Preparation process of centella asiatica extract
By preparing a molecularly imprinted polymer resin for Centella asiatica extract, the problem of low efficiency in existing Centella asiatica extraction methods has been solved, and high-purity Centella asiatica extract has been prepared. This extract has cell proliferation-promoting and soothing effects and is suitable for medical preparations and cosmetics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI HUAZHI WO BIOMEDICAL TECHNOLOGY CO LTD
- Filing Date
- 2026-01-12
- Publication Date
- 2026-06-23
AI Technical Summary
Existing methods for extracting Centella asiatica have long operating cycles, require large amounts of solvent, and are difficult to efficiently separate structurally similar substances. Furthermore, the application of molecularly imprinted polymers in the separation of Centella asiatica extracts has not been fully utilized.
Using self-made organic-inorganic hybrid nanomaterials chloropropylhepta(triethoxysilane-urea)POSS and octa(triethoxysilane-urea)POSS as functional monomers, molecularly imprinted polymer resins of Centella asiatica extract were prepared by combining molecular imprinting technology. The purification efficiency was improved by enzyme-assisted hot ethanol extraction and resin purification technology.
Achieving high-purity separation of Centella asiatica extract with a purity of 99.8% was demonstrated, exhibiting good effects in promoting cell proliferation, cell migration, and soothing, making it suitable for medical formulations and cosmetics.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of medical preparations or cosmetics, specifically to the field of preparation technology of plant-derived active ingredients in medical preparations or cosmetics, and more specifically to the preparation process of Centella asiatica extract. Background Technology
[0002] Studies have found that Centella asiatica extract can be used as an active ingredient in medical formulations or cosmetics, for preparing gel formulations to treat skin damage, or added to repairing skin care products.
[0003] In industry, the main extraction methods for Centella asiatica extract include ethanol extraction, water extraction, and enzymatic extraction. The most common purification method is column chromatography using macroporous resin, which often requires multiple chromatography steps, resulting in long operation cycles, large solvent consumption, and difficulty in efficiently separating structurally similar substances. Molecularly imprinted polymers, on the other hand, possess the ability to remember the size, shape, functional groups, and spatial structure of template molecules and have been applied to the separation research of Centella asiatica extract. For example, CN119591787 A discloses the separation and purification of asiaticoside molecules using molecular imprinting technology.
[0004] The study also found that polyhedral oligosiloxanes (POSSs), as functional monomers or crosslinking agents, can improve the adsorption properties of molecularly imprinted polymers and their selectivity for target analytes during polymerization.
[0005] This invention cites the following references:
[0006] Anhui University published Nie Wangyan's doctoral dissertation, "Preparation and Performance Study of POSS-based Low Dielectric Constant Nanoporous Silica Thin Films," in October 2010, which disclosed the chemical structure and preparation method of hydrogen-containing POSS (T8H8).
[0007] The journal *Journal of Southwest Forestry University* published an article titled "Research on DOPO Derivatives / Octaaminopropyl POSS Flame-Retardant Wood-Plastic Composites" by Zhang Ling et al. in its May 2023 issue (Volume 43, Issue 3, pp. 136-144), which disclosed the chemical structure and preparation method of octaaminopropyl POSS (OA-POSS). Summary of the Invention
[0008] This invention uses self-made organic-inorganic hybrid nanomaterials chloropropylhepta(triethoxysilane-urea)POSS, octa(triethoxysilane-urea)POSS, and 3-aminopropyltriethoxysilane as functional monomers. Utilizing the synergistic effect of the three, molecular imprinting technology is used to prepare a molecularly imprinted polymer resin for Centella asiatica extract, which is used for the purification of Centella asiatica extract and to improve the separation purity of Centella asiatica extract.
[0009] The preparation process of Centella asiatica extract includes the following steps:
[0010] Step 1: Extract concentrated Centella asiatica product from Centella asiatica using enzyme-assisted hot ethanol extraction combined with resin purification technology.
[0011] Step 2: Prepare a molecularly imprinted polymer resin for Centella asiatica extract and fill it into a solid-phase extraction column to obtain a resin column. Purify the concentrated Centella asiatica product through the resin column to obtain Centella asiatica extract, which is hydroxyasiaticoside.
[0012] The method for preparing the imprinted polymer resin is as follows: using AB-8 styrene-type macroporous adsorption resin as a carrier and chloropropyl hepta(triethoxysilane-urea)POSS as raw material, the hepta(triethoxysilane-urea)POSS structure is anchored onto the carrier by Friedel-Crafts alkylation.
[0013] Using the hepta(triethoxysilane-urea)POSS structure on the support, along with octa(triethoxysilane-urea)POSS and 3-aminopropyltriethoxysilane as functional monomers, the functional monomers pre-assemble with the template molecule asiaticoside via hydrogen bonding. Under the action of an acid catalyst and a crosslinking agent, in-situ polymerization occurs on the support surface via a sol-gel method. The template molecule is then removed with an eluent to obtain the product.
[0014] Preferably, the preparation method of the chloropropylhepta(triethoxysilane-ureido)POSS is as follows:
[0015] Using hydrogen-containing POSS (T8H8) as raw material and tetraethylammonium hydroxide as the opening reagent, the product T7H7(SiOH)3 was synthesized by the vertex-opening method.
[0016] Using product T7H7(SiOH)3 as raw material, chloropropyltrimethoxysilane was used as a capping reagent to synthesize chloropropylT8H7 via the vertex-capping method.
[0017] One molar equivalent of propyl isocyanate triethoxysilane reacts with one molar equivalent of allylamine in an amino-isocyanate addition reaction to produce allylureopropyl isocyanate triethoxysilane.
[0018] One molar equivalent of chloropropyl T8H7 undergoes a hydrosilylation reaction with 7.05-7.1 molar equivalents of allyl ureapropyltriethoxysilane to produce chloropropyl hepta(triethoxysilane-urea)POSS.
[0019] Preferably, the method for preparing the octa(triethoxysilane-urea)POSS is as follows: octa(triethoxysilane-urea)POSS is generated by an amino-isocyanate addition reaction between 1 molar equivalent of octaaminopropylPOSS and 8.05-8.1 molar equivalents of propyltriethoxysilane.
[0020] Preferably, the crosslinking agent is one of methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, and butyl orthosilicate;
[0021] Preferably, the acid catalyst is acetic acid or hydrochloric acid;
[0022] Preferably, the eluent is a 20-50 wt% methanol aqueous solution.
[0023] Preferably, the imprinted polymer resin is formulated as follows: 18-22 parts by weight of AB-8 styrene-type macroporous adsorption resin, 2-3 parts by weight of chloropropyl hepta(triethoxysilane-urea)POSS, 3-4 parts by weight of 3-aminopropyltriethoxysilane, 2-4 parts by weight of octa(triethoxysilane-urea)POSS, 4.5-5.5 parts by weight of hydroxyascorbic acid glycoside, and 8-9 parts by weight of tetraethyl orthosilicate.
[0024] Preferably, the extraction method for the Centella asiatica concentrate in step one is as follows:
[0025] Add 90-110 parts by weight of Centella asiatica powder to 900-1100 parts by volume of 85-95 wt% ethanol aqueous solution, stir and disperse at room temperature, then add 0.8-1.5 parts by weight of cellulase and 0.5-1.0 parts by weight of pectinase, stir and extract at 55-65℃ for 2-3 hours, incubate at 90-105℃ for 3-8 minutes to inactivate the enzymes, and obtain Centella asiatica extract. The supernatant obtained by centrifugation is purified by tandem passing through AB-8 styrene macroporous adsorption resin column and D101 styrene macroporous adsorption resin column, eluted with 80-90 wt% ethanol aqueous solution, and concentrated by rotary evaporation to obtain Centella asiatica concentrated product.
[0026] Preferably, in step two, when purifying the Centella asiatica concentrate with a resin column, a 20-50 wt% methanol aqueous solution is used as the eluent.
[0027] The purity of the Centella asiatica extract prepared according to the above process is >99%.
[0028] Preferably, the Centella asiatica extract can be used as an active ingredient in medical preparations or cosmetics, for preparing gel formulations to treat skin damage, or added to repairing skin care products.
[0029] Beneficial effects:
[0030] Synthesize chloropropylhepta(triethoxysilane-ureido)POSS and octa(triethoxysilane-ureido)POSS;
[0031] Chloropropylhepta(triethoxysilane-urea)POSS was anchored on the surface of the AB-8 styrene macroporous adsorption resin support via a Friedel-Crafts alkylation reaction of chloro-phenyl.
[0032] Using the hepta(triethoxysilane-urea)POSS structure on the surface of the carrier, octa(triethoxysilane-urea)POSS and 3-aminopropyltriethoxysilane as functional monomers, Centella asiatica extract (hydroxyasiaticoside) as template molecules, and tetraethyl orthosilicate as a crosslinking agent, an in-situ polymerization reaction was carried out on the surface of the resin carrier via a sol-gel method under acid catalysis. After removing the template molecules, a Centella asiatica extract molecularly imprinted polymer resin was prepared. The imprinted polymer resin was then packed into a solid-phase extraction column to obtain a resin column. The purity of the Centella asiatica extract purified by this resin column reached 99.8%.
[0033] The experimental results show that the Centella asiatica extract prepared by this invention has a good ability to promote cell proliferation and cell migration, that is, it has good repair and soothing effects. Detailed Implementation Example 1:
[0034] The preparation process of Centella asiatica extract includes the following steps:
[0035] Step 1: Select the whole plant of Centella asiatica harvested before flowering, rinse it with clean water to remove surface dirt and sand, and place it in a cool and ventilated place at 25℃ to air dry until the moisture has fully evaporated.
[0036] Step 2: Put the air-dried Centella asiatica into a grinder and grind it. Then pass it through a 65-mesh sieve to obtain Centella asiatica powder.
[0037] Step 3: Conduct quality inspection on the Centella asiatica powder, as detailed below:
[0038] The moisture content of Centella asiatica powder was determined to be 7.2% according to GB / T 6283-2008 "Determination of Moisture Content in Chemical Products - Karl Fischer Method (General Method)".
[0039] According to GB 5009.4-2016 "National Food Safety Standard - Determination of Ash Content in Food", the ash content of Centella asiatica powder was determined to be 5.3%.
[0040] According to GB / T 35828-2018 "Determination of Chromium, Arsenic, Cadmium, Antimony and Lead in Cosmetics by Inductively Coupled Plasma Mass Spectrometry", the lead content of Centella asiatica powder was determined to be 4.1 ppm and the arsenic content was 1.6 ppm.
[0041] The mercury content of Centella asiatica powder was determined to be 0.08 ppm according to GB 7917.1-1987 "Standard Test Methods for Hygienic Chemicals in Cosmetics".
[0042] According to the "Cosmetic Safety Technical Specifications", the limits for lead (Pb) are ≤10ppm; the limits for arsenic (As) are ≤2ppm; and the limits for mercury (Hg) are ≤1ppm. Therefore, the Centella asiatica powder prepared in this embodiment meets the safety standards for cosmetic raw materials.
[0043] Step 4: Add 100g of Centella asiatica powder prepared in Step 2 to 1000mL of 90wt% ethanol aqueous solution, stir and disperse at room temperature for 30min, then add 1.2g of cellulase (CAS No. 9012-54-8) and 0.8g of pectinase (CAS No. 9032-75-1), heat to 60℃ and stir to extract for 2.5h, place in a 100℃ water bath for 5min and then perform enzyme inactivation treatment to obtain Centella asiatica extract;
[0044] Step 5: Centrifuge the Centella asiatica extract prepared in Step 4 at 4000 r / min for 15 min to remove precipitate impurities and obtain the supernatant of Centella asiatica extract.
[0045] Step 6: The supernatant of the Centella asiatica extract prepared in Step 5 is purified by sequentially passing it through a nonpolar AB-8 styrene macroporous adsorption resin column (particle size 0.3-1.25 mm) and a weakly polar D101 styrene macroporous adsorption resin column (particle size 0.3-1.25 mm). The sample loading amount is 19.8 mg / g, the sample loading flow rate is controlled at 0.8 BV / h, 85 wt% ethanol aqueous solution is used as the eluent, the eluent volume is 5 BV, and the resin column diameter-to-height ratio is 1:7, to obtain the permeate of Centella asiatica extract.
[0046] Step 7: The permeate of the Centella asiatica extract prepared in Step 6 is concentrated by rotary evaporation at 40°C and 100 r / min to obtain the concentrated product.
[0047] Step 8: The concentrated product was dissolved in acetone and purified by a self-made resin column. The loading amount was 118 mg / g, and the loading flow rate was controlled at 0.8 BV / h. Elution was performed using 40 wt% methanol aqueous solution at a volume of 5 BV. The permeate was concentrated by rotary evaporation at 40℃ and 100 r / min to obtain 2.76 g of Centella asiatica extract. Based on this, the content of Centella asiatica extract in Centella asiatica was calculated to be 2.76 g / 100 g = 27.6 mg / g.
[0048] (I) Experiment 1 on the repair efficacy of Centella asiatica extract:
[0049] The Centella asiatica extract prepared in this embodiment was administered at eight different concentrations, and cytotoxicity tests were conducted on human dermal fibroblasts. The specific experimental methods and steps are as follows:
[0050] Cell resuscitation: Human dermal fibroblasts (provided by Boxi Biotechnology Co., Ltd.) were resuscitated at a rate of 2 × 10⁻⁶. 6 / bottle inoculated into T75 bottle, incubated at 37℃ in 5% CO2 incubator for 3 days;
[0051] Plating: Culture until cell confluence reaches approximately 90%, digest cells with 0.25% trypsin, collect by centrifugation, resuspend in DMEM low-glucose medium for counting, and take one 96-well plate, taking 4.8 × 10⁶ cells. 6 Each cell was resuspended in 12 mL of LDM low-glucose medium, at a concentration of 4 × 10⁶ cells / well. 4 Cells were plated in 3 replicates for each test concentration and incubated at 37°C in a 5% CO2 incubator for 24 hours.
[0052] Grouping: Set up negative control, positive control, test sample and blank control;
[0053] Solution preparation: Prepare Centella asiatica extract samples of different concentrations according to the test concentration using complete DMEM low-glucose medium, and filter to sterilize;
[0054] Drug administration: When the cell confluence rate in the 96-well plate reaches 40-60%, administer the drug, discard the original culture medium, add 100 μL of DMEM low-glucose medium to each well for the negative control; add 10% dimethyl sulfoxide (DMSO) DMEM low-glucose medium to each well for the positive control; add 100 μL of DMEM low-glucose medium containing the corresponding concentration of the sample to each well for the sample group; no cells are seeded in the blank control, only 100 μL of DMEM low-glucose medium is added; after drug administration, incubate the 96-well plate at 37℃ in a 5% CO2 incubator for 20 h;
[0055] Assay: After 20 h of cell culture, 20 μL / well of thiazolyl blue (MTT) solution (5 mg / mL) was added to a 96-well plate and cultured for another 4 h at 37 °C in a 5% CO2 incubator. After 4 h, the cells were removed, the supernatant was discarded, and 150 μL / well of dimethyl sulfoxide (DMSO) was added and pipetted until thoroughly mixed. The cells were then incubated at room temperature in the dark for 30 min. The cell suspension was then pipetted and mixed again. The absorbance of each well was measured at OD492 nm using an enzyme-linked immunosorbent assay (ELISA) monitor.
[0056] Cell relative viability calculation: Calculated using the formula: Cell relative viability % = [(Sample OD - Blank control OD) / (Negative control OD - Blank control OD)] × 100%;
[0057] The specific experimental results are shown in Table 1 below;
[0058] Table 1. Survival rate of human dermal fibroblasts after treatment with different concentrations of Centella asiatica extract.
[0059]
[0060] Conclusion: The relative cell viability of the Centella asiatica extract prepared in this embodiment was greater than 100% at concentrations of 0.002%, 0.007%, 0.021%, 0.062%, and 0.185%; and compared with the negative control group, the p value was less than 0.05, showing a significant difference, indicating that the Centella asiatica extract prepared in this embodiment has a certain ability to promote cell proliferation at certain concentrations.
[0061] (II) Experiment 2 on the repair efficacy of Centella asiatica extract:
[0062] The Centella asiatica extract prepared in this embodiment was administered at three different concentrations, and cytotoxicity tests were conducted on human dermal fibroblasts (the specific experimental methods and procedures are described in Experiment 1 on the repair efficacy of Centella asiatica extract). The MTT assay results are shown in Table 2 below.
[0063] Table 2. Effects of Centella asiatica extract on the viability of human dermal fibroblasts (MTT assay)
[0064]
[0065] Based on the toxicity test results in Table 2, the test concentration was selected, and the cell scratch migration distance was determined on human dermal fibroblasts. The specific experimental procedures are as follows:
[0066] Cell seeding: Draw parallel lines evenly on the bottom of a 6-well plate using a marker pen, with 3 parallel lines drawn vertically in each well; seed cells at a rate of 1×10⁶ cells / well. 6 1 cell / well seeded into a 6-well plate, 1 well for each test concentration, 3 mL per well, and incubated at 37°C in a 5% CO2 incubator for 72 h;
[0067] Scraping and drug administration: 72 hours later, cell scratching was performed. Using a 200µL pipette tip, the cells were scratched vertically, perpendicular to the three previously drawn parallel lines. One scratch was made in the middle of the well, and another in the middle of the upper and lower halves, for a total of three scratches. After scratching, the culture medium in the 6-well plate was aspirated, and the cells were washed three times with 3mL L PBS to remove the scratched cells. 3mL of culture medium (1% serum) containing the corresponding concentration of the test substance was added to the test substance wells, and normal cell culture medium (1% serum) was added to the negative control wells. After drug administration, the 6-well plate was photographed after scratching and placed in a CO2 incubator for incubation.
[0068] Photograph observation: Take photographs 6 hours after culture to analyze the cell scratch migration distance;
[0069] Migration distance calculation: Cell migration distance = initial scratch distance - scratch distance after 6 hours of culture;
[0070] The specific experimental results are shown in Table 3 below;
[0071] Table 3 Summary of cell scratch migration distance
[0072]
[0073] Conclusion: Compared with the negative control, the Centella asiatica extracts (concentrations of 0.1%, 0.05%, and 0.01%) prepared in this example showed significantly longer cell migration distances after 6 hours of cell scratching, indicating that the Centella asiatica extracts (concentrations of 0.1%, 0.05%, and 0.01%) prepared in this example have a cell migration-promoting effect, which can be used as one of the pieces of evidence for the repair efficacy of the Centella asiatica extracts prepared in this example.
[0074] (III) Experiment 1 on the soothing effects of Centella asiatica extract:
[0075] This experiment was conducted according to the experimental methods in the group standard TSHRH033-2020 "Test of Soothing Efficacy of Cosmetics - In Vitro Determination of TNF-α Inflammatory Factor Content - Test Method for Lipopolysaccharide-Induced Macrophages RAW264.7".
[0076] The Centella asiatica extract prepared in this embodiment was administered at eight different concentrations, and cytotoxicity tests were conducted on mouse mononuclear macrophage leukocytes (RAW264.7). The specific experimental procedures are as follows:
[0077] Cell resuscitation: Mouse mononuclear macrophage leukocytes (RAW264.7) (provided by Bokxi Biotechnology Co., Ltd.) were resuscitated at a rate of 8 × 10⁻⁶ cells / year. 6 / bottle was inoculated into T75 bottle and incubated at 37℃ in a 5% CO2 incubator for 2 days, with the medium changed daily;
[0078] Plating: Culture until cell confluence reaches approximately 90%. Use a cell scraper to scrape cells from the culture flask, collect them by centrifugation, resuspend them in DMEM high-glucose medium, and count them. Take one 96-well plate and collect 3.6 × 10⁶ cells. 6 Each cell was resuspended in 12 mL L DMEM high-glucose medium at a concentration of 3 × 10⁶ cells / well. 4 Cells were plated in 3 replicates for each test concentration and incubated at 37°C in a 5% CO2 incubator for 24 hours.
[0079] Grouping: Set up negative control, positive control, test sample and blank control;
[0080] Solution preparation: Prepare Centella asiatica extract samples of different concentrations according to the test concentration using complete DMEM high-glucose medium, and filter to sterilize;
[0081] Drug administration: When the cell confluence rate in the 96-well plate reaches 40-60%, drug administration is performed. The original culture medium is discarded. For the negative control, 100 μL of DMEM high-glucose medium is added to each well; for the positive control, 100 μL of DMEM high-glucose medium containing 10% dimethyl sulfoxide (DMSO) is added to each well; for the sample group, 100 μL of DMEM high-glucose medium containing the corresponding concentration of the sample is added to each well; for the blank control, no cells are seeded, only 100 μL of DMEM high-glucose medium is added; for all groups, lipopolysaccharide (LPS) is added to a final concentration of 10 μg / mL; after drug administration, the 96-well plate is placed in a 37°C, 5% CO2 incubator for 20 h.
[0082] Assay: After 20 h of cell culture, 20 μL / well of thiazolyl blue (MTT) solution (5 mg / mL) was added to a 96-well plate and cultured for another 4 h at 37 °C in a 5% CO2 incubator. After 4 h, the cells were removed, the supernatant was discarded, 150 μL / well of DMSO was added and the cells were thoroughly mixed by pipetting. The cells were then placed at room temperature in the dark for 30 min. The cell suspension was then mixed again by pipetting, and the absorbance of each well was measured at OD492 nm using an enzyme-linked immunosorbent assay (ELISA) monitor.
[0083] Cell relative viability calculation: Calculated using the formula: Cell relative viability % = [(Sample OD - Blank control OD) / (Negative control OD - Blank control OD)] × 100%;
[0084] The specific experimental results are shown in Table 4 below;
[0085] Table 4. Survival rate of mouse mononuclear macrophage leukocytes (RAW264.7) after treatment with Centella asiatica extract.
[0086]
[0087] Based on the toxicity test results in Table 4, the test concentration was selected, and the content of the inflammatory factor TNF-α was measured in mouse monocyte / macrophage leukocytes (RAW264.7). The specific experimental procedures are as follows:
[0088] Cell seeding: Cells at 3 × 10⁻⁶ 4 100 μL of cells / well was seeded into a 96-well plate, with 3 replicates for each test concentration. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h.
[0089] Drug administration: 24 h later, the culture medium in the 96-well plate was aspirated. 200 μL of culture medium containing the test substance was added to the test substance wells, culture medium containing the positive control was added to the positive control wells, and normal complete cell culture medium was added to the negative control and blank control wells. After drug administration, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 2 h for pretreatment. LPS stimulation: After the 2 h pretreatment, except for the blank control wells, LPS was added to each well at a final concentration of 10 μg / mL, and the plate was incubated at 37°C, 5% CO2 for another 24 h.
[0090] Collect the supernatant: After 24 hours of incubation, collect 200 μL of cell culture supernatant from each well into a 1.5 mL sterile centrifuge tube and store it at -80°C for later use.
[0091] ELISA kit for detecting TNF-α levels: ELISA testing should be performed according to the instructions for use of the mouse tumor necrosis factor α (TNF-α) enzyme-linked immunosorbent assay kit. The specific steps are as follows:
[0092] Adding standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard sample of different concentrations to each standard sample well;
[0093] Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent, all other steps are the same) and sample wells; add 40 μL of sample diluent to the sample wells on the enzyme-labeled plate, and then add 10 μL of sample to be tested (the final sample dilution concentration is 5 times); add the sample to the bottom of the wells of the enzyme-labeled plate, trying not to touch the well wall, and gently shake to mix;
[0094] Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank wells;
[0095] Incubation: After sealing with sealing film, incubate at 37℃ for 1 hour;
[0096] Solution preparation: Dilute the 20-fold concentrated washing solution with 20 times distilled water and set aside.
[0097] Washing: Remove the sealing film, discard the liquid, spin dry, fill each hole with washing liquid, let stand for 30 seconds, then discard.
[0098] Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop color at 37°C in the dark for 15 min.
[0099] Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).
[0100] Measurement: Zero the instrument with the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm;
[0101] The specific experimental results are shown in Table 5 below;
[0102] Table 5 Summary of TNF-α levels (inflammatory factor)
[0103]
[0104] Note: Compared with the negative control group, a p-value < 0.05 indicates a significant difference, indicated by "#"; a p-value < 0.01 indicates a highly significant difference, indicated by "##"; compared with the negative control group, a p-value < 0.05 indicates a significant difference, indicated by "*", and a p-value < 0.01 indicates a highly significant difference, indicated by "**".
[0105] Conclusion: Compared with the negative control group, the content of the inflammatory factor TNF-α in the Centella asiatica extracts (concentrations of 0.556%, 0.185%, and 0.062%) prepared in this example was significantly downregulated, indicating that Centella asiatica extract can inhibit the production of TNF-α at the tested concentrations, which can serve as one of the pieces of evidence for the soothing effect of Centella asiatica extract.
[0106] (iv) Experiment 2 on the soothing effects of Centella asiatica extract:
[0107] To investigate the inhibitory effect of Centella asiatica extract on hyaluronidase, the following experiment was conducted, with the specific steps as follows:
[0108] The experiment was set up with a sample group, a sample background group, an enzyme reaction group, and a solvent background group. Each sample group for each test concentration needed to have 3 replicates, and the enzyme reaction group also needed to have 3 replicates. Refer to Table 6 below, add the reagents to the test tubes in order, and measure the absorbance at around 530 nm after the reaction.
[0109] Table 6 Sample Addition Requirements
[0110]
[0111] Hyaluronidase inhibition rate (%) = {[(A A -A B )-(A C -A D )] / (A A -A B )}×100%;
[0112] The specific experimental results are shown in Table 7 below;
[0113] Table 7 Summary of Hyaluronidase Inhibition Rate
[0114]
[0115] Note: Compared with the negative control group, the positive control group and the sample group, a p-value <0.05 indicates a significant difference, indicated by "*", and a p-value <0.01 indicates a highly significant difference, indicated by "**".
[0116] Conclusion: According to the test results, the hyaluronidase inhibition rate of the positive control group (2 mg / mL dipotassium glycyrrhizate) was 17.87%, which is within the standard range, verifying the effectiveness of the experimental system; at the test concentration, the average inhibition rate of Centella asiatica extract on hyaluronidase activity prepared in this example was 18.20%, with a standard deviation of <15%, and compared with the negative control group, the p value was <0.05, indicating that the Centella asiatica extract sample has a certain soothing effect;
[0117] (v) The Centella asiatica extract was quantitatively analyzed using an Agilent 1260 high-performance liquid chromatograph. The specific parameters are as follows:
[0118] Configure a Neptune C18 column (250 mm × 4.6 mm, 5 μm);
[0119] The column temperature is 25℃;
[0120] The detection wavelength is 206nm;
[0121] The column flow rate is 1 mL / min;
[0122] The injection volume was set to 10 µL;
[0123] Acetonitrile was used as mobile phase A and water as mobile phase B. Gradient elution was performed with the following elution program: 0-30 min 22% A, 30-40 min 30% A, 40-50 min 40% A.
[0124] The specific analysis results are as follows: 2.76g of Centella asiatica extract contains 99.8% hydroxyasiaticoside (CAS number 18449-41-7);
[0125] (vi) The preparation method of the above-mentioned self-made resin column is as follows:
[0126] Step 1: Set the resin formulation for the resin column. The specific formulation is as follows: 20g AB-8 styrene-type macroporous adsorption resin, 2.6g chloropropylhepta(triethoxysilane-urea)POSS (0.001mol), 3.1g 3-aminopropyltriethoxysilane (0.014mol), 2.9g octa(triethoxysilane-urea)POSS (0.001mol), 4.9g hydroxyasiaticoside (0.005mol), and 8.3g tetraethyl orthosilicate (0.04mol).
[0127] Step 2: Under the catalysis of Lewis acid, the chlorine functional group of chloropropylhepta(triethoxysilane-urea)POSS and the phenyl group on the surface of AB-8 styrene-type macroporous adsorption resin microspheres undergo a Friedel-Crafts alkylation reaction to achieve surface grafting modification of styrene-type macroporous adsorption resin by chloropropylhepta(triethoxysilane-urea)POSS. This results in the uniform formation of an organic-inorganic hybrid layer rich in triethoxysilane structure on the surface of the styrene-type macroporous adsorption resin, thus obtaining a functional monomer resin.
[0128] The Lewis acid can be selected from one of aluminum trichloride, ferric trichloride, tin tetrachloride, and zinc dichloride; in this embodiment, aluminum trichloride is selected.
[0129] The experimental steps for functionalized monomer resin are as follows: 20g of AB-8 styrene-type macroporous adsorption resin (particle size 0.3-1.25mm) and 200mL of anhydrous acetone are added to a three-necked flask and stirred at room temperature for 30min to allow swelling. Then, 3.0g of anhydrous aluminum trichloride and 2.6g of chloropropylhepta(triethoxysilane-urea)POSS are added to the three-necked flask in sequence. The mixture is heated to 60℃ and stirred for 20h. After cooling to room temperature, the mixture is filtered, washed repeatedly with anhydrous ethanol, and air-dried naturally. After the ethanol odor has basically disappeared, the mixture is transferred to a vacuum drying oven and vacuum dried at 40℃ to obtain the functionalized monomer resin.
[0130] Step 3: Using hydroxyascorbic acid as a template molecule, and chloropropylhepta(triethoxysilane-urea)POSS, 3-aminopropyltriethoxysilane and octa(triethoxysilane-urea)POSS on the surface of the functional monomer resin as composite functional monomers, the composite functional monomers and the template molecule are pre-assembled on the resin surface through hydrogen bonding to obtain pre-assembled polymerized monomers loaded on the resin.
[0131] The specific experimental steps for the pre-assembled polymer monomer loaded on the resin are as follows: 4.9g of asiaticoside, 3.1g of 3-aminopropyltriethoxysilane, 2.9g of octa(triethoxysilane-ureido)POSS and the functionalized monomer resin prepared in step two are added to 200mL of anhydrous acetone, stirred at room temperature for 2h and allowed to stand for 24h to obtain the pre-assembled polymer monomer loaded on the resin.
[0132] Step 4: Add tetraethyl orthosilicate crosslinking agent to the pre-assembled polymer monomer loaded on the resin. Under the action of acetic acid catalyst, the polymer monomer undergoes in-situ polymerization reaction on the surface of the resin carrier by sol-gel method. Then, the template molecules are removed by methanol aqueous solution as eluent to obtain imprinted resin.
[0133] The specific experimental steps for preparing the imprinted resin are as follows: 8.3 g of tetraethyl orthosilicate is added to the pre-assembled polymer monomer loaded on the resin, ultrasonically degassed for 5 min, stirred and mixed at room temperature for 2 h, then 1 mL of 1 mol / L acetic acid aqueous solution is added, stirred and reacted at room temperature for 24 h, filtered, and repeatedly washed with 40 wt% methanol aqueous solution. The hydroxyasiaticoside in the washing solution is detected by an Agilent 1260 high performance liquid chromatograph (the detection conditions are the same as those in (V)) until the hydroxyasiaticoside template molecules are completely removed. The resin is air-dried for 24 h and then dried in a vacuum drying oven at 40 ℃ for 6 h to obtain the imprinted resin.
[0134] The adsorption capacity of the imprinted resin was tested using the following method:
[0135] Prepare 100 mL of a 5 mg / mL asiaticoside acetone solution, add 1 g of imprinting resin, stir at room temperature for 12 h, filter, and use an Agilent 1260 high-performance liquid chromatograph to determine the final mass concentration of asiaticoside in the solution. Calculate the adsorption capacity using the following method:
[0136] Adsorption capacity (mg / g) = [(C0-C1)×V]÷M;
[0137] Wherein, C0 is the initial mass concentration of the hydroxyasiaticoside acetone solution, and its unit is mg / mL. At this time, C0 = 10 mg / mL.
[0138] C1 is the final mass concentration of the hydroxyasiaticoside acetone solution, and its unit is mg / mL;
[0139] V is the volume of the hydroxyasiaticoside acetone solution, and its unit is mL. In this case, V = 100 mL.
[0140] M is the mass of the imprinting resin, and its unit is g. In this case, M=1g.
[0141] Test results showed that the adsorption capacity of the imprinted resin for asiaticoside was 118 mg / g;
[0142] Step 5: Pack 2.5g of imprinting resin into a 20mL solid-phase extraction empty column, and rinse with 60mL of methanol to obtain a resin column. Example 2:
[0143] Preparation of octa(triethoxysilane-urea)POSS: An amino-isocyanate addition reaction was carried out between 1 molar equivalent of octaaminopropylPOSS and 8.08 molar equivalents of propyltriethoxysilane isocyanate (CAS No. 24801-88-5) to generate octa(triethoxysilane-urea)POSS, whose chemical structural formula is as follows:
[0144] ;
[0145] The chemical structure of R is as follows: ;
[0146] The experimental procedure for octa(triethoxysilane-urea)POSS is as follows: Under nitrogen protection, 8.9 g of octaaminopropylPOSS and 30 mL of anhydrous tetrahydrofuran were added to a three-necked flask and stirred at room temperature until completely dissolved. Then, 30 mL of anhydrous tetrahydrofuran solution containing 20 g of isocyanatetriethoxysilane was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 4 h, heated to 40 °C and stirred for 4 h, then evaporated under reduced pressure and dried under vacuum to obtain octa(triethoxysilane-urea)POSS.
[0147] The 1H NMR spectrum of octa(triethoxysilane-urea)POSS is characterized as follows: 1 H NMR (DMSO-d6, 400MHz) δ: 0.80-0.88 (m, 32H), 1.25-1.29 (t, 72H), 1.82-2.07 (m, 32H), 3.12-3.26 (m, 32H), 3.61-3.79 (m, 48H), 6.01-6.04 (m, 16H);
[0148] The synthetic route for preparing chloropropylhepta(triethoxysilane-ureido)POSS is as follows:
[0149] The first step involved synthesizing product T7H7(SiOH)3 via a vertex-opening method using hydrogen-containing POSS (T8H8) as the raw material and tetraethylammonium hydroxide as the opening reagent. The experimental steps were as follows: Under nitrogen protection, 4.2 g of hydrogen-containing POSS (T8H8) and 80 mL of anhydrous tetrahydrofuran were added to a three-necked flask and stirred at room temperature until completely dissolved. Then, 4 mL of a 35 wt% tetraethylammonium hydroxide aqueous solution was added to the flask, and the mixture was heated to 70 °C and refluxed for 6 h. After cooling to room temperature, the pH was adjusted to neutral using 0.1 mol / L dilute hydrochloric acid. The product was then dissolved in diethyl ether by rotary evaporation under reduced pressure, dried over anhydrous magnesium sulfate, filtered, and the diethyl ether was removed by rotary evaporation. The product T7H7(SiOH)3 was obtained after drying, and its chemical structural formula is as follows:
[0150] ;
[0151] The second step involved using product T7H7(SiOH)3 as a raw material and chloropropyltrimethoxysilane (CAS No. 2530-87-2) as a capping reagent to synthesize chloropropylT8H7 via a vertex-capping method. The experimental procedure was as follows: Under nitrogen protection, 4.0 g of product T7H7(SiOH)3 and 40 mL of anhydrous tetrahydrofuran were added to a three-necked flask and stirred at room temperature until completely dissolved. The flask was then placed in an ice-water bath, and 10 mL of an anhydrous tetrahydrofuran solution containing 1.7 mL of chloropropyltrimethoxysilane was added dropwise. The mixture was stirred in the ice-water bath for 30 min, then the ice-water bath was removed, and the reaction was continued at room temperature for 12 h. The solution was then concentrated under reduced pressure by rotary evaporation to a saturated state and dried to obtain chloropropylT8H7, whose chemical structural formula is:
[0152] ;
[0153] The third step involves an amino-isocyanate addition reaction between 1 molar equivalent of propyltriethoxysilane (CAS No. 24801-88-5) and 1 molar equivalent of allylamine to produce allylureapropyltriethoxysilane. The experimental procedure is as follows: Under nitrogen protection, 25 mL of propyltriethoxysilane and 80 mL of anhydrous tetrahydrofuran are added to a three-necked flask and cooled in an ice bath. Then, 30 mL of anhydrous tetrahydrofuran solution containing 8.2 mL of allylamine is added dropwise. After the addition is complete, the mixture is stirred at room temperature for 2 hours, then heated to 40°C and stirred for another 2 hours. The tetrahydrofuran is removed by rotary evaporation under reduced pressure. The mixture is washed twice with 50 mL of n-hexane and dried under vacuum to obtain allylureapropyltriethoxysilane, whose chemical structural formula is:
[0154] ;
[0155] Step 4: 1 molar equivalent of chloropropyl T8H7 reacts with 7.06 molar equivalents of allylureapropyltriethoxysilane in a hydrosilylation reaction to generate chloropropylhepta(triethoxysilane-ureo)POSS. The experimental procedure is as follows: 1.5 g of chloropropyl T8H7 and 80 mL of anhydrous tetrahydrofuran are added to a three-necked flask and stirred to dissolve. 6.2 g of allylureapropyltriethoxysilane and 30 μL of Karstedt catalyst (2% Pt) are added, and the mixture is refluxed at 70 °C for 12 h. After cooling to room temperature, 15 mg of triphenylphosphine is added, and the mixture is stirred for 1 h. The catalyst is removed by filtration, and the solvent is removed by rotary evaporation of the filtrate. After drying, chloropropylhepta(triethoxysilane-ureo)POSS is obtained, with the following chemical structural formula:
[0156] ;
[0157] The chemical structure of R is as follows: ;
[0158] The 1H NMR spectrum of chloropropylhepta(triethoxysilane-ureo)POSS is characterized as follows: 1 H NMR (DMSO-d6, 400MHz) δ: 0.78-0.94 (m, 30H), 1.26-1.30 (t, 63H), 1.84-2.08 (m, 30H), 3.09-3.26 (m, 28H), 3.61-3.79 (m, 44H), 6.01-6.04 (m, 14H).
Claims
1. A preparation process for Centella asiatica extract, characterized in that, Includes the following steps: Step 1: Add 90-110 parts by weight of Centella asiatica powder to 900-1100 parts by volume of 85-95 wt% ethanol aqueous solution, stir and disperse at room temperature, then add 0.8-1.5 parts by weight of cellulase and 0.5-1.0 parts by weight of pectinase, stir and extract at 55-65℃ for 2-3 hours, incubate at 90-105℃ for 3-8 minutes to inactivate the enzymes, and obtain Centella asiatica extract. The supernatant obtained by centrifugation is purified by tandem passing through AB-8 styrene macroporous adsorption resin column and D101 styrene macroporous adsorption resin column, eluted with 80-90 wt% ethanol aqueous solution, and concentrated by rotary evaporation to obtain Centella asiatica concentrated product. Step 2: Prepare a molecularly imprinted polymer resin for Centella asiatica extract and fill it into a solid-phase extraction column to obtain a resin column. Purify the concentrated Centella asiatica product through the resin column to obtain Centella asiatica extract, which is hydroxyasiaticoside. The formulation of the imprinted polymer resin is as follows: by weight, 18-22 parts of AB-8 styrene-type macroporous adsorption resin, 2-3 parts of chloropropyl hepta(triethoxysilane-urea)POSS, 3-4 parts of 3-aminopropyltriethoxysilane, 2-4 parts of octa(triethoxysilane-urea)POSS, 4.5-5.5 parts of asiaticoside and 8-9 parts of tetraethyl orthosilicate; The method for preparing the imprinted polymer resin is as follows: using AB-8 styrene-type macroporous adsorption resin as a carrier and chloropropyl hepta(triethoxysilane-urea)POSS as a raw material, the hepta(triethoxysilane-urea)POSS structure is anchored onto the carrier by Friedel-Crafts alkylation; the hepta(triethoxysilane-urea)POSS structure on the carrier, along with octa(triethoxysilane-urea)POSS and 3-aminopropyltriethoxysilane, serve as functional monomers. The functional monomers are pre-assembled with the template molecule asiaticoside through hydrogen bonding. Under the action of an acid catalyst and tetraethyl orthosilicate, an in-situ polymerization reaction occurs on the carrier surface via a sol-gel method. The template molecule is removed with an eluent to obtain the product. The chemical structural formula of chloropropylhepta(triethoxysilane-ureido)POSS is: ； The chemical structural formula of octa(triethoxysilane-urea)POSS is: ； The chemical structure of R is as follows: .
2. The preparation process of Centella asiatica extract according to claim 1, characterized in that, The preparation method of the chloropropylhepta(triethoxysilane-ureido)POSS is as follows: Using hydrogen-containing POSS (T8H8) as raw material and tetraethylammonium hydroxide as the opening reagent, the product T7H7(SiOH)3 was synthesized by the vertex-opening method. Using product T7H7(SiOH)3 as raw material, chloropropyltrimethoxysilane was used as a capping reagent to synthesize chloropropylT8H7 via the vertex-capping method. One molar equivalent of propyl isocyanate triethoxysilane reacts with one molar equivalent of allylamine in an amino-isocyanate addition reaction to produce allylureopropyl isocyanate triethoxysilane. One molar equivalent of chloropropyl T8H7 undergoes a hydrosilylation reaction with 7.05-7.1 molar equivalents of allyl ureapropyltriethoxysilane to produce chloropropyl hepta(triethoxysilane-urea)POSS.
3. The preparation process of Centella asiatica extract according to claim 1, characterized in that, The method for preparing the octa(triethoxysilane-urea)POSS is as follows: octa(triethoxysilane-urea)POSS is generated by an amino-isocyanate addition reaction between 1 molar equivalent of octaaminopropylPOSS and 8.05-8.1 molar equivalents of propyltriethoxysilane.
4. The preparation process of Centella asiatica extract according to claim 1, characterized in that, The acid catalyst is acetic acid or hydrochloric acid; The eluent is a 20-50 wt% methanol aqueous solution.
5. The preparation process of Centella asiatica extract according to claim 1, characterized in that, In step two, when purifying the concentrated Centella asiatica product using a resin column, a 20-50 wt% methanol aqueous solution is used as the eluent.
6. The Centella asiatica extract prepared according to any one of claims 1-5, characterized in that, The purity of the Centella asiatica extract is >99%.