CN111053744A - Baicalin liposome and application thereof - Google Patents
Baicalin liposome and application thereof Download PDFInfo
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- CN111053744A CN111053744A CN201911351878.9A CN201911351878A CN111053744A CN 111053744 A CN111053744 A CN 111053744A CN 201911351878 A CN201911351878 A CN 201911351878A CN 111053744 A CN111053744 A CN 111053744A
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Abstract
The invention discloses a baicalin liposome, which comprises baicalin, ceramide and a lipid material, wherein the baicalin is used as a medicinal component, the ceramide is simultaneously used as one of the medicinal component and the lipid material component, and the baicalin liposome is prepared by adopting an active medicament carrying method. The invention is formed by catalyzing the active encapsulation of the baicalin liposome through the pH difference, solves the problems of poor stability and solubility of the baicalin and difficult single use to exert curative effect, simultaneously overcomes the problems of low encapsulation efficiency of the baicalin liposome and poor stability of ceramide, and expands the application range and the use mode of the baicalin. The baicalin liposome has the functions of moisturizing and resisting oxidation, and can be applied to cosmetics as an active part.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a baicalin liposome and application thereof.
Background
Baicalin (BCL) is a flavonoid compound extracted and separated from the dried root of Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi) which is a dicotyledonous Labiatae plant, has remarkable biological activity, and comprises anti-inflammatory, immune regulation, anti-allergic reaction, anti-tumor, antibacterial, antiviral, antihypertensive and the like, wherein the anti-inflammatory and immune regulation effects are particularly prominent. Baicalin shows clear anti-inflammatory and immune modulating effects in a variety of animal models of inflammation and autoimmunity, including: asthma, acute pancreatitis, acute lung injury, hepatic fibrosis, and dermatitis solaris.
The baicalin structure contains phenolic hydroxyl, is unstable and is easy to be oxidized and discolored by air. Moreover, baicalin is difficult to dissolve, is almost insoluble in water, and is insoluble in conventional solvents such as methanol, ethanol, acetone, and slightly soluble in chloroform. The instability and poor solubility of baicalin make the product having baicalin alone as the effective ingredient have a very high index of refraction, while the use of scutellaria baicalensis extract as the effective ingredient is common. The main drug effect component of the scutellaria baicalensis extract is baicalin, and in addition, the scutellaria baicalensis extract also contains a plurality of other components and even impurities, the components are mixed together for use, the elucidation of the action mechanism is difficult, whether the scutellaria baicalensis extract contains antagonism or not and even the toxicity is difficult to explain, and the risk is higher compared with the single use of the baicalin. In view of the above properties of baicalin, its application is greatly limited.
People research the baicalin liposome and investigate the preparation process and stability of the baicalin liposome. For example, in the research on the preparation and stability of baicalin liposome (Schisandra chinensis, etc., in the No. 25, No. 1 of the J.Pharmacopeia (2005)) the baicalin liposome is prepared by reverse phase evaporation, ether injection and thin film ultrasound. The liposome prepared by reverse phase evaporation method has average particle diameter of 360 + -42 nm and entrapment rate of 56.02 + -5.3%. The average particle diameter of the liposome prepared by the injection method is 120nm, and the entrapment rate is 26.14 +/-1.5%. The average particle diameter of the liposome prepared by the film method is 260nm, and the entrapment rate is 53.65 +/-4.8%. For example, in the study of baicalin liposome preparation and research on in vivo and in vitro pharmaceutics (Xuli Kun, Hebei medical university, Master thesis), four methods, namely a film dispersion method, an active drug loading method, an ether injection method and a reverse phase evaporation method, are adopted to prepare the baicalin liposome. The conditions are not easy to control when ether injection method is adopted, and the obtained lipidThe liposome is thinner, and the encapsulation efficiency of the prepared liposome is poorer in measurement result. The entrapment rate of the liposome obtained by adopting an active drug loading method is not high, and is only 26.9 +/-7.4 percent, because baicalin is poor in lipophilicity and hydrophilicity and is almost insoluble in water. The baicalin liposome is prepared by a reverse phase evaporation method, and the prepared liposome is more uniform, and the entrapment rate is higher than 45.4%. Research on technology for preparing baicalin liposome by reverse phase evaporation (Lvfengjiao, China pharmaceutical journal, No. 54, No. 3 of 2.2019) discloses preparation of baicalin liposome modified by combination of vitamin E and polysorbate 80 by reverse phase evaporation, wherein L is used16(45) The orthogonal experimental design method inspects the influence of various factors on the encapsulation rate of the liposome, determines the optimal process of the baicalin liposome, and the encapsulation rate is 70.22 percent and the drug-loading rate is 3.18 percent.
At present, the preparation method of the baicalin liposome mainly comprises a film method and a reverse phase evaporation method. As baicalin is almost insoluble in water, in the preparation process of the baicalin liposome, the baicalin is mainly added as a lipid material in a powder form and is wrapped in a phospholipid bilayer, the liposome is unstable and easy to separate out, the degradation of the liposome is accelerated, and the uniformity and the particle size of the liposome are greatly challenged.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel baicalin liposome, which promotes the active encapsulation formation of the baicalin liposome through the pH difference, and greatly improves the uniformity and encapsulation efficiency of the baicalin liposome.
The specific technical scheme of the invention is as follows:
a baicalin liposome comprises baicalin, ceramide and a lipid material, wherein the baicalin is used as a medicinal component, the ceramide has the functions of a medicinal active component and the lipid material, and is prepared by adopting an active medicine carrying method, and the baicalin liposome comprises the following steps:
(1) weighing ceramide and lipid materials to prepare blank ceramide liposome;
(2) adding ammonium sulfate buffer solution, wrapping ammonium sulfate in liposome internal water phase, and removing ammonium sulfate in liposome external water phase;
(3) adding baicalin water solution to obtain baicalin liposome.
The aqueous baicalin solution is acidic, and preferably pH of the aqueous baicalin solution is adjusted to 5-9 with alkali such as NaOH, more preferably the aqueous baicalin solution is neutral or nearly neutral (pH 6.5-7.5).
One specific operation is:
(1) precisely weighing ceramide and lipid materials to prepare blank ceramide liposome;
(2) adding ammonium sulfate buffer solution, hydrating for 20 minutes at 40 ℃, and dissolving with ultrasonic assistance;
(3) filtering, homogenizing the filtrate for 5-15 times by high pressure homogenizer;
(4) HBS is dialyzed for 24 hours;
(5) adding baicalin water solution, stirring in water bath at 40 deg.C for 20min to obtain baicalin liposome.
The method comprises preparing blank ceramide liposome, dialyzing with ammonium sulfate solution to form inner and outer pH difference of liposome membrane, adding baicalin water concentrated solution to make baicalin molecule enter inner water phase of liposome actively due to the difference of inner and outer concentration of baicalin, and forming colloidal precipitate under weak acid condition of inner water phase. Specifically, the above method removes ammonium sulfate from the aqueous solution outside liposome by dialysis so that the concentration of ammonium sulfate inside and outside liposome forms a concentration gradient, thereby generating a pH gradient, i.e., NH in the internal aqueous phase4 +Is easy to decompose into NH3And H+And different ions have different permeability coefficients to the bilayer, and the permeability performance of the ions to the bilayer has the following rule, Namely (NH)4)2SO4<SO4 2-<NH4 +<H+<NH3. As the neutral ammonia molecules leave the liposomes, protons are retained in the inner aqueous phase of the liposomes, resulting in a difference in H + concentration between the inner and outer aqueous phases of the liposomes, i.e. a pH gradient is created. After the baicalin compound enters the internal water phase, the baicalin compound changes into an ionic state when meeting an acid environment, and is prevented from returning to the external water phase and reacting with SO4 2-The formed sulfate has very low permeability coefficient to the bilayer, and the encapsulation efficiency of the liposome is improved. At the same time, NH3The escape of molecules creates a diffusion potential for ammonium ions, which becomes the driving force for the drug to cross the phospholipid bilayer into the internal aqueous phase and to accumulate in the internal aqueous phase. Finally, the baicalin compound can be stored in the water phase in the liposome to complete the active encapsulation process.
The preferable mass ratio of the ceramide to the lipid material of the baicalin liposome of the invention is 2-4: 20-28. In the baicalin liposome, preferably, the lipid material comprises one or more of lecithin, hydrogenated lecithin, cholesterol oleate, glycerol trilaurate, glycerol monostearate, stearic acid, glycerol palmitate stearate, glycerol behenate, glycerol trioleate, medium-chain fatty acid ester, medium-chain triglyceride, polyethylene glycol oleate glyceride and caprylic/capric glyceride. More preferably cholesterol and hydrogenated lecithin and/or lecithin.
The baicalin liposome preferably comprises the following components in percentage by mass: ceramide: baicalin: lipid material 2-4: 1-2: 20-28.
Preferably, the mass ratio of the ceramide to the baicalin is 2: 1. the mass ratio of the ceramide to the hydrogenated lecithin and/or the lecithin to the cholesterol is 2-4: 20-28: 2-6.
The baicalin liposome has the functions of moisturizing, oxidation resistance, inflammation resistance, aging resistance and photoaging resistance, can be applied to treatment of skin diseases, skin beautifying and skin care, is particularly suitable for treatment of dermatitis, eczema, acne, rosacea, sensitive skin diseases, photo-dermatosis, psoriasis and other diseases, and is particularly suitable for wrinkle removal, freckle removal and scar removal in beautifying and nursing.
The invention has the advantages that:
(1) the invention is formed by active encapsulation of the baicalin liposome under the catalysis of pH difference, and greatly improves the uniformity and encapsulation efficiency of the baicalin liposome. The encapsulation efficiency of the baicalin liposome can reach 94.6%, the particle size is small and uniform, and the average particle size is 90 nm.
(2) Ceramides are a major part of the intercellular matrix and play an important role in maintaining the water balance of the stratum corneum. However, when ceramide is compounded with other auxiliary materials as an active substance, the phenomenon of precipitation is easy to occur, and the application of ceramide is limited. According to the invention, ceramide is added into lipid materials to prepare the baicalin liposome, so that the stability of ceramide in a compound preparation can be obviously improved, the encapsulation efficiency of baicalin is further increased, and the baicalin liposome is free from leakage after being placed for a long time. In addition, ceramide can improve the thickness of phospholipid bimolecules, increase the elasticity of plasma membranes and be beneficial to maintaining the water phase in the liposome.
(3) The invention systematically characterizes the baicalin liposome through various physicochemical characterization ways, including atomic force scanning probe microscope, transmission electron microscope, laser particle size scattering instrument, high performance liquid chromatography and other means, and researches show that the liposome has the advantages of round appearance, uniform particle size, high encapsulation rate and good stability at normal temperature. The safety, moisture retention and oxidation resistance of the liposome are examined by using human skin fibroblasts, melanocytes and animal experimental technology. The comprehensive experiment result shows that the compound has good safety and better moisturizing performance, and can effectively remove the oxidation free radicals.
The invention solves the problems of poor stability and solubility of the baicalin and difficulty in single use to exert curative effect, simultaneously overcomes the problems of low encapsulation rate of the baicalin liposome and poor stability of ceramide, and widens the application range and the use mode of the baicalin. The baicalin liposome provided by the invention has the functions of moisturizing and resisting oxidation, and can be applied to cosmetics as an active part.
Drawings
FIG. 1 is an atomic force microscope image of the baicalin liposome of the present invention.
FIG. 2 is a transmission electron microscope image of the baicalin liposome of the present invention.
FIG. 3 shows the particle size and PDI of the baicalin liposome of the present invention.
FIG. 4 shows the results of the encapsulation efficiency measurement of baicalin liposome of the present invention.
FIG. 5 shows the survival rate of human dermal fibroblasts after administration of the baicalin liposome of the present invention.
Figure 6 is a microscopic picture of human skin fibroblasts after administration of the baicalin liposome of the present invention.
FIG. 7 shows 435S cell survival rate after administration of baicalin liposome of the present invention.
FIG. 8 is a 435S cell microscopic image of the baicalin liposome of the present invention after administration.
FIG. 9. mouse skin irritation experiment HE staining results of mouse skin sections after administration of baicalin liposome of the present invention.
FIG. 10 is a result of the moisture content of the mouse skin after administration of the baicalin liposome according to the present invention.
FIG. 11 shows the results of measuring the antioxidant SOD expression of baicalin liposome of the present invention.
FIG. 12 shows the measurement results of antioxidant ROS expression of baicalin liposome.
Detailed Description
The following examples are provided to illustrate specific steps of the present invention, but are not intended to limit the scope of the examples.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
EXAMPLE 1 preparation of baicalin Liposome I of the invention
200mg of phospholipid, 20mg of cholesterol and 20mg of ceramide are precisely weighed respectively and are simultaneously placed in a 500mL round-bottom flask, and a proper amount of ethanol is added for complete dissolution under ultrasonic assistance. The mixture is placed in a water bath at 50 ℃ for rotary evaporation to remove ethanol, and a light yellow film is formed on the inner wall of the flask. Adding 250ml-1000ml of 250mM ammonium sulfate buffer solution, hydrating for 20 minutes at 40 ℃, and dissolving with the aid of ultrasound; then filtering, homogenizing the filtrate for 5-15 times by a high pressure homogenizer, dialyzing with HBS for 24h to obtain baicalin liposome I, wherein the baicalin aqueous solution (5mg/ml, pH6.5-7.5)1ml is stirred in 40 deg.C water bath for 20 min.
EXAMPLE 2 preparation of baicalin liposome II of the present invention
200mg of phospholipid, 16mg of cholesterol, 2mg of triolein, 2mg of cholesterol oleate and 20mg of ceramide are precisely weighed respectively and are simultaneously placed in a 500mL round-bottom flask, and a proper amount of ethanol is added for complete dissolution under the assistance of ultrasound. The flask was placed in a water bath at 50 ℃ and rotary evaporated to remove ethanol, and a yellow film was formed on the inner wall of the flask. Adding 250ml-1000ml of 250mM ammonium sulfate buffer solution, hydrating in water bath at 40 ℃ for 20 minutes, and dissolving with the aid of ultrasound; then filtering, homogenizing the filtrate for 5-15 times by a high pressure homogenizer, dialyzing with HBS for 24h to obtain baicalin liposome II, wherein the baicalin aqueous solution (10mg/ml, pH6.5-7.5)1ml is stirred in 40 deg.C water bath for 20 min.
EXAMPLE 3 preparation of baicalin Liposome III of the invention
200mg of phospholipid, 14mg of cholesterol, 2mg of triolein, 2mg of medium-chain triglyceride, 2mg of polyethylene glycol oleate and 20mg of ceramide are precisely weighed and placed in a 500mL round-bottom flask simultaneously, and a proper amount of ethanol is added for complete dissolution under the assistance of ultrasound. The flask was placed in a water bath at 50 ℃ and rotary evaporated to remove ethanol, and a yellow film was formed on the inner wall of the flask. Adding 250ml-1000ml of 250mM ammonium sulfate buffer solution, hydrating in water bath at 40 ℃ for 20 minutes, and dissolving with the aid of ultrasound; then filtering, homogenizing the filtrate for 5-15 times by high pressure homogenizer, dialyzing with HBS for 24 hr to obtain baicalin liposome III with baicalin water solution (20mg/ml, pH6.5-7.5)1ml, and stirring in 40 deg.C water bath for 20 min.
EXAMPLE 4 preparation of ceramide-free baicalin liposomes
200mg of phospholipid and 14mg of cholesterol are precisely weighed respectively and are placed in a 500mL round-bottom flask simultaneously, and a proper amount of ethanol is added for complete dissolution under the assistance of ultrasound. The flask was placed in a water bath at 50 ℃ and rotary evaporated to remove ethanol, and a yellow film was formed on the inner wall of the flask. Adding 250ml-1000ml of 250mM ammonium sulfate buffer solution, hydrating in water bath at 40 ℃ for 20 minutes, and dissolving with the aid of ultrasound; then filtering, homogenizing the filtrate for 5-15 times by high pressure homogenizer, dialyzing with HBS for 24 hr to obtain baicalin liposome with baicalin water solution (5mg/ml, pH6.5-7.5)1ml, and stirring in 40 deg.C water bath for 20 min. The encapsulation efficiency detection result shows that the encapsulation efficiency of the liposome is lower than 60%, and the encapsulation performance of the liposome on baicalin is poor. The results show that ceramide is very critical for the preparation of baicalin liposome.
Example 4 preparation of liposomes with ceramide in combination with other lipid materials
Accurately weighing 14mg of cholesterol, 2mg of triolein, 2mg of medium-chain triglyceride and 2mg of polyethylene glycol oleate, respectively adding 20mg of ceramide, placing in a 500mL round-bottom flask, adding a proper amount of ethanol, and performing ultrasonic assistance to completely dissolve the ceramide. The flask was placed in a water bath at 50 ℃ and rotary evaporated to remove ethanol, and a yellow film was formed on the inner wall of the flask. Adding 250ml-1000ml of 250mM ammonium sulfate buffer solution, hydrating in water bath at 40 ℃ for 20 minutes, and dissolving with the aid of ultrasound; then filtering, and homogenizing the filtrate for 5-15 times by a high-pressure homogenizer. In addition, the Tyndall effect is not generated by laser irradiation, which indicates that no liposome is formed, and the subsequent measurement of the particle size of the emulsion also proves that no nano-scale liposome is formed. The results show that the ceramide-based two-component lipid material alone cannot form liposomes.
Example 6 physicochemical characterization of the baicalin liposomes of the invention
1. Atomic force scanning probe microscope
The liposome I was evaluated morphologically using an atomic force scanning probe microscope. Diluting the liposome with deionized water as dispersion medium to 100 μ g/mL phospholipid concentration, filtering with 0.22 μm microporous membrane, coating 10 μ L of the liposome onto the surface of silicon wafer, standing at room temperature, drying, and detecting with atomic force microscope. The results are shown in FIG. 1. The results show that: the liposome has a particle size of about 100nm, is in a regular spherical shape, and has a smooth surface.
2. Transmission electron microscope
The morphology of liposome I was further studied using transmission electron microscopy. Diluting liposome with deionized water as dispersion medium, filtering with 0.22 μm microporous membrane, floating copper net coated with carbon film on liposome solution for 1min, taking out, drying with filter paper, floating copper net with trapped liposome particles on 1% uranyl acetate aqueous solution for 1min, taking out, and drying with filter paper. After standing overnight at room temperature, observation was carried out using a transmission electron microscope at 120 kV. The results are shown in FIG. 2. The results show that: the transmission electron microscope of the liposome presents an obvious phospholipid bilayer structure, and the particle size of the liposome is about 100nm and is in a similar circular structure.
3 measurement of particle diameter
The baicalin liposomes prepared in examples 1 to 3 were each diluted to 1mL with PBS, and after being mixed uniformly, the particle size of the liposomes was measured with a light scattering particle size meter. The wavelength of the laser beam of the instrument is set to 633nm, and the included angle between the incident light and the scattered light beam is 90 degrees. The equilibration time was 30s for each sample, 20 cycle times were measured, and the measurement temperature was set at 25 ℃. The final measurement for each sample is the average of 3 measurements. The Polydispersity (PDI) indicates the degree of uniformity of particle size, with particles being more uniform as the polydispersity decreases. The results are shown in FIG. 3. The results showed that the average particle size of each group of liposomes was between 92.74-100.08nm and PDI was between 0.17-0.21, indicating that the 3 liposomes had uniform particle size.
3. Encapsulation efficiency determination
Taking the baicalin liposome prepared in example 1-3, filtering with 0.45 μm microporous membrane, demulsifying with methanol at a ratio of 1:1, ultrasonic treating for 5min, and measuring with HPLC. Calculating the formula: EE% ═ drug loading/drug loading x 100% by HPLC. The results are shown in FIG. 4, where 1 is the encapsulation efficiency of liposome I ceramide; 2 is the entrapment rate of the liposome II ceramide; 3 is the entrapment rate of liposome III ceramide; 4 is the encapsulation rate of the liposome I baicalin; 5 is the encapsulation rate of liposome II baicalin; 6 is the encapsulation rate of liposome III baicalin. The results show that the encapsulation efficiency of each group of liposome is more than 81%, which indicates that the liposome can better encapsulate baicalin and the encapsulation conditions are relatively suitable.
5. In vitro Release study
2mL of the baicalin liposome prepared in example 1-3 was placed in a dialysis bag (MWCO,12000-14000 Da), both ends of the liposome were tied up and placed in 20.0mL of release medium, shaking was carried out at 37 ℃ and 100rpm for 12, 24, 48 and 72 hours, 0.5mL of release medium was taken out, and fresh release medium with the same volume was immediately added after each sampling. And adding equal volume of methanol into each sample, demulsifying, and measuring the contents of ceramide and baicalin by using HPLC.
Calculating the formula: in vitro release rate ═ 100% (amount of drug in release medium/dose measured at i h).
TABLE 1 ceramide in vitro Release
TABLE 2 in vitro release of baicalin
The results are shown in tables 1 and 2. The result shows that the in vitro release rate of each group of liposome ceramide is less than 40% and the in vitro release rate of baicalin is not more than 20% at 72h, which indicates that the prepared liposome has the characteristic of slowly releasing the drug and can effectively prevent the drug in the liposome from leaking in the using process.
6. Storage stability
Taking 5mL of the baicalin liposome prepared in the example 1-3 into a penicillin bottle, and sealing. The sealed liposome is respectively stored in a refrigerator at normal temperature and 4 ℃, and is respectively taken out at 0, 10, 20, 30 and 60d to observe the appearance, the particle size and the encapsulation efficiency change of the liposome.
TABLE 3 change in ceramide encapsulation efficiency
TABLE 4 baicalin encapsulation efficiency variation
TABLE 5 particle size variation
Note: 1 is 'I liposome'; 2 is 'II liposome'; 3 is "liposome III".
The results are shown in tables 3, 4 and 5. The results show that the entrapment efficiency, particle size and PDI change within reasonable ranges after each liposome is stored for 30 days at room temperature and 4 ℃, and show that 3 kinds of liposomes have good stability.
Example 7 evaluation of safety of the baicalin liposomes of the present invention
1. Evaluation of cytotoxicity
The culture condition of human skin fibroblast is DMEM medium containing 10% fetal calf serum and double antibody (penicillin 100U/ml and streptomycin 100 ug/ml), and passage is carried out for 1 time in 4-5 days. The culture condition of 435S cells was DMEM medium containing 10% fetal bovine serum, diabody (penicillin 100U/ml and streptomycin 100. mu.g/ml), and passaged 1 time for 2-3 days. Both cells contained 5% CO at 37 deg.C2Culturing in a cell culture box, and taking cells in logarithmic growth phase for experiment. Respectively preparing human skin fibroblast and 435S cell in logarithmic growth phase into single cell suspension, adding 200 μ L of cell suspension into 96-well plate, each well is 5 × 103And (3) adding liposome I-III groups and blank liposome groups after the cells adhere to the wall, setting 7 concentration gradients in each experimental group, wherein the concentration of 1-7 is 5, 2.5, 1.25, 0.63, 0.32, 0.16 and 0.08 mu M in sequence, continuously culturing for 40h, and taking out the culture plate. The culture medium was discarded, 200. mu.L of a precooled trichloroacetic acid solution (TCA, 10%) was added to each well, and the cells were fixed for 1 hour in a 4 ℃ refrigerator. And then washed 5 times with distilled water to remove trichloroacetic acid. After drying in air, 100. mu.L of 0.4% SRB dye solution was added to each well, and the mixture was dyed at room temperature for 20min, after which the dye solution was discarded, and washed 5 times with 1% acetic acid to remove unbound dye. Air conditionerAfter air drying, 200. mu.L of Tris solution (pH 10.5, 10mmol/L) was added to each well for solubilization, and shaken on a plate shaker for 30min until all of the SRB dye was dissolved. The 96-well plate was placed in a microplate reader and the Optical Density (OD) per well was measured at a wavelength of 540 nm. The percent cell survival (percent) was used to evaluate the cytotoxic effect of each formulation group on cells. Percent cell survival was calculated according to the following formula:
the results are shown in FIG. 5. The results show that the survival rate of human skin fibroblasts in the measured concentration range of each liposome preparation has no obvious influence, and the results show that each liposome preparation hardly generates cytotoxicity to human skin fibroblasts in the measured concentration range.
A micrograph of human skin fibroblasts after administration is shown in figure 6. The results show that the human skin fibroblasts of each liposome preparation have normal morphology and good growth state, and show that each liposome preparation has almost no cytotoxic effect on human skin fibroblasts.
The 435S cell survival results are shown in fig. 7. The results show that the survival rate of 435S cells in each liposome preparation group was between 75-100% over the measured concentration range, indicating that each liposome preparation had no significant cytotoxic effect on 435S cells.
A micrograph of 435S cells after administration is shown in figure 8. The results showed that 435S cells grew well and the morphology was normal in each liposome preparation group in the measured concentration range, indicating that each liposome preparation had almost no cytotoxic effect on 435S cells.
2. Skin irritation test in mice
30 mice, 3 experimental groups, 1 blank group and 1 saline group, each group was randomly assigned 6. Removing hair from abdomen with 10% sodium sulfide depilatory, and depilating area of 2 × 2cm2. Respectively smearing baicalin liposome I, II, III (baicalin content of 0.2mg/mL), blank liposome and physiological saline 0.5mL for 10 days, and smearing for 1 time per day. The mice were then sacrificed, conventional methodFrozen sections were made and HE stained to investigate skin condition.
The results are shown in FIG. 9. Wherein 1 is a normal saline group; 2 is blank liposome group; 3 is liposome group I "; 4 is liposome group II; liposome No. 5, group III. The results show that the skin structure of each group of mice is complete, the epidermis thickness is uniform, the cells are arranged closely, the cell nucleus morphology is normal, the skin appendage morphology is normal and the number is large, and the results show that various liposomes have no irritation to the skin of the mice.
Example 8 evaluation of the Experimental Effect of the baicalin liposomes of the present invention
1. Evaluation of skin moisturizing Effect of mice
30 mice, 3 experimental groups, 1 blank group and 1 saline group, each group was randomly assigned 6. Removing hair from abdomen with 10% sodium sulfide depilatory, and depilating area of 2 × 2cm2. The preparation is applied with ceramide liposome, blank liposome and physiological saline 0.5mL with different prescriptions for 10 days for 1 time per day. And abdominal moisture values of the mice were measured with a skin moisture content tester before administration, 2h, 6h and 12h after application, respectively, and the values were recorded (each value was measured 3 times repeatedly and averaged).
The results are shown in FIG. 10. Wherein 1 is a normal saline group; 2 is blank liposome group; 3 is liposome group I; 4 is liposome group II; liposome No. 5, group III. The results show that the skin moisture content of each group of mice has no significant difference, and show that the skin moisture content of the mice is not obviously affected by various liposomes.
2. Test for antioxidant free radical
Reactive Oxygen Species (ROS) are oxygen-containing chemically reactive chemical species, and during ambient pressure (e.g., ultraviolet light), ROS levels increase dramatically. This can cause severe damage to the cell structure, which is referred to as oxidative stress. Superoxide dismutase (SOD) is a metalloenzyme that can scavenge free radicals in the body, the more active it is, the more the organism is relatively more capable of scavenging free radicals.
Inoculating logarithmic growth phase skin fibroblast in 24-well culture plate at 37 deg.C and 5% CO2Incubate overnight in the environment. Ultraviolet (UVA) dose (5J/cm)2). Are respectively provided forBaicalin liposome I, II, III (baicalin content of 0.1mg/ml), blank liposome (control group) were added. Changes in SOD and ROS were measured after 3 h.
SOD expression measurement results are shown in FIG. 11, where 1 is blank liposome group; 2 is liposome group I; 3 is liposome group II; liposome No. 4, group III. The results show that the liposome II and the liposome III can obviously increase the SOD activity in skin fibroblasts, and the liposome I has no obvious influence on the SOD activity, so that the liposome II and the liposome III can improve the SOD activity in the skin fibroblasts and enhance the antioxidant capacity of the skin fibroblasts.
The results of ROS expression assay are shown in FIG. 12, 1 is blank liposomes; 2 is a positive control (provided by the kit, Rosu 50 mg/ml); 3 is liposome I; 4 is liposome II; liposome No. 5. The results show that the liposomes I-III can obviously reduce the ROS content in skin fibroblasts, and have no significant difference from each other, which indicates that 3 liposomes can inhibit the generation of ROS and reduce the oxidative stress level of skin fibroblasts.
Claims (10)
1. A baicalin liposome is characterized by comprising baicalin, ceramide and lipid materials, and is prepared by adopting an active drug-loading method.
2. The baicalin liposome of claim 1, which is prepared by the following method:
(1) weighing ceramide and lipid materials to prepare blank ceramide liposome;
(2) adding ammonium sulfate buffer solution, wrapping ammonium sulfate in liposome internal water phase, and removing ammonium sulfate in liposome external water phase;
(3) adding baicalin water solution to obtain baicalin liposome.
3. The baicalin liposome according to claim 2, characterized in that the aqueous solution of baicalin has a pH of 5-9.
4. The baicalin liposome of claim 1, characterized in that the mass ratio of ceramide to lipid material is 2-4: 20-28.
5. The baicalin liposome of claim 1, characterized in that the lipid material comprises one or more of lecithin, hydrogenated lecithin, cholesterol oleate, glyceryl trilaurate, glyceryl monostearate, stearic acid, glyceryl palmitostearate, glyceryl behenate, glyceryl trioleate, medium chain fatty acid esters, medium chain triglycerides, polyethylene glycol glyceryl oleate and glyceryl caprylate-caprate.
6. Baicalin liposomes according to claim 4 characterised in that the lipid material comprises cholesterol, lecithin and/or hydrogenated lecithin.
7. The baicalin liposome of claim 1, wherein the baicalin liposome comprises the following components by mass: ceramide: baicalin: lipid material 2-4: 1-2: 20-28.
8. The baicalin liposome of claim 6, characterized in that the mass ratio of ceramide to baicalin is 2: 1.
9. the baicalin liposome of claim 5, characterized in that the mass ratio of ceramide, lecithin and/or hydrogenated lecithin and cholesterol is 2-4: 20-28: 2-6.
10. Use of the baicalin liposomes of any one of claims 1-8 in the treatment of skin diseases, skin cosmetology, skin care.
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| CN114642610A (en) * | 2022-02-17 | 2022-06-21 | 广东丸美生物技术股份有限公司 | Moisturizing composition and preparation method and application thereof |
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