CN111228220B - Naringin nano lipid carrier and preparation method and application thereof - Google Patents

Naringin nano lipid carrier and preparation method and application thereof Download PDF

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CN111228220B
CN111228220B CN202010038269.4A CN202010038269A CN111228220B CN 111228220 B CN111228220 B CN 111228220B CN 202010038269 A CN202010038269 A CN 202010038269A CN 111228220 B CN111228220 B CN 111228220B
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naringenin
lipid carrier
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ngn
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祁荣
胡睿
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Peking University
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Abstract

The invention provides a naringenin nano lipid carrier, a preparation method and application thereof. The naringenin nanometer lipid carrier comprises naringenin and nanometer lipid carrier; when the naringenin nanometer lipid carrier is prepared by adopting a rotary evaporation method, the nanometer lipid carrier comprises phospholipid, trilaurin, medium chain triglyceride and polyoxyethylene castor oil; when the naringenin nano lipid carrier is prepared by adopting an emulsification evaporation-low temperature solidification method, the nano lipid carrier comprises phospholipid, glyceryl monostearate, stearic acid, oleic acid and pluronic F68. The naringenin nanometer lipid carrier has high encapsulation rate and drug-loading rate, and can improve the oral bioavailability of naringenin. Compared with free naringenin and naringenin nanoliposomes in the comparison document 3, the naringenin nanoliposome prepared by the emulsification evaporation-low temperature solidification method has stronger trans-intestinal epithelial cell monolayer transport efficiency and higher small intestine absorption effect. Therefore, the medicine has stronger inhibiting effect on the nonalcoholic fatty liver under lower dosage.

Description

Naringin nano lipid carrier and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a naringenin nano lipid carrier, a preparation method thereof and application thereof in preparation of a medicine for preventing and/or treating non-alcoholic fatty liver disease.
Background
Naringenin (NGN) is a natural flavonoid compound widely existing in plants of Rutaceae and the like, has the function of resisting non-alcoholic fatty liver, but is an insoluble drug, so that the Naringenin is low in dissolution in gastrointestinal tracts after oral administration, so that the loss and waste of the drug are caused, and the clinical curative effect and application of the Naringenin are influenced. Pharmaceutical researchers have been working on the improvement of naringenin solubility by formulation techniques. The reported dosage forms comprise solid dispersing agents, multi-chamber liposomes, beta-cyclodextrin complexes, phospholipid complexes, nano self-emulsifying drug delivery systems, degradable nanoparticles and the like. The above dosage forms all improve the solubility and oral bioavailability of NGN to some extent, but still have many problems to be solved. Such as: organic solvent residue in the preparation process; the storage stability of the liquid formulation; the surfactant has high oral safety, large particle size, etc.
Nanometer Lipid Carriers (NLC) are novel nanometer preparations developed in 90 s of the 20 th century, and are nanometer-scale drug delivery systems prepared by taking solid and liquid lipids with physiological compatibility, biodegradability and high melting point as framework materials. NLCs have been developed based on Solid Lipid Nanoparticles (SLNs). SLN is only composed of solid lipid, a single lipid component forms a neat and compact crystal lattice, and the drug loading is low; during storage, solid lipids are susceptible to transformation from the alpha to beta crystalline form, resulting in drug leakage. The improvement of NLC is that the liquid lipid at normal temperature is added into the solid lipid, which increases the crystal disorder degree of the carrier, thereby improving the drug-loading rate of the drug.
Based on the excellent characteristics of the nano lipid carrier, a nano lipid carrier preparation (NGN-NLC) of naringenin is constructed, so that the dissolution and absorption characteristics of naringenin are improved, the dosage of a medicament is reduced, and the same treatment effect is achieved.
Disclosure of Invention
The invention aims to provide a naringenin nano lipid carrier and a preparation method and application thereof. The invention can improve the oral bioavailability of naringenin by preparing the naringenin into the naringenin nano lipid carrier.
The naringenin nano lipid carrier provided by the invention comprises naringenin and a nano lipid carrier;
when the naringenin nano lipid carrier is prepared by the first method (method 1) of rotary evaporation, the nano lipid carrier comprises phospholipid, glycerol trilaurate, medium chain triglyceride and polyoxyethylene castor oil;
the phospholipid is at least one of egg lecithin, soybean phospholipid, cephalin, hydrogenated lecithin, 1, 2-dioleoyloxypropyl-N, N, N-trimethyl ammonium bromide, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, sodium taurocholate, beta-sitosterol and cholesterol acetyl ester;
the mass ratio of naringenin to the phospholipids, the medium chain triglycerides, glycerol trilaurate and polyoxyethylene castor oil may be, in order, 21: 60-240: 20-80: 4-16: 90-230, specifically 21: 60: 20:4: 230;
when the second method (method 2) is adopted to prepare the naringenin nano lipid carrier by an emulsification evaporation-low temperature solidification method, the nano lipid carrier comprises phospholipid, glyceryl monostearate, stearic acid, oleic acid and pluronic F68;
the phospholipid is at least one of egg lecithin, soybean phospholipid, cephalin, hydrogenated lecithin, 1, 2-dioleoyloxypropyl-N, N, N-trimethyl ammonium bromide, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, sodium taurocholate, beta-sitosterol and cholesterol acetyl ester;
the mass ratio of naringenin to phospholipid, glyceryl monostearate, stearic acid, oleic acid and pluronic F68 can be 25: 40-160: 20-80: 20-80: 20-80: 200-600, specifically 25: 40: 20: 20: 20: 600, preparing a mixture;
the invention also provides a method (method 1) for preparing the naringenin nano lipid carrier by adopting a rotary evaporation method, which comprises the following steps:
1) dissolving naringenin, phospholipid, glycerol trilaurate and medium chain triglyceride in a solvent, mixing, and then removing the solvent to obtain a mixture;
2) hydrating the mixture obtained in the step 1) with an aqueous medium and polyoxyethylene castor oil, and carrying out ice bath probe ultrasound to obtain a naringenin nano lipid carrier;
in the above preparation method, the solvent may be chloroform and/or methanol;
the ratio of the mass of naringenin to the volume of the solvent may be 1 g: 100-400 mL, specifically 1 g: 238-310 mL or 1 g: 150-350 mL or 1 g: 238 mL.
In the present invention, the solvent may be a mixed solvent of chloroform and methanol. Wherein the volume ratio of the chloroform to the methanol can be 1: 0.5-2, and specifically can be 2: 1.
In the preparation method, the solvent is removed in the step 1) by adopting a reduced pressure rotary evaporation method, and the temperature of the reduced pressure rotary evaporation can be 37-40 ℃, specifically 37 ℃, 40 ℃ or 37-40 ℃.
In the above preparation method, the aqueous medium may be pure water or a buffer solution, and the buffer solution includes a phosphate buffer solution and a physiological saline;
the pH value of the phosphate buffer solution can be 7.4, and the concentration of the phosphate buffer solution can be 0.002-0.02M, specifically 0.01M;
the ratio of the mass of naringenin to the volume of the aqueous medium may be 1 g: 100-300 mL.
In the preparation method, the probe ultrasound is carried out in an ice-water bath;
the ultrasonic time of the probe can be 15-20 min, specifically 15min, 20min or 15-20 min;
the amplitude of the ultrasonic wave of the probe can be 80-100% of the amplitude of the full output power; specifically, it may be 80%, 100% or 80-100%.
When the second method (method 2) is adopted to prepare the naringenin nano lipid carrier by an emulsification evaporation-low temperature solidification method, the nano lipid carrier comprises solid lipid (phospholipid, glyceryl monostearate and stearic acid), liquid lipid (oleic acid) and pluronic F68;
the phospholipid is at least one of egg lecithin, soybean phospholipid, cephalin, hydrogenated lecithin, 1, 2-dioleoyloxypropyl-N, N, N-trimethyl ammonium bromide, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, sodium taurocholate, beta-sitosterol and cholesterol acetyl ester;
the mass ratio of naringenin to phospholipid, glyceryl monostearate, stearic acid, oleic acid and pluronic F68 can be 25: 40-160: 20-80: 20-80: 20-80: 200-600, specifically 25: 40: 20: 20: 20: 600.
the invention also provides a method (method 2) for preparing the naringenin nano lipid carrier by adopting an emulsification evaporation-low temperature curing method, wherein the method comprises the following steps:
1') dissolving naringenin, phospholipid, glyceryl monostearate, stearic acid and oleic acid in a solvent, and mixing to obtain a mixture;
2') mixing the mixture obtained in the step 1) with an aqueous medium containing pluronic F68 to form an emulsion, and performing ice-bath deposition and solidification to obtain a naringenin nano lipid carrier;
in the preparation method, the solvent is absolute ethyl alcohol;
the ratio of the mass of naringenin to the volume of the solvent may be 1 g: 100-400 mL, specifically 1 g: 238-313 mL or 1 g: 150-350 mL.
In the preparation method, in the step 2'), the emulsion can be formed by a mechanical method-magnetic stirring method, and the temperature is 70-75 ℃, specifically 75 ℃, 70 ℃ or 70-75 ℃.
In the above preparation method, the aqueous medium may be pure water or a buffer solution,
the buffer solution comprises phosphate buffer solution and normal saline;
the pH value of the phosphate buffer solution is 7.4, and the concentration of the phosphate buffer solution is 0.002-0.02M, and specifically can be 0.01M;
the ratio of the mass of naringenin to the volume of the aqueous medium may be 1 g: 100-800 mL.
The mass percentage content of the pluronic F68 in the aqueous medium can be 1-3%;
the encapsulation rate of the naringenin nano lipid carrier can be 92-100%, and specifically can be 94.5 +/-0.5% or 99.9 +/-0.1%;
the medicine carrying amount of the naringenin nano lipid carrier is higher than 20%, and specifically can be 22.5 +/-1.7% or 23.7 +/-0.4%.
The application of the naringenin nanometer lipid carrier in preparing the medicine for treating the non-alcoholic fatty liver disease (NAFLD) also belongs to the protection scope of the invention.
The minimum effective dose of the naringenin nanolipid carrier for resisting NAFLD is less than or equal to 50 mg/kg-1·day-1And more specifically mayIs 12.5 mg/kg-1·day-1Or 50 mg/kg-1·day-1。
In the application, the non-alcoholic fatty liver disease is inflammatory non-alcoholic fatty liver disease, such as: MCD diet-induced non-alcoholic fatty liver disease.
The invention has the following advantages:
the preparation method of the naringenin nanometer lipid carrier is simple and convenient, easy to control and operate, clean and safe, free of toxic organic solvent residues, and capable of realizing continuous batch production;
the prepared naringenin nanometer lipid carrier has uniform average particle size, high drug loading capacity and good stability, and is compared with two articles of naringenin nanometer lipid carriers reported before (comparison document 1)[1]And reference 2[2]) The formulations of the present invention have higher drug encapsulation efficiencies and drug loadings than formulations set forth in previously published articles, depending on the lipid composition or method of preparation.
Figure BDA0002366798390000041
[1] Plum silence naringenin nanostructure lipid carrier prescription optimization and preliminary evaluation [ J ] Chinese herbal medicine, 2015,46(2):211-215.
[2]Raeisi S,Chavoshi H,Mohammadi M,et al.Naringenin-loaded nano-structured lipid carrier fortifies oxaliplatin-dependent apoptosis in HT-29 cell line[J].Process Biochemistry,2019,83:168-175.
The naringenin nanometer lipid carrier improves the dissolution and absorption characteristics of naringenin, can reduce the dosage of the medicine, improves the oral bioavailability of the medicine, and achieves better treatment effect. Compared with our earlier granted patent of naringenin nanoliposome (NGN-NL)[3]NGN-NLC2 has higher encapsulation efficiency and drug loading rate, and has better effect of resisting NAFLD.
Figure BDA0002366798390000042
Figure BDA0002366798390000051
[3] The application of naringenin, naringenin nanoliposome and the preparation method and application thereof are disclosed in patent No. ZL 201610353469.2, inventor: qirong and old clever.
Drawings
Fig. 1 is a particle size distribution diagram of naringenin nanolipid carrier. FIG. 1A is a particle size distribution diagram of naringenin nanolipid carrier prepared by the inventive method 1; fig. 1B is a particle size distribution diagram of the naringenin nanolipid carrier prepared by the method 2 of the present invention.
Fig. 2 is an in vitro release curve of naringenin, naringenin nanoliposome carriers prepared by methods 1 and 2 of the present invention.
FIG. 3 is a graph of plasma total naringenin concentration-time curve of naringenin, naringenin nanoliposome carriers prepared by methods 1 and 2 of the present invention in C57BL/6 mice.
FIG. 4 is the mouse serum ALT and AST levels. Figure 4A is ALT levels; fig. 4B shows AST level. n is 5.***Representation compared to Control group, p<0.001; # #, ######indicatecomparison with MCD group, p<0.05，p<0.01，p<0.001。
FIG. 5 is a graph showing the results of oil-red O staining of mouse liver morphological sections. FIG. 5A is a Control group; FIG. 5B is a MCD model set; FIG. 5C is the MCD + NGN 50 set; FIG. 5D shows the MCD + NGN 100 set; FIG. 5E shows MCD + NGN-NLC 112.5 group; FIG. 5F is the MCD + NGN-NLC 150 group; FIG. 5G is set MCD + NGN-NLC 212.5.
FIG. 6 shows the results of lipid extraction from mouse liver. n is 5.***Representation compared to Control group, p<0.001; # denotes comparison with the MCD group, p<0.05。
Fig. 7 shows the results of the drug delivery experiment for MDCK cells. n is 3.*，**，***Representing comparison with the NGN group, p<0.05，p<0.01，p<0.001; # denotes comparison with the NGN-NLC1 group, p<0.05；&For comparison with the NGN-NLC2 group, p<0.05。
FIG. 8 shows the small intestine of ratResults of balloon inversion experiments. Fig. 5A is the ileum group; fig. 5B is the jejunum group. n is 3.*，**，***Representing comparison with the NGN group, p<0.05，p<0.01，p<0.001; # and # indicates p compared to NGN-NLC1 group<0.05，p<0.01；&For comparison with the NGN-NLC2 group, p<0.05。
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of naringenin nanoliposome Carrier (method 1)
Accurately weighing 0.021g naringenin, 0.060g soybean phospholipid, 0.020g medium chain triglyceride and 0.004g trilaurin, adding 3.3mL chloroform and 1.7mL methanol, fully dissolving, and performing rotary evaporation under reduced pressure at 37 ℃ in a water bath condition to remove the solvent until a uniform lipid film is formed on the wall of the eggplant-shaped bottle. 3mL of 0.01M phosphate buffer (pH 7.4) containing 230mg of polyoxyethylated castor oil was added, and sonication was performed for 15min (80% of full amplitude) in an ice bath to obtain naringenin nanolipid carrier 1(NGN-NLC1) of the present invention.
Example 2 preparation of naringenin nanoliposome Carrier (method 2)
Accurately weighing 0.025g naringenin, 0.040g soybean phospholipid, 0.020g stearic acid, 0.020g glyceryl monostearate, 0.020g oleic acid, adding 2mL ethanol, dissolving, adding 20mL 0.01M phosphate buffer solution (pH 7.4) containing 3% Pluronic F68, and forming emulsion at 75 deg.C until naringenin concentration is 8.3 mg/mL-1. Depositing and curing for 1h under ice bath to obtain the naringenin nano lipid carrier 2(NGN-NLC 2).
Example 3 application of naringenin nanoliposome Carrier
1. Quality evaluation of naringenin nano lipid carrier
And (3) particle size measurement: taking a proper amount of NGN-NLC1 obtained in the embodiment 1 of the invention, and measuring the particle size of the NGN-NLC1 by a Malvern Zeta potentiometer to obtain the particle size of (120.8 +/-1.9) nm; the particle size of the NGN-NLC2 with a proper amount in the invention example 2 is measured by a Malvern Zeta potentiometer, the measured particle size is (147.5 +/-7.4) nm, and the particle size distribution diagram is shown in figure 1.
Determination of the encapsulation efficiency EE (%):
(1) establishing a naringenin standard curve by an HPLC method:
chromatographic conditions are as follows: the measuring instrument: model LC-15 high performance liquid chromatograph (shimadzu, japan) (equipped with an ultraviolet detector); a chromatographic column: c18(250 mm. times.4.6 mm,5 μm); mobile phase: methanol/0.2% phosphoric acid water 71:29 (V/V); the volume flow rate is 0.7 mL/min-1(ii) a The sample injection amount is 20 μ L, and the detection wavelength is 289 nm.
Establishing a standard curve to obtain a regression equation as follows:
A＝127956C+38999，R2is a mixture of a water-soluble polymer and a water-soluble polymer, wherein the water-soluble polymer is 0.9999%,
in the above formula, A is the peak area, and C is the naringenin concentration (. mu.g.mL)-1)。
(2) Encapsulation efficiency and drug loading measurements
10 mu L of the prepared NGN-NLC1 and NGN-NLC2 solutions are taken respectively, methanol is added, and ultrasonic (frequency: 40kHz, power: 100W) is carried out to demulsify for 10 min. Diluting by a certain multiple, injecting 20 μ L of sample under the chromatographic condition, and calculating the total dosage of the nano lipid carrier NGN according to the peak area and the standard curve. Respectively taking 200 mu L of the prepared NGN-NLC1 and NGN-NLC2 solutions, performing ultracentrifugation (21130 Xg, 0.5h and 4 ℃), precisely sucking 10 mu L of supernate, diluting the supernate to a certain concentration by using a mobile phase, injecting 20 mu L of the supernate, and calculating the drug amount encapsulated in the nano lipid carrier prescription according to a peak area and a standard curve. Can be determined according to the formula: the encapsulation efficiency of the naringenin nano lipid carrier is calculated by encapsulated drug quantity/total drug quantity multiplied by 100%. According to the formula: the drug loading rate is [ amount of the nano lipid carrier traditional Chinese medicine/total carrier amount ] x 100%, and the drug loading rate of the naringenin nano lipid carrier is calculated. The encapsulation efficiency of NGN-NLC1 in example 1 of the invention is 99.9 +/-0.1%, and the drug loading is 23.7 +/-0.4%; the encapsulation efficiency of NGN-NLC2 is 94.5 +/-0.5%, and the drug loading rate is 22.5 +/-1.7%.
2. In-vitro drug release curve of naringenin nano lipid carrier
The concentrations of the compounds of the present invention in example 1 and example 2 were set to 0.5 mg/mL, respectively-12mL of NGN-NLC1 and NGN-NLC2 solutions were placed in dialysis bags, both ends of which were fastened, and placed in 100mL of release medium at a constant temperature of 37 ℃ with a shaker speed of 100 rpm. 1.5mL of the release medium outside the dialysis bag was taken at 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0h, respectively, and simultaneously supplemented with an equal amount of fresh release medium at the same temperature. The content of NGN in the release medium was determined by HPLC method at each time point. In vitro release profiles of naringenin nanolipid carriers were plotted with cumulative release percentage as ordinate and time as abscissa, as shown in fig. 2.
As can be seen from figure 2, the in-vitro drug release result of the naringenin nanolipid carrier 1 shows that the release rate of NGN-NLC1 reaches 68.57 +/-0.15% in the first 4h, and then the NGN-NLC1 is slowly released, and the cumulative release reaches 86.2 +/-0.36% in 24 h; the in-vitro drug release result of the naringenin nanolipid carrier 2 shows that the drug release rate of NGN-NLC2 in the first 4 hours reaches 66.1 +/-6.06%, and then the NGN-NLC2 is slowly released, and the accumulated release in 24 hours reaches 85.5 +/-5.4%.
3. Research on pharmacokinetics of naringenin nano lipid carrier
25 + -2 g Male C57BL/6 mice 18, divided into three groups of 6 mice each. Fasting was performed for 12h before administration, and water was freely available. The one-time gavage of two groups of mice is equivalent to 50 mg/kg-1Naringenin or 12.5 mg/kg-1NGN-NLC1 or 12.5mg kg-1NGN-NLC 2. Two groups of mice were bled about 0.1mL at 0.083, 0.167, 0.25, 0.5, 1,2, 4, 6, 8, 12 and 24h orbital, placed in heparin centrifuge tubes, centrifuged at 4000rpm for 10min, the supernatant was separated and the plasma concentration was determined. The time-of-drug curves are shown in fig. 3, the pharmacokinetic parameters were calculated, and the pharmacokinetic parameter results are shown in table 1.
TABLE 1 pharmacokinetic parameters of single gavage of mouse naringenin or its nano-lipid carrier (Mean + -SD, n ═ 3)
Figure BDA0002366798390000071
P <0.05, p <0.01, indicating comparison to NGN drug substance; # p <0.05, which is compared to NGN-NLC 1.
As is clear from Table 1, the amounts of NGN-NLC1 and NGN-NLC2 of the present invention were 12.5 mg/kg-1Then the water content reaches 50 mg/kg with the free naringenin-1The same or better bioavailability shows that the naringenin nano liposome carrier can improve the oral bioavailability of naringenin; t of NGN-NLC21/2The significant difference of/h compared with NGN-NLC1 shows that NGN-NLC2 prolongs the half-life of NGN in vivo significantly.
4. Inhibitory effect of naringenin nano lipid carrier on non-alcoholic fatty liver disease of mouse induced by methionine choline deficiency diet (MCD diet)
(1) Male C57BL/6 mice of 6 weeks old, average weight 20g, were randomly divided into four groups, and normal group (Control group) was fed with normal breeding feed for 7 days while gavage PBS 200. mu.L per day; the model group was given a pure MCD feed for 7 days to induce a non-alcoholic fatty liver model while gavage was performed daily with 200 μ L of PBS (MCD group); the drug group is fed with MCD feed for 7 days, and 50 mg/kg is administered by gavage-1Or 100 mg/kg-1NGN bulk drugs (MCD + NGN 50 group or MCD + NGN 100 group); 12.5 mg/kg-1Or 50 mg/kg-1NGN-NLC1(MCD + NGN-NLC 112.5 group or MCD + NGN-NLC 150 group); 12.5 mg/kg-1NGN-NLC2(MCD + NGN-NLC 212.5 group). After 7 days, all mice were sacrificed and harvested.
(2) Mouse selection
After 7 days of continuous administration, the mice in (1) above were fasted overnight, weighed, and blood was taken from the inner canthus of capillary, and serum was isolated for measurement of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST). Anesthetized mice were lethal, mice livers were completely isolated, and total liver weight was weighed. Then the liver is taken for morphological analysis and liver lipid extraction.
The results of liver serological tests show (as shown in fig. 4) that the serum AST and ALT levels of the mice are obviously increased under the MCD diet induction, indicating that the livers of the mice are damaged. Administration of naringenin 100 mg/kg-1Has certain inhibition effect on the increase of ALT and AST of the liver, and the naringenin encapsulated in the nano lipid carrier has the effect of protecting the liverMore remarkable.
(3) Tissue morphology experimental method
Taking the material liver in the step (2), carefully taking a part of fresh liver tissue, putting the fresh liver tissue into 4% paraformaldehyde solution for fixation for 2h, taking out the liver tissue, transferring the liver tissue into 20% sucrose solution, wrapping the liver tissue in OCT after overnight, putting the OCT into liquid nitrogen for freezing, and carrying out frozen section on the liver tissue, wherein the thickness of each liver tissue is 7 microns. Carrying out oil red O staining on the frozen section of the mouse liver, and qualitatively judging the amount of lipid deposition in the liver cells according to the staining depth; the TG content of the liver tissue is determined by lipid extraction of the liver tissue, and the lipid level in the liver is quantified.
(4) Oil red O staining of mouse liver frozen section
Washing frozen section with PBS for 3min, 2 times; fixing with 4% paraformaldehyde for 10 min; soaking in double distilled water for 3 times for 2 min; 60% isopropanol 10min (dehydration); dyeing for 30min by using oil red O; quickly washing in 60% isopropanol for several times to remove loose color; washing with double distilled water for several times (storing in water); staining cell nucleus with hematoxylin for 1 min; washing with double distilled water for several times; 90% glycerol was mounted and photographed under microscope.
The preparation method of the oil red O dye comprises the following steps: a0.5% stock solution (mother liquor) was made from 1g of oil red O powder in 200mL of isopropanol. The 60% working solution is prepared by adding 6 parts of mother liquor into 4 parts of double distilled water, shaking vigorously, standing for 10min, and filtering. (ready-to-use, used up in 2 h)
The preparation method of 4% paraformaldehyde comprises the following steps: 4g of paraformaldehyde was weighed, added to 0.01M 100mL of PBS, and dissolved in a water bath at 60 ℃ overnight.
As shown in fig. 5, the result of oil-red-O staining of liver slices shows that compared with the normal group, lipid deposition in the liver of mice in the MCD model group is significant, naringenin drug substance has a certain reduction effect on liver lipid deposition, and NGN-NLC1 and NGN-NLC2 increase the inhibitory effect of naringenin on liver lipid deposition, which indicates that NGN-NLC1 and NGN-NLC2 increase the inhibitory effect of naringenin on liver lipid deposition by improving the oral bioavailability of naringenin.
(5) Mouse liver lipid extraction
Another fresh piece of liver tissue was weighed, homogenized in 1mL of pre-chilled PBS, transferred to a 10mL clean, dry glass tube, and added 2:1 in 4mL of chloroform/methanol, vortexed thoroughly for 30s, and then centrifuged at 2000rpm for 30min at 4 ℃. The upper aqueous phase is transferred to a new glass tube called an aqueous phase tube, and the lower organic phase is transferred to another new glass tube called an organic phase tube. To the aqueous tube was added 3mL of chloroform/methanol, and the mixture was vortexed thoroughly for 30s at 4 ℃ and 2000rpm, and centrifuged for 30 min. The lower organic phase was transferred to an organic phase tube and dried with nitrogen in a fume hood. Adding 3% TritonX-100(v-v)500 μ L, dissolving, repeatedly beating, shaking at constant temperature of 50 deg.C for 30min, dissolving lipid, and determining total Triglyceride (TG) content. The content of TG in the liver was determined as the weight of the lipid-extracted liver.
As shown in FIG. 6, the results of lipid extraction from mouse liver were consistent with the results of oil red O staining, the level of TG in mouse liver was significantly increased in the model group, and naringenin crude drug 100 mg/kg-1Has obvious effect of reducing TG content, 50 mg/kg- 1NGN-NLC1 and 12.5mg kg-1NGN-NLC2 significantly reduced the TG content in mouse liver.
4. In vitro absorption experiment of naringenin nano lipid carrier
(1) MDCK cell drug transport assay
In a Transwell chamber (polycarbonate membrane filter (pore size 3.0 μm, 1.12 cm)2Growth area of (d)) 0.5mL of seed having a density of 4X 105Cells/cm2The MDCK cell of (1). Determining whether the cell has formed a monolayer by measuring trans-epithelial cell membrane resistance (TEER) until the TEER value reaches 350-400 (omega cm)2) And can be used for epithelial cell transport research.
NGN, NGN-NLC1, NGN-NLC2 or naringenin nanoliposome (NGN-NL) were prepared into 500. mu.L of 200. mu.M test solution with HBSS as a solvent, and added to the chamber, respectively, and then 1.5mL of HBSS was added to the outside of the chamber. Transwell plates at 37 ℃ and 5% CO2Medium incubation, 0.2mL of liquid was aspirated outside the chamber at 1 and 3h, respectively, while the corresponding volume of blank HBSS was replenished. The concentration of NGN in the collected samples was then analyzed by HPLC, with triplicate determinations for each sample.
The transmembrane transport coefficient (Papp) is calculated as: papp ═ cm × s (Q/t)/(a × C0)-1
Wherein Q/t is the amount of drug transported per second across an MDCK monolayer (mol. s)-1) And A is the surface area (1.13 cm) of the polycarbonate film2) And C0 is the initial drug concentration in the cell.
As shown in FIG. 7, the MDCK cell drug transport experiment results show that the efficiency of transport absorption across epithelial cell monolayers NGN-NLC2 > NGN-NL > NGN-NLC1> NGN.
(2) Rat small intestine enterocyst overturning and absorbing experiment
Taking about 300g of healthy SD male rats, fasting for 12h before experiment, anesthetizing with 10% chloral hydrate, opening the abdominal cavity, taking 24cm downwards from 10cm below the pylorus of the stomach to be jejunum, and taking 24cm upwards from 5cm above ileocecal valve to be ileum. Flushing with 0 deg.C Taiwan liquor until the effluent is no longer turbid and has no intestinal content. The mesentery and fat on the surface of the intestine section were peeled off, and the jejunum and ileum were divided into 8cm sections each. Ligating one end of the intestinal canal on a self-made plastic sleeve, turning the intestinal canal by fingers gently to enable the mucous membrane to face outwards, washing the intestinal canal by a 4 ℃ Taiwan liquor, and ligating the other end to form a saccular intestinal canal.
Placing it into a test tube containing 2mL of blank Taiwan liquid, placing the tube in 37 deg.C constant temperature water bath, balancing for 5min, injecting 1mL of blank Taiwan liquid into different intestinal tubes respectively with a syringe, and replacing 2mL of Taiwan liquid outside the intestinal tubes with 2 mg/mL of blank Taiwan liquid prepared respectively with Taiwan liquid as solvent-1Simultaneously with the start of the timing, 0.2mL of the test solution of NGN, NGN-NLC1, NGN-NLC2 or NGN-NL was sampled from the intestinal tube at 15, 30, 45 and 60min, respectively, while the same volume of blank tai chi solution was supplemented, and the concentration of NGN in the collected sample was analyzed by HPLC, and each sample was assayed in triplicate.
As shown in fig. 8, the results of the rat intestinal tract pouch inversion experiment show that the absorption amounts of different dosage forms of NGN in the ileum segment and jejunum segment of the small intestine are both: NGN-NLC2 > NGN-NL > NGN-NLC1> NGN. It is shown that NGN-NLC2 has the best intestinal absorption, better than NGN-NLC1 and better than our previously granted NGN-NL patent.

Claims (10)

1. A naringenin nanometer lipid carrier comprises naringenin and nanometer lipid carrier;
when the naringenin nano lipid carrier is prepared by adopting a rotary evaporation method, the nano lipid carrier comprises phospholipid, trilaurin, medium chain triglyceride and polyoxyethylene castor oil;
the phospholipid is at least one of egg lecithin, soybean phospholipid, cephalin, hydrogenated lecithin, 1, 2-dioleoyloxypropyl-N, N, N-trimethyl ammonium bromide, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, sodium taurocholate, beta-sitosterol and cholesterol acetyl ester;
the mass ratio of naringenin to the phospholipids, the medium chain triglyceride, the trilaurin and the polyoxyethylene castor oil is 21: 60-240: 20-80: 4-16: 90-230;
when the naringenin nano lipid carrier is prepared by adopting an emulsification evaporation-low temperature solidification method, the nano lipid carrier comprises phospholipid, glyceryl monostearate, stearic acid, oleic acid and pluronic F68;
the phospholipid is at least one of egg lecithin, soybean phospholipid, cephalin, hydrogenated lecithin, 1, 2-dioleoyloxypropyl-N, N, N-trimethyl ammonium bromide, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, sodium taurocholate, beta-sitosterol and cholesterol acetyl ester;
the mass ratio of naringenin to phospholipid, glyceryl monostearate, stearic acid, oleic acid and pluronic F68 is 25: 40-160: 20-80: 20-80: 20-80: 200-600.
2. A method for preparing the naringenin nanoliposome carrier of claim 1 by rotary evaporation, comprising the following steps:
1) dissolving naringenin, phospholipid, glycerol trilaurate and medium chain triglyceride in a solvent, mixing, and then removing the solvent to obtain a mixture;
2) hydrating the mixture obtained in the step 1) with an aqueous medium and polyoxyethylene castor oil, and carrying out ice bath probe ultrasound to obtain the naringenin nano lipid carrier.
3. The method of claim 2, further comprising: in the step 1), the solvent is chloroform and/or methanol;
the mass ratio of the naringenin to the solvent is 1 g: 100-400 mL;
removing the solvent in the step 1) by adopting a reduced pressure rotary evaporation method, wherein the temperature of the reduced pressure rotary evaporation is 37-40 ℃.
4. The method of claim 2, further comprising: in the step 2), the aqueous medium is pure water or a buffer solution, and the buffer solution comprises a phosphate buffer solution;
the mass ratio of the naringenin to the volume of the aqueous medium is 1 g: 100-300 mL;
the ultrasonic time of the probe is 15-20 min;
the amplitude of the ultrasonic wave of the probe is 80-100% of the amplitude of the full output power.
5. A method for preparing the naringenin nanolipid carrier of claim 1 by an emulsification evaporation-low temperature solidification method comprises the following steps:
1') dissolving naringenin, phospholipid, glyceryl monostearate, stearic acid and oleic acid in a solvent to obtain a mixture;
2') mixing the mixture obtained in the step 1) with an aqueous medium containing pluronic F68 to form an emulsion, and performing ice-bath deposition and solidification to obtain the naringenin nano lipid carrier.
6. The method of claim 5, further comprising: in step 1'), the solvent is absolute ethyl alcohol;
the mass ratio of the naringenin to the solvent is 1 g: 100-400 mL;
the mass percentage of the pluronic F68 in the aqueous medium is 1-3%.
7. The method of claim 5, further comprising: in the step 2'), the forming method of the emulsion is a mechanical method-magnetic stirring method, and the temperature is 70-75 ℃;
the aqueous medium is pure water or a buffer solution,
the buffer solution comprises a phosphate buffer solution;
the mass ratio of the naringenin to the volume of the aqueous medium is 1 g: 100-800 mL.
8. The method according to claim 4 or 7, characterized in that: the pH value of the phosphate buffer solution is 7.4, and the concentration of the phosphate buffer solution is 0.002-0.02M.
9. The use of the naringenin nanoliposome carrier of claim 1 in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease.
10. Use according to claim 9, characterized in that: the non-alcoholic fatty liver is inflammatory non-alcoholic fatty liver.
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