A centella asiatica extract, a preparation method thereof, and an application thereof in preparing cosmetics with soothing and repairing effects

Abstract

The present application belongs to the technical field of cosmetics, and particularly relates to a Centella asiatica extract, a preparation method thereof and application of the Centella asiatica extract in preparation of cosmetics with soothing and repairing effects. Water or an ethanol aqueous solution is selected as an extraction solvent, an extract obtained through extraction is subjected to MCI Gel column chromatography for enrichment, elution is performed with water or the ethanol aqueous solution, and the Centella asiatica extract containing total glycosides and aglycone is obtained after concentration and drying. The Centella asiatica extract provided by the present application can extract total glycosides and aglycone at the same time, the preparation method is simple, the obtained Centella asiatica extract has a protective effect on H2O2 damaged HSF cells and HaCaT cells, has an effect of promoting secretion of hyaluronic acid (HA) and collagen type I (Col I) of the HaCaT cells, has DPPH free radical scavenging activity, can be used as a cosmetic component with soothing and repairing effects, and can be applied to various cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a Centella asiatica extract, its preparation method, and its application in the preparation of cosmetics with soothing and repairing effects. Background Technology

[0002] Sensitive skin is a skin condition characterized by high sensitivity, poor tolerance, and susceptibility to allergies, which can change with environmental factors and physical conditions. While not considered a skin disease medically, symptoms can be alleviated or eliminated through the use of cosmetics. Sensitive skin primarily affects the face, scalp, and back of the hands, and its main characteristics include dryness, tightness, redness, itching, stinging, and flaking. In severe cases, it can negatively impact a person's mood and mental state. The causes of sensitive skin are complex, and there is currently no specific cure. Using cosmetics with specific functions is an effective way to alleviate symptoms.

[0003] The causes of sensitive skin include impaired skin barrier function, neurological factors, and inflammation, with impaired skin barrier function being a significant cause of skin sensitivity. Skin damage is mainly manifested in intracellular water loss, oxidative free radical damage, reduced secretion of the cellular matrix, and a decrease in keratinocytes. Therefore, skin sensitivity can be improved by repairing damaged keratinocytes, promoting the secretion of the cellular matrix, and reducing inflammatory responses.

[0004] Centella asiatica is a plant of the Apiaceae family (Apiaceae family). Centella asiaticaThe dried whole herb of Centella asiatica has a bitter and pungent taste and is cold in nature. It has the effects of clearing heat and dampness, detoxifying and relieving pain, and is often used for damp-heat jaundice, heatstroke diarrhea, carbuncles and boils, and traumatic injuries. Studies have shown that Centella asiatica has the effects of removing blemishes, anti-inflammation, antibacterial and repairing. The main active ingredients in Centella asiatica are triterpenoid saponins and their aglycones, mainly including asiaticoside, hydroxyasiaticoside, asiatic acid and hydroxyasiatic acid. Patent CN109320580A discloses a method for extracting asiatic acid from Centella asiatica, which includes multiple steps such as alkalization, macroporous resin column chromatography, solvent extraction, silica gel column chromatography, and activated carbon decolorization. Patent CN111518859A discloses a method for preparing asiatic aglycones by enzymatic hydrolysis. Patent CN112957764A discloses a method for extracting asiaticoside from Centella asiatica by supercritical fractionation. Patent CN112773745A discloses a method for extracting asiaticoside from Centella asiatica, involving multiple extractions with solvents such as acetone, water, methyl isobutyl ketone, and methanol, and steps such as macroporous adsorption resin column chromatography. Patent CN113171321A discloses a method for extracting total asiaticosides from Centella asiatica, with main steps including defatting, ethanol solution extraction, activated carbon decolorization, and chitosan flocculation for impurity removal. The obtained extract mainly contains asiaticoside and hydroxyasiaticoside, but no aglycone components. Patent CN113307842A discloses a method for extracting asiatic acid, involving extraction with petroleum ether, chloroform, and ethyl acetate, followed by silica gel column chromatography and methanol recrystallization. Patent CN111285916B discloses a method for extracting total asiaticosides from Centella asiatica, involving extraction with alkaline ethanol, concentration, macroporous adsorption resin chromatography, alumina decolorization, and anion exchange resin column chromatography.

[0005] In summary, existing technologies mainly involve extraction processes of total glycosides or aglycones from Centella asiatica. However, there are no reports in the existing technologies of simultaneously extracting both total glycosides and aglycones, the two main active ingredients, from Centella asiatica. Summary of the Invention

[0006] The purpose of this invention is to provide a Centella asiatica extract, its preparation method, and its application. The preparation method provided by this invention can simultaneously obtain two major active ingredients, total glycosides and aglycones, from Centella asiatica, and the preparation method is simple and easy for industrial production.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing Centella asiatica extract, comprising the following steps: Centella asiatica is soaked in a polar solvent to extract Centella asiatica extract; the polar solvent is water or an aqueous solution of primary ethanol. The Centella asiatica extract was subjected to MCI Gel column chromatography to obtain a first Centella asiatica purified solution; the eluent used in the MCI Gel column chromatography was water or a second aqueous ethanol solution. The first Centella asiatica purified solution was mixed with ethanol and subjected to alcohol precipitation. After solid-liquid separation, the second Centella asiatica purified solution was obtained. The solvent was removed from the second Centella asiatica purified solution to obtain the Centella asiatica extract; the Centella asiatica extract includes asiaticoside, hydroxyasiaticoside, asiaticoside, and hydroxyasiaticoside.

[0008] Preferably, the volume percentage of ethanol in the first aqueous ethanol solution is 20-80%.

[0009] Preferably, the extraction is performed 1 to 4 times; the ratio of the mass of Centella asiatica to the volume of the polar solvent used in each extraction is 1 kg: (4 to 10) L.

[0010] Preferably, the extraction includes one or more of room temperature extraction, ultrasonic extraction, and reflux extraction; Each extraction was performed at room temperature for 24 to 72 hours. During each extraction, the ultrasonic power of the ultrasonic extraction is 500~5000W, the ultrasonic frequency is 20~60kHz, and the ultrasonic time is 0.5~2h. During each extraction, the reflux extraction temperature is 60~100℃, and the reflux time is 1~5h.

[0011] Preferably, the packing material used in the MCI Gel column is MCI GEL CHP20P, CHP20 / P20, CHP20 / P120, CHP50 / P20 or CHP07 / P120.

[0012] Preferably, the ratio of the volume of the filler to the mass of the Centella asiatica is (0.25~1)L:1kg.

[0013] Preferably, the volume of the eluent is 3 to 4 column volumes; and the volume percentage of ethanol in the second aqueous ethanol solution is 20 to 80%.

[0014] Preferably, before the alcohol precipitation, the first Centella asiatica purified solution is concentrated to obtain a Centella asiatica concentrate; the relative density of the Centella asiatica concentrate is 1.3~1.5; the volume ratio of the Centella asiatica concentrate to the ethanol is 1:(1~5).

[0015] The present invention provides a Centella asiatica extract prepared by the preparation method described above, wherein the Centella asiatica extract comprises asiaticoside, hydroxyasiaticoside, asiaticoside, and hydroxyasiaticoside.

[0016] This invention provides the application of the Centella asiatica extract described in the above technical solution in the preparation of cosmetics with soothing and repairing effects.

[0017] This invention provides a method for preparing Centella asiatica extract, comprising the following steps: soaking Centella asiatica in a polar solvent to obtain a Centella asiatica extract; the polar solvent is water or a first aqueous ethanol solution; performing MCI column chromatography on the Centella asiatica extract to obtain a first purified Centella asiatica solution; the eluent used in the MCI column chromatography is water or a second aqueous ethanol solution; mixing the first purified Centella asiatica solution with ethanol for alcohol precipitation, and obtaining a second purified Centella asiatica solution after solid-liquid separation; removing the solvent from the second purified Centella asiatica solution to obtain the Centella asiatica extract; the Centella asiatica extract comprises asiaticoside, hydroxyasiaticoside, asiaticopic acid, and hydroxyasiaticopic acid. This invention uses water or an aqueous ethanol solution as the extraction solvent. The extract obtained by soaking is enriched by MCI column chromatography, eluted with water or an aqueous ethanol solution, and then purified a second time by alcohol precipitation to obtain a Centella asiatica extract rich in asiaticoside, hydroxyasiaticoside, asiaticopic acid, and hydroxyasiaticopic acid. Compared with existing technologies, this invention can simultaneously extract total glycosides and aglycone active components from Centella asiatica. Only one column chromatography step is needed to enrich triterpenoid active components represented by asiaticoside, hydroxyasiaticoside, asiatic acid, and hydroxyasiatic acid. The extracted parts have significant soothing and repairing effects. The operation is simple, highly operable, and suitable for industrial production.

[0018] This invention provides the application of the Centella asiatica extract described in the above-mentioned technical solution in the preparation of cosmetics with soothing and repairing effects. The Centella asiatica extract prepared by this invention protects HSF cells and HaCaT cells damaged by H2O2, promotes the secretion of hyaluronic acid and type I collagen by HaCaT cells, and possesses DPPH free radical scavenging activity, thereby achieving the effects of soothing and repairing the skin. Attached Figure Description

[0019] Figure 1 Effects of Centella asiatica extract prepared in Example 1 on the viability of HaCaT and HSF cells; Figure 2 The repair activity of Centella asiatica extract prepared in Example 1 on H2O2-damaged HSF and HaCaT cells; Figure 3 The Centella asiatica extract prepared in Example 1 promotes the secretion activity of HA and ColI in HaCaT cells; Figure 4 The HPLC chromatogram of the Centella asiatica extract prepared in Example 1 is shown. Detailed Implementation

[0020] This invention provides a method for preparing Centella asiatica extract, comprising the following steps: Centella asiatica is soaked in a polar solvent to extract Centella asiatica extract; the polar solvent is water or an aqueous solution of primary ethanol. The Centella asiatica extract was subjected to MCI Gel column chromatography to obtain a first Centella asiatica purified solution; the eluent used in the MCI Gel column chromatography was water or a second aqueous ethanol solution. The first Centella asiatica purified solution was mixed with ethanol and subjected to alcohol precipitation. After solid-liquid separation, the second Centella asiatica purified solution was obtained. The solvent was removed from the second Centella asiatica purified solution to obtain the Centella asiatica extract; the Centella asiatica extract includes asiaticoside, hydroxyasiaticoside, asiaticoside, and hydroxyasiaticoside.

[0021] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0022] Centella asiatica is soaked in a polar solvent to extract Centella asiatica extract; the polar solvent is water or an aqueous solution of first ethanol.

[0023] In this invention, the Centella asiatica is preferably the whole plant.

[0024] In this invention, prior to the extraction, the Centella asiatica is preferably pretreated, which preferably includes the following steps: drying and pulverizing the Centella asiatica sequentially. This invention does not have specific requirements regarding the exact process of drying and pulverizing.

[0025] In this invention, the polar solvent is preferably a first aqueous ethanol solution.

[0026] In this invention, when the polar solvent used in the extraction is preferably a first ethanol aqueous solution, the volume percentage of ethanol in the first ethanol aqueous solution is preferably 20-80%, more preferably 25-75%, and even more preferably 50-70%.

[0027] In this invention, the extraction is preferably performed 1 to 4 times, more preferably 2 to 4 times.

[0028] In this invention, the ratio of the mass of the Centella asiatica to the volume of the polar solvent used in each extraction is preferably 1 kg:(4~10) L, more preferably 1 kg:(5~9) L, and even more preferably 1 kg:(6~8) L.

[0029] In this invention, the extraction preferably includes room temperature extraction, ultrasonic extraction, or reflux extraction.

[0030] In this invention, the extraction is preferably carried out at room temperature, and the extraction time at room temperature is preferably 24-72 hours, and more preferably 36-48 hours, for each extraction.

[0031] In this invention, the extraction is preferably performed using ultrasonic extraction. During each extraction, the ultrasonic power is preferably 500~5000W, more preferably 1000~4500W, the ultrasonic frequency is preferably 20~60kHz, more preferably 30~50kHz, and the ultrasonic time is preferably 0.5~2h, more preferably 1~1.5h.

[0032] In this invention, the extraction is preferably carried out during reflux extraction. During each extraction, the temperature of the reflux extraction is preferably 60~100℃, more preferably 65~100℃; the reflux time of the reflux extraction is preferably 1~5h.

[0033] In this invention, when the number of extractions is greater than 1, the extracts obtained from each extraction are combined to obtain the Centella asiatica extract.

[0034] After obtaining the Centella asiatica extract, the present invention performs MCI Gel column chromatography to separate the Centella asiatica extract to obtain a purified Centella asiatica solution; the eluent used in the MCI Gel column chromatography is water or a second ethanol aqueous solution.

[0035] In this invention, prior to the MCI Gel column chromatography separation, the Centella asiatica extract is preferably subjected to solid-liquid separation, and the resulting filtrate is then subjected to MCI Gel column chromatography separation. In this invention, the solid-liquid separation is preferably performed by centrifugation, and the specific implementation of the centrifugation method is not particularly required.

[0036] In this invention, the packing material used in the MCI Gel column is preferably MCI GEL CHP20P, CHP20 / P20, CHP20 / P120, CHP50 / P20 or CHP07 / P120, more preferably MCI GEL CHP20P or CHP20 / P120, and even more preferably MCI GEL CHP20P.

[0037] In this invention, the eluent used in the MCI Gel column chromatography separation is water or a second ethanol aqueous solution, more preferably a second ethanol aqueous solution. When the eluent used in the MCI Gel column chromatography separation is a second ethanol aqueous solution, the volume percentage of ethanol in the second ethanol aqueous solution is preferably 20-80%, more preferably 25-75%, and even more preferably 30-70%.

[0038] In this invention, the volume ratio of the packing material used in the MCI Gel column chromatography to the mass of the Centella asiatica is preferably (0.25~1)L:1kg, more preferably (0.3~0.9)L:1kg.

[0039] In this invention, the volume of the eluent is preferably 3 to 4 column volumes, more preferably 3 column volumes.

[0040] After obtaining the first Centella asiatica purified solution, the present invention mixes the first Centella asiatica purified solution with ethanol for alcohol precipitation, and then performs solid-liquid separation to obtain the second Centella asiatica purified solution. In the present invention, the solid-liquid separation is preferably performed by centrifugation, and the supernatant obtained by centrifugation is the second Centella asiatica purified solution.

[0041] In this invention, the ethanol is preferably used in the form of an aqueous ethanol solution, wherein the volume percentage of ethanol in the aqueous ethanol solution is 90-94%.

[0042] In this invention, after obtaining the first Centella asiatica purified solution and before performing the alcohol precipitation, the invention preferably further includes concentrating the first Centella asiatica purified solution to obtain a Centella asiatica concentrate; and subjecting the Centella asiatica concentrate to the alcohol precipitation. In this invention, the concentration is preferably vacuum concentration, the relative density of the Centella asiatica concentrate is preferably 1.3~1.5; the volume ratio of the Centella asiatica concentrate to the ethanol is preferably 1:(1~5), more preferably 1:(1.5~4.5), and even more preferably 1:(2~4).

[0043] After obtaining the second Centella Asiatica purified solution, the present invention removes the solvent from the second Centella Asiatica purified solution to obtain the Centella Asiatica extract; the Centella Asiatica extract includes asiaticoside, asiaticoside, asiaticoside, and asiaticoside.

[0044] In this invention, the solvent removal method is preferably concentration drying, which preferably includes vacuum distillation, vacuum drying, freeze drying or spray drying, and more preferably freeze drying.

[0045] The present invention provides a Centella asiatica extract prepared by the preparation method described above, wherein the Centella asiatica extract comprises asiaticoside, hydroxyasiaticoside, asiaticoside, and hydroxyasiaticoside.

[0046] In this invention, the Centella asiatica extract contains 1.18% asiaticoside, 2.72% asiaticoside, 0.44% asiatic acid, and 1.90% asiatic acid by mass.

[0047] This invention provides the application of the Centella asiatica extract described in the above technical solution in the preparation of cosmetics with soothing and repairing effects.

[0048] In this invention, the Centella asiatica extract protects HSF cells and HaCaT cells damaged by H2O2, promotes the secretion of hyaluronic acid and type I collagen by HaCaT cells, and has DPPH free radical scavenging activity to achieve soothing and repairing effects.

[0049] This invention obtains total glycosides and aglycone active components (including asiaticoside, asiaticoside, asiatic acid, and asiaticoside) from Centella asiatica. The invention uses an ethanol-water solution as the extraction solvent, employing room temperature extraction, ultrasonic extraction, or reflux extraction, performed once or multiple times. The extract is enriched by MCI Gel column chromatography using any one of the following packing materials: MCI Gel CHP, CHP20 / P20, CHP20 / P120, CHP50 / P20, CHP07 / P120, etc. After elution with water or an ethanol-water solution, the extract is concentrated and dried to obtain the total glycosides and aglycone fractions. The total glycosides and aglycone fractions provided by this invention have a protective effect against H2O2-damaged HSF cells and HaCaT cells, promote the secretion of hyaluronic acid (HA) and type I collagen (ColⅠ) by HaCaT cells, and exhibit DPPH free radical scavenging activity. They can be used as soothing and repairing cosmetic components in various cosmetics.

[0050] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1 Take 2 kg of dried Centella asiatica, pulverize it, and extract it twice by reflux with 6 times the volume (v / w) of 70% ethanol aqueous solution. Combine the ethanol extracts, centrifuge and collect the supernatant, and perform MCI column chromatography (MCI GEL CHP20P, 1L). Then elute with 4L of 70% ethanol aqueous solution. Combine all the eluted ethanol solutions, concentrate under reduced pressure to a small volume (relative density of 1.35), add 4 times the volume of ethanol (94%), stir continuously, centrifuge and collect the supernatant, recover under reduced pressure to obtain Centella asiatica extract, which contains asiaticoside, hydroxyasiaticoside, asiaticopic acid and hydroxyasiatic acid.

[0052] The components of the Centella asiatica extract prepared in this embodiment were analyzed by high performance liquid chromatography.

[0053] HPLC conditions for asiaticoside and hydroxyasiaticoside: Column: vp-ODS 4.6×150 mm; Mobile phase: Acetonitrile-2 mmol / L betacyclodextrin (24:76); Detection wavelength: 205 nm; Flow rate: 1 mL / min; Column temperature: 30℃; HPLC conditions for asiatic acid and hydroxyasiatic acid: Column: Eclipse XDB-C18 4.6×150 mm; Mobile phase: Acetonitrile-water-0.3wt% acetic acid; 0 min: 10⁻⁸⁵⁻⁵; 38min: 55-35-5; 45min: 55-35-5; 55min: 10-85-5; Detection wavelength: 210 nm; Flow rate: 1 mL / min; Column temperature: 30℃; The results are as follows Figure 4 As shown, the retention time of asiaticoside was 15.67 min and the peak area was 1758; the retention time of hydroxyasiaticoside was 7.90 min and the peak area was 8099; the retention time of hydroxyasiatic acid was 27.70 min and the peak area was 23471; and the retention time of asiatic acid was 31.85 min and the peak area was 5554.

[0054] According to the liquid phase data, the mass percentage of asiaticoside in the Centella asiatica extract prepared by this invention is 1.18%, the mass percentage of hydroxyasiaticoside is 2.72%, the mass percentage of asiaticoside is 0.44%, and the mass percentage of hydroxyasiaticoside is 1.90%.

[0055] Example 2 Take 2 kg of dried Centella asiatica, pulverize it, and extract it three times by reflux with 8 times the volume (v / w) of 70% ethanol aqueous solution. Combine the ethanol extracts, centrifuge and collect the supernatant, and perform MCI column chromatography (MCI GEL CHP20P, 1L). Then elute with 4L of 70% ethanol aqueous solution. Combine all the eluted ethanol solutions, concentrate under reduced pressure to a small volume (relative density 1.35), add it to 3 times the volume of ethanol (94%), stir continuously, centrifuge and collect the supernatant, freeze dry to obtain Centella asiatica extract, which contains asiaticoside, hydroxyasiaticoside, asiaticopic acid and hydroxyasiatic acid.

[0056] Example 3 Take 2 kg of dried Centella asiatica, pulverize it, and extract it three times by reflux with 10 times the volume (v / w) of 70% ethanol aqueous solution. Combine the ethanol extracts, centrifuge and collect the supernatant, and perform MCI column chromatography (CHP20 / P120, 1 L). Then elute with 4 L of 70% ethanol aqueous solution. Combine all the eluted ethanol solutions, concentrate under reduced pressure to a small volume (relative density 1.35), add it to 3 times the volume of ethanol (94%), stir continuously, centrifuge and collect the supernatant, freeze dry to obtain Centella asiatica extract, which contains asiaticoside, hydroxyasiaticoside, asiaticopic acid and hydroxyasiatic acid.

[0057] Test case (1) DPPH free radical scavenging activity test: Material: 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; anhydrous ethanol (Ethanol) was purchased from Sichuan Xilong Scientific Co., Ltd. instrument: Varioskan LUX benchtop multi-functional microplate reader (Thermo Scientific, USA); analytical balance (AG135, Metler Toledo, China); Experimental procedure: 1) Prepare a 100 μM working solution of DPPH using anhydrous ethanol; 2) In a 96-well plate, add 100 μL of DPPH working solution and 100 μL of the sample to be tested to each well, and set up 3 replicate wells for each group. 3) Let it sit at room temperature for 30 minutes; 4) Measure the absorbance at 517nm using a microplate reader. Calculate the inhibition rate using the formula: Inhibition rate = (Control group - Experimental group) / Control group × 100%. Calculate the IC50 using the statistical software GraphPad Prism 6. 50 ; The results are shown in Table 1. As can be seen from Table 1, the Centella asiatica extract prepared in Example 1 has DPPH free radical scavenging activity, with a scavenging rate of 21.8% at 200 μg / mL and 36.2% at 400 μg / mL. Table 1. Scavenging rate of DPPH by Centella asiatica extract prepared in Example 1

[0058] Note: Vitamin C is a positive control, IC 50 It is 3.65 μg / mL.

[0059] (2) Protective effect on HSF and HaCaT cells damaged by H2O2: Material: Human skin fibroblasts (HSF) and immortalized human epidermal cells (HaCaT) were purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd.; culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); hydrogen peroxide (H2O2) was purchased from Xilong Scientific (Sichuan, China). instrument: Full-wavelength scanning multi-functional microplate reader (Thermofisher Varioskan LUX, USA); CO2 incubator (Thermo 3111, USA); Biosafety cabinet (Thermo 1300 series A2, USA).

[0060] Experimental procedure: 1) Take cells in the logarithmic growth phase, discard the old culture medium, wash twice with PBS, and discard the PBS; 2) Digest the cells with 0.25wt% trypsin. When the cell outlines are observed to darken and become rounded under a microscope, quickly remove the trypsin. 3) Stop digestion with DMEM complete medium containing 10 wt% FBS and resuspend the cells. Take 10 μL of the cell suspension, count the cells using a cell counter, and adjust the cell concentration to 3 × 10⁻⁶ cells with culture medium. 4 / mL, seeded into 96-well plates, add 100μL of cell suspension to each well, and incubate in a 37℃, 5vol% CO2 incubator for 24h to allow the cells to adhere; 4) Discard the culture medium and add 1mM H2O2 prepared with complete culture medium to the model damage wells, adding 100μL to each well. Add an equal amount of complete culture medium to the control group and continue incubation in the incubator for 1h. 5) Remove H2O2, wash twice with PBS, discard the PBS, add the diluted sample to the plate, add 100 μL to each well, set 3 replicates for each concentration, and continue to incubate in the incubator for 24 h. 6) Remove the culture medium, add the prepared MTT solution (1 mg / mL), add 100 μL to each well, and incubate in an incubator for 4 h; 7) Remove the MTT solution, add DMSO, 100 μL per well, and incubate in an incubator for 10 min; 8) Use an ELISA reader to measure the absorbance value at a wavelength of 490nm. Calculate the repair rate using the formula: Repair rate = (Experimental group - Model group) / (Control group - Model group) × 100%, and plot the result using the statistical software GraphPad Prism 5. Repeat the experiment 3 times.

[0061] result: like Figure 1 As shown, the Centella asiatica extract prepared in Example 1 had no significant effect on the cell viability of HaCaT and HSF cells at concentrations of 3.2-200 μg / mL, and showed no obvious cytotoxicity.

[0062] like Figure 2 As shown, the Centella asiatica extract prepared in Example 1, at concentrations of 3.2-50 μg / mL, exhibited a certain protective effect against H2O2-damaged HaCaT and HSF cells, showing a dose-response relationship. At concentrations of 12.5-50 μg / mL, the Centella asiatica extract prepared in Example 1 achieved a repair rate of 19.9-34.9% for H2O2-damaged HaCaT cells and a repair rate of 31.7-35.0% for HSF cells.

[0063] (3) Activity test to promote the secretion of hyaluronic acid (HA) and type I collagen (Co1 I) by HaCaT cells: Material: Human immortalized epidermal cells (HaCaT) were purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd.; culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); hyaluronic acid quantitative detection kit (HA ELISA Kit) was purchased from Wuhan Huamei Biotechnology Co., Ltd. (Wuhan, China); type I collagen quantitative detection kit (Col I ELISA Kit) was purchased from Wuhan Huamei Biotechnology Co., Ltd. (Wuhan, China).

[0064] instrument: Full-wavelength scanning multi-functional microplate reader (Thermofisher Varioskan LUX, USA); CO2 incubator (Thermo 3111, USA); biosafety cabinet (Thermo 1300 series A2, USA); low-temperature centrifuge (Eppendorf centrifuge 5415 R, USA); Experimental procedure: 1) Take cells in the logarithmic growth phase, discard the old culture medium, wash twice with PBS, and discard the PBS; 2) Digest the cells with 0.25% trypsin. When the cell outlines are observed to darken and become rounded under a microscope, quickly remove the trypsin. 3) Stop digestion with DMEM complete medium containing 10 wt% FBS and resuspend the cells. Take 10 μL of the cell suspension, count the cells using a cell counter, and adjust the cell concentration to 1 × 10⁻⁶ cells with culture medium. 4 / mL, seeded into 96-well plates, add 100μL of cell suspension to each well, and incubate in a 37℃, 5vol% CO2 incubator for 24h to allow the cells to adhere; 4) Discard the culture medium, add the diluted sample to the plate, add 100 μL to each well, set 3 replicates for each concentration, and continue to incubate in the incubator for 48 h; 5) Take the supernatant culture medium, centrifuge at 1000g for 20 min at 4℃, and collect the supernatant for testing; 6) Follow the instructions of the HA and Col I assay kit to detect the content of Col I and HA; 8) Use an ELISA reader to measure the absorbance value at a wavelength of 450nm. Calculate the growth rate using the formula: Growth rate = (Experimental group - Negative) / Negative × 100%, and plot the result using the statistical software GraphPad Prism 5. Repeat the experiment 3 times.

[0065] result: like Figure 3 As shown, the Centella asiatica extract prepared in Example 1 can promote the secretion of type I collagen (Col I) and hyaluronic acid (HA) by HaCaT cells at concentrations of 3.2-50 μg / mL. At concentrations of 12.5-50 μg / mL, the promotion rate of Col I secretion is 17.8-29.5%, and the promotion rate of HA secretion is 11.2-20.0%.

[0066] The products prepared in Examples 2 and 3 have similar effects to the product prepared in Example 1.

[0067] In summary, the Centella asiatica extract prepared in Examples 1-3 of this invention has a protective effect on HSF cells and HaCaT cells damaged by H2O2, can promote the secretion of hyaluronic acid (HA) and type I collagen (CoI I) by HaCaT cells, has DPPH free radical scavenging activity, and can be used as a component of cosmetics with soothing and repairing effects in various cosmetics.

[0068] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing Centella asiatica extract, characterized in that, Includes the following steps: Centella asiatica is soaked in a polar solvent to extract Centella asiatica extract; the polar solvent is water or an aqueous solution of primary ethanol. The Centella asiatica extract was subjected to MCI Gel column chromatography to obtain the first Centella asiatica purified solution. The eluent used in the MCI Gel column chromatography separation is water or a second ethanol aqueous solution; The first Centella asiatica purified solution was mixed with ethanol and subjected to alcohol precipitation. After solid-liquid separation, the second Centella asiatica purified solution was obtained. The solvent was removed from the second Centella asiatica purified solution to obtain the Centella asiatica extract; the Centella asiatica extract includes asiaticoside, hydroxyasiaticoside, asiaticoside, and hydroxyasiaticoside.

2. The preparation method according to claim 1, characterized in that, The volume percentage of ethanol in the first aqueous ethanol solution is 20-80%.

3. The preparation method according to claim 1 or 2, characterized in that, The extraction is performed 1 to 4 times; the ratio of the mass of Centella asiatica to the volume of the polar solvent used in each extraction is 1 kg: (4 to 10) L.

4. The preparation method according to claim 3, characterized in that, The extraction includes one or more of room temperature extraction, ultrasonic extraction, and reflux extraction; Each extraction was performed at room temperature for 24 to 72 hours. During each extraction, the ultrasonic power of the ultrasonic extraction is 500~5000W, the ultrasonic frequency is 20~60kHz, and the ultrasonic time is 0.5~2h. During each extraction, the reflux extraction temperature is 60~100℃, and the reflux time is 1~5h.

5. The preparation method according to claim 1, characterized in that, The packing material used in the MCI Gel column is MCI GELCHP20P, CHP20 / P20, CHP20 / P120, CHP50 / P20 or CHP07 / P120.

6. The preparation method according to claim 5, characterized in that, The ratio of the volume of the filler to the mass of the Centella asiatica is (0.25~1)L:1kg.

7. The preparation method according to claim 1, characterized in that, The eluent has a volume of 3 to 4 column volumes; the second aqueous ethanol solution has a volume percentage of ethanol of 20 to 80%.

8. The preparation method according to claim 1, characterized in that, Before the alcohol precipitation, the first Centella asiatica purified solution is concentrated to obtain a Centella asiatica concentrate; the relative density of the Centella asiatica concentrate is 1.3~1.5; the volume ratio of the Centella asiatica concentrate to the ethanol is 1:(1~5).

9. The Centella asiatica extract prepared by the preparation method according to any one of claims 1 to 8, characterized in that, The Centella asiatica extract includes asiaticoside, hydroxyasiaticoside, asiaticoside, and hydroxyasiaticoside.

10. The use of the Centella asiatica extract according to claim 9 in the preparation of cosmetics with soothing and repairing effects.

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