Ginsenoside Rd liposome as well as green preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a ginsenoside Rd liposome as well as a green preparation method and application thereof. The method comprises the following steps: (a) adding a wrapping material into an absolute ethyl alcohol solution to prepare a wrapping material solution; (b) impregnating the solid-phase particles in a wrapping material solution, removing absolute ethyl alcohol, and combining the soluble solid-phase particles with a wrapping material to obtain a no-load liposome; and (c) mixing the no-load liposome with a ginsenoside Rd solution to obtain the ginsenoside Rd liposome. According to the invention, a novel liposome preparation method is adopted for the first time by utilizing the characteristics that solid-phase particles are of a sphere-like structure and are easy to dissolve in water, ginsenoside Rd is embedded in liposome, and a novel liposome preparation process which is simple in process, small in organic solvent dosage and rapid is constructed. And the prepared liposome particles are high in embedding rate, good in particle uniformity and high in stability.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a ginsenoside Rd liposome and a green preparation method and application thereof. Background Art

[0002] Ginsenoside Rd has multiple pharmacological activities and significant biological activities. It has important applications in the treatment of cardiovascular and cerebrovascular diseases, immune system diseases, central nervous system diseases, anti-tumor, and kidney protection. Ginsenoside Rd can play an anti-tumor role by affecting ubiquitination and downregulating the expression of MDR1 protein; it can inhibit free radical-mediated lipid peroxidation reactions, protect cell membranes from the effects of oxygen free radicals, reduce oxidative damage, protect the kidneys, and delay aging. In addition, ginsenoside Rd also has biological activities such as analgesia, improving memory, regulating immunity, anti-inflammatory, anti-fatigue, regulating the central nervous system, regulating vasodilation and lowering blood pressure, and preventing arteriosclerosis. The multiple pharmacological activities of ginsenoside Rd have attracted widespread attention in the medical field. However, the low oral bioavailability, poor membrane permeability and metabolic instability of ginsenoside Rd have seriously restricted the clinical application of ginsenoside Rd.

[0003] The hydrophilic environment inside the liposome can load hydrophilic drugs, while the lipophilic drugs can be loaded between the phospholipid molecular layers that constitute the liposome, which prolongs the drug's retention time in the body, improves its bioavailability and has good biocompatibility and cell affinity. Encapsulating ginsenoside Rd inside the liposome can effectively solve the problems of low oral bioavailability, poor membrane permeability and metabolic instability of ginsenoside Rd. At present, the main methods for preparing liposomes include thin film dispersion method, organic solvent injection method, reverse evaporation method, multiple emulsion method, microfluidic technology, etc. Liu Junmei et al. used thin film dispersion method to embed ginsenosides with soybean lecithin and cholesterol as liposome membrane raw materials, and the liposome embedding rate was about 90%, with small particle size and good dispersibility. Liang Xinmiao et al. used organic solvent injection method to embed ginsenosides in the membrane composed of phospholipids and cholesterol. The obtained liposomes had uniform particle size distribution and an encapsulation rate of about 80%, which can enhance the cell uptake function and improve the transdermal absorption ability of ginsenosides. Existing liposome preparation methods generally have problems such as organic solvents being difficult to completely remove and taking a long time to remove, and the industrial technology for mass production of liposomes has few selectivities and is immature.

[0004] Chinese patent document CN117017921A also discloses a ginsenoside (S)-Rh2 and (R)-Rh2 liposome and a preparation method thereof, wherein the encapsulation material solution is first subjected to rotary evaporation to form a film, and then a ginsenoside solution is added to obtain a ginsenoside liposome, wherein the encapsulation efficiency is above 90%, the particle size is between 100-120 nm, and the polydispersity index PDI of the liposome is less than 0.2. However, the patent uses a large amount of organic solvents such as methanol, ethanol, and ether in the film-forming process, which requires a large amount of organic solvents and requires a rotary evaporation process, making it difficult to prepare on a large scale.

[0005] In order to increase the deposition surface of phospholipids or prepare thinner films, organic solutions of lipids and drugs can be sprayed onto fine granular carriers, such as powdered sodium chloride, sorbitol, lactose or polysaccharides, to produce a product called proliposomes. When the dry powder is hydrated while mixing with a rotary mixer, the phospholipids swell and the carrier dissolves rapidly, thereby obtaining a liposome suspension in an aqueous solution. However, the existing proliposomes are all drugs and lipids that are co-loaded on soluble solid phase particles through organic solvents, and then dissolved in water to obtain a liposome suspension. This method has the following shortcomings: in order to remove the solvent, it is inevitable to perform heat treatment, which will have a certain impact on the stability of the drug, especially for thermosensitive drugs, increasing the time and difficulty of heat treatment. In addition, the proliposomes loaded with drugs must take into account the performance of each material component during transportation and storage, which will inevitably have a certain impact on the performance of the drug and increase the cost of transportation and storage. In addition, the solid phase particles used in the existing proliposomes are all soluble particles and cannot be used to prepare powdered liposomes. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a ginsenoside Rd liposome and a green preparation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A green preparation method of ginsenoside Rd glycoside liposomes, comprising the following steps:

[0009] (a) adding a wrapping material into an anhydrous ethanol solution to prepare a wrapping material solution;

[0010] (b) immersing the solid phase particles in a coating material solution, removing the anhydrous ethanol, and combining the solid phase particles with the coating material to obtain empty liposomes;

[0011] (c) mixing the empty liposomes with the ginsenoside Rd solution to obtain ginsenoside Rd liposomes.

[0012] Preferably, the encapsulating material in step (a) is one of lecithin and lecithin-cholesterol;

[0013] The mass ratio of lecithin to cholesterol is 6: 1. The mass concentration of the encapsulating material in the encapsulating solution is 0.02-0.2 g / mL, preferably 0.1-0.2 g / mL, and more preferably 0.15-0.2 g / mL.

[0014] Preferably, the solid phase particles in step (b) are sucrose, salt, or silica gel. The mechanism of action of the solid phase particles is to utilize their huge specific surface area to make the coating material solution volatilize on the particle surface to form a film of the coating material with a huge specific surface area.

[0015] The particle size of the sucrose solid phase particles is 150-1250 μm, preferably 550-650 μm.

[0016] The particle size of the salt solid phase particles is 150-1250 μm, preferably 600-700 μm.

[0017] The particle size of the silica gel solid phase particles is 48 to 150 μm, preferably 48 to 74 μm.

[0018] The method for removing the anhydrous ethanol is one of evaporating in a water bath, blowing dry, and standing at room temperature.

[0019] The mass ratio of the solid phase particles to the wrapping material is (2.08-21.16):1; the mass ratio of the sucrose solid phase particles to the wrapping material is (2.5-21.16):1; preferably (2.5-6.25):1. The wrapping material is calculated in lecithin. The wrapping material is calculated in lecithin.

[0020] The mass ratio of the salt solid phase particles to the encapsulating material is (2.08-6.25):1, preferably 2.5:1.

[0021] The mass ratio of the silica gel solid phase particles to the encapsulating material is (2.08-4.17):1, preferably 2.08:1.

[0022] Preferably, in step (c), the mixing method of the empty liposomes and the ginsenoside Rd solution includes: vortex oscillation and shaking.

[0023] When the solid phase particles are sucrose, the mass ratio of the ginsenoside Rd to the encapsulating material is (8.4-106):100, preferably (8.4-17.4):100; the solvent of the ginsenoside Rd solution is water, and the concentration of the ginsenoside Rd solution is 0.6-4.8 mg / mL, preferably 0.6 mg / mL.

[0024] When the solid phase particles are salt, the mass ratio of ginsenoside Rd to empty liposomes is converted to a mass ratio of ginsenoside Rd to lecithin of (8.4-34.8):100, preferably 8.4:100. The concentration of the ginsenoside Rd solution is 0.6-4.8 mg / mL.

[0025] When the solid phase particles are silica gel, the mass ratio of the ginsenoside Rd drug to the empty liposomes is converted to a mass ratio of the ginsenoside Rd drug to lecithin of (18.5-62):100, preferably (18.5-37):100. The concentration of the ginsenoside Rd solution is 0.3-4.8 mg / mL, preferably 0.6-4.8 mg / mL.

[0026] The present invention also provides a ginsenoside Rd liposome prepared by the above preparation method.

[0027] When the solid phase particles are sugars, the particle size of the ginsenoside Rd liposomes of the ginsenoside Rd liposomes is 137.16-249.8 nm, and the aggregation index (PDI) is 0.121-0.319.

[0028] When the solid phase particles are salt, the particle size of ginsenoside Rd liposomes is 111.5-130.7 nm, and the aggregation index (PDI) is 0.247-0.308.

[0029] Preferably, the method further comprises a finishing method in step (d), wherein the granulation method comprises subjecting the ginsenoside Rd liposomes to high-speed shearing, ultrasound, homogenization or extrusion treatment, preferably extrusion, more preferably microfiltration extrusion.

[0030] The present invention also provides an application of ginsenoside Rd liposomes prepared by the above-mentioned preparation method, especially in the preparation of medical cosmetics, which can inhibit tyrosinase.

[0031] Beneficial effects of the present invention:

[0032] 1. The present invention utilizes the spherical structure of solid phase particles and their water-soluble characteristics, adopts a new liposome preparation method for the first time, embeds ginsenoside Rd in liposomes, and constructs a new liposome preparation process with simple process, small amount of organic solvent, and rapidity. The obtained liposome particles have high embedding rate, good particle uniformity, and high stability. It provides a reference for the innovation of liposome preparation technology and more ideal liposome preparation, and also facilitates the clinical application and further research of ginsenosides.

[0033] 2. The present invention utilizes the large specific surface area of ​​solid phase particles, and forms a wrapping material film with a large specific surface area by volatilizing the wrapping material solution on the surface of the solid phase particles to obtain empty liposomes. The empty liposomes provided by the present invention are convenient for transportation, storage, and use. The drug-loaded liposomes can be obtained by uniformly mixing the aqueous solution of the drug carried with the empty liposomes.

[0034] 3. The present invention uses silica gel as a solid phase carrier, and the obtained liposomes have the advantages of high surface area and strong stability. During the drying process, only a short time and low speed centrifugation operation is required to completely separate the epimedium extract liposomes from the dispersed system, which greatly reduces the risk of instability of the epimedium fat liposomes. For unstable liposomes, the method of the present invention can be conveniently prepared into a solid form for storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 HPLC spectra of the permeate and concentrate of the ginsenoside Rd samples of Examples 1-3 of the present invention;

[0036] Figure 2 HPLC spectra of the permeate and concentrate of the ginsenoside Rd samples of Examples 4-6 of the present invention;

[0037] Figure 3 HPLC spectra of the permeate and concentrate of the ginsenoside Rd samples of Examples 7-10 of the present invention;

[0038] Figure 4 HPLC spectra of the permeate and concentrate of the ginsenoside Rd samples of Examples 11-15 of the present invention;

[0039] Figure 5 This is a liposome particle size distribution diagram after microfiltration of the liposome solution in Example 2 of the present invention;

[0040] Figure 6 This is a diagram showing the particle size distribution of liposomes after microfiltration of the liposome solution in Example 9 of the present invention. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below through specific implementation examples, but it should not be understood that the present invention is limited to the following examples, and all technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0042] The ginsenoside Rd (ginseng root extract, 60% total saponins) described in the examples was purchased from Shaanxi Kepler Biology, lecithin was purchased from Beijing Aoboxing Biology Co., Ltd.; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; sucrose was purchased directly from the store in regular sizes, and cholesterol was purchased from Sinopharm Chemical Reagent Co., Ltd. Salt: Salt was purchased directly from the store in regular sizes. Silica gel: manufactured by Haihua Desiccant Factory, Shibei District, Qingdao City, model HG / T2354-92, particle size is 48-74μm. Artificial skin is Strat-MTM Membrane (25mm Discs) purchased from Merck Millipore Ltd.

[0043] Example 1

[0044] Weigh lecithin and add it to anhydrous ethanol solution, sonicate for 10 minutes, prepare 0.2g / mL lecithin solution, and store it in dark conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat it in a water bath at 50°C, then add sucrose to the lecithin solution at a mass ratio of 2.5:1 of sucrose solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes.

[0045] Take the empty liposomes, add 0.6 mg / mL ginsenoside Rd solution according to the mass ratio of ginsenoside to lecithin of 8.4:100, and shake for 10 minutes to obtain the ginsenoside liposome solution.

[0046] Example 2

[0047] Weigh lecithin and add it to anhydrous ethanol solution, sonicate for 10 minutes, prepare 0.2g / mL lecithin solution, and store it in dark conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat it in a water bath at 50°C, then add sucrose to the lecithin solution at a mass ratio of 6.25:1 of sucrose solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes.

[0048] Take the empty liposomes, add 0.6 mg / mL ginsenoside Rd solution according to the mass ratio of ginsenoside to lecithin of 17.4:100, and shake for 10 minutes to obtain the ginsenoside liposome solution.

[0049] Example 3

[0050] Weigh lecithin and add it to anhydrous ethanol solution, sonicate for 10 minutes, prepare 0.15g / mL lecithin solution, and store it in dark conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat it in a water bath at 50°C, then add sucrose to the lecithin solution at a mass ratio of 2.5:1 of sucrose solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes.

[0051] Take the empty liposomes, add 0.6 mg / mL ginsenoside Rd solution according to the mass ratio of ginsenoside to lecithin of 8.4:100, and shake for 10 minutes to obtain the ginsenoside liposome solution.

[0052] Example 4

[0053] Weigh lecithin and add it to anhydrous ethanol solution, sonicate for 10 minutes, prepare 0.2g / mL lecithin solution, and store it in dark conditions. Place sucrose (average particle size of 600μm) in an evaporating dish, heat it in a water bath at 50°C, then add sucrose to the lecithin solution at a mass ratio of 2.5:1 of sucrose solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes.

[0054] Take the empty liposomes, add 4.8 mg / mL ginsenoside Rd solution according to the mass ratio of ginsenoside to lecithin of 8.4:100, shake for 10 minutes, and obtain ginsenoside liposome solution. The encapsulation efficiency is calculated to be 61.32%.

[0055] Example 5

[0056] Weigh lecithin and add it to anhydrous ethanol solution, sonicate for 10 minutes, prepare 0.02363 g / mL lecithin solution, and store it in dark conditions. Place sucrose (average particle size of 600 μm) in an evaporating dish, heat it in a water bath at 50°C, then add sucrose to the lecithin solution at a mass ratio of sucrose solid phase particles to lecithin of 21.16:1, stir while adding, evaporate in a water bath, and obtain empty liposomes.

[0057] Take the empty liposomes, add 1.2 mg / mL ginsenoside Rd solution according to the mass ratio of ginsenoside to lecithin of 106:100, and shake for 10 minutes to obtain the ginsenoside liposome solution.

[0058] Example 6

[0059] The preparation conditions of this embodiment are the same as those of embodiment 2, except that cholesterol is added to lecithin, and the mass ratio of lecithin to cholesterol is 6:1.

[0060] Example 7

[0061] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat in a water bath at 50°C, then add salt to the lecithin solution at a mass ratio of 2.08:1 of salt solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is converted into a mass ratio of 14.8:100 of the solid content of ginsenoside Rd solution to lecithin, and shake for 10min to obtain ginsenoside Rd liposome solution.

[0062] Example 8

[0063] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat in a water bath at 50°C, then add salt to the lecithin solution at a mass ratio of 6.25:1 of salt solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 1.2mg / mL ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is equivalent to a mass ratio of 34.8:100 of the solid content of ginsenoside Rd solution to lecithin, and shake for 10min to obtain ginsenoside Rd liposome solution.

[0064] Example 9

[0065] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat in a water bath at 50°C, then add salt solid phase particles to the lecithin solution at a mass ratio of 2.5:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is equivalent to the mass ratio of the solid content of ginsenoside Rd solution to lecithin of 8.4:100, shake for 10 minutes, and obtain ginsenoside Rd liposome solution.

[0066] Example 10

[0067] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place salt (average particle size of 650μm) in an evaporating dish, heat in a water bath at 50°C, then add salt solid phase particles to the lecithin solution at a mass ratio of 2.5:1, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 4.8mg / mL ginsenoside Rd solution according to the mass ratio of the solid content of the empty liposomes to the ginsenoside Rd solution, which is equivalent to the mass ratio of the solid content of the ginsenoside Rd solution to the mass ratio of lecithin of 8.4:100, shake for 10 minutes, and obtain ginsenoside Rd liposome solution.

[0068] Embodiment 11

[0069] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place silica gel in an evaporating dish, heat in a water bath at 50°C, then add silica gel particles to the lecithin solution at a mass ratio of 2.08:1 of silica gel solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 0.3mg / mL of ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is converted into a mass ratio of 18.5:100 of the solid content of ginsenoside Rd solution to lecithin, and shake for 10 minutes to obtain a ginsenoside Rd liposome solution.

[0070] Example 12

[0071] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place silica gel in an evaporating dish, heat in a water bath at 50°C, then add silica gel particles to the lecithin solution at a mass ratio of 2.08:1 of silica gel solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 4.8mg / mL of ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is converted into a mass ratio of 18.5:100 of the solid content of ginsenoside Rd solution to lecithin, and shake for 10 minutes to obtain a ginsenoside Rd liposome solution.

[0072] Example 13

[0073] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place silica gel in an evaporating dish and heat it in a water bath at 50°C, then add silica gel particles to the lecithin solution at a mass ratio of 2.08:1 of silica gel solid phase particles to lecithin, stir while adding, and evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 1.2mg / mL ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is converted into a mass ratio of 37:100 of the solid content of ginsenoside Rd solution to lecithin, and shake for 10 minutes to obtain ginsenoside Rd liposome solution.

[0074] Embodiment 14

[0075] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place silica gel in an evaporating dish and heat it in a water bath at 50°C. Then add silica gel particles to the lecithin solution at a mass ratio of 4.17:1 of silica gel solid phase particles to lecithin, stir while adding, and evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 1.2mg / mL ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is converted into a mass ratio of 62:100 of the solid content of ginsenoside Rd solution to lecithin, and shake for 10 minutes to obtain ginsenoside Rd liposome solution. The 48-hour transmittance is 57.99.

[0076] Embodiment 15

[0077] Weigh lecithin and add anhydrous ethanol solution to prepare 0.2g / mL lecithin solution, ultrasonicate for 10 minutes, and store in dark conditions. Place silica gel in an evaporating dish, heat in a water bath at 50°C, then add silica gel particles to the lecithin solution at a mass ratio of 3.125:1 of silica gel solid phase particles to lecithin, stir while adding, evaporate in a water bath to obtain empty liposomes. Take empty liposomes, add 1.2mg / mL of ginsenoside Rd solution according to the mass ratio of the solid content of empty liposomes to ginsenoside Rd solution, which is converted to a mass ratio of 49.5:100 of the solid content of ginsenoside Rd solution to lecithin, and shake for 10 minutes to obtain a ginsenoside Rd liposome solution.

[0078] Comparative Example 1

[0079] The preparation conditions of this comparative example are the same as those of Example 2, except that no sucrose solid phase particles are added.

[0080] Comparative Example 2

[0081] Weigh lecithin and ginsenoside, add anhydrous ethanol, ultrasonically dissolve, centrifuge to obtain an organic solution containing lecithin and ginsenoside (wherein the mass ratio of ginsenoside to lecithin is 17.4:100, and the mass concentration of lecithin in the solution is 0.2 g / mL), ultrasonically for 10 minutes, and store in the dark. Take sucrose particles and place them in an evaporating dish. Under the condition of heating in a water bath at 50°C, add the organic solution containing lecithin and ginsenoside at a mass ratio of 6.25:1 of sucrose particles to the encapsulating material, stir while adding, evaporate in a water bath, and then add deionized water to obtain a ginsenoside extract liposome emulsion (wherein the mass ratio of deionized water to ginsenoside extract is 1000:0.6).

[0082] Comparative Example 3

[0083] Weigh lecithin and add anhydrous ethanol solution, ultrasonicate for 10 minutes, prepare 0.2g / mL lecithin solution, and store in dark. Take sucrose granules and place them in a rotary evaporator, then spray lecithin solution into the rotary evaporator at a mass ratio of 6.25:1 of sucrose granules to packaging material, and the rotary evaporation temperature is 50°C to obtain empty liposomes. Take empty liposomes, add 0.6mg / mL ginsenoside solution according to the mass ratio of ginsenoside to lecithin of 17.4:100, shake for 10 minutes, and obtain ginsenoside liposome suspension.

[0084] Test example 1:

[0085] Encapsulation efficiency test:

[0086] In Examples 1 to 10, 0.1 mL of the liposome emulsion prepared in each example was placed in a centrifuge tube (CF), centrifuged at 8000 rpm for 10 min, the supernatant was taken out and added to an ultrafiltration centrifuge tube (UF-CF, the pore size of the ultrafiltration membrane is 30 KD), and then 0.3 mL of pure water was added, and the centrifugation time at 8000 rpm was 10 min. The permeate was taken as the test solution for free component detection (the present invention refers to the permeate). 0.4 mL of anhydrous ethanol was added to the above CF, weighed, ultrasonically dissolved, added to the above UF-CF, ultrafiltration centrifuged at 8000 rpm for 10 min, and the permeate was taken to make up the lost weight with anhydrous ethanol as the test solution for the detection of the encapsulated components (the present invention refers to the concentrate). The permeate and the concentrate were respectively subjected to liquid chromatography detection.

[0087] In Examples 11 to 15, 0.1 mL of ginsenoside liposome suspension was taken into a centrifuge tube, 0.3 mL of purified water was added, and after centrifugation at 8000 rpm for 10 min, the supernatant was taken out and microfiltered through a 0.45 μm filter membrane to obtain a test solution for free component detection. 0.4 mL of anhydrous ethanol was added to the centrifugal sediment, and after ultrasound and centrifugation at 8000 rpm for 10 min, the supernatant was taken out and microfiltered through a 0.45 μm filter membrane to obtain a test solution for encapsulated component detection. Liquid chromatography analysis was performed to detect the amount of ginsenoside Rd in the test solution of free components and encapsulated components, and the encapsulation rate was calculated according to the following formula.

[0088] Here, encapsulation rate (%) = amount of encapsulated components / (amount of free components + amount of encapsulated components) × 100%. That is, encapsulation rate (%) = peak area of ​​concentrated solution / (peak area of ​​permeate + peak area of ​​concentrated solution) × 100%.

[0089] Chromatographic conditions: bonded silica gel C18 (4.6 mm*250 mm, 5 μm), acetonitrile (A)-aqueous solution (B) as mobile phase, gradient elution (0-10 min, B 21%-30%; 10-28 min, B 30%-31%; 28-65 min, B 31%-32%; 65-125 min, B 32%-50%; 125-145 min, B 50%-56%; 145-155 min, B 100%), flow rate 1.0 ml / min, column temperature 35 °C, detection wavelength 203 nm, injection volume: 20 μL.

[0090] Table 1 Liposome encapsulation efficiency of ginsenoside Rd prepared in the examples of the present invention and the comparative examples (%)

[0091]

[0092]

[0093] As can be seen from Table 1: lecithin is the raw material for forming the liposome shell. If the amount of lecithin is too little, the number of liposomes formed is small, and the encapsulation rate is poor; if the amount of lecithin is too much, the lecithin will adhere to each other and cannot form a spherical shape with good morphology, which will have a small capacity for ginsenosides or form a large liposome shell, making it difficult for a small amount of drugs to enter, and also making the encapsulation rate small. The concentration of ginsenosides has a greater influence on the embedding rate. When the concentration is high, the solution volume is small, resulting in a larger lecithin concentration. At a lower ginsenoside concentration, the embedding rate is higher. The present invention has significant advantages over the prior art, and a higher embedding rate can be obtained within a wider parameter range, and the preparation process is simple, the amount of organic solvent used is small, and the preparation time is short, and it can be prepared on a large scale.

[0094] like Figure 1-4As shown in the figure, they are the HPLC spectra of the permeate and the concentrated solution of the ginsenoside liposome prepared in Examples 1-15, respectively. It can be seen from the figure that both the permeate and the concentrated solution have peaks at the ginsenoside Rd chromatographic peak (the retention time of the ginsenoside Rd chromatographic peak is about 70 min), but the peak of the permeate is smaller and close to a straight line, while the concentrated solution has a stronger peak, indicating that the encapsulated drug leaks very little and has strong encapsulation.

[0095] Test Example 2:

[0096] Particle size determination of ginsenoside liposomes:

[0097] 0.1 mL of the ginsenoside Rd liposome solution prepared in Example 2 was taken and diluted to 1 mL with water. The average particle size of the ginsenoside Rd liposome was 220.86 nm, the average PDI was 0.267, and the average potential was -48.36 mV by three measurements using a laser particle size analyzer Zetasizer Nano ZS90.

[0098] 0.1 mL of the ginsenoside Rd liposome solution prepared in Example 2 was taken and diluted to 1 mL with water. After grinding for 10 min using a cryo-grinder, the average particle size of the ginsenoside Rd liposomes was measured three times using a laser particle size analyzer Zetasizer Nano ZS90, and the average particle size was 252.53 nm and the average PDI was 0.218.

[0099] 0.1 mL of the ginsenoside Rd liposome solution prepared in Example 2 was taken and diluted to 1 mL with water. After being crushed by an ultrasonic crusher for 10 min, the average particle size of the ginsenoside Rd liposome was measured three times by a laser particle size analyzer Zetasizer Nano ZS90, and the average particle size was 236.36 nm and the average PDI was 0.649.

[0100] 0.1 mL of the ginsenoside Rd liposome solution prepared in Example 2 was taken and diluted to 1 mL with water. After filtering with a filter membrane, the average particle size of the ginsenoside Rd liposome was measured three times by a laser particle size analyzer Zetasizer Nano ZS90, and the average particle size was 137.16 nm, and the average PDI was 0.212. Figure 5 shown. Figure 6 This is the particle size distribution diagram of liposomes detected three times after filtration through the filter membrane in Example 9.

[0101] Analysis shows that under the four treatment methods, the liposome particle size after microfiltration treatment is the smallest, about 100nm, and the PDI is the smallest. The sample is stable and evenly dispersed. The potential of the liposomes without special treatment was measured to be -48.36mV. The surface of the liposome particles is negatively charged, and the absolute value of the potential is between 30 and 70mV, with good stability.

[0102] Table 2 Average particle size and potential of ginsenoside Rd liposomes prepared in Examples and Comparative Examples

[0103] Liposomes Average particle size / nm PDI Potential / mV Example 1 213.1 0.221 -46.0 Example 2 137.16 0.212 -46.7 Example 3 247.0 0.246 -55.9 Example 4 209.1 0.260 -51.7 Example 5 249.8 0.319 -20.6 Example 6 211.9 0.121 -50.6 Example 7 111.5 0.256 -24.0 Example 8 128.1 0.264 -34.8 Example 9 130.7 0.247 -24.2 Example 10 114.6 0.308 -32.6 Comparative Example 1 265.3 0.328 -43.2 Comparative Example 2 125.1 0.279 -46.6 Comparative Example 3 138.8 0.266 -43.2

[0104] The results of Table 2 show that compared with sucrose solid phase particles as carriers, the liposomes prepared with sodium chloride as solid phase particles have smaller particle sizes, and the average potential of the obtained liposomes is negative, indicating that the surface of the liposome particles is negatively charged and has good stability. Ginseng saponin aqueous solution is difficult to be absorbed through the skin. A solution with a volume fraction of 20% propylene glycol was prepared. Due to the penetration-promoting effect of propylene glycol, its transdermal absorption effect can be detected. The particle size of liposomes prepared with silica gel as solid phase particles depends on the particle size of the silica gel particles used. Under the same conditions, due to the strong adsorption performance of silica gel, its skin permeability was not detected. A solution containing 5% Tween-20 was prepared, and its certain transdermal absorption ability was detected.

[0105] Test Example 3

[0106] Transdermal diffusion experiment:

[0107] Utilize Strat-MTM Membrane (Merck Millipore Ltd) transdermal diffusion test device, add PEG400 aqueous solution with mass concentration of 40% into diffusion cell, take ginsenoside liposome emulsion obtained in embodiment or comparative example, add propylene glycol, make its volume fraction be 20%, then take 0.3mL and evenly disperse on the smooth surface of artificial skin, permeate at room temperature, take out 0.2mL permeation solution from diffusion cell after every 24h, carry out liquid chromatography determination as skin permeation solution, and make up the volume with PEG400 aqueous solution with mass concentration of 40%. After removing the residual material on the smooth surface of artificial skin, soak it with 60% ethanol with the same volume as diffusion cell for half an hour, take out 0.2ml solution as skin retention solution, carry out liquid chromatography determination, and calculate skin permeation rate. Skin permeation rate=amount of ginsenoside Rd in skin permeation solution / amount of ginsenoside Rd in ginsenoside liposome emulsion×100%.

[0108] Skin retention rate = the amount of ginsenoside Rd in the skin retention solution / the amount of ginsenoside Rd in the ginsenoside liposome emulsion × 100%.

[0109] 1 ml of the permeated solution in the diffusion cell and 1 ml of the artificial skin immersion solution were mixed, concentrated by evaporation, and dissolved in 0.5 mL of DMSO for tyrosinase activity inhibition detection.

[0110] Test Example 4

[0111] Tyrosinase inhibitory activity

[0112] 1. Solution preparation

[0113] L-Tyrosine: Prepare a 1 mg / ml solution using phosphate buffer;

[0114] Levodopa: Prepare a 1 mg / ml solution using phosphate buffer;

[0115] Phosphate buffer (0.1 mol / L, pH 7.0): Take 0.68 g of potassium dihydrogen phosphate, add 29.1 ml of 0.1 mol / L sodium hydroxide solution, and dilute with water to 100 ml.

[0116] Tyrosinase solution: Prepare a 0.4 mg / ml solution using phosphate buffer;

[0117] Sample solution: Use phosphate buffer to prepare a ginsenoside sample solution with a concentration of 1.5 mg / ml;

[0118] Vitamin C: Prepare a 0.5 mg / ml solution in PBS buffer.

[0119] 2. Experimental Procedure

[0120] The experiment set up a blank reference group (BR), a blank control group (BC), a sample reference group (SR), a sample group (S), and a positive control group (C).

[0121] Group S: 70 μl of phosphate buffer (0.1 mol / L, pH 7.0), 20 μl of DMSO, 10 μl of L-tyrosine solution, 10 μl of L-DOPA solution, 20 μl of test solution and 10 μl of tyrosinase solution were added to the wells in sequence, and the wells were incubated at 30°C in an ELISA reader for 10 min. After the incubation, the absorbance was measured at 492 nm.

[0122] SR group: 20 μl of PBS solution replaced 10 μl of L-DOPA and 10 μl of L-tyrosine solution, and other steps remained unchanged.

[0123] BR group: 90 μl of PBS solution, 20 μl of DMSO solution and 10 μl of tyrosinase solution were added to the wells.

[0124] BC group: 70 μl of PBS solution, 20 μl of DMSO, 10 μl of L-tyrosine solution, 10 μl of L-DOPA, and 10 μl of tyrosinase solution were added to the wells.

[0125] Group C: The sample was replaced with 20 μl of vitamin C solution, and the other steps remained unchanged.

[0126] Add the solutions to the small holes in sequence and keep them in a constant temperature vibration plate at 30°C for 10 minutes in an ELISA reader. After the end, measure the absorbance at 492nm and calculate the inhibition rate according to the following formula.

[0127]

[0128] Table 3 Tyrosinase inhibitory activity

[0129]

[0130] The results in Table 3 show that both the ginsenoside sample solution and the ginsenoside liposome suspension were detected to have a certain artificial skin membrane penetration concentration and a certain tyrosinase inhibitory activity. No tyrosinase inhibitory activity was detected in the permeated solution of the ginsenoside aqueous solution after transdermal diffusion, and the measured permeability data was also 0, indicating that the artificial skin penetration ability of ginsenosides can be effectively improved by encapsulating liposomes with lecithin.

Claims

1. A green preparation method of ginsenoside Rd glycoside liposomes, characterized in that: The steps include: (a) adding a wrapping material into an anhydrous ethanol solution to prepare a wrapping material solution; (b) immersing the solid phase particles in a coating material solution, removing the anhydrous ethanol, and combining the solid phase particles with the coating material to obtain empty liposomes; (c) mixing the empty liposomes with the ginsenoside Rd solution to obtain ginsenoside Rd liposomes.

2. The preparation method according to claim 1, characterized in that: The encapsulating material in step (a) is one of lecithin and lecithin-cholesterol; The mass concentration of the encapsulating material in the encapsulating material solution is 0.1-0.2 g / mL.

3. The preparation method according to claim 1, characterized in that: The solid phase particles in step (b) are sucrose, salt, or silica gel. The particle size of the sucrose solid phase particles is 150-1250 μm; the particle size of the salt solid phase particles is 150-1250 μm; and the particle size of the silica gel solid phase particles is 48-150 μm.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the solid phase particles to the encapsulating material is (2.08-21.16):

1. Preferably, the mass ratio of the sucrose solid phase particles to the encapsulating material is (2.5-21.16): 1; the mass ratio of the salt solid phase particles to the encapsulating material is (2.08-6.25): 1; the mass ratio of the silica gel solid phase particles to the encapsulating material is (2.08-4.17):

1.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the sucrose solid phase particles to the packaging material is (2.5-6.25):1, the mass ratio of the salt solid phase particles to the packaging material is 2.5:1, and the mass ratio of the silica gel solid phase particles to the packaging material is 2.08:

1.

6. The preparation method according to claim 1, characterized in that: When the solid phase particles are sucrose, the mass ratio of the ginsenoside Rd to the encapsulating material is (8.4-106):100, preferably (8.4-17.4):100; the solvent of the ginsenoside Rd solution is water, and the concentration of the ginsenoside Rd solution is 0.6-4.8 mg / mL, preferably 0.6 mg / mL. When the solid phase particles are salt, the mass ratio of ginsenoside Rd to empty liposomes is converted to a mass ratio of ginsenoside Rd to lecithin of (8.4-34.8):100, preferably 8.4:

100. The concentration of the ginsenoside Rd solution is 0.6-4.8 mg / mL. When the solid phase particles are silica gel, the mass ratio of the ginsenoside Rd drug to the empty liposomes is converted to a mass ratio of the ginsenoside Rd drug to lecithin of (18.5-62):100, preferably (18.5-37):

100. The concentration of the ginsenoside Rd solution is 0.3-4.8 mg / mL, preferably 0.6-4.8 mg / mL.

7. The preparation method according to claim 1, characterized in that: The method also includes a finishing method in step (d), wherein the granulation method comprises subjecting the ginsenoside Rd liposomes to high-speed shearing, ultrasound, homogenization or extrusion treatment, preferably extrusion, more preferably microfiltration extrusion.

8. A ginsenoside Rd liposome prepared by the method according to any one of claims 1 to 7.

9. The liposome according to claim 8, characterized in that When the solid phase particles are sugars, the particle size of the ginsenoside Rd liposomes of the ginsenoside Rd liposomes is 137.16-249.8 nm, and the aggregation index (PDI) is 0.121-0.

319. When the solid phase particles are salt, the particle size of ginsenoside Rd liposomes is 111.5-130.7 nm, and the aggregation index (PDI) is 0.247-0.

308.

10. Use of the ginsenoside Rd liposome according to any one of claims 8 to 9 in the preparation of medical cosmetics.

Images