significantly improve the extraction efficiency and obtain a Anomatis extract with high content of active ingredients and stable properties.

Step 103, filter.

Filter the crude extract of Anomatis to remove residues and obtain a filtrate.

Step 104: Concentrate to volume.

The obtained filtrate is subjected to membrane separation and/or concentration under reduced pressure to remove ethanol, and the volume is adjusted to obtain a concentrated solution.

In one embodiment, the volume is adjusted to 10-20 times the mass of Anomatis dry powder to obtain a concentrated solution.

Step 105, dissolve and decolorize.

A co-solvent and a stabilizer are added to the obtained concentrated liquid, and then decolorized to obtain a Anomatis decolorizing liquid.

Since in step 104, a trace amount of water-insoluble precipitate will precipitate out of the extract, adding a co-solvent can dissolve the precipitate, and adding a stabilizer can stabilize the heating and concentration process, resulting in physical changes in the extract such as color. This step improves the stability of the extract, improves quality such as color, improves solubility, etc.

In one embodiment, 20%-50% w/w co-solvent and

0.1%-5% w/w stabilizer.

In one embodiment, the co-solvent is one or more of glycerol, 1,3-propanediol, and 1,3-butanediol.

In one embodiment, the stabilizer is one or more of 1,2-hexanediol, 1,2-pentanediol, and p-hydroxyacetophenone.

In one embodiment, macroporous adsorption resin is used for adsorption and decolorization, and the amount of resin used is 4%-40% w/v.

In one embodiment, the macroporous adsorption resin includes one or more of D101, AB-8, XAD-2, HDP-100, HP-10, and LSD-20.

In one embodiment, the adsorption method is static adsorption, and the adsorption time is 0.5-8 hours.

Step 106, sterilization.

Filter and sterilize the Anoectochilus decolorizing solution.

Step 107, add preservative.

Add preservatives to the sterilized solution to obtain anomatis extract.

In one embodiment, 0.1%-0.5% w/w preservative is added to the sterilized solution; the preservative is sodium metabisulfite, phenoxyethanol, benzyl alcohol, potassium sorbate, and sodium sorbate. One or several.

In the second aspect, the embodiments of the present application provide an Anomatis extract, which is prepared by a Anomatis extract preparation process.

In a third aspect, the embodiments of the present application provide an application of a Anomatis extract, which is an application of the Anomatis extract in skin care products for soothing, anti-inflammation, anti-wrinkle, or repair.

Example 1

S1. Grinding: Take the dried whole plant of Anomatis and crush it with a grinder and then pass it through the No. 1 sieve to obtain 20g of Anomatis dry powder;

S2. Extraction: Add 30% ethanol solvent to the dry powder obtained in S1 at a material-to-liquid ratio of 1:15 for ultrasonic extraction once. The ultrasonic frequency is 480W and the ultrasonic extraction time is 30 minutes to obtain a crude extract of Anoectochilus;

S3. Filtration: Filter the crude extract of Anomatis obtained in S2 to remove residue;

S4. Concentration: Concentrate the filtrate obtained in S3 under reduced pressure to remove ethanol, and dilute the volume with water to 10 times the mass of Anoectochilus dry powder;

S5. Dissolve: Add glycerol, 1,2-hexanediol, and p-hydroxyacetophenone to the concentrated solution obtained in S4. The final solution is a crude drug: solution = 1:20 mass ratio, and the composition is 48.6% w/w concentrated Liquid, 50% w/w glycerol, 0.7% w/w 1,2-hexanediol and 0.7% w/w p-hydroxyacetophenone;

S6. Decolorization: Use 20% w/w XAD-2 macroporous adsorption resin for adsorption and decolorization of the solution obtained in S5 for 2 hours to obtain a Anomatis decolorization liquid;

S7. Sterilization: filter and sterilize the decolorizing liquid described in S6;

S8. Antiseptic compounding: Add 0.1% w/w sodium metabisulfite to the sterilized solution of S7 to obtain anomatis extract.

Glycerol, 1,3-propanediol, and 1,3-butanediol are used as co-solvents, all of which are miscible with water or ethanol.

In the preparation process of Anoectochilus extract, after concentration and removal of ethanol, water-insoluble active substances precipitate, which can be dissolved by adding glycerin, 1,3-propylene glycol, and 1,3-butanediol as co-solvents.

1,2-hexanediol and 1,2-pentanediol have oil-water amphipathic properties because their molecular structures contain two hydroxyl hydrophilic groups and linear alkyl hydrophobic groups. During the preparation process of the Anomatis extract, after concentration and removal of ethanol, a trace amount of water-insoluble precipitate precipitates, which can be dissolved by adding 1,2-hexanediol and 1,2-pentanediol as co-solvents. The verification experiment results are as follows:

Table 1 Effect of the addition of 1,2-hexanediol and 1,2-pentanediol on the clarity of Anomatis extract

In this embodiment, p-hydroxyacetophenone is selected as the stabilizer. Parahydroxyacetophenone is an antioxidant that can inhibit the oxidative discoloration of phenols and other pigments in plant extracts. During the preparation process of Anomatis extract, the addition of p-hydroxyacetophenone can inhibit the discoloration of Anomatis extract. The verification experiment results are as follows:

Table 2 Effect of the added amount of p-hydroxyacetophenone on the color of Anomatis extract

Example 2

S1. Crushing: Take the dried whole plant of Anomatis and grind it with a grinder and then pass it through the No. 4 sieve to obtain 50g of Anoectochilus dry powder;

S2. Extraction: Add 10% ethanol solvent to the dry powder obtained in S1 according to the material-to-liquid ratio of 1:20 for ultrasonic extraction twice. The ultrasonic frequency is 480W and the ultrasonic extraction time is 30 minutes. Combine the crude extract of Anoectochilus;

S3. Filtration: Filter the crude extract of Anomatis obtained in S2 to remove residue;

S4. Concentration: Concentrate the filtrate obtained in S3 under reduced pressure to remove ethanol, and dilute the volume with water to 15 times the mass of Anoectochilus dry powder;

S5. Dissolution: Add 1,3-propanediol, 1,2-pentanediol, and p-hydroxyacetophenone to the concentrated solution obtained in S4. The final solution is a crude drug: solution = 1:20 mass ratio, composition is 68.6% w/w concentrate, 30% w/w 1,3-propanediol, 0.7% w/w 1,2-pentanediol and 0.7% w/w p-hydroxyacetophenone;

S6. Decolorization: Use 20% w/w D101 macroporous adsorption resin to adsorb and decolorize the solution obtained in S5 for 4 hours to obtain a Anomatis decolorizing liquid;

S7. Sterilization: filter and sterilize the decolorizing liquid described in S6;

S8. Preservative compounding: Add 0.5% w/w potassium sorbate to the sterilized solution of S7 to obtain anomatis extract.

Example 3

S1. Crushing: Take the dried whole plant of Anomatis and crush it with a grinder and then pass it through the No. 2 sieve to obtain 100g of Anoectochilus dry powder;

S2. Extraction: Add 50% ethanol solvent to the dry powder obtained in S1 at a material-to-liquid ratio of 1:10 for ultrasonic extraction twice. The ultrasonic frequency is 480W and the ultrasonic extraction time is 10 minutes. Combine the crude extract of Anoectochilus;

S3. Filtration: Filter the crude extract of Anomatis obtained in S2 to remove residue;

S4. Concentration: Concentrate the filtrate obtained in S3 under reduced pressure to remove ethanol, and dilute the volume with water to 10 times the mass of Anoectochilus dry powder;

S5. Dissolution: Add 1,3-butanediol, 1,2-hexanediol, and p-hydroxyacetophenone to the concentrated solution obtained in S4. The final solution is a crude drug: solution = 1:20 mass ratio, and the composition is: 68.6% w/w concentrate, 30% w/w 1,3-butanediol, 0.7% w/w 1,2-hexanediol and 0.7% w/w p-hydroxyacetophenone;

S6. Decolorization: Use 10% w/w AB-8 and HP-10 macroporous adsorption resin for adsorption and decolorization of the solution obtained in S5 for 2 hours to obtain a Anomatis decolorization solution;

S7. Sterilization: filter and sterilize the decolorizing liquid described in S6;

S8. Preservative compounding: Add 0.5% w/w benzoic acid to the sterilized solution of S7 to obtain anomatis extract.

Comparative example

This comparative example is different from Example 1 except for the ultrasonic extraction in S2 and the addition of co-solvent and stabilizer in S5. The other processes are the same as Example 1.

S1. Grinding: Take the dried whole plant of Anomatis and crush it with a grinder and then pass it through the No. 1 sieve to obtain 20g of Anomatis dry powder;

S2. Extraction: Add the dry powder obtained in S1 to 30% ethanol solvent at a material-to-liquid ratio of 1:15 and soak it for extraction once to obtain a crude extract of Anoectochilus;

S3. Filtration: Filter the crude extract of Anomatis obtained in S2 to remove residue;

S4. Concentration: Concentrate the filtrate obtained in S3 under reduced pressure to remove ethanol, and dilute the volume with water to 10 times the mass of Anoectochilus dry powder;

S5. Dissolution: Add pure water to the concentrated solution obtained in S4. The final solution is a crude drug: solution = 1:20 mass ratio, composed of 50% w/w concentrated solution and 50% w/w water;

S6. Decolorization: Use 20% w/w XAD-2 macroporous adsorption resin for adsorption and decolorization of the solution obtained in S5 for 2 hours to obtain a Anomatis decolorization liquid;

S7. Sterilization: Filter and sterilize the decolorizing liquid described in S6 to obtain Anomatis extract;

S8. Antiseptic compounding: Add 0.1% w/w sodium metabisulfite to the sterilized solution of S7 to obtain anomatis extract.

Comparative experiment on the polysaccharide content of the Anomatis extracts of Examples 1-3 and the comparative example

Table 3 Comparison of polysaccharide content of Anoectochilus extracts between Examples 1-3 and Comparative Examples

It can be seen from Table 3 that the polysaccharide content of Anoectochilus extracted using ultrasonic method is relatively high.

Stability test of Anomatis extracts in Examples 1-3 and Comparative Examples

The stability test of the Anomatis extract obtained by the process of this application was carried out with reference to the TSHFCA002-2021 Cosmetic Stability Test Guidelines. The test was carried out for 6 months under the conditions of 45℃±2℃ and relative humidity of 45%RH±5%RH. The inspection items were appearance, smell, pH value, and Anoectochus polysaccharide content. The test standards are shown in Table 4 below:

Table 4 Stability test of Anomatis extract

According to the designed stability test plan, an accelerated test was conducted, and the results are the 6-month accelerated test results (Table 5).

Table 5 Stability test results of Anomatis extract

As can be seen from Table 5, the quality control indicators of the Anoectochus extract obtained by the process of the present application are within the scope of the standard requirements in terms of raw material appearance, odor, pH value, relative density, microorganisms, and Anoectochus polysaccharide content, and are consistent with the control. Compared with other examples, its physical and chemical properties and active ingredient content will not change significantly over time, thus ensuring the activity of the Anoectochilus extract itself.

Test of soothing and anti-inflammatory effects of Anomatis extract

Through lipopolysaccharide (LPS)-induced macrophage RAW264.7 inflammation model, the levels of inflammatory factors TNF-α and IL-1α in RAW264.7 cells were detected to observe the anti-inflammatory effect of Anoectochilus extract. Inoculate the cultured cells in a 6-well plate at 7 × 10 ⁵ cells/well, 2 mL per well, place in a 5% CO ₂ incubator, and incubate at 37°C for 24 hours; remove the supernatant and administer 2 mL to the model group ; 5 μg/mL lipopolysaccharide (LPS); sample group: 5 μg/mL lipopolysaccharide (LPS) + anoectochilus extract of the present application; blank control group: add serum-free culture medium; place in CO ₂ incubator (37°C , 5% CO ₂ ) and continue culturing for 24 hours; aspirate the supernatant and operate according to the instructions of the kit.

As shown in the results in Figure 1 and Figure 2, Control: Blank control group; Model: 5 μg/mL LPS induction model group; Positive control group: 100 μg/mL DXMS (dexamethasone); Experimental group: Anoectochilus extract at high, medium and low doses Materials, JXL-H, 5μg/mLLPS + 10% Anoectochilus extract; JXL-M, 5μg/mL LPS + 5% Anomatis extract; JXL-L, 5μg/mL LPS + 2.5% Anomatis extract things. # indicates a significant difference compared with Control (P<0.05); * indicates a significant difference compared with Model (P<0.05).

Control: Blank control group; Model: 5 μg/mL LPS induction group; Positive control group: 100 μg/mL DXMS (dexamethasone); Experimental group: High, high, and low-dose Anomatis extract, JXL-H, 5 μg/mL LPS+1 % Anomatis extract; JXL-M, 5 μg/mL LPS + 0.5% Anomatis extract; JXL-L, 5 μg/mLLPS + 0.1% Anomatis extract. # indicates a significant difference compared with Control (P<0.05); * indicates a significant difference compared with Model (P<0.05).

Compared with the blank control group (Control), the content levels of TNF-α and IL-1α in the model group (Model) were significantly increased, indicating that the inflammatory cell model was successfully established. Anoectochilus extract has a significant inhibitory effect on the TNF-α secretion of RAW264.7 cells induced by LPS; at the same time, Anoectochilus extract also has a significant inhibitory effect on the IL-1α secretion of RAW264.7 cells induced by LPS. And it is dose-dependent. This shows that the anoectochilus extract of the present application has good anti-inflammatory effects and is suitable for use in soothing skin care products.

Test on the anti-wrinkle effect of Anomatis extract

After the human skin fibroblasts (HSF) in the logarithmic growth phase were resuspended and counted, they were seeded into a 6-well cell culture plate at a density of 2.5×10 ⁵ /well, placed in a CO ₂ incubator, and cultured at 37°C for 24 hours; After the cells adhere to the wall, remove the original culture medium and add 2 mL of medicine to each well; the experimental group design is Control (blank control group), add culture medium containing 1% serum without any treatment; add DMEM containing 1% serum to the experimental group The anoectochilus extract of this application diluted in the culture medium; put it into a CO2 incubator and culture it at 37°C for 24 hours; then collect the cell supernatant, centrifuge it at 8000 rpm for 10 minutes, and take the supernatant according to the instructions of the Human COL1α1 (Collagen Type I Alpha 1) ELISA Kit Perform operations.

The experimental results are shown in Figure 3. Control: blank control group; experimental group: high, medium and low-dose Alematophila anomatis extract, JXL-H, 1% (v/v) Anoectochilus arunchus extract; JXL-M, 0.5% ( v/v) Anoectochus extract; JXL-L, 0.25% (v/v) Anomatis extract. * indicates significant difference compared with Control (P<0.05).

The anoectochilus extract of the present application at concentrations of 1% and 0.5% significantly promotes the synthesis of type I collagen by human skin fibroblasts (HSF). Compared with the blank control group, the synthesis of type I collagen is increased by more than 20%. This shows that the anoectochilus extract of the present application is suitable for use in anti-wrinkle skin care products.

Test on the repairing effect of Anomatis extract

(1) Cell proliferation test

Human keratinocytes (HaCaT) cells were seeded into a 96-well plate at 5.0×10 ³ /well, 100uL per well, and cultured in a CO ₂ incubator for 12 hours; discard the supernatant, add test drugs, and add the blank control group (Control) Containing 1% serum culture medium without any treatment, the competitive product control group is 0.1 mg/mL NMN, and the sample group is the Anomatis extract of the present application (concentrations are 1%, 0.75%, 0.5%, 0.25%); culture 24h, discard the supernatant, add 100uL of complete culture medium containing 10% CCK-8 reagent to each well, incubate for 1.5h, and detect at 450nm with a microplate reader.

The experimental results are shown in Figure 4. Compared with the blank control group, both the competitive control group and the sample group have a certain effect on promoting the proliferation of HaCaT cells, but the 1% Anoectochilus extract has a stronger effect on promoting the proliferation of HaCaT cells.

(2) Cell migration test

Human keratinocytes (HaCaT) cells were seeded into a 6-well plate at 3 × 10 ⁵ /well, 2 mL per well, and cultured in a CO ₂ incubator for 24 hours; scratch each well with a 200 μL pipette tip, and remove the culture medium. Wash excess cells with PBS and remove the solution; add test drugs, add culture medium containing 1% serum to the blank control group (Control) without any treatment, add 10ng/mL EGF to the positive control group, and add the gold thread of this application to the sample group Lotus extract (concentration: 1%, 0.25%) was cultured in a CO ₂ incubator, and photos were taken and recorded at 0h, 12h, 24h, and 48h respectively; imageJ quantitative analysis was performed to obtain the migration area.

As shown in Figure 5-6, the migration rate of HaCaT cells has a positive correlation with time. At 48 hours after adding the drug, the migration rate of the positive control group was 30.5%; while in the sample group, treated with 1% Anoectochilus extract, The migration rate of HaCaT cells is 23.9%. When treated with 0.5% Anoectochilus extract, the migration rate of HaCaT cells is 16.6%; the migration rate of the blank control group (Control) is 14.7%. Compared with the blank control group (Control), Anomatis extracts all promote the migration of HaCaT cells, which shows that the Anomatis extracts of the present application have a certain repairing effect and are suitable for use in repairing skin care products.

In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the present application. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of this application, "plurality" means two or more than two, unless otherwise explicitly and specifically limited.

The above are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person familiar with the technical field can easily think of various changes or modifications within the technical scope disclosed in the present application. Replacements, these should all be covered by the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claims.

Claims (10)

1. A process for preparing anoectochilus roxburghii extract, which is characterized by comprising the following steps:

pulverizing dried whole strain of Anoectochilus roxburghii with pulverizer, and sieving to obtain dry powder;

adding the dry powder into an ethanol solvent, and performing normal-temperature ultrasonic extraction to obtain a crude anoectochilus roxburghii extract;

filtering the anoectochilus formosanus crude extract to remove dregs, thereby obtaining filtrate;

membrane separation and/or reduced pressure concentration are carried out on the obtained filtrate to remove ethanol, and the volume is fixed to obtain concentrated solution;

adding a cosolvent and a stabilizer into the obtained concentrated solution, and decoloring to obtain a anoectochilus roxburghii decoloring solution;

filtering and sterilizing the anoectochilus formosanus decolorization solution;

adding antiseptic into the sterilized solution to obtain anoectochilus roxburghii extract.

2. The process for preparing anoectochilus roxburghii extract according to claim 1, wherein the dry powder is prepared by the following steps of: 4-1:40, adding the extract into an ethanol solvent, and performing ultrasonic extraction at normal temperature; wherein the volume fraction of the ethanol solvent is 5-70%, the ultrasonic frequency is 480W, the ultrasonic extraction time is 10-60min, and the extraction times are 1-2.

3. The process for preparing the anoectochilus roxburghii extract according to claim 1, wherein the volume is fixed to 10-20 times of the mass of the anoectochilus roxburghii dry powder, so as to obtain a concentrated solution.

4. The process for preparing anoectochilus roxburghii extract according to claim 1, wherein 20-50% w/w of cosolvent and 0.1-5% w/w of stabilizer are added into the obtained concentrated solution.

5. The process for preparing anoectochilus roxburghii extract according to any one of claims 1 to 4, wherein the cosolvent is one or more of glycerol, 1, 3-propanediol and 1, 3-butanediol;

the stabilizer is one or more of 1, 2-hexanediol, 1, 2-pentanediol and p-hydroxyacetophenone.

6. The process for preparing anoectochilus roxburghii extract according to any one of claims 1 to 4, wherein the adsorption decolorization is carried out by using macroporous adsorption resin, and the resin usage amount is 4% -40% w/v.

7. The process for preparing anoectochilus roxburghii extract according to claim 6, wherein the macroporous adsorption resin comprises one or more of D101, AB-8, XAD-2, HDP-100, HP-10 and LSD-20;

the adsorption mode is static adsorption, and the adsorption time is 0.5-8h.

8. The process for preparing anoectochilus roxburghii extract according to any one of claims 1-4, wherein the sterilized solution is added with 0.1% -0.5% w/w preservative; the preservative is one or more of sodium metabisulfite, phenoxyethanol, benzyl alcohol, potassium sorbate and sodium sorbate.

9. An anoectochilus roxburghii extract, which is prepared by the preparation process of the anoectochilus roxburghii extract according to any one of claims 1-8.

10. Use of the anoectochilus roxburghii extract according to claim 9 in a skin care product for relieving inflammation, or reducing wrinkles, or repairing.