Asiaticoside nanostructure lipid carrier and preparation method thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to an asiaticoside nanostructure lipid carrier and a preparation method thereof. The nanostructure lipid carrier is prepared by uniformly mixing and homogenizing a water phase and an oil phase, the water phase is a solution containing an emulsifier, the oil phase is a mixed solution containing asiaticoside, solid lipid and liquid lipid, and the nanostructure lipid carrier is prepared by a melt emulsification-high pressure homogenization method. The prepared asiaticoside nanostructure lipid carrier has the advantages of being small in particle size, high in encapsulation efficiency and good in stability, the preparation method is simple and controllable, repeatability is good, the problems that asiaticoside is not prone to being dissolved in water and low in bioavailability are solved, and an alternative delivery system is provided for asiaticoside.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to an asiatica glycoside nanostructure lipid carrier and a preparation method thereof. Background Art

[0002] As the largest organ in the human body, the skin is not only the first line of defense for the body, preventing the invasion of pathogens and harmful substances from the outside world, but also plays a role in regulating body temperature, sensing touch, pressure and temperature changes. However, in daily life, the skin is also one of the most vulnerable tissues. In particular, major skin injuries caused by burns, trauma and surgery are increasing year by year. These severe open wounds are prone to form hypertrophic scars during the healing process, resulting in a decline in normal skin function, which restricts the patient's movement and causes aesthetic and psychological trauma. Wound healing is a complex and dynamic process involving the synthesis of various cells, growth factors, cytokines, signaling pathways and extracellular matrix. The imbalance of these factors can lead to scar formation. Different clinical treatments and non-surgical methods include laser therapy, radiotherapy and cryosurgery, etc., but have failed to achieve functional recovery.

[0003] Asiaticoside (AS) is one of the main triterpenoid compounds contained in the plant Centella asiatica. It is clinically used to promote wound healing and reduce scar formation. It has multiple biological functions, such as anti-inflammatory, antioxidant activity, stimulation of collagen synthesis and promotion of angiogenesis, promotion of fibroblast proliferation and inhibition of scar hyperplasia. However, its insolubility in water limits its clinical application as an external drug or in wound dressings and scaffolds.

[0004] Nanostructured lipid carrier (NLC) uses mixed lipids as carriers, and its composition includes solid lipids, liquid lipids, emulsifiers, embedding substances and dispersion medium water. Liquid lipids at room temperature are added to solid lipids, thereby increasing the disorder of the crystals and making the carrier have higher crystal defects, so that more drug molecules can be carried and the drug leakage rate during storage can be reduced. This special nanostructure can accommodate more drug molecules, increase the drug loading and encapsulation rate, avoid the leakage of encapsulated drugs during storage, increase the stability of the system, and better control the release effect of the drug. Summary of the invention

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

[0006] The purpose of the present invention is to provide a nanostructured lipid carrier of Centella asiatica, which is formed by homogenizing an aqueous phase and an oil phase after mixing. The aqueous phase is a solution containing a surfactant; the oil phase is a mixed solution containing Centella asiatica, solid lipids and liquid lipids.

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

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

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

[0010] In an optional embodiment, the liquid lipid is selected from one or two of fish oil, oleic acid macrogol glyceride or monolinoleic acid glyceride.

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

[0012] Another object of the present invention is to provide a method for preparing a nanostructured lipid carrier of Centella asiatica, comprising the following steps: step 1, heating and melting Centella asiatica, solid lipids and liquid lipids to mix and prepare 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 prepare colostrum; step 4, homogenizing the colostrum with a high-pressure microfluidizer, cooling it, and filtering it to prepare the nanostructured lipid carrier of Centella asiatica.

[0013] In an optional embodiment, in step 1, the heating temperature is 65-85°C.

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

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

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

[0017] The asiatica glycoside nanostructured lipid carrier prepared by the present invention has the advantages of small particle size, high encapsulation rate and good stability, and the preparation method is simple and controllable with good repeatability, which solves the difficult problems of asiatica glycoside being poorly soluble in water and having low bioavailability, increases the absorption of asiatica glycoside, improves its therapeutic effect, provides an alternative delivery system for asiatica glycoside, and provides a new preparation for clinical wound treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The solubilization ability of different surfactants on solid and liquid lipids, including 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 taurodeoxycholate; 14: Polyether F127.

[0019] Figure 2 This is the appearance diagram of the AS-NLC of the present invention, wherein the left side shows ultrapure water, and the right side shows AS-NLC.

[0020] Figure 3 Graph showing the particle size of the AS-NLC of the present invention.

[0021] Figure 4 TEM image of the AS-NLC of the present invention.

[0022] Figure 5 Representative images of wounds at days 0, 3, 7, 11, and 14.

[0023] Figure 6 The figure shows 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 images of Masson staining of skin wounds on day 14.

[0026] Fig. 9 H&E staining of the main organs (heart, liver, spleen, lung, and kidney) of the three groups of rats on day 14. DETAILED DESCRIPTION

[0027] The present invention is further described below with reference to the examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] Example 1

[0029] Accurately weigh 0.18 g of laurin and 0.12 g of monolinolein, heat them in a water bath to 80°C, and dissolve them under stirring. Weigh 100 mg of asiatica glycoside and add them to the mixed lipids to fully mix and melt them to obtain an oil phase. Weigh 1.0 g of Tween-20 and place it in a beaker. Add 20 ml of ultrapure water and stir to dissolve it. Heat it to the temperature of the oil phase to obtain an aqueous phase. Under a magnetic stirring condition of 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 20 minutes. The mixed solution is homogenized by a high-pressure microfluidizer for 3 cycles (500 bar, power 30%), then cooled to room temperature, and filtered through a 0.45 μm microporous filter membrane to obtain AS-NLC (asiatica glycoside nanostructured 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 lauric acid glyceride and 0.35 g of monolinoleic acid glyceride, heat them in a water bath to 65° C., and dissolve them under stirring. Weigh another 60 mg of Centella asiatica glycoside and add them to the mixed lipids to fully mix and melt them to obtain an oil phase. Weigh 0.6 g of Tween-20 and place it in a beaker, add 20 ml of ultrapure water and stir to dissolve, and heat to the temperature of the oil phase to obtain an aqueous phase. Under a magnetic stirring condition of 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 10 minutes. The mixed solution is homogenized by a high-pressure microfluidizer for one cycle (500 bar, power 50%), then cooled to room temperature, and filtered through a 0.45 μm microporous filter membrane to obtain AS-NLC (Centella asiatica glycoside nanostructured 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 lauric acid glyceride and 0.4 g of monolinoleic acid glyceride, heat them in a water bath to 85° C., and dissolve them under stirring. Weigh another 140 mg of asiatica glycoside and add them to the mixed lipids to fully mix and melt them to obtain an oil phase. Weigh 1.4 g of Tween-20 and place it in a beaker, add 20 ml of ultrapure water and stir to dissolve, and heat to the temperature of the oil phase to obtain an aqueous phase. Under a magnetic stirring condition of 1000 r / min, slowly add the isothermal aqueous phase to the oil phase and stir for 30 minutes. The mixed solution is homogenized by a high-pressure microfluidizer for 9 cycles (500 bar, power 10%), then cooled to room temperature, and filtered through a 0.45 μm microporous filter membrane to obtain AS-NLC (asiatica glycoside nanostructured 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 embodiment 1 is that the solid lipid is glyceryl monostearate, and the added amount is 0.18 g. Other details are the same as those in embodiment 1. The obtained AS-NLC encapsulation efficiency is 88.12%, and the average particle size is 44.92±0.704 nm.

[0036] Example 5

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

[0038] Example 6

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

[0040] Example 7

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

[0042] Example 8

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

[0044] Example 9

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

[0046] Study on the preparation technology of lipid nanostructured carrier of Centella asiatica

[0047] 1. Screening of AS-NLC preparation methods

[0048] 1. Melt emulsification-ultrasonic dispersion method: Preparation of AS-NLC: Weigh 0.125g of lauric acid glyceride and monolinoleic acid glyceride, heat to 80°C in a water bath, dissolve under stirring, weigh 50mg of asiaticoside, add to the mixed lipids to fully mix and melt to obtain an oil phase; place 0.5g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir and dissolve to form an aqueous solution with a content of 5% (w / v), and heat to the temperature of the oil phase to obtain an aqueous phase. Under 1000r / min magnetic stirring, slowly add the isothermal aqueous phase dropwise to the oil phase, stir for 20min to form colostrum. Quickly place the colostrum in an ultrasonic cell crusher for ultrasonic dispersion for 10min (probe No. 2, power 80%, ultrasonic 5s. Interval 5s), cool and solidify at room temperature for 30min, and filter through a 0.45μm microporous filter membrane to obtain AS-NLC.

[0049] 2. High-speed shear-ultrasound method: Preparation of AS-NLC: Weigh 0.125 g of lauric acid glyceride and monolinoleic acid glyceride, heat to 80°C in a water bath, dissolve under stirring, weigh 50 mg of asiaticoside, add to the mixed lipids to fully mix and melt to obtain an oil phase; put 0.5 g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir and dissolve to form an aqueous solution with a content of 5% (w / v), and heat to the temperature of the oil phase to obtain an aqueous phase. Under 1000 r / min magnetic stirring conditions, slowly add the isothermal aqueous phase to the oil phase and stir for 20 minutes, the mixed solution is subjected to high-speed shear (1st gear) for 2 minutes to form a pre-emulsified product, and then the pre-emulsified product is ultrasonically dispersed by an ultrasonic cell crusher for 10 minutes (probe No. 2, power 80%, ultrasonic 5s. Interval 5s), cooled and solidified at room temperature for 30 minutes, and filtered through a 0.45 μm microporous filter membrane to obtain AS-NLC.

[0050] 3. Thin film hydration-ultrasound method: Preparation of AS-NLC: Weigh 50 mg of asiaticoside, 125 mg of lauric acid glyceride, and 125 mg of monolinoleyl glyceride, place them in an EP tube containing 10 mL of anhydrous ethanol and heat them to make a uniform solution, transfer them to an eggplant-shaped bottle, and rotary evaporate them at 45°C and 60 r / min to form a uniform thin film layer; place 0.5 g of Tween-20 in a beaker, add an appropriate amount of distilled water and stir to dissolve it into an aqueous solution with a content of 5% (w / v), and heat it to the same temperature as the oil phase, add it to the eggplant-shaped bottle and hydrate it for 20 minutes to fully mix the thin film layer with the aqueous phase; the hydrated mixed liquid is ultrasonically dispersed for 10 minutes using an ultrasonic cell crusher (probe No. 2, power 80%, ultrasound 5s. Interval 5s), and after cooling and solidification at room temperature for 30 minutes, it is filtered through a 0.45 μm microporous filter membrane to obtain AS-NLC.

[0051] 4. High-speed shear-high-pressure homogenization method: Preparation of AS-NLC: Weigh 0.125 g of lauric acid glyceride and monolinoleic acid glyceride, heat to 80°C in a water bath, dissolve under stirring, weigh 50 mg of asiatica glycoside, add to the mixed lipids to fully mix and melt to obtain an oil phase; put 0.5 g of Tween-20 in a beaker, add an appropriate amount of distilled water, stir and dissolve to form an aqueous solution with a content of 5% (w / v), and heat to the temperature of the oil phase to obtain an aqueous phase. Under 1000 r / min magnetic stirring conditions, slowly add the isothermal aqueous phase to the oil phase and stir for 20 minutes. The mixed solution is subjected to high-speed shear (1st gear) for 2 minutes to form a pre-emulsified product. Subsequently, the pre-emulsified product is homogenized by a high-pressure microfluidizer for 3 cycles (500 bar, power 30%) and then cooled to room temperature, and filtered through a 0.45 μm microporous filter membrane to obtain AS-NLC.

[0052] 5. Melt emulsification-high pressure homogenization method: Preparation of AS-NLC: Weigh 0.125g of lauric acid glyceride and monolinoleic acid glyceride, heat to 80°C in a water bath, dissolve under stirring, weigh 50mg of asiatica glycoside, add to the mixed lipids to fully mix and melt to obtain an oil phase; put 0.5g of Tween-20 in a beaker, add appropriate amount of distilled water, stir and dissolve to form an aqueous solution with a content of 5% (w / v), and heat to the temperature of the oil phase to obtain an aqueous phase. Under 1000r / min magnetic stirring, slowly add the isothermal aqueous phase to the oil phase and stir for 20min, homogenize the mixture by a high-pressure microfluidizer for 3 cycles (500bar, power 30%), cool to room temperature, and filter through a 0.45μm microporous filter membrane to obtain AS-NLC.

[0053] AS-NLC was prepared by five methods, including melt emulsification-ultrasonic dispersion, high-speed shear-ultrasonic method, thin film hydration-ultrasonic method, high-speed shear-high-pressure homogenization method, and melt emulsification-high-pressure homogenization method. The obtained preparations were evaluated using appearance, average particle size and aggregation index (PDI) as indicators, and the most suitable preparation method for AS-NLC was selected.

[0054] The obtained preparations were evaluated by appearance, average particle size and PDI, and the results are shown in Table 1. The particle sizes of the emulsions prepared by high-speed shear-ultrasound method, emulsification-ultrasound method and thin film hydration-ultrasound method were all above 150nm, and the particle sizes of the emulsions prepared by high-speed shear-high pressure homogenization method, melt emulsification-high pressure homogenization method and were between 59-67nm, among which the particle size of the emulsion prepared by melt emulsification-high pressure homogenization method was the smallest, about 59.51nm. The PDI value was 0.25, indicating that the emulsion was uniformly dispersed and clear and transparent. Compared with other preparation methods, the preparation process of emulsification-ultrasound method and melt emulsification-high pressure homogenization method was relatively simple. Since the particle size of the emulsion prepared by melt emulsification-high pressure homogenization method was smaller than that measured by emulsification-ultrasound method, after comprehensive consideration, melt emulsification-high pressure homogenization method was finally selected for subsequent single factor investigation and response surface optimization.

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

[0056]

[0057] 2. Screening of solid lipids

[0058] Solid lipids were screened by the solubility method. 1 g of each of 13 solid lipids including monostearate, distearate, tristearate, behenate, stearic acid polyethylene glycol glyceride, lauroyl polyoxyethylene glyceride, mono- and distearate glycerides, lauric acid, palmitic acid, stearic acid, hexadecanol, cetearyl alcohol, and lauric acid glyceride were accurately weighed and melted in a water bath at 80°C. 0.01 g of asiaticoside was added to each solid lipid in small amounts and multiple times. The mixture was heated at 80°C and vortexed appropriately to fully dissolve until saturated solubility was reached. No more asiaticoside could be dissolved and a small amount of precipitation occurred. The solubility of asiaticoside in different solid lipids was observed by visual inspection and the amount of asiaticoside added in different solid lipids was counted. Three solid lipids with better solubility for asiaticoside were selected for subsequent screening.

[0059] The addition amount and solubility of asiaticoside in different solid lipids are shown in Tables 2 and 3. Through visual observation, when the first addition amount of asiaticoside was 10 mg, lauric acid glyceryl and monostearic acid glyceryl could completely dissolve asiaticoside, and the solubility was the best. Except for these two solid lipids, the rest reached saturation, among which mono- and distearic acid glyceryl could dissolve most of asiaticoside, and the solubility was second. Therefore, lauric acid glyceryl, monostearic acid glyceryl and mono- and distearic acid glyceryl were selected as solid lipids.

[0060] Table 2 Addition amount of Centella asiatica in different solid lipids

[0061]

[0062] Table 3 Solubility of Centella asiatica in different solid lipids

[0063]

[0064] (Note: Completely dissolved: ++++; Dissolved a lot: +++; Dissolved half: ++; Not dissolved at all: +)

[0065] 3. Screening of Liquid Lipids

[0066] Liquid lipids were screened by the solubility method. 2 ml of each of 16 liquid lipids including caprylic acid laurate macrogol glyceride, propylene glycol monolaurate, isopropyl myristate, fish oil, castor oil polyoxyl esters, medium-chain triglycerides, triolein, oleoyl polyoxyethylene glyceride, oleic acid macrogol glyceride, palmitic acid glyceride, monolinoleic acid glyceride, diethylene glycol monoethyl ether acetate, monocaprylic acid propylene glycol ester, linoleoyl polyoxyethylene glyceride, oleic acid, and triacetin were placed in a 5 ml EP tube. 0.01 g of asiatica sideroside was added to each liquid lipid in turn. The EP tube was placed in a constant temperature oscillator at 37°C for 3 days to observe its dissolution and fully dissolve until the saturated solubility was reached. No more asiatica sideroside could be dissolved and a small amount of precipitation was observed. The solubility of asiaticaoside in different liquid lipids was observed by visual inspection and the amount of asiaticaoside added in different liquid lipids was counted. Three liquid lipids with better solubility for asiaticaoside were selected for subsequent 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 is not much different. Fish oil, oleic acid macrogol glyceride, and monolinoleic acid glyceride can dissolve a little asiaticoside, and the rest can hardly be dissolved. Therefore, the liquid lipids screened out are fish oil, oleic acid macrogol glyceride, and monolinoleic acid glyceride.

[0068] Table 4 Addition amount of asiaticaside in different liquid lipids

[0069]

[0070] Table 5 Solubility of Centella asiatica in different liquid lipids

[0071]

[0072] (Note: Completely dissolved: ++++; Dissolved a lot: +++; Dissolved half: ++; Almost not dissolved: +)

[0073] 4. Screening of surfactants

[0074] Screen surfactants based on their solubilizing ability, accurately weigh 0.5 g of 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 taurodeoxycholate, and polyether F-127, and add appropriate amount of ultrapure water to prepare a 5% (w / v) surfactant solution; accurately weigh 50 mg of solid lipid lauric acid glyceride and liquid lipid oleic acid polyethylene glycol glyceride to prepare a 100 mg solid-liquid lipid mixture, add 3 ml of ether and stir to mix; respectively add the 14 surfactant solutions to the solid-liquid lipid mixture, vortex to mix it thoroughly, place it in a constant temperature water bath at 40°C, heat it to evaporate and remove the ether, and obtain an emulsion. An appropriate amount of emulsion was measured and diluted 10 times with ultrapure water to obtain a test solution. The absorbance of the test solution was measured at 510 nm using a spectrophotometer. Ultrapure water was used as a calibration solution. Three surfactants with better solubility for asiatica glycoside were selected for subsequent screening.

[0075] The solubilization ability of different surfactants on solid and liquid lipids is shown in Figure 2. Figure 1 As shown. The emulsifying ability of the surfactant is determined by observing the transmittance. The smaller the absorbance, the higher the transmittance, the smaller the particle size, and the better the emulsifying effect. The solubilizing ability of different surfactants for solid and liquid lipids is from large to small: Tween-20>EL-60>EL-35>EL-80>sodium taurodeoxycholate sodium cholate>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 the same type of surfactants as EL-60, only EL-60 with the smallest absorbance among the three was selected. Therefore, the three surfactants screened out are Tween-20, EL-60 and sodium taurodeoxycholate sodium cholate.

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

[0077] The appearance of the AS-NLC prepared in Example 1 was observed visually, and an appropriate amount of AS-NLC was dropped on the surface of the copper mesh containing the carbon film, and left at room temperature for 15 minutes. The excess liquid was absorbed with filter paper, 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 measured using a high-sensitivity Zeta potential and particle size analyzer.

[0078] The appearance of the prepared AS-NLC is shown in the attached Figure 2 , particle size see attached Figure 3 , transmission electron microscopy (TEM) see attached Figure 4AS-NLC is clear and transparent, with a light blue fluorescence; the average particle size is 44.417±1.752nm, and the PDI value is 0.222±0.014; TEM shows that AS-NLC is a regular spherical shape with uniform size, no adhesion, small particle size and good dispersion.

[0079] Effect Example 2 AS-NLC Pharmacological Efficacy Study

[0080] 1. Experimental Methods

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

[0082] 1.1 Establishment of wound model

[0083] A full-thickness skin wound model was established in the experiment. All animals were anesthetized with 10% chloral hydrate injected intraperitoneally 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 applied to the wound, respectively. 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 was recorded by imaging on days 0, 3, 7, 10, and 14, respectively. The wound closure behavior was measured and analyzed using Image J software.

[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 major organs such as the heart, liver, spleen, lung, and kidney were collected. The samples were fixed with 4% paraformaldehyde, then inserted into paraffin and sliced ​​vertically into 5 μm thick longitudinal sections. Hematoxylin and eosin (H&E) staining was used to perform histological analysis of the skin wound regeneration and the five organs, and to observe whether there was inflammation or other damage in each group of organs and skin wounds. Masson staining was used to analyze the regeneration of the skin wound collagen accumulation state.

[0087] 2 Results and analysis

[0088] 2.1 Wound healing

[0089] Wound closure is the most critical factor in wound repair because the skin barrier prevents bacteria from invading the skin. Therefore, we evaluated the wound healing properties of AS-NLC on full-thickness wounds in rats. KM mice were randomly divided into Control, Blank-NLC, and AS-NLC groups. The drugs were administered every three days after surgery. Figure 5 The wound images of each group at 0, 3, 7, 10, and 14 days. After 14 days of treatment, the AS-NLC group had the highest recovery quality, 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 showed a significant rapid closure rate of nearly 80% on day 7. On day 14, the AS-NLC-treated group almost achieved 93% wound closure (Figure Figure 6 ). These results indicate that AS-NLC has a good effect on wound repair and skin regeneration by accelerating wound closure.

[0090] 2.2 HE and Masson staining

[0091] To further explore the specific treatment effects in different groups, pathological changes in wound healing were evaluated. Tissue samples were stained using H&E staining on day 14. Figure 7 As shown in the figure, the treatment effects of the Blank-NLC group and the AS-NLC group were significantly better than those of the Control group. The Control group still had a large wound defect, and new epidermis had grown at the edge of the granulation tissue in the Blank-NLC group and the AS-NLC group. In contrast, in the AS-NLC group, there was a continuous epidermal layer, well-organized dermis, and mature skin appendages (hair follicles), indicating complete healing. The wounds treated with AS-NLC had smaller epidermal regeneration and scar width than those in the Blank-NLC group. At the same time, the Masson trichrome staining (Masson) staining results (see Appendix Figure 8 ) showed that after 14 days of healing, the skin tissue of the AS-NLC group showed highly regular collagen deposition and smaller scar width, which was almost the same as that of normal skin tissue. This was consistent with the results of H&E staining. In addition, after 14 days, H&E staining of important organs (heart, spleen, liver, kidney, lung) showed that there were no signs of inflammation or pathological changes in these organs (see Appendix Fig. 9 ). The results showed that it exhibited good safety.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A nanostructured lipid carrier of Centella asiatica, characterized in that: The nanostructured lipid carrier is formed by homogenizing a water phase and an oil phase after mixing them evenly, wherein the water phase is a solution containing a surfactant, and the oil phase is a mixed solution containing asiatica glycoside, solid lipids and liquid lipids.

2. The asiatica glycoside nanostructured lipid carrier according to claim 1, characterized in that: The mass volume ratio concentration of the surfactant in the water phase is 3% to 7%; the mass volume ratio concentration of the solid-liquid lipid in the oil phase is 1.5% to 3.5%, and the mass volume ratio concentration of the amount of asiaticaside is 0.3% to 0.7%.

3. The asiatica glycoside nanostructured lipid carrier according to claim 1, characterized in that: The mass ratio of solid lipid to liquid lipid in the oil phase is 1:(0.6-4).

4. The asiatica glycoside nanostructured lipid carrier according to claim 1, characterized in that: The solid lipid is selected from one or two of lauric acid glyceryl, monostearic acid glyceryl or mono- and distearic acid glyceryl.

5. The asiatica glycoside nanostructured lipid carrier according to claim 1, characterized in that: The liquid lipid is selected from one or two of fish oil, oleic acid macrogol glyceride or monolinoleic acid glyceride.

6. The asiatica glycoside nanostructured lipid carrier according to claim 1, characterized in that: The surfactant includes any one or two of Tween-20, polyoxyethylene castor oil or sodium taurodeoxycholate.

7. The method for preparing the asiaticoside nanostructured lipid carrier according to claim 1, characterized in that: The following steps are involved: Step 1: Heat and melt centella asiatica, solid lipid and liquid lipid to mix well to prepare an oil phase; Step 2: dissolving the surfactant in water to obtain an aqueous solution as the aqueous phase; Step 3: Add the water phase to the oil phase to prepare colostrum; Step 4: homogenize the colostrum with a high-pressure microfluidizer, cool it, and filter it to obtain the asiatica glycoside nanostructured lipid carrier.

8. The method for preparing the asiaticoside nanostructured lipid carrier according to claim 7, characterized in that: In the step 1, the heating temperature is 65-85°C.

9. The method for preparing the asiaticoside nanostructured lipid carrier according to claim 7, characterized in that: In the step 3, the water phase is slowly added to the oil phase under magnetic stirring conditions and stirred for a certain period of time to form colostrum, wherein the speed of the magnetic stirring is 500 to 1500 r / min and the stirring time is 10 to 30 min.

10. The method for preparing the asiaticoside nanostructured lipid carrier according to claim 7, characterized in that: In step 4, the homogenization pressure is 500 bar, the number of homogenization cycles is 1 to 9 times, and the homogenization power is 10% to 50%.

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