Asiaticoside gel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine gel preparation, in particular to asiaticoside gel and a preparation method and application thereof. The asiaticoside gel is used for preparing a preparation for wound healing and comprises the following components in parts by weight: 0.2-2 parts of an asiaticoside extract or lipidosome with the content of total asiaticoside in an active ingredient being 70-80wt%, 0.2-2 parts of a p-hydroxybenzoate compound, 2-10 parts of glycerol and 5-20 parts of carbomer. The preparation method comprises the following steps: S1, swelling carbomer, and adjusting the pH value to obtain carbomer gel; s2, dissolving the asiaticoside extract or lipidosome and a p-hydroxybenzoate compound into glycerol to obtain a glycerol solution; and S3, adding a glycerol solution into the carbomer gel, and uniformly stirring to obtain the asiaticoside gel. The asiaticoside gel prepared by the invention has the advantages of no toxic or side effect, freshness, transparency, no greasiness and good wound healing effect in the wound healing process.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine gel preparation technology, specifically to a asiaticoside gel, its preparation method, and its application. Background Technology

[0002] Centella asiatica is a plant belonging to the genus Centella of the family Apiaceae. It is widely distributed throughout the world, and in my country, it is mainly found south of the Yangtze River. The whole plant is used medicinally. The Chinese Pharmacopoeia describes its functions and indications as: clearing heat and promoting diuresis, detoxifying and reducing swelling. It is used for damp-heat jaundice, heatstroke diarrhea, urinary stones and hematuria, carbuncles and boils, and injuries from falls and blows.

[0003] Centella asiatica mainly contains polyyne compounds, triterpenes and their glycosides, volatile oils, flavonoids, alkaloids, and other compounds. Among them, asiaticoside, a triterpenoid, is considered the main active ingredient responsible for Centella asiatica's skin-repairing effects. The main asiaticoside compounds include asiaticoside-B, hydroxyasiaticoside, and asiaticoside. In wound healing, asiaticoside can regulate inflammation, prevent excessive reactions, stimulate fibroblast proliferation and migration, and inhibit excessive scar hyperplasia.

[0004] Currently, most products on the market contain complex Centella Asiatica ingredients, with low levels of asiaticoside, unclear biotoxicity, and ambiguous therapeutic effects. Summary of the Invention

[0005] In view of the technical problems pointed out in the background art, the purpose of this invention is to prepare a asiaticoside gel and to verify its toxicity and performance through relevant cell and animal experiments on high-content asiaticoside. The preparation method of the gel of this invention is simple to operate.

[0006] The asiaticoside gel comprises: carbomer, asiaticoside extract or liposomes, glycerin, and parabens. The asiaticoside gel, by weight, consists of: 0.2-2 parts asiaticoside extract or liposomes, 0.2-2 parts parabens, 2-10 parts glycerin, and 5-20 parts carbomer.

[0007] Among them, the total asiaticoside content in the asiaticoside extract or liposomes is 70-80 wt%; the parabens are methylparaben, ethylparaben, and butylparaben.

[0008] The preparation steps of asiaticoside gel are as follows:

[0009] 1) Disperse carbomer powder into water with constant stirring, let stand overnight to allow it to fully swell, then add triethanolamine or NaOH to adjust the pH to 7-8 to obtain carbomer gel;

[0010] 2) Add parabens and asiaticoside extract or liposomes with a total asiaticoside content of 70-80 wt% to glycerol to prepare a glycerol solution;

[0011] 3) Add the glycerol solution to the carbomer gel and stir well to obtain the asiaticoside gel.

[0012] The above-mentioned asiaticoside gel is used to prepare a gel for wound healing.

[0013] Beneficial effects:

[0014] Carbomer gel, as a sustained-release agent, encapsulates the active ingredient asiaticoside. If asiaticoside liposomes are used, the two work synergistically to more effectively and precisely control the release rate, avoid the rapid loss of the active ingredient asiaticoside, and ensure that asiaticoside continues to act on the wound.

[0015] When Centella asiatica liposomes are applied to the skin, the phospholipids in the liposomes fuse with the intercellular lipids of the stratum corneum, expanding lipid channels. Both the gel and glycerin help the skin hydrate better; fully hydrated stratum corneum cells swell, and intercellular spaces increase. The combined effect of these three factors increases permeability, allowing Centella asiatica liposomes to penetrate the stratum corneum more easily and enhancing transdermal absorption.

[0016] The asiaticoside gel prepared by this invention has the advantages of being non-toxic and non-greasy, refreshing and transparent, and having a good wound healing effect during the wound healing process. It is a novel wound healing preparation with great development potential. Attached Figure Description

[0017] Figure 1 The results of the hemolysis test of asiaticoside in Example 5 of the present invention are shown.

[0018] Figure 2 The results of the cytotoxicity test of asiaticoside L929 in Example 6 of the present invention are shown.

[0019] Figure 3 The results of the cytotoxicity test of asiaticoside 3T3 in Example 6 of this invention are shown.

[0020] Figure 4 The image shown is a migration diagram of asiaticoside L929 cells in Example 7 of this invention.

[0021] Figure 5 The figure shows the cell migration rate of asiaticoside L929 cells in Example 7 of the present invention.

[0022] Figure 6 The image shown is a migration diagram of asiaticoside 3T3 cells in Example 7 of this invention.

[0023] Figure 7 The figure shows the migration rate of asiaticoside 3T3 cells in Example 7 of this invention.

[0024] Figure 8 The image shown is a staining diagram of the live and dead cells of asiaticoside 3T3 cells in Example 8 of this invention.

[0025] Figure 9 The figure shows the survival rate of asiaticoside 3T3 cells in Example 8 of this invention.

[0026] Figure 10 The image shown is a wound healing diagram of asiaticoside in Example 9 of the present invention.

[0027] Figure 11 The figure shows the wound healing rate of asiaticoside in Example 9 of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0029] Example 1: Extraction of asiaticoside compounds from crude Centella asiatica and determination of total asiaticoside content

[0030] Weigh 100g of crude Centella asiatica (from Xi'an Shengqing Biotechnology Co., Ltd.) containing approximately 10% (mass fraction) total glycosides. Add 1000mL of 95% (volume fraction) ethanol. Stir at 200r / min for 60min at room temperature. Filter under reduced pressure. Concentrate the filtrate to dryness using rotary evaporation. Dissolve in 60mL of water saturated with n-pentanol, then add 200mL of water-saturated n-pentanol for liquid-liquid extraction. Collect the supernatant, evaporate to dryness using rotary evaporation, dissolve in 50mL of methanol, and add dropwise to 250mL of dichloromethane while stirring continuously. The precipitate is filtered through a glass frit funnel and dried to obtain a light yellow solid.

[0031] Content determination of samples: The determination of total asiaticoside content was performed according to the method in Part I of the Chinese Pharmacopoeia 2025. Accurately weigh 50±5 mg of asiaticoside extract (according to the Chinese Pharmacopoeia 2025), place it in a 50 ml volumetric flask, dissolve it in methanol, dilute to the mark, and shake well to obtain the test solution. Accurately weigh 10 mg each of hydroxyasiaticoside reference standard and asiaticoside reference standard, place them in a 50 ml volumetric flask, dissolve them in methanol, dilute to the mark, and shake well to obtain the reference solution. An Agilent 1260 high-performance liquid chromatograph was used; octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-2 mmol / L betacyclodextrin solution (24:76) was used as the mobile phase; the detection wavelength was 205 nm; the flow rate was 1 mL / min; and the injection volume was 10 μL. The theoretical plate number, calculated based on the asiaticoside peak, should not be less than 4000. The average content of total asiaticoside compounds in the asiaticoside extract was determined to be 77%.

[0032] Example 2

[0033] Measure 95 mL of pure water and stir at 1000 rpm to form a vortex. Continue stirring and slowly add 1 g of carbomer 940 powder, dispersing it evenly. After the powder is added, continue stirring for 30 minutes and let it stand overnight. After sufficient swelling, add triethanolamine solution dropwise until the pH reaches 7 and stir evenly. Dissolve 0.1 g of methylparaben and 0.1 g of the asiaticoside extract from Example 1 in 5 g of glycerol to obtain a glycerol solution. Mix the carbomer gel and the glycerol solution and stir evenly to obtain the asiaticoside gel, which has a relatively high viscosity.

[0034] Example 3

[0035] Measure 95 mL of pure water and stir at 800 rpm to form a vortex. Continue stirring and slowly add 0.8 g of carbomer 941 powder, dispersing it evenly. After the powder is added, continue stirring for 20 minutes and let it stand overnight. After sufficient swelling, add sodium hydroxide solution dropwise until the pH reaches 6, and stir until homogeneous. Dissolve 0.1 g of butylparaben and 0.2 g of asiaticoside extract in 2 g of glycerol to obtain a glycerol solution. Mix the carbomer gel and the glycerol solution and stir until homogeneous to obtain the asiaticoside gel. This gel has low viscosity and good flowability.

[0036] Example 4

[0037] Measure 95 mL of pure water and stir at 1200 rpm to form a vortex. Continue stirring and slowly add 1.5 g of carbomer ETD2020 powder, dispersing it evenly. After adding the powder, continue stirring for 40 minutes and let it stand overnight. After sufficient swelling, add triethanolamine dropwise until the pH reaches 8 and stir evenly. Dissolve 20 mg of ethylparaben and 0.4 g of asiaticoside liposomes in 10 g of glycerol to obtain a glycerol solution. Mix the carbomer gel and the glycerol solution and stir evenly to obtain the asiaticoside gel. This gel has a moderate viscosity and a smooth, refreshing feel.

[0038] Example 5 Hemolysis Test

[0039] The hemolysis test is based on the integrity of the red blood cell membrane and the release of hemoglobin. In an isotonic environment (such as physiological saline), normal red blood cells maintain their intact membranes, with a high concentration of hemoglobin trapped within the cell. Through centrifugation, intact red blood cells precipitate to the bottom of the tube, and the supernatant becomes clear and colorless or slightly yellow.

[0040] When red blood cells are exposed to harmful substances, they may be damaged through the following mechanisms: 1. Osmotic pressure: Hypotonic solutions allow a large amount of water to enter the cells, causing them to swell and rupture. 2. Membrane damage: The test substance directly damages the lipid bilayer of the red blood cell membrane or attacks membrane proteins, causing membrane perforation or tearing. After the red blood cell membrane ruptures, hemoglobin is released into the surrounding fluid. The released hemoglobin will make the supernatant appear red, and the intensity of the red color is directly proportional to the degree of hemolysis. By centrifuging to remove unruptured cells and debris, the absorbance of the supernatant can be measured using a spectrophotometer at approximately 540 nm (the characteristic absorption wavelength of hemoglobin) to quantitatively calculate the hemolysis rate.

[0041] Operating steps

[0042] Fresh blood was obtained from the orbital cavity of healthy mice, and erythrocytes were separated by cryocentrifugation. A PBS solution (phosphate buffer saline) containing 20% ​​(v / v) erythrocytes was mixed with serum-free asiaticoside culture medium solutions prepared in Example 1 at different concentrations (0.25, 0.5, 1, 2, 4 mg / mL). PBS and Triton X-100 (polyethylene glycol octylphenyl ether) were used as negative and positive controls, respectively, and the mixture was co-cultured at 37°C for 2 h. The mixture was separated by cryocentrifugation, and the absorbance of the supernatant at 540 nm was measured to investigate the hematologic toxicity of asiaticoside. Hemolysis rate (%) = (sample absorbance − negative control absorbance) / (positive control absorbance − negative control absorbance) × 100%.

[0043] in conclusion

[0044] When the concentration of asiaticoside is less than 2 mg / mL, the hemolysis rate is <5%, indicating no hemolytic activity and meeting biosafety requirements. See details below. Figure 1 .

[0045] Example 6 Cytotoxicity test

[0046] The core principle of the MTT assay is based on the activity of succinate dehydrogenase in the mitochondria of living cells. MTT is a yellow, water-soluble thiazolyl blue salt. When added to a cell culture system, it can cross the cell membrane and enter living cells. Dehydrogenases such as succinate dehydrogenase present in the mitochondria of living cells catalyze the reduction of yellow MTT to form formazan, an insoluble blue-purple crystal, which is deposited in the cells, especially around the mitochondria. This reaction only occurs in living cells; dead cells lose this ability. Adding a specific organic solvent (such as dimethyl sulfoxide (DMSO), isopropanol, or acidified SDS solution) dissolves the formazan crystals within the cells, forming a homogeneous blue-purple solution. This solution has characteristic absorption peaks near 490 nm or 570 nm. The absorbance (OD value) is measured using a microplate reader. Within a certain cell number range, the amount of formazan produced is directly proportional to the number of living cells (or metabolic activity). Therefore, by comparing the absorbance values ​​of the experimental group and the control group, the relative cell viability or inhibition rate can be calculated.

[0047] Operating steps

[0048] The cytotoxicity of asiaticoside prepared in Example 1 was detected using the MTT assay, employing mouse fibroblasts (L929) and mouse embryonic fibroblasts (3T3), respectively. Cells were seeded in 96-well plates (10... 4 Cells were incubated at 37°C with 5% CO2 for 24 hours to allow for full cell adhesion and entry into the logarithmic growth phase. Different working concentrations of asiaticoside (0.25, 0.5, 1, 2, 4 mg / mL) were prepared using DMEM medium. After sterilization, cells were added, and the cells were incubated at 37°C with 5% CO2 for 24 hours. Five replicates were set up for each group. A control group was prepared with only DMEM medium and no cell seeding. A negative control group was prepared with DMEM medium and cell seeding but no asiaticoside. Finally, MTT solution was added, the culture plate was gently shaken to mix, and the plate was returned to the incubator for another 4 hours in the dark. A small amount of blue-purple precipitate appeared at the bottom of the wells. The supernatant was carefully aspirated, 150 µL of DMSO was added, and the culture plate was placed on a shaker and shaken at low speed for 10-15 minutes until the purple crystals were completely dissolved and the solution was homogeneous. Immediately use a microplate reader, set the detection wavelength to 490 nm, zero the instrument using a blank control group (wells containing only culture medium and DMSO), and measure the absorbance. Calculate cell viability: Cell viability (%) = (OD0.05)2 实验组 - OD空白组 ) / (OD 阴性对照组 - OD 空白组 )×100%.

[0049] in conclusion

[0050] After treatment with 0.25, 0.5, 1, 2, and 4 mg / mL asiaticoside solutions, the survival rate of mouse fibroblasts (L929) was over 100%, indicating virtually no toxicity. After treatment with 0.25, 0.5, and 1 mg / mL asiaticoside solutions, the survival rate of mouse embryonic fibroblasts (3T3) was over 100%, indicating virtually no toxicity. However, after treatment with 2 and 4 mg / mL asiaticoside solutions, the survival rate of mouse embryonic fibroblasts (3T3) was below 20%, showing some toxicity. For detailed results, see [link to results]. Figure 2 , Figure 3 .

[0051] Example 7 Cell migration rate

[0052] The purpose of cell migration assays is to simulate and quantitatively assess the migration / motility of cells in vitro. This experiment investigates the potential of different concentrations of asiaticoside for wound re-epithelialization. The scratch assay is one of the most classic and intuitive methods for studying lateral cell migration. Its basic principle is to artificially create a cell-free "scratch" area on a monolayer of adherent cells, and then observe and record the process of peripheral cells migrating towards this blank area until it covers the "scratch." Using a sterile pipette tip, scraper, or specialized scratcher, a straight line is drawn in a culture well evenly covered with cells, and the cells in that area are removed. The scraped cell debris is washed away with PBS, leaving a clearly defined "cell-free area." The culture medium is changed, and the cells sense the change in space and surrounding environment, initiating directional migration from the scratch edge. Images are taken periodically (e.g., 0, 6, 12, 24, 48 hours) at fixed locations under an inverted microscope. Image analysis software is used to measure the width or area of ​​the scratch at different time points, calculating the distance of cell migration or the percentage of scratch closure, thereby quantifying the cell migration speed.

[0053] Operating steps

[0054] In vitro scratch assays were performed using mouse embryonic fibroblasts (3T3) and mouse fibroblasts (L929). Both cell lines were seeded into 6-well plates, with each well containing 5 × 10⁶ cells. 5Cells were cultured overnight in serum-containing DMEM cell culture medium to obtain cell layers. Straight lines were drawn on the formed cell layers using a sterile 10 μL pipette tip, and the drawn cells were then washed away with PBS. Immediately, solutions containing 0, 0.25, 0.5, 1, 2, and 4 mg / mL of the asiaticoside solution prepared in Example 1, prepared in serum-free DMEM cell culture medium, were added. Six-well plates were placed in a 37°C, 5% CO2 cell culture incubator for incubation. Five replicates were set up for each group. Cell migration was measured using an inverted microscope at 0, 12, and 24 hours, and the migration rate was calculated. Cell migration rate (%) = ((initial width - final width) / initial width) × 100%.

[0055] in conclusion

[0056] Under the influence of different concentrations of asiaticoside, the cell migration rates of L929 cells at 12 h, from highest to lowest, were 1 mg / mL > 2 mg / mL > 4 mg / mL > 0.5 mg / mL > 0.25 mg / mL > 0 mg / mL; and at 24 h, the cell migration rates, from highest to lowest, were 1 mg / mL > 0.5 mg / mL > 0.25 mg / mL > 2 mg / mL > 4 mg / mL > 0 mg / mL.

[0057] Under different concentrations of asiaticoside, the cell migration rates of 3T3 cells at 12 h decreased as follows: 4 mg / mL > 2 mg / mL > 0.5 mg / mL > 1 mg / mL > 0 mg / mL > 0.25 mg / mL, with no significant difference in cell migration rate. At 24 h, the cell migration rates decreased as follows: 0.5 mg / mL > 1 mg / mL > 0.25 mg / mL > 2 mg / mL > 4 mg / mL > 0 mg / mL. See details below. Figure 4 , Figure 5 , Figure 6 , Figure 7 .

[0058] Example 8 Live / Dead Staining Method

[0059] Live / Dead cell staining is a commonly used technique that provides a direct and rapid way to simultaneously identify and quantify the ratio of live to dead cells in a cell population. It serves as an auxiliary tool for evaluating cytotoxicity, directly assessing cell viability, monitoring cell membrane integrity, observing cell morphology, and distinguishing between apoptosis and necrosis. This example demonstrates the use of asiaticoside to detect its acute toxic effects on cells, investigating the survival of 3T3 cells before and after exposure to different concentrations of asiaticoside.

[0060] This method is based on the different responses of two fluorescent dyes to cell membrane integrity and intracellular enzyme activity. Common staining agents for live cells include SYTO 9 and calcein-AM; staining for dead cells typically involves propidium iodide (PI) or ethidium homodimer-1 (EthD-1). SYTO 9 can freely pass through the cell membranes of all cells (live and dead), and once inside the cell, it binds to both DNA and RNA double strands. Upon binding to nucleic acids, it emits a bright 500 nm green fluorescence when excited by 480 nm blue light. PI or EthD-1 are water-soluble, positively charged nucleic acid dyes that normally cannot penetrate the intact cell membrane of live cells. When cells die, cell membrane integrity is lost, resulting in pores or ruptures. The dye enters the cell and binds strongly to DNA / RNA in the cell nucleus and cytoplasm, as well as to nucleic acids. It competitively replaces the already bound SYTO 9, or quenches the green fluorescence of SYTO 9 through fluorescence resonance energy transfer. Under 535 nm excitation light, the nucleus / cytoplasm of dead cells emits a bright 617 nm red fluorescence.

[0061] Operating steps

[0062] Serum-free culture medium solutions containing 0.25, 0.5, 1, 2, and 4 mg / mL of asiaticoside prepared in Example 1 were co-incubated with 3T3 cells for 4 h, and then the cells were resuspended in PBS. Five replicates were set up for each group. Cell pellets were obtained by centrifugation (4℃, 2000 rpm, 3 min), and 20 μL of a mixed working solution of SYTO 9 and PI was added for staining. The pellets were then thoroughly mixed and incubated at room temperature in the dark for 20 min. 5-10 μL of the stained suspension was then dropped onto a glass slide and covered with a coverslip. Excess liquid was blotted away with filter paper. Imaging was performed using an inverted fluorescence microscope. Live bacteria were stained green with SYTO 9, while dead bacteria were stained red with PI due to damage to the bacterial cell wall and cell membrane. The viability rate was calculated as follows: Viability rate (%) = [Number of green fluorescent cells / (Number of green fluorescent cells + Number of red fluorescent cells)] × 100%

[0063] in conclusion

[0064] Using the live / dead staining method, the cell viability of 3T3 cells after co-incubation with different concentrations of asiaticoside solution for 4 hours ranged from 34% to 48%. See details below. Figure 8 , Figure 9 .

[0065] Example 9: Animal in vivo wound healing model

[0066] A standardized full-thickness skin defect was created on the skin of live animals (rats and rabbits) to simulate acute clinical trauma. By monitoring the wound closure process and combining it with histological analysis, the physiological and pathological processes of wound healing and their influencing factors were studied. The animal's own repair system consists of three phases: the first is the inflammatory phase, where platelets aggregate to stop bleeding, and neutrophils and macrophages infiltrate and clear necrotic tissue and pathogens; the second is the proliferative phase, where fibroblasts proliferate and secrete collagen to form granulation tissue to fill the wound; endothelial cells form new capillaries; and epidermal keratinocytes migrate and proliferate from the wound edge to the center, re-epithelializing. The third is the remodeling phase, where collagen fibers remodel and arrange themselves, and the granulation tissue gradually transforms into scar tissue with increased strength.

[0067] This embodiment evaluates the comprehensive effects of different concentrations of asiaticoside on the wound closure process through an in vivo wound healing experiment in animals.

[0068] Implementation steps

[0069] Seven-week-old SPF-grade Balb / c female mice (weighing approximately 20g) purchased from Nanjing Qingzilan Technology Co., Ltd. were used and housed in the laboratory for one week to acclimatize. The mice were randomly divided into six groups of three. Before modeling, the backs of the mice were shaved to expose the skin. The next day, the mice were anesthetized by an intraperitoneal injection of 4% chloral hydrate. Subsequently, an 8×8 mm circular wound was created on the shaved back of the mice using surgical scissors. One group served as the PBS control group, while the remaining five groups received daily drips (100 μL) of a saline solution of asiaticoside prepared in Example 1 at concentrations of 0.25, 0.5, 1, 2, and 4 mg / mL, respectively, at 10, 2, and 14 days. Wound healing was observed and recorded on days 0, 2, 4, 6, 8, 10, 12, and 14.

[0070] in conclusion

[0071] The 1 mg / mL asiaticoside showed the best effect in wound healing, with good healing achieved by day 8 and a mostly smooth scar by day 12. See details below. Figure 10 , Figure 11 .

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A asiaticoside gel, characterized in that, The asiaticoside gel is composed of the following components by weight: 0.2-2 parts asiaticoside extract or liposomes, 0.2-2 parts parabens, 2-10 parts glycerol, and 5-20 parts carbomer.

2. The asiaticoside gel as described in claim 1, characterized in that, The content of total asiaticoside, an active ingredient, in the asiaticoside extract or liposome is 70-80 wt%.

3. The asiaticoside gel as described in claim 1, characterized in that, The parabens are methylparaben, ethylparaben, and butylparaben.

4. A method for preparing asiaticoside gel as described in claim 1, characterized in that, The preparation method steps are as follows: 1) Add carbomer powder to water with constant stirring, let stand overnight to allow it to fully swell, then add triethanolamine or NaOH to adjust the pH to obtain carbomer gel; 2) Prepare a glycerol solution by adding parabens and asiaticoside extract or liposomes to glycerol; 3) Add the glycerol solution to the carbomer gel and stir well to obtain the asiaticoside gel.

5. The method for preparing asiaticoside gel as described in claim 4, characterized in that, Step 1) Adjust the pH to 7-8.

6. An application of the asiaticoside gel as described in claim 1, characterized in that, The asiaticoside gel is used to prepare a gel for wound healing.

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