Gypenoside nanostructured lipid carrier and preparation method thereof

Abstract

The present application relates to the technical field of pharmaceutical preparation, in particular to a kind of asiaticoside nanostructured lipid carrier and preparation method thereof.The nanostructured lipid carrier is formed by homogenization after water phase and oil phase are mixed, wherein the water phase is a solution containing emulsifier, the oil phase is a mixed solution containing asiaticoside, solid lipid and liquid lipid, and is prepared by melt emulsification-high pressure homogenization method.The asiaticoside nanostructured lipid carrier prepared by the present application has the advantages of small particle size, high encapsulation efficiency and good stability, and the preparation method is simple and controllable, with good repeatability, solving the problems of asiaticoside not easily soluble in water and low bioavailability, and providing a selectable delivery system for asiaticoside.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a asiaticoside nanostructure lipid carrier and its preparation method. Background Technology

[0002] As the largest organ in the human body, the skin is not only the body's first line of defense, preventing the invasion of external pathogens and harmful substances, but also plays a role in regulating body temperature and sensing tactile, pressure, and temperature changes. However, in daily life, the skin is also one of the most easily damaged tissues, especially with the increasing number of major skin injuries caused by burns, trauma, and surgery each year. These severe open wounds are prone to forming hypertrophic scars during the healing process, leading to a decline in normal skin function, thus restricting the patient's movement and causing aesthetic and psychological trauma. Wound healing is a complex and dynamic process involving the synthesis of various cells, growth factors, cytokines, signaling pathways, and the extracellular matrix. Imbalances in these factors can lead to scar formation. Various clinical treatments and non-surgical methods, including laser therapy, radiotherapy, and cryosurgery, have failed to achieve functional restoration.

[0003] Asiaticoside (AS) is one of the main triterpenoid compounds contained in the plant Centella asiatica. Clinically, it is used to promote wound healing and reduce scar formation, possessing various biological functions such as anti-inflammatory and antioxidant activities, collagen synthesis stimulation and angiogenesis promotion, fibroblast proliferation promotion, and inhibition of excessive scar hyperplasia. However, its insolubility in water limits its clinical application as a topical medication or in wound dressings and scaffolds.

[0004] Nanostructured lipid carriers (NLCs) utilize a mixture of lipids as a carrier, comprising solid lipids, liquid lipids, emulsifiers, encapsulating substances, and a dispersion medium of water. By adding liquid lipids (which are liquid at room temperature) to solid lipids, the disorder of the crystal structure increases, giving the carrier a higher degree of crystal defects. This allows it to carry more drug molecules and reduces drug leakage during storage. This unique nanostructure can accommodate more drug molecules, improving drug loading and encapsulation efficiency, preventing leakage of encapsulated drugs during storage, increasing system stability, and better controlling drug release. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to prepare asiaticoside into a nanostructured lipid carrier to increase the absorption of asiaticoside and improve its therapeutic effect. The nanostructured lipid carrier has small particle size, high encapsulation efficiency and good stability, while improving the water solubility and bioavailability of asiaticoside, providing a new formulation for clinical wound treatment.

[0006] The purpose of this invention is to provide a asiaticoside nanostructured lipid carrier, which is formed by homogenizing an aqueous phase and an oil phase. The aqueous phase is a solution containing a surfactant, and the oil phase is a mixture containing asiaticoside, solid lipids, and liquid lipids.

[0007] In one optional embodiment, the amount of surfactant in the aqueous phase (W / V) is 3% to 7%; the amount of solid-liquid lipids in the oil phase (W / V) is 1.5% to 3.5%, and the amount of asiaticoside (W / V) is 0.3% to 0.7%.

[0008] In one optional embodiment, the mass ratio of solid lipids to liquid lipids in the oil phase is 1:(0.6-4).

[0009] In one alternative embodiment, the solid lipid is selected from one or two of glyceryl laurate, glyceryl monostearate, or glyceryl monostearate and glyceryl distearate.

[0010] In one alternative embodiment, the liquid lipid is selected from one or two of fish oil, polyethylene glycol oleate, or glyceryl monolinoleate.

[0011] In one alternative embodiment, the surfactant includes any one or two of Tween-20, polyoxyethylene castor oil (EL-60), or sodium taurine deoxycholate.

[0012] Another objective of this invention is to provide a method for preparing a asiaticoside nanostructured lipid carrier, comprising the following steps: Step 1, heating and melting asiaticoside, solid lipids and liquid lipids to form an oil phase; Step 2, using an aqueous solution of a surfactant as an aqueous phase; Step 3, adding the aqueous phase to the oil phase to obtain a pre-emulsion; Step 4, homogenizing the pre-emulsion using a high-pressure microfluidic homogenizer, cooling, and filtering to obtain the asiaticoside nanostructured lipid carrier.

[0013] In one alternative embodiment, the heating temperature in step 1 is 65–85°C.

[0014] In one optional embodiment, in step 3, the aqueous phase is slowly added to the oil phase and stirred for a certain period of time under magnetic stirring to form a primary emulsion, wherein the magnetic stirring speed is 500-1500 r / min and the stirring time is 10-30 min.

[0015] In one optional embodiment, in step 4, the homogenization pressure is 500 bar, the number of homogenization cycles is 1 to 9, and the homogenization power is 10% to 50%.

[0016] The present invention has the following beneficial effects:

[0017] The asiaticoside nanostructure lipid carrier prepared by this invention has the advantages of small particle size, high encapsulation efficiency and good stability. Moreover, the preparation method is simple, controllable and reproducible, solving the problems of asiaticoside's poor water solubility and low bioavailability. It increases the absorption of asiaticoside and improves its therapeutic effect, providing an alternative delivery system for asiaticoside and a new formulation for clinical wound treatment. Attached Figure Description

[0018] Figure 1 The solubilizing abilities of different surfactants on solid-liquid lipids, where 1: Tween-20; 2: Tween-60; 3: EL-20; 4: EL-35; 5: EL-40; 6: EL-60; 7: EL-80; 8: CO-40; 9: CO-60; 10: RH-40; 11: SP-80; 12: Poloxamer 188; 13: Sodium taurine deoxycholate; 14: Polyether F127.

[0019] Figure 2 This is an external view of the AS-NLC of the present invention, where left: ultrapure water, right: AS-NLC.

[0020] Figure 3 This is a particle size distribution diagram of the AS-NLC of the present invention.

[0021] Figure 4 This is a transmission electron microscope image of the AS-NLC of the present invention.

[0022] Figure 5 Representative images of the wound on days 0, 3, 7, 11, and 14.

[0023] Figure 6 The curves show the wound healing rate on days 0, 3, 7, 11, and 14.

[0024] Figure 7 Representative images of H&E staining of skin wounds on day 14.

[0025] Figure 8 Representative image of a skin wound stained with Masson stain on day 14.

[0026] Figure 9 H&E staining of major organs (heart, liver, spleen, lung, and kidney) of three groups of rats after 14 days. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Example 1

[0029] Accurately weigh 0.18 g of glyceryl laurate and 0.12 g of glyceryl monolinoleate, heat to 80 °C in a water bath, and dissolve under stirring. Separately weigh 100 mg of asiaticoside and add it to the mixed lipids to fully mix and melt to obtain the oil phase. Weigh 1.0 g of Tween-20 and place it in a beaker, add 20 ml of ultrapure water, stir to dissolve, and heat to the temperature of the oil phase to obtain the aqueous phase. Under magnetic stirring at 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 20 min. Homogenize the mixture three times using a high-pressure microfluidic homogenizer (500 bar, 30% power), cool to room temperature, and filter through a 0.45 μm microporous membrane to obtain AS-NLC (asiaticoside nanostructure lipid carrier), with an encapsulation efficiency of 89.16% and an average particle size of 44.417 ± 1.752 nm.

[0030] Example 2

[0031] Accurately weigh 0.35 g of glyceryl laurate and 0.35 g of glyceryl monolinoleate, heat to 65°C in a water bath, and dissolve under stirring. Separately weigh 60 mg of asiaticoside and add it to the mixed lipids to fully mix and melt to obtain the oil phase. Weigh 0.6 g of Tween-20 and place it in a beaker, add 20 ml of ultrapure water, stir to dissolve, and heat to the temperature of the oil phase to obtain the aqueous phase. Under magnetic stirring at 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 10 min. Homogenize the mixture once using a high-pressure microfluidic homogenizer (500 bar, 50% power), cool to room temperature, and filter through a 0.45 μm microporous membrane to obtain AS-NLC (asiaticoside nanostructure lipid carrier), with an encapsulation efficiency of 74.49% and an average particle size of 42.37 ± 2.242 nm.

[0032] Example 3

[0033] Accurately weigh 0.1 g of glyceryl laurate and 0.4 g of glyceryl monolinoleate, heat to 85°C in a water bath, and dissolve under stirring. Separately weigh 140 mg of asiaticoside and add to the mixed lipids to fully mix and melt to obtain the oil phase. Weigh 1.4 g of Tween-20 and place it in a beaker, add 20 ml of ultrapure water, stir to dissolve, and heat to the temperature of the oil phase to obtain the aqueous phase. Under magnetic stirring at 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 30 min. Homogenize the mixture 9 times using a high-pressure microfluidic homogenizer (500 bar, 10% power), cool to room temperature, and filter through a 0.45 μm microporous membrane to obtain AS-NLC (asiaticoside nanostructure lipid carrier), with an encapsulation efficiency of 75.58% and an average particle size of 36.26 ± 1.526 nm.

[0034] Example 4

[0035] The difference between this embodiment and Example 1 is that the solid lipid is glyceryl monostearate, and the amount added is 0.18g. Other specific details are the same as in Example 1. The resulting AS-NLC encapsulation efficiency is 88.12%, and the average particle size is 44.92±0.704nm.

[0036] Example 5

[0037] The difference between this embodiment and Example 1 is that the solid lipid is glyceryl monostearate and glyceryl distearate, and the amount added is 0.35g. Other specific details are the same as in Example 1. The resulting AS-NLC encapsulation efficiency is 86.31%, and the average particle size is 47.64±1.482nm.

[0038] Example 6

[0039] The difference between this embodiment and Example 1 is that the liquid lipid is polyethylene glycol glyceride oleate, and the amount added is 0.12g. Other specific details are the same as in Example 1. The resulting AS-NLC encapsulation efficiency is 82.32%, and the average particle size is 41.78±0.675nm.

[0040] Example 7

[0041] The difference between this embodiment and Example 1 is that the liquid lipid is fish oil, and the amount added is 0.12g. Other specific details are the same as in Example 1. The resulting AS-NLC encapsulation efficiency is 86.53%, and the average particle size is 47.41±0.892nm.

[0042] Example 8

[0043] The difference between this embodiment and Example 1 is that the surfactant used is EL-60, and the amount added is 1.0g. Other specific details are the same as in Example 1. The resulting AS-NLC encapsulation efficiency is 87.61%, and the average particle size is 41.78±0.675nm.

[0044] Example 9

[0045] The difference between this embodiment and Example 1 is that the surfactant is sodium taurine deoxycholate, and the amount added is 1.0g. Other specific details are the same as in Example 1. The resulting AS-NLC encapsulation efficiency is 86.45%, and the average particle size is 42.13±0.921nm.

[0046] Study on the preparation process of asiaticoside lipid nanostructure carrier

[0047] I. Screening of AS-NLC preparation methods

[0048] 1. Melt Emulsification-Ultrasonic Dispersion Method: Preparation of AS-NLC: Weigh 0.125 g each of glyceryl laurate and glyceryl monolinoleate, heat to 80°C in a water bath, and dissolve under stirring. Separately weigh 50 mg of asiaticoside and add it to the mixed lipids to fully mix and melt, obtaining the oil phase. Place 0.5 g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir and dissolve to form a 5% (w / v) aqueous solution, and heat to the temperature of the oil phase to obtain the aqueous phase. Under magnetic stirring at 1000 r / min, slowly add the isothermal aqueous phase dropwise to the oil phase, stirring for 20 min to form the primary emulsion. Quickly place the primary emulsion into an ultrasonic cell disruptor and ultrasonically disperse for 10 min (probe No. 2, power 80%, 5 s sonication, 5 s interval). After cooling and solidifying at room temperature for 30 min, filter through a 0.45 μm microporous membrane to obtain AS-NLC.

[0049] 2. High-speed shear-ultrasonic method: Preparation of AS-NLC: Weigh 0.125g each of glyceryl laurate and glyceryl monolinoleate, heat to 80℃ in a water bath, and dissolve under stirring. Separately weigh 50mg of asiaticoside and add it to the mixed lipids to fully mix and melt, obtaining the oil phase. Place 0.5g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir and dissolve to form a 5% (w / v) aqueous solution, and heat to the temperature of the oil phase to obtain the aqueous phase. Under magnetic stirring at 1000r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 20min. The mixture is then subjected to high-speed shear (level 1) for 2min to form a pre-emulsified product. Subsequently, the pre-emulsified product is ultrasonically dispersed for 10min using an ultrasonic cell disruptor (probe 2, power 80%, 5s sonication, 5s interval). After cooling and solidifying at room temperature for 30min, it is filtered through a 0.45μm microporous membrane to obtain AS-NLC.

[0050] 3. Thin-film hydration-ultrasonic method: Preparation of AS-NLC: Weigh 50 mg of asiaticoside, 125 mg of glyceryl laurate, and 125 mg of glyceryl monolinoleate, place them in an EP tube containing 10 mL of anhydrous ethanol, and heat to form a homogeneous solution. Transfer to a flask and rotary evaporate at 45 °C and 60 r / min until a uniform thin film layer is formed. Place 0.5 g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir to dissolve into a 5% (w / v) aqueous solution, and heat to the same temperature as the oil phase. Add this solution to the flask and hydrate for 20 min to ensure thorough mixing of the thin film layer with the aqueous phase. The hydrated mixture is then ultrasonically dispersed for 10 min using an ultrasonic cell disruptor (probe No. 2, power 80%, 5 s sonication, 5 s interval). After cooling and solidifying at room temperature for 30 min, filter through a 0.45 μm microporous membrane to obtain AS-NLC.

[0051] 4. High-speed shear-high pressure homogenization method: Preparation of AS-NLC: Weigh 0.125 g each of glyceryl laurate and glyceryl monolinoleate, heat to 80°C in a water bath, and dissolve under stirring. Separately weigh 50 mg of asiaticoside and add it to the mixed lipids to fully mix and melt, obtaining the oil phase. Place 0.5 g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir and dissolve to form a 5% (w / v) aqueous solution, and heat to the temperature of the oil phase to obtain the aqueous phase. Under magnetic stirring at 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 20 min. The mixture is then subjected to high-speed shear (level 1) for 2 min to form a pre-emulsified product. Subsequently, the pre-emulsified product is homogenized for 3 cycles (500 bar, 30% power) in a high-pressure microfluidic homogenizer, cooled to room temperature, and filtered through a 0.45 μm microporous membrane to obtain AS-NLC.

[0052] 5. Melt Emulsification-High Pressure Homogenization Method: Preparation of AS-NLC: Weigh 0.125 g each of glyceryl laurate and glyceryl monolinoleate, heat to 80°C in a water bath, and dissolve under stirring. Separately weigh 50 mg of asiaticoside and add it to the mixed lipids to fully mix and melt, obtaining the oil phase. Place 0.5 g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir and dissolve to form a 5% (w / v) aqueous solution, and heat to the temperature of the oil phase to obtain the aqueous phase. Under magnetic stirring at 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 20 min. Homogenize the mixture for 3 cycles (500 bar, 30% power) using a high-pressure microfluidic homogenizer, cool to room temperature, and filter through a 0.45 μm microporous membrane to obtain AS-NLC.

[0053] AS-NLCs were prepared using five methods: melt emulsification-ultrasonic dispersion, high-speed shear-ultrasonic dispersion, thin-film hydration-ultrasonic dispersion, high-speed shear-high-pressure homogenization, and melt emulsification-high-pressure homogenization. The resulting formulations were evaluated using appearance, average particle size, and particle density index (PDI) to select the most suitable preparation method for AS-NLCs.

[0054] The obtained formulations were evaluated using appearance, average particle size, and PDI as indicators, and the results are shown in Table 1. The emulsions prepared by high-speed shear-ultrasound, emulsification-ultrasound, and thin-film hydration-ultrasound methods all had particle sizes above 150 nm. The emulsions prepared by high-speed shear-high-pressure homogenization, melt emulsification-high-pressure homogenization, and other methods had particle sizes between 59 and 67 nm. Among these, the emulsion prepared by melt emulsification-high-pressure homogenization had the smallest measured particle size, approximately 59.51 nm. The PDI value was 0.25, indicating that the emulsion was uniformly dispersed and clear. Compared with other preparation methods, the emulsification-ultrasound and melt emulsification-high-pressure homogenization methods were relatively simple. However, since the emulsions obtained by melt emulsification-high-pressure homogenization had smaller particle sizes than those obtained by emulsification-ultrasound, the melt emulsification-high-pressure homogenization method was ultimately selected for subsequent single-factor investigations and response surface methodology optimization.

[0055] Table 1. Screening of preparation methods (n=3)

[0056]

[0057] II. Screening of solid lipids

[0058] Solid lipids were screened using a solubility method. 1g each of 13 different solid lipids, including glyceryl monostearate, glyceryl distearate, glyceryl tristearate, glyceryl behenate, polyethylene glycol stearate, glyceryl lauroyl polyoxyethylene glycerate, glyceryl monostearate and glyceryl distearate, lauric acid, palmitic acid, stearic acid, hexadecyl alcohol, cetearyl alcohol, and glyceryl lauryl ester, were accurately weighed and melted in a water bath at 80°C. 0.01g of asiaticoside was added to each solid lipid in small, repeated additions, and the mixture was heated at 80°C with appropriate vortexing until saturation was achieved, meaning no more asiaticoside could be dissolved and a small amount precipitated. The solubility of asiaticoside in different solid lipids was observed visually, and the amount of asiaticoside added in each solid lipid was counted. The three solid lipids with the best solubility for asiaticoside were selected for further screening.

[0059] The addition amounts and solubilities of asiaticoside in different solid lipids are shown in Tables 2 and 3. Visual observation revealed that when 10 mg of asiaticoside was added initially, glyceryl laurate and glyceryl monostearate completely dissolved it, exhibiting the best solubility. All other solid lipids except these two reached saturation, with glyceryl monostearate and glyceryl distearate showing the second-best solubility, capable of dissolving most of the asiaticoside. Therefore, glyceryl laurate, glyceryl monostearate, and glyceryl monostearate and glyceryl distearate were selected as the solid lipids.

[0060] Table 2. Amount of Centella asiatica extract added to different solid lipids

[0061]

[0062] Table 3 Solubility of asiaticoside in different solid lipids

[0063]

[0064] (Note: Completely dissolved: ++++; Dissolves a large amount: +++; Dissolves half: ++; Completely insoluble: +)

[0065] III. Screening of Liquid Lipids

[0066] Liquid lipids were screened using a solubility method. 2 ml of each of the following 16 liquid lipids—caprylic / capric glyceride, propylene glycol monolaurate, isopropyl myristate, fish oil, castor oil polyoxyethylene ester, medium-chain triglycerides, trioleic acid glycerides, oleyl polyoxyethylene glycerides, oleic acid propylene glycol glycerides, palmitic acid glycerides, linoleic acid glycerides, diethylene glycol monoethyl ether acetate, propylene glycol monocaprylic acid glycerides, linoleyl polyoxyethylene glycerides, oleic acid, and triacetyl glyceride—was placed in a 5 ml EP tube. A small amount of 0.01 g of asiaticoside was added to each liquid lipid sequentially. The EP tubes were placed in a 37°C constant-temperature shaker and shaken for 3 days to observe the solubility. The solution was allowed to fully dissolve until saturation was reached, at which point no more asiaticoside could be dissolved and a small amount precipitated out. The solubility of asiaticoside in different liquid lipids was observed visually, and the amount of asiaticoside added in different liquid lipids was counted. Three liquid lipids with better solubility of asiaticoside were selected for further screening.

[0067] The addition amount and solubility of asiaticoside in different liquid lipids are shown in Tables 4 and 5. The solubility of asiaticoside in different solid lipids does not differ much. Fish oil, polyethylene glycol oleate, and glyceryl monooleate can dissolve a small amount of asiaticoside, while the rest are almost insoluble. Therefore, the selected liquid lipids are fish oil, polyethylene glycol oleate, and glyceryl monooleate.

[0068] Table 4. Amount of Centella asiatica extract added to different liquid lipids

[0069]

[0070] Table 5. Solubility of asiaticoside in different liquid lipids

[0071]

[0072] (Note: Completely dissolved: ++++; Dissolves a large amount: +++; Dissolves half: ++; Almost not dissolved: +)

[0073] IV. Screening of Surfactants

[0074] Surfactants were screened based on their solubilizing ability. 0.5g each of 14 surfactants, including Tween-20, Tween-60, EL-20, EL-35, EL-40, EL-60, EL-80, CO-40, CO-60, RH-40, SP-80, poloxamer-188, sodium taurine deoxycholate, and polyether F-127, were accurately weighed and mixed with an appropriate amount of ultrapure water to prepare a 5% (w / v) surfactant solution. 50mg each of solid lipid glyceryl laurate and liquid lipid polyethylene glycol oleate were accurately weighed to prepare a 100mg solid-liquid lipid mixture. 3ml of diethyl ether was added and stirred until homogeneous. The solutions of the 14 surfactants were added to the solid-liquid lipid mixture, vortexed to ensure thorough mixing, and then heated in a 40℃ constant temperature water bath to evaporate and remove the diethyl ether, yielding an emulsion. A suitable amount of emulsion was diluted 10 times with ultrapure water to obtain the test solution. The absorbance of the test solution was measured at 510 nm using a spectrophotometer. Ultrapure water was used as the calibration solution. Three surfactants with good solubility for asiaticoside were selected for further screening.

[0075] The solubilizing abilities of different surfactants on solids, liquids, and lipids are shown in the attached table. Figure 1 As shown. The emulsifying ability of surfactants is determined by observing transmittance. The lower the absorbance, the higher the transmittance, the smaller the particle size, and the better the emulsification effect. The solubilizing ability of different surfactants for solid-liquid lipids, from largest to smallest, is as follows: Tween-20 > EL-60 > EL-35 > EL-80 > sodium taurodeoxycholate > RH-40 > EL-40 > Tween-60 > CO-40 > CO-60 > EL-20 > polyether F127 > poloxamer 188 > Span-80. Since EL-35 and EL-80 are surfactants of the same type as EL-60, only EL-60 with the lowest absorbance was selected. Therefore, the three surfactants selected are Tween-20, EL-60, and sodium taurodeoxycholate.

[0076] Example 1: morphology and particle size of AS-NLC

[0077] The morphology of the AS-NLC prepared in Example 1 was observed visually. An appropriate amount of AS-NLC was dropped onto the surface of a copper mesh containing a carbon film, left at room temperature for 15 minutes, excess liquid was absorbed with filter paper, and the solution was dried and observed under a transmission electron microscope. An appropriate amount of AS-NLC solution was placed in a cuvette, and the particle size of the AS-NLC was determined using a high-sensitivity zeta potential and particle size analyzer.

[0078] The appearance of the prepared AS-NLC is shown in the appendix. Figure 2 See attached document for particle size. Figure 3 See attached transmission electron microscope (TEM) image. Figure 4The AS-NLC was clear and transparent, showing a faint blue fluorescence; the average particle size was 44.417 ± 1.752 nm, and the PDI value was 0.222 ± 0.014; TEM showed that the AS-NLC was in an overall regular spherical shape, with uniform size, no adhesion, small particle size and good dispersibility.

[0079] Effect Example 2 Pharmacodynamic Study of AS-NLC

[0080] 1. Experimental Method

[0081] KM mice (male, 18 - 22 g) were purchased from Hunan Slack Jingda Experimental Co., Ltd. (License No.: SCXK(Xiang)2021 - 0002), and the protocol involving animals was approved by the Animal Care and Use Committee of Hainan Medical University.

[0082] 1.1 Establishment of Wound Model

[0083] An experimental full-thickness skin wound model was established. All animals were anesthetized by intraperitoneal injection of 10% chloral hydrate before surgery. The back skin of each mouse was shaved and disinfected with 75% ethanol. A full-thickness circular wound with a diameter of 10 mm was made. After the full-thickness skin wound model was established, the mice were randomly divided into three groups (n = 5), including the Control group, the Blank-NLC group and the AS-NLC group. 100 μl of PBS, Blank-NLC and AS-NLC were respectively applied to the wound surface. After that, the wound was covered with a transparent film bandage to avoid scratching or biting the specimen until the end of the test. The drug was administered once every 3 days, and the wound closure situation was imaged and recorded on days 0, 3, 7, 10, and 14. The Image J software was used to measure and analyze the wound closure behavior.

[0084] Wound healing rate (%) = (Initial wound area - Indicated wound area) / Initial wound area × 100%.

[0085] 1.2 H&E Staining and Masson Staining

[0086] On the 14th day, the skin wound repair area and the main organs such as the heart, liver, spleen, lungs, and kidneys were collected. Fixed with 4% paraformaldehyde, then embedded in paraffin, and vertically sectioned into 5-μm-thick longitudinal sections. Histological analysis of skin wound healing and regeneration and five organs was performed using hematoxylin and eosin (H&E) staining to observe whether there was inflammation or other damage in each group of organs and skin wounds, and Masson staining was used to analyze the regeneration of collagen accumulation status in skin wounds.

[0087] 2 Results and Analysis

[0088] 2.1 Wound Healing

[0089] Wound closure is the most critical factor in wound healing because the skin barrier prevents bacterial invasion. Therefore, we evaluated the wound healing properties of AS-NLC on full-thickness wounds in rats. KM mice were randomly assigned to Control, Blank-NLC, and AS-NLC groups. Administered the medication every three days post-surgery. Figure 5 Images of the wounds at days 0, 3, 7, 10, and 14 for each group are shown. After 14 days of treatment, the AS-NLC group showed the highest quality of healing, while the other two groups still had unhealed wounds. We then quantified the wound closure rate by calculating the repair area. The AS-NLC group exhibited a significantly rapid closure rate, approaching 80%, on day 7. By day 14, the AS-NLC treatment group had achieved almost 93% wound closure (see attached figures). Figure 6 These results indicate that AS-NLC has good wound repair and skin regeneration effects by accelerating wound closure.

[0090] 2.2 HE and Masson staining

[0091] To further investigate the specific treatment effects in different groups, the pathological changes in wound healing were assessed. Tissue samples were stained using H&E staining on day 14. (See attached image.) Figure 7 As shown, the treatment effects of the Blank-NLC and AS-NLC groups were significantly better than those of the Control group. The Control group still retained a large wound defect, while new epidermis had grown at the edges of the granulation tissue in the Blank-NLC and AS-NLC groups. In contrast, the AS-NLC group exhibited a continuous epidermis, well-tissue dermis, and mature skin appendages (hair follicles), indicating complete healing. Wounds treated with AS-NLC showed less epidermal regeneration and scar width compared to the Blank-NLC group. Furthermore, Masson trichrome staining results (see attached image) also demonstrate this. Figure 8 The results showed that, 14 days after healing, the skin tissue in the AS-NLC group exhibited highly regular collagen deposition and a smaller scar width, almost identical to normal skin tissue. This was consistent with the H&E staining results. Furthermore, H&E staining of vital organs (heart, spleen, liver, kidney, lung) 14 days later showed no signs of inflammation or pathological changes in these organs (see appendix). Figure 9 The results showed that it exhibited good safety.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Centella asiatica glycosides nanostructured lipid carrier, characterized in that, The nanostructured lipid carrier is formed by homogenizing a water phase and an oil phase, the water phase is a solution containing a surfactant, the oil phase is a mixture containing asiaticoside, solid lipid and liquid lipid, The mass-volume concentration of the surfactant is 3% to 7%, the mass-volume concentration of the solid-liquid lipid is 1.5% to 3.5%, and the mass-volume concentration of the amount of asiaticoside is 0.3% to 0.7%; The mass ratio of the solid lipid to the liquid lipid in the oil phase is 1: (0.6-4); The solid lipid is selected from one or two of glyceryl laurate, glyceryl monostearate or glyceryl mono-diestearate; The liquid lipid is selected from one or two of fish oil, polyethylene glycol glyceryl oleate or glyceryl monolinoleate; The surfactant includes any one or two of Tween-20, polyoxyethylene castor oil or sodium taurodeoxycholate.

2. The process for the preparation of Gynostemma pentaphyllum glycosides nanostructured lipid carriers as claimed in claim 1 wherein, The method comprises the following steps: Step 1: melt and mix asiaticoside, solid lipid and liquid lipid to prepare an oil phase; Step 2: dissolve a surfactant in water to obtain an aqueous solution as a water phase; Step 3: add the water phase to the oil phase to prepare a preliminary emulsion; Step 4: homogenize the preliminary emulsion by a high-pressure microfluidizer, cool, filter and obtain an asiaticoside nanostructured lipid carrier; In step 1, the heating temperature is 65-85℃; in step 3, the water phase is slowly added to the oil phase under magnetic stirring for a certain period of time to form a preliminary emulsion, wherein the stirring speed of the magnetic stirring is 500-1500r / min, and the stirring time is 10-30min; in step 4, the homogenization pressure is 500bar, the homogenization cycle number is 1-9 times, and the homogenization power is 10%-50%.

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