CN108309939B - Nano-structure lipid carrier containing schisandra lignan compounds and preparation method and determination method thereof - Google Patents
Nano-structure lipid carrier containing schisandra lignan compounds and preparation method and determination method thereof Download PDFInfo
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- G01N30/02—Column chromatography
Abstract
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a nano-structure lipid carrier containing a schisandra lignan compound, and a preparation method and a determination method thereof. The nanostructured lipid carrier comprises: 10-24 parts of schisandra lignan compound, 240-360 parts of glycerin monostearate, 40-120 parts of medium-chain triglyceride, 188300-500 parts of poloxamer and 10 parts of water. In the nano-structure lipid carrier, the components and the contents thereof supplement each other and have synergistic effect, so that the solubility of the schisandra lignan compound can be increased, the action time of the schisandra lignan compound is prolonged, the nano-structure lipid carrier has good stability, higher bioavailability and lower toxic and side effects of medicaments, and meanwhile, the nano-structure lipid carrier has good indexes of average particle size, encapsulation efficiency, medicament-loading rate and the like, is not easy to isomerize and degrade, and has good stability.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a nano-structure lipid carrier containing a schisandra lignan compound, and a preparation method and a determination method thereof.
Background
Senile dementia is also called Alzheimer's disease, and is a chronic progressive mental decline disease with cognitive dysfunction, affective disorder and psychobehavioral abnormality as main clinical features. With the progress of aging of the population accelerating, the incidence of senile dementia increases year by year. At present, 600 million people are in China, and the number of the senile dementia patients is predicted to be increased to 1009 million people in 2025. Meanwhile, liver diseases are a common disease in the world, and the incidence rate is high, such as alcoholic fatty liver, alcoholic hepatitis, liver cirrhosis, liver fibrosis, liver cancer and the like. Both of the above diseases seriously affect the health level and the quality of life of people, and simultaneously bring great burden to families and society of patients, which has become a non-negligible social problem.
Nano-structured Lipid Carriers (NLC) are a new generation of Lipid nanoparticles, and liquid oil or mixed matrix in a certain proportion is used to replace solid matrix in solid Lipid nanoparticles, so that the regular lattice structure of solid Lipid is disturbed, the proportion of irregular crystal forms in the nanoparticles is increased, more drug molecules can be accommodated, the encapsulation efficiency and drug loading capacity of the drug are improved, and the encapsulated drug is prevented from being extruded and leaked in the storage process.
Schisandrol A, a kind of lignans compound of Schisandra chinensis (Schisandrol A), is a lignans compound separated from Schisandra chinensis (Schisandra chinensis) of south China and north China, and has a molecular formula of C24H32O7Relative to molecular mass 432.5, it is insoluble in water, has low bioavailability and is metabolized quickly, which limits its clinical application. Modern pharmacological research shows that schizandrol A has neurotrophic and protective effects on central nervous system diseases through various ways of relieving brain tissue neuron degeneration and loss, improving synaptic function, inhibiting microglial cell activation, resisting oxidative damage and the like, has obvious treatment effects on diseases such as senile dementia, epilepsy and Parkinson's disease and the like, and is a medicament with great potential for treating Alzheimer's disease。
Through patent and literature inquiry, no research report on a nano-structure lipid carrier containing schisandra lignan compounds (such as schizandrin A) is found at present.
Disclosure of Invention
Aiming at the defects that the schisandra lignan compounds (particularly schizandrol A) are difficult to dissolve in water, low oral bioavailability and the like, the invention provides a nano-structure lipid carrier containing the schisandra lignan compounds and a preparation method thereof.
Accordingly, it is a primary object of the present invention to provide a nanostructured lipid carrier containing schisandra lignan compounds.
The invention also mainly aims to provide a preparation method of the nano-structured lipid carrier containing the schisandra lignan compound.
The invention provides a nano-structure lipid carrier containing a schisandra lignan compound, which comprises the following components and contents:
the schisandra lignan compound comprises: one or more of schizandrol A, schizandrin B, schizandrin C, schisantherin A, schisanhenol, gomisin D, gomisin J, gomisin G, and angeloylgomisin H.
Preferably, the schisandra lignan compound is schisandrin.
Wherein the glyceryl monostearate is solid lipid, the medium-chain triglyceride is liquid lipid, and the poloxamer188 is emulsifier. Through a great deal of research, the inventor discovers that the three components are combined to use, and the prepared nano-structure lipid carrier is uniform, clear and transparent blue-light latex and has excellent performance.
Preferably, the nano-structured lipid carrier containing the schisandra lignan compound comprises the following components and contents thereof:
at this level, the morphology, average particle size, encapsulation efficiency and drug loading of the nanostructured lipid carrier are further improved.
The nano-structure lipid carrier containing the schisandra lignan compound is round and regular spherical, has small average particle size, is uniformly distributed, and has high encapsulation efficiency and drug-loading rate, namely the average particle size is 88-128 nm, the PDI value is 0.20-0.22, the encapsulation efficiency is 87-90%, and the drug-loading rate is 2.1-2.8%.
The nano-structure lipid carrier containing the schisandra lignan compound has an accumulated release rate of 52-56% in 0-4 h and a release rate of 64-68% in 24 h.
In a specific embodiment, the nanostructured lipid carrier containing schisandra lignan compound comprises the following components and contents thereof:
at this level, the morphology, average particle size, encapsulation efficiency, and drug loading of the nanostructured lipid carrier are optimal.
The invention provides a preparation method of a nano-structure lipid carrier containing a schisandra lignan compound, which comprises the following steps:
(1) mixing and heating schisandra lignan compound, glyceryl monostearate and medium-chain triglyceride to 50-90 ℃ to be used as an oil phase;
(2) adding water into poloxamer188, uniformly stirring by ultrasonic waves, and heating to 50-90 ℃ to be used as a water phase;
(3) adding the water phase into the oil phase at the same temperature dropwise under stirring; the stirring time is 5min to 25 min; stirring at the speed of 300 r/min-900 r/min, and stirring at constant temperature to prepare primary emulsion; and ultrasonically dispersing the primary emulsion for 3-20 min by using an ultrasonic cell disruptor while the primary emulsion is hot, and naturally cooling and solidifying at room temperature to obtain the colostrum.
Preferably, the heating temperature of the oil phase is 75 +/-1 ℃; the heating temperature of the water phase is 75 +/-1 ℃; stirring for 10 min; the stirring speed is 900 r/min; the ultrasonic dispersion time was 10min, power was 750W, amplitude was 100%, 3 seconds apart per 3 seconds of ultrasound.
In addition, the invention provides a pharmaceutical composition, which comprises the nanostructure lipid carrier containing the schisandra lignan compound and pharmaceutically acceptable auxiliary materials.
Preferably, the pharmaceutical composition can be tablets, capsules, granules, freeze-dried powder and the like.
A freeze-dried preparation comprises the nano-structure lipid carrier containing the schisandra lignan compound and 5-10% of trehalose based on the weight of the nano-structure lipid carrier. The freeze-dried preparation has smooth and full surface, good redispersibility and good stability.
Preferably, the pre-freezing temperature of the freeze-dried preparation is-80 ℃, and the pre-freezing time is 12 h; the freeze-drying time is 24 h.
In addition, the invention provides application of the nano-structure lipid carrier containing the schisandra lignan compound in preparing a medicament for treating Alzheimer's disease or liver disease.
In addition, the invention provides a method for determining schizandrol A in the nanostructure lipid carrier containing the schizandrol lignan compounds, which comprises the following steps:
chromatographic conditions are as follows: a chromatographic column: c18Column, 5 μm, 4.6 × 250 nm; mobile phase: 65:35(v/v) methanol-water; flow rate: 1.0 mL/min; detection wavelength: 250 nm; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution;
precisely weighing appropriate amount of schizandrol A reference substance, placing in a measuring flask, adding methanol for dissolving, and fixing volume to scale mark to obtain schizandrol A reference substance stock solution, and storing in a refrigerator at 4 deg.C;
precisely measuring a schisandrin A nanostructured lipid carrier into a measuring flask, adding methanol for ultrasonic demulsification, fixing volume to scale, centrifuging the solution at 10000r/min for 10min, and filtering with 0.22 μm microporous membrane to obtain a test solution;
and (4) respectively taking the reference substance solution and the test solution, and respectively injecting samples under the chromatographic conditions for determination.
In the nanostructure lipid carrier containing the schisandra lignan compound, the components and the content thereof supplement and cooperate with each other, so that the solubility of the schisandra lignan compound can be improved, the action time of the schisandra lignan compound can be prolonged, the nano structure lipid carrier has good stability, high bioavailability and low toxic and side effects of medicines, and the nano structure lipid carrier has good indexes of average particle size, encapsulation efficiency, drug loading rate and the like, is not easy to isomerize and degrade, and has good stability.
Drawings
FIG. 1-1 is an HPLC assay profile of a blank nanostructured lipid carrier;
FIG. 1-2 is an HPLC determination profile of a schizandrol A control solution;
FIGS. 1-3 are HPLC assay profiles of schisandrin A nanostructured lipid carrier;
FIG. 2 is a schizandrol A standard curve;
FIG. 3 is a transmission electron micrograph of a schisandrin nanostructured lipid carrier;
FIG. 4 is a particle size distribution diagram of a schisandrin A nanostructured lipid carrier;
fig. 5 is an in vitro release profile of schizandrin a nanostructured lipid carrier.
Detailed Description
The invention is explained below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way.
Abbreviations: schizandrol a (schiscandrin, SCH); nanostructured Lipid Carriers (NLC); polydispersion index (PDI); x-ray diffraction (XRD); glycerol Monostearate (glycoryl monostarate, GMS); medium Chain Triglycerides (MCT); poloxamer188 (Poloxamer 188); schisandrin A nanostructured lipid carrier (SCH-NLC).
The instrument comprises the following steps: high performance liquid chromatograph LC-20AT (Shimadzu, Japan), BT125D electronic balance (Sadoris scientific instruments, Beijing GmbH), Milli-Q type ultrapure water preparation instruments (Millipore, France), electric constant temperature water bath (Tianjin Tester instruments, Ltd.), KQ5200DE type ultrasonic cleaner (ultrasonic instruments, Ltd. of Kunshan city), HJ-3 digital display constant temperature magnetic stirrer (Tokyo instruments, Ltd.), ultrasonic cell disruptor sound-proof box (Biotech Co., Ltd. of Nanjing star), centrifuge (eppendorf, Centrigugge 5424), Nano-ZS type Zetasizer laser granulometer (Malvern, UK), transmission electron microscope (JEM-1400, Japan JEOL, 80 kv); lyophilizer (Alpha2-4 LDplus); bruker D8Advance X-ray diffractometer, germany.
Example 1: preparation of nanostructured lipid carrier containing schisandrin
The prescription is as follows:
mixing schizandrol A and lipid (glyceryl monostearate and medium chain triglyceride), heating in water bath to 75 + -1 deg.C to obtain oil phase; adding water for injection into poloxamer188, stirring with ultrasound, heating to 75 + -1 deg.C, and making into water phase; dropwise adding the water phase into the oil phase at the same temperature under magnetic stirring, stirring at constant temperature for 10min at stirring speed of 900r/min to obtain primary emulsion, ultrasonically dispersing with ultrasonic cell disruptor for 10min (power of 750W, amplitude of 100%, 3 s/s interval of 3 s/s), and naturally cooling at room temperature to solidify to obtain nanometer structure lipid carrier (SCH-NLC) containing schizandrol A.
Example 2: preparation of nanostructured lipid carrier containing schisandrin
The prescription is as follows:
the preparation method is the same as example 1.
Example 3: preparation of nanostructured lipid carrier containing schisandrin
The prescription is as follows:
the preparation method is the same as example 1.
Example 4: preparation of freeze-dried preparation containing nano-structure lipid carrier of schisandrin
Taking SCH-NLC prepared in example 1, adding 10 wt% of mannitol as a freeze-drying protective agent, and then pre-freezing at-80 ℃ for 12 h; and then freeze-drying for 24h to obtain the freeze-dried preparation of the nano-structure lipid carrier containing the schisandrin.
Example 5: establishment of HPLC analytical method
Chromatographic conditions are as follows: a chromatographic column: ZORBAX SB-C18(5 μm, 4.6X 250 nm); mobile phase: methanol-water (65: 35); flow rate: 1.0 mL/min; detection wavelength: 250 nm; column temperature: 40 ℃; sample introduction amount: 10 μ L.
And (3) special investigation: accurately weighing an appropriate amount of SCH reference substance, placing in a 10mL measuring flask, adding methanol to dissolve, and fixing the volume to the scale mark to obtain 0.3mg/mL SCH reference substance stock solution, and storing in a refrigerator at 4 deg.C for later use. Precisely measuring 0.5mL of SCH-Nanostructured Lipid Carrier (NLC) into a 10mL measuring flask, adding methanol, performing ultrasonic demulsification, fixing the volume to scale, centrifuging the solution at 10000r/min for 10min, and filtering with a 0.22 μm microporous filter membrane to obtain SCH-NLC. Preparing blank NLC test solution by the same method. Respectively sampling reference solution and sample solution under the above chromatographic conditions, respectively, introducing 10 μ L sample, and recording chromatogram, the results are shown in FIGS. 1-3. FIG. 1-1 is an HPLC assay profile of a blank nanostructured lipid carrier; FIG. 1-2 is an HPLC determination profile of a schizandrol A control solution; FIGS. 1-3 are HPLC determination profiles of schisandrin A nanostructured lipid carrier.
It can be seen that: the retention time of SCH-NLC is consistent with that of a reference substance, under the detection condition, blank NLC has no interference to the content determination of SCH, and the method has good specificity.
And (3) linear relation investigation: accurately measuring appropriate amount of SCH reference stock solution, and preparing with methanol to obtain solutions with concentrations of 7.73 μ g-m L-1、15.45μg·m L-1、30.90μg·m L-1、61.80μg·m L-1、92.70μg·m L-1The SCH standard series of solutions was analyzed under the chromatographic conditions described above, and the peak areas were recorded. Drawing a standard curve by taking the mass concentration of SCH as an abscissa (X) and taking a peak area as an ordinate (Y) to obtain a linear regression equation Y21468X +8548.6, R2The standard curve is shown in fig. 2, which is 0.9998. FIG. 2 is a schizandrin A standard curve. The results show that: SCH was 7.73 to 92.70 μ g m L-1The linear relationship is good.
And (3) precision test: the precision absorption mass concentration is 7.73 mu g-m L-1、30.90μg·m L-1、92.70μg·m L-1Measuring the standard solutions with low, medium and high concentrations for five times in a day, and calculating the precision in the day; the samples are injected once a day, continuously measured for five days, and the precision in the day is calculated. The results are shown in tables 1 and 2.
TABLE 1 results of precision in day (n ═ 5)
TABLE 2 day precision results (n ═ 5)
As can be seen from the table, the HPLC detection method established by the invention has good precision in day and day (RSD is less than 3 percent) and meets the requirement of methodology.
And (3) stability test: and (3) placing the newly prepared test solution at room temperature, carrying out sample injection measurement at 0, 4, 8, 12 and 24 hours, and inspecting the stability of the sample. The results are shown in Table 3. The result shows that the schizandrol A test solution is stable within 24 h.
TABLE 3 stability test results
And (3) repeatability test: taking the same batch of samples, preparing 6 parts of sample solution in parallel, carrying out sample injection measurement, and calculating the content. The results are shown in Table 4. The results show that the method has good repeatability.
TABLE 4 results of the repeatability tests
Recovery rate test: precisely sucking 0.25mL, 1mL and 2mL of SCH reference solution with known concentration into a 10mL volumetric flask, respectively adding 0.5mL of blank NLC and 3mL of mobile phase, performing ultrasonic treatment until NLCs are completely dissolved, adding the mobile phase to a constant volume to scale, preparing sample solutions with low, medium and high concentration gradients of 7.73, 30.90 and 61.80 mu g/mL, filtering with a 0.22 mu m filter membrane, detecting samples according to the method, repeating for 3 times, and calculating the recovery rate (see Table 5). According to the measurement result, the average recovery rate is over 96 percent, the RSD is less than 3 percent, the method shows that the recovery rate meets the requirement of methodology, and the preparation auxiliary materials have no influence on the measurement of the schizandrol A.
TABLE 5 recovery test results
Test example 1: morphological observation
An appropriate amount of SCH-NLC prepared in example 1 was dropped on the surface of a carbon-sprayed copper mesh to spread the liquid over the entire copper mesh as much as possible, stained with 1.0% uranyl acetate, and then the excess liquid was removed with filter paper, dried naturally, and then placed under a transmission electron microscope to observe the morphology, as shown in FIG. 3. FIG. 3 is a transmission electron micrograph of SCH-NLC. As can be seen, the nanostructured lipid carrier is a rounded, regular spherical shape.
SCH-NLC prepared in examples 2 and 3 had a morphology similar to that of SCH-NLC prepared in example 1.
Test example 2: measurement of particle diameter
An appropriate amount of SCH-NLC prepared in example 1 was diluted at room temperature, and the particle size and distribution of the prepared SCH-NLC were measured using a Nano-ZS Nano laser particle sizer at a measurement temperature of 25 ℃ (n-3), and the results are shown in fig. 4. The results show that the particle size distribution of the nanoparticles is unimodal, the average particle size is 88nm, the PDI is 0.20, and the range of the particle size distribution is narrow.
According to the measurement, the SCH-NLC prepared in the example 2 has unimodal particle size distribution, the average particle size of 96nm, PDI of 0.21 and narrow particle size distribution range.
According to the SCH-NLC prepared in example 3, the particle size distribution of the nanoparticles is unimodal, the average particle size is 128nm, the PDI is 0.22, and the particle size distribution range is narrow.
Test example 3: encapsulation efficiency and drug loading rate measurement
In this experiment, the entrapment rate and drug loading of SCH-NLC prepared in example 1 were determined by ultrafiltration centrifugation. Accurately measuring 0.5mL of SCH-NLC dispersion, placing in an inner tube of a centrifugal ultrafiltration tube with 3 ten thousand of intercepted relative molecular mass, centrifuging at 10000r/min for 10min, placing the filtrate of an outer tube in a 5mL measuring flask, fixing the volume of methanol to the scale, passing through a 0.22 mu m microporous filter membrane, and measuring the drug content according to HPLC conditions to obtain free W. And putting 0.5mL of dispersion liquid into a 10mL measuring flask, ultrasonically demulsifying with methanol for 30min, fixing the volume to a scale, centrifuging, taking supernate, filtering the supernate through a 0.22 mu m microporous filter membrane, and measuring the drug content according to the HPLC condition, wherein the drug content is W. The encapsulation efficiency and drug loading were calculated according to the following formula:
EE% (-W dose-free)/W dose × 100%
DL% (W addition amount-W free)/(M-W free) × 100%
In the formula, EE is the encapsulation efficiency, DL is the drug loading capacity, the addition amount of W is the addition amount (g) of schizandrol A in the nanoparticle lipid carrier dispersion liquid, the amount (g) of W free drug measured by an HPLC method, and M is the total mass (g) of the nanoparticle lipid carrier. The results are shown in Table 5.
TABLE 5 measurement of SCH-NLC encapsulation efficiency
As a result: the encapsulation efficiency of SCH-NLC prepared in example 1 was 89.13%, and the drug loading was 2.74%.
Through determination: the encapsulation efficiency of SCH-NLC prepared in example 2 was 87.40%, and the drug loading was 2.46%.
Through determination: the encapsulation efficiency of SCH-NLC prepared in example 3 was 88.90%, with a drug loading of 2.11%.
Test example 4: in vitro Release study
The study carried out on the in vitro drug release characteristics of SCH-LC prepared in example 1 using a dialysis bag method. The specific method comprises the following steps: the dialysis bags were soaked for 12h before the experiment until diffusion equilibrium. 2mL of SCH-NLC solution is precisely measured, the SCH-NLC solution is filled into a dialysis bag, the two ends of the dialysis bag are tightly tied, the dialysis bag is placed into 200mL of pH7.4PBS release medium containing 0.3% (w/v) Tween80, the dialysis bag is shaken at 37 ℃ and 75rpm/min, 2mL of samples are taken at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours respectively, and the release medium with the same amount and temperature is quickly supplemented. After the sample passes through the membrane, the content is measured, the cumulative release rate of the SCH at each time point is calculated, and the release curve is drawn by plotting the cumulative release percentage against the time. In addition, the drug solution with the same drug content is precisely sucked as a reference, and the operation is the same as above. The in vitro release profile is shown in figure 5. As can be seen from FIG. 5, SCH-NLC is released rapidly within the first 4h, nearly 52.1% of the drug is released from NLC, SCH bulk drug is released relatively rapidly, and 94% of the drug is released within 10 h; SCH-NLC is accumulated and released within 24h to reach 64.4%, burst release phenomenon exists in the initial stage, and the drug is continuously and slowly released later, so that the SCH-NLC has good slow release characteristic compared with SCH bulk drug, and the SCH-NLC can be prepared into a nano-structure lipid carrier to have a certain slow release effect.
Through determination: the SCH-NLC prepared in example 2 has an accumulated release rate of 53.6% in 0-4 h and a release rate of 66.2% in 24 h.
Through determination: the SCH-NLC prepared in example 3 has an accumulated release rate of 55.3% in 0-4 h and a release rate of 67.8% in 24 h.
Based on the above description of the summary of the invention, a person skilled in the art can apply the invention in its entirety, and all changes that are the same principle or similar are to be considered as included in the scope of the invention.
Claims (10)
1. A nanostructured lipid carrier containing schisandrin comprises the following components and contents:
2. the nanostructured lipid carrier containing schisandrin according to claim 1, consisting of the following components and their contents:
3. the nanostructured lipid carrier containing schisandrin according to claim 1 or 2, wherein the nanostructured lipid carrier is round and regular spherical, the average particle diameter is 88-128 nm, the PDI value is 0.20-0.22, the encapsulation efficiency is 87-90%, and the drug loading rate is 2.1-2.8%.
4. The nanostructured lipid carrier containing schizandrol A according to claim 1 or 2, wherein the release rate of the nanostructured lipid carrier containing schizandrol A is 52-56% in 0-4 h and 64-68% in 24 h.
5. The method for preparing the nanostructured lipid carrier containing schisandrin according to any one of claims 1 to 4, comprising the following steps:
(1) mixing schisandrin, glycerin monostearate and medium-chain triglyceride, and heating to 50-90 deg.C to obtain oil phase;
(2) adding water into poloxamer188, uniformly stirring by ultrasonic waves, and heating to 50-90 ℃ to be used as a water phase;
(3) adding the water phase into the oil phase at the same temperature dropwise under stirring; the stirring time is 5min to 25 min; stirring at the speed of 300 r/min-900 r/min, and stirring at constant temperature to prepare primary emulsion; and ultrasonically dispersing the primary emulsion for 3-20 min by using an ultrasonic cell disruptor while the primary emulsion is hot, and naturally cooling and solidifying at room temperature to obtain the colostrum.
6. The method for preparing the nanostructured lipid carrier containing schizandrol A according to claim 5, wherein the heating temperature of the oil phase is 75 ± 1 ℃; the heating temperature of the water phase is 75 +/-1 ℃; stirring for 10 min; the stirring speed is 900 r/min; the ultrasonic dispersion time was 10min, power was 750W, amplitude was 100%, 3 seconds apart per 3 seconds of ultrasound.
7. A pharmaceutical composition, which comprises the nanostructured lipid carrier containing schisandrin of any one of claims 1 to 4 or the nanostructured lipid carrier containing schisandrin prepared by the preparation method of the nanostructured lipid carrier containing schisandrin of any one of claims 5 to 6, and pharmaceutically acceptable excipients.
8. A lyophilized preparation comprising the nanostructured lipid carrier containing schisandrin according to any one of claims 1 to 4 or prepared by the method of preparing the nanostructured lipid carrier containing schisandrin according to any one of claims 5 to 6, and trehalose in an amount of 5% to 10% by weight of the nanostructured lipid carrier.
9. Use of the nanostructured lipid carrier containing schisandrin according to any one of claims 1 to 4, the nanostructured lipid carrier containing schisandrin prepared by the method for preparing the nanostructured lipid carrier containing schisandrin according to any one of claims 5 to 6, the pharmaceutical composition according to claim 7 or the lyophilized formulation according to claim 8 for the preparation of a medicament for the treatment of alzheimer's disease or liver disease.
10. A method for determining schisandrin in the nanostructured lipid carrier containing schisandrin according to any one of claims 1 to 4, the nanostructured lipid carrier containing schisandrin prepared by the method for preparing the nanostructured lipid carrier containing schisandrin according to any one of claims 5 to 6, the pharmaceutical composition according to claim 7 or the lyophilized preparation according to claim 8, comprising the steps of:
chromatographic conditions are as follows: a chromatographic column: c18Column, 5 μm, 4.6 × 250 nm; mobile phase: 65:35(v/v) methanol-water; flow rate: 1.0 mL/min; detection wavelength: 250 nm; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution;
precisely weighing appropriate amount of schizandrol A reference substance, placing in a measuring flask, adding methanol for dissolving, and fixing volume to scale mark to obtain schizandrol A reference substance stock solution, and storing in a refrigerator at 4 deg.C;
precisely measuring a schisandrin A nanostructured lipid carrier into a measuring flask, adding methanol for ultrasonic demulsification, fixing volume to scale, centrifuging the solution at 10000r/min for 10min, and filtering with 0.22 μm microporous membrane to obtain a test solution;
and (4) respectively taking the reference substance solution and the test solution, and respectively injecting samples under the chromatographic conditions for determination.
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