CN109157515B - Coenzyme Q10Clathrate self-assembly liposome precursor and preparation method thereof - Google Patents
Coenzyme Q10Clathrate self-assembly liposome precursor and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a coenzyme Q10A self-assembly liposome precursor of inclusion compound and a preparation method thereof belong to the technical field of medicine and health care products. Coenzyme Q10The self-assembled liposome precursor of the inclusion compound is prepared by the following raw materials by weight: coenzyme Q10: cyclodextrin: lecithin: mannitol 1: 5-15: 0.5-1.5: 5-15. The liposome precursor prepared by the invention can be self-assembled into liposome solution when encountering intestinal juice containing cholic acid, and has the characteristics of higher dissolution rate and higher bioavailability.
Description
Technical Field
The invention relates to coenzyme Q10The self-assembled liposome precursor of the inclusion compound has better treatment and health care effects on cardiovascular diseases, high mountain anoxia, diabetes, low immunity and other diseases, and belongs to the technical field of medicines and health care products.
Background
Coenzyme Q10(Coenzyme Q10) Is a compound synthesized by human body, is named because the polymerization degree of the side chain-polyisoprene on the six-position of the mother nucleus is 10, and has a structure similar to vitamin K. Coenzyme Q10Divided into reduced coenzyme Q10(Co Q10H2Ubiquinol, panthenol) and oxidized coenzyme Q10(Co Q10Ubiquinone, Ubiquinone), CoQ10H2Is Co Q10The two-electron reduction of (1). In vivo, Co Q10H2And Co Q10The compound is used as a hydrogen/electron transfer body to jointly participate in transmembrane electron transport systems such as mitochondrial inner membrane respiratory chain and the like and the process of cell oxidative phosphorylation, and plays an important role in the synthesis of Adenosine Triphosphate (ATP).
Coenzyme Q10Is an indispensable important physiological substance in human body, has many important physiological functions in human body due to the chemical structure characteristics, is an important energy transfer substance in life activities, is a key substance for the rate-limiting reaction of the mitochondrial respiratory chain, and plays an important role in the aspects of cell energy generation, oxidation resistance, free radical elimination and biological activity enhancement. Coenzyme Q contained in human body10The total amount is 500-1500mg, the peak is reached after 20 years old, then the peak is rapidly reduced, and the peak is reduced by 57% compared with the young people of 20 years old after 70 years old. It has been found that coenzyme Q is found in many patients10The physiological level is reduced remarkably, such as coenzyme Q in heart failure patients (HF), cardiac muscle and blood of patients10The content is obviously reduced, and coenzyme Q is supplemented from an external source10After that, the symptoms of the patient are obviously improved. The research finds that the coenzyme of a plurality of patients with low immunity and tumor patientsQ10The physiological level is also low, and coenzyme Q is supplemented10Thereafter, the symptoms were all improved. Therefore, the human body is supplemented with coenzyme Q from an external source10For the treatment of various coenzyme Q10The physiological level is low, which becomes a recognized clinical treatment method.
Coenzyme Q10Is fat-soluble substance, is insoluble in water, belongs to the class II substance in the biological pharmacy, is the absorption rate-limiting step when dissolved, has low bioavailability when orally taken, and has the absolute bioavailability less than 5 percent. Wherein the coenzyme Q10Coenzyme Q due to instability to light, heat and water10Since the problems of bioavailability and stability limit the application, in response to this situation, various methods of improving the bioavailability and stability of oral administration, such as inclusion techniques, emulsification techniques, liposomes, etc., have been developed in recent years. The published documents at present are searched for the following patents and documents related to the present patent: preparation of coenzyme Q by stirring is described in Journal ActaPoloniae Pharmaceutica (1995), vol.52, No.5, pp.379-386 and 1996, vol.53, No.3, pp.193-19610The gamma-cyclodextrin inclusion compound. ② U.S. patent 6861447, which discloses a method for preparing a gamma-cyclodextrin inclusion compound. ③ world patent WO2005/111224 discloses a new coenzyme Q10The beta-cyclodextrin inclusion compound is prepared by a stirring method. The Chinese patent CN200510048010.3 discloses a water-soluble coenzyme Q10Preparation technology of the composition. International patent No. 03802446.2 discloses reduced coenzyme Q10The stabilizing method and composition of (1). Sixthly, Chinese patent ZL200610046307.0 discloses a water-soluble coenzyme Q10Hydroxypropyl-beta-cyclodextrin inclusion compound and its preparation method. Seventhly, Chinese patent CN200510137887.X discloses coenzyme Q10Pharmaceutical preparation of liposome and its preparation method, wherein the food science of the eight people's literature, 2007, vol28, no3, describes coenzyme Q10A method for preparing liposome. The document ninthly 2015, vol.36, No.21 describes coenzyme Q10Liposome precursor preparation and quality studies. The above documents mainly disclose coenzyme Q10The cyclodextrin inclusion compound and liposome, and their preparation method, inclusion technique and lipidThe product is coenzyme Q10A better approach to solubility and bioavailability, although cyclodextrin inclusion compounds and liposomes all increase coenzyme Q10Solubility and bioavailability, but coenzyme Q10The cyclodextrin inclusion compound has low solubility and incomplete dissolution, and coenzyme Q10The liposome is basically in a liquid form, has unstable physicochemical properties, cannot be placed for a long time, needs an organic solvent and cholesterol in the preparation process, and contains cholesterol and solvent residues. The bioavailability of the two rats is generally reported to be 1-3 times, and the ideal level is not reached. The inclusion compound self-assembly liposome technology is a novel technology for fusing the two, can play the advantages of the two and overcome the defects of the two technologies, and the preparation method of the inclusion compound self-assembly liposome comprises a one-step method and a two-step method, wherein the two-step method is to prepare the inclusion compound firstly and then dissolve lipid and an additive by using an organic solvent to prepare the liposome; the one-step method is that the main drug, cyclodextrin, lipid and additive are dissolved in organic solvent together for preparation; in both methods, an organic solvent is used to dissolve the main drug, lipid and an additive, the additive is usually cholesterol, and the additive has certain influence on the safety of the human body. At present, the self-assembled liposome of the inclusion compound is generally liquid and has poor stability. The liposome solid precursor reported at present is basically prepared by preparing a liposome solution and then freeze-drying the liposome solution into a solid, the liposome reconstructed by covering water has poor stability, a method for self-assembling the liposome precursor without preparing the liposome is not shown, the report and the patent of the inclusion compound self-assembling liposome precursor are not shown at present, and coenzyme Q is not shown in the report and the patent of the inclusion compound self-assembling liposome precursor10Clathrate self-assembly liposome and coenzyme Q10Inclusion compound self-assembly liposome precursor literature and patent. None of the above patent documents relate to the present invention, and coenzyme Q has not been searched for at present10The patent and literature research reports of inclusion compound self-assembly liposome and inclusion compound liposome thereof.
Disclosure of Invention
The present invention provides a coenzyme Q for solving the above problems10The invention relates to a clathrate self-assembly liposome precursor and a preparation method thereof, wherein cyclodextrin is utilized to prepare coenzyme Q10Inclusion and liposome biphasic drug loadingForm coenzyme Q10The inclusion compound self-assembly liposome precursor can be self-assembled into liposome in artificial intestinal juice simulating cholic acid, and can be combined with coenzyme Q10Clathrate and coenzyme Q10Compared with the liposome, the liposome has more remarkable solubility, dissolution rate and bioavailability, has better stability, and is easier to realize and more stable in actual industrial production.
In order to solve the technical problems, the invention is realized by the following technical scheme: coenzyme Q10The self-assembled liposome precursor of the inclusion compound is prepared by the following raw materials by weight: coenzyme Q10: cyclodextrin: lecithin: mannitol 1: 5-15: 0.5-1.5: 5-15.
The above coenzyme Q10The self-assembled liposome precursor of the inclusion compound is prepared by the following raw materials by weight: coenzyme Q10: cyclodextrin: lecithin: mannitol 1: 7-10: 0.8-1.5: 5-10.
The cyclodextrin is gamma-cyclodextrin, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin.
The lecithin is soybean lecithin or egg yolk lecithin.
The above-mentioned coenzyme Q10The preparation method of the clathrate self-assembly liposome precursor comprises the following process steps: taking cyclodextrin and coenzyme Q according to the weight ratio10Adding water in 5-8 times of cyclodextrin, mixing, stirring to obtain paste, heating to 50-60 deg.C, grinding in colloid mill for 5-10 times, adding water, diluting to solid concentration of 10% to obtain cyclodextrin coenzyme Q10Grinding fluid is reserved; taking phospholipid according to the weight ratio, adding water which is 5-8 times of the weight of the cyclodextrin, mixing and dispersing uniformly to obtain phospholipid dispersion liquid for later use; taking mannitol according to the weight ratio, putting the mannitol into a fluidized bed as a mother nucleus, spraying the phospholipid dispersion liquid to coat a phospholipid membrane on the surface of mannitol particles, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 90-110 ℃, and the air outlet temperature of the fluidized bed is 35-45 ℃ to obtain the coenzyme Q10The inclusion compound self-assembled liposome precursor.
Due to the adoption of the technical scheme, the invention has the following characteristics and effects:
coenzyme Q obtained by the invention10The clathrate self-assembly liposome precursor has the following characteristics: it is a coenzyme Q-containing10Liposome precursors of cyclodextrin inclusion compounds, coenzyme Q10The clathrate formed with cyclodextrin can self-assemble into clathrate liposome, clathrate coenzyme Q and non-clathrate coenzyme Q when meeting water in the presence of phospholipid membrane10The liposome is wrapped between the inner phase and the double-layer phospholipid by phospholipid double-layer molecules, and in the environment of the artificial intestinal juice containing the cholic acid, the cholic acid in the intestinal juice is beneficial to the formation of the liposome, is similar to a cholic acid mixed micelle, has smaller particle size and promotes the absorption of a biological membrane. Surprisingly, the inclusion compound self-assembled liposome precursor and coenzyme Q10Compared with the prototype drug or the inclusion compound thereof or the simple liposome, the drug has extremely obvious dissolution rate and solubility in the artificial intestinal fluid containing cholic acid, and the bioavailability test of rats shows that the bioavailability of the drug in 12 hours is 6.8 times that of the prototype drug and is obviously 3.7 times that of the prototype drug of the inclusion compound and 4.2 times that of the prototype drug of the simple liposome, thereby proving that the drug has obvious bioabsorption characteristic. Analyzing the absorption mechanism, which can form liposome to promote absorption, and other absorption mechanisms can exist, and analyzing coenzyme Q included by phospholipid and cyclodextrin after the precursor meets intestinal juice10Partial displacement may occur because the binding constant of phospholipids to cyclodextrins is much greater than that of coenzyme Q10Coenzyme Q in monomolecular state displaced by cyclodextrin10Can be captured by cholic acid molecules to form liposome cholic acid mixed micelles, and the cholic acid micelles have strong capability of transporting and passing through mucous membranes. In vitro dissolution tests prove that the dissolution rate of the cyclodextrin inclusion compound self-assembled liposome precursor artificial intestinal juice reaches more than 90% in 60 minutes, the dissolution rate of a prototype drug is less than 5%, the dissolution rate of an inclusion compound is 30%, and the dissolution rate of a simple liposome is 40%; rat bioavailability test the cyclodextrin inclusion compound self-assembled liposome precursor C of the present inventionmaxThe original drug C reaches 30.78mg/lmax2.39mg/l, C of clathratemax16.64mg/l, lipid onlyC of bodymax18.84 mg/l; the bioavailability of the clathrate self-assembly liposome precursor in 12 hours is 6.8 times of that of the prototype drug, and the bioavailability of the clathrate self-assembly liposome precursor, the clathrate and the simple liposome in 24 hours is respectively 4.8 times, 2.7 times and 2.9 times of that of the prototype drug. Coenzyme Q10The in vitro dissolution and bioavailability of the clathrate self-assembly liposome precursor are obviously superior to those of the simple inclusion and liposome technology, and are more obvious compared with the original medicine. Compared with the traditional liposome technology, the cyclodextrin inclusion compound self-assembly liposome precursor can be self-assembled in intestinal juice to form inclusion compound liposome, does not use organic solvent and cholesterol, does not have a process of forming liposome in advance and then drying, is easy to carry out industrial production, and has higher preparation stability and safer administration.
Coenzyme Q obtained by the invention10Clathrate self-assembly liposome precursor, which is formed in coenzyme Q10Based on the cyclodextrin inclusion compound, a phospholipid membrane coated on mannitol particles meets intestinal fluid medium of cholic acid and is self-assembled with the inclusion compound to form an inclusion compound liposome, and the particle size of the reconstructed liposome liquid is about 170-210 nm. This was confirmed mainly by the following experiment in which the clathrate compound of the present invention was reconstituted with an aqueous medium to prepare a lyophilizate and coenzyme Q10The physical mixture of cyclodextrin, lecithin and mannitol is subjected to thermal analysis (DSC) and high performance liquid chromatography, and the result shows that the DSC spectrum of the freeze-dried substance of the clathrate self-assembly liposome precursor reconstructed liquid is different from the DSC spectrum of the physical mixture, and the coenzyme Q in the DSC spectrum of the freeze-dried substance of the novel clathrate self-assembly liposome precursor reconstructed liquid10The characteristic peak of melting point peak disappears, the cyclodextrin inclusion compound self-assembly liposome is built in cholic acid-containing artificial intestinal fluid before weight, the average particle size is 170-210 nm, the Zeta potential is-40-70 mV, the liposome entrapment rate of the reconstruction fluid is more than 63%, the formed liposome is round as shown by a transmission electron microscope, the liposome is proved to be formed, and in-vitro dissolution rate measurement and bioavailability tests show that the cyclodextrin inclusion compound self-assembly liposome is obviously superior to prototypes such as medicine, inclusion compound and liposome. Coenzyme Q of the invention10Clathrate self-assembly liposome precursor has higher dissolution rate, so that the clathrate self-assembly liposome precursor is used in free radical scavenging testHas better effect, and the capability of the self-assembled liposome precursor of the coenzyme Q10 clathrate compound of the invention for removing free radicals is obviously superior to that of the coenzyme Q10Inclusion compound, simple liposome and prototype drug.
Coenzyme Q of the invention10Coenzyme Q in clathrate self-assembly liposome precursor10The content determination method comprises taking coenzyme Q10Adding a small amount of water into a proper amount of the self-assembly liposome precursor of the inclusion compound for dissolving, heating and ultrasonically treating by using ethanol, cooling and fixing the volume to a specified concentration, filtering, taking a subsequent filtrate as a test solution, injecting a sample, and performing chromatographic conditions: coenzyme Q10: and (3) a C18 column, wherein the mobile phase is acetonitrile, tetrahydrofuran and water (volume ratio) is 55: 40:5, the detection wavelength is 280nm, and the quantification is carried out by an external standard method.
Coenzyme Q of the invention10And (3) measuring the encapsulation efficiency of the clathrate self-assembly liposome precursor. Taking a proper amount of the clathrate self-assembly liposome precursor sample, dispersing in a proper amount of cholic acid-containing artificial intestinal fluid to fully dissolve, filtering with a 0.45 μm filter membrane, taking the subsequent filtrate as a sample solution, injecting sample, and measuring coenzyme Q containing no free drug in the clathrate self-assembly liposome precursor10Content (c); taking coenzyme Q10Adding a small amount of water into a proper amount of the clathrate self-assembly liposome precursor sample for dissolving, heating and ultrasonically treating with ethanol, cooling to a constant volume to a specified concentration, filtering, taking a subsequent filtrate as a test solution, injecting a sample, and measuring coenzyme Q in the clathrate self-assembly liposome precursor10And (4) calculating the encapsulation efficiency according to the total content.
Coenzyme Q of the invention10And (3) measuring the particle size and the Zeta potential of the clathrate self-assembly liposome precursor after being coated with water. Taking coenzyme Q10The self-assembly inclusion compound liposome precursor is dispersed by a proper amount of cholic acid-containing artificial intestinal juice, the test temperature is 25 ℃, the test times are 3 times, and the particle size and the Zeta potential are measured by adopting a dynamic laser light scattering particle size tester.
Coenzyme Q of the invention10And (3) carrying out transmission electron microscope determination on the clathrate self-assembly liposome precursor after being coated with water. Taking coenzyme Q10Adding appropriate amount of cholic acid-containing artificial intestinal juice into appropriate amount of clathrate self-assembly liposome precursor, dispersing, spreading appropriate amount of dispersion on copper mesh, dripping 2% phosphotungstic acid aqueous solution to negatively dye 15min, after natural drying, taking out the copper mesh, observing the surface morphology and structure of the sample under a transmission electron microscope, and obtaining coenzyme Q10The self-assembly liposome precursor of the inclusion compound is a round or round-like solid sphere, has uniform size and good dispersion, and basically has no adhesion.
Thermal analysis (DSC) determination: taking coenzyme Q10Lecithin, cyclodextrin, mannitol, and physical mixtures and inclusion compounds thereof, and the self-assembled liposome precursor lyophilizate was coated with 5mg each, tabletted, and subjected to thermal analysis (DSC) measurement, and the results are shown in fig. 1, fig. 2, fig. 3, fig. 4, fig. 5, fig. 6, fig. 7, fig. 8, fig. 9, fig. 10, fig. 11, and fig. 12.
Drawings
FIG. 1 is Q10DSC spectrogram of raw materials.
FIG. 2 is a DSC spectrum of lecithin.
FIG. 3 is a DSC spectrum of beta-cyclodextrin.
FIG. 4 is a gamma-cyclodextrin DSC spectrum.
FIG. 5 is a DSC of hydroxypropyl-beta-cyclodextrin.
FIG. 6 is a mannitol DSC profile.
FIG. 7 shows coenzyme Q10DSC spectrogram of physical mixture of lecithin, beta-cyclodextrin and mannitol.
FIG. 8 shows coenzyme Q10DSC spectrogram of physical mixture of lecithin, hydroxypropyl-beta-cyclodextrin and mannitol.
FIG. 9 shows coenzyme Q10DSC of physical mixture of lecithin, gamma-cyclodextrin and mannitol.
FIG. 10 shows coenzyme Q of example 510DSC spectrum of the precursor of the self-assembly liposome of the beta-cyclodextrin inclusion compound.
FIG. 11 shows coenzyme Q in example 1010DSC spectrogram of self-assembled liposome precursor of hydroxypropyl-beta-cyclodextrin inclusion compound.
FIG. 12 is coenzyme Q of example 1510DSC spectrum of self-assembly liposome precursor of gamma-cyclodextrin inclusion compound.
Fig. 13 is a particle size diagram of a self-assembly liposome precursor dispersion of inclusion compound.
FIG. 14 is Zeta potential diagram of the dispersion of clathrate self-assembly liposome precursor.
FIG. 15 is a transmission electron microscope image of a dispersion liquid of clathrate self-assembly liposome precursor.
Figure 16 is a dissolution curve for inclusion compound self-assembled liposome precursors, inclusion compounds, simple liposomes, physical mixtures.
FIG. 17 shows coenzyme Q of clathrate self-assembly liposome precursor, clathrate, simple liposome, and prototype drug10Plasma concentration-time profile.
Detailed Description
The present invention will be further described with reference to the following examples. The following examples are only a few specific examples of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by the design concept should fall within the scope of infringing on the protection scope of the present invention.
The methods in the following examples are conventional methods unless otherwise specified.
The percentages in the following examples are by mass unless otherwise specified.
Example 1
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including oxidized coenzyme Q10: beta-cyclodextrin: soybean lecithin: mannitol 1: 5: 0.5: 5.
taking 50 parts by mass of beta-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 250 parts by mass of water, mixing and dispersing uniformly, stirring into paste, heating to 50 ℃, putting into a colloid mill, circularly grinding for 5 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 5 parts by mass of soybean lecithin into 250 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; adding 50 weight parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on mannitol microparticle surface, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 100 ℃ and the air outlet temperature of the fluidized bed is 40 ℃, and the oxidized coenzyme Q is obtained10Clathrate self-assembly liposome precursor。
Example 2
Reduced coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including reduced coenzyme Q in certain weight proportion10: beta-cyclodextrin: egg yolk lecithin: mannitol 1: 7: 0.8: 8.
taking 70 parts by mass of beta-cyclodextrin and 10 parts by mass of reduced coenzyme Q10Adding 560 parts by mass of water, mixing, dispersing, stirring to paste, heating to 55 ℃, putting into a colloid mill, circularly grinding for 7 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 8 parts by mass of egg yolk lecithin into 560 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; taking 80 parts by mass of mannitol, placing into a fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat the surface of mannitol particles with phospholipid membrane, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the fluidized bed, wherein the air inlet temperature of the fluidized bed is 110 ℃, and the air outlet temperature of the fluidized bed is 45 ℃ to obtain the reduced coenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 3
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including oxidized coenzyme Q10: beta-cyclodextrin: soybean lecithin: mannitol 1: 10: 1.5: 10.
taking 100 parts by mass of beta-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 700 parts by mass of water, mixing and dispersing uniformly, stirring into paste, heating to 60 ℃, putting into a colloid mill, circularly grinding for 10 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; taking 15 parts by mass of soybean lecithin, adding 700 parts by mass of water, mixing and dispersing uniformly to obtain a phospholipid dispersion liquid for later use; adding 100 mass parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on mannitol microparticle surface, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 105 ℃ and the air outlet temperature of the fluidized bed is 43 ℃, and the oxidation is obtainedCoenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 4
Reduced coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including reduced coenzyme Q in certain weight proportion10: beta-cyclodextrin: egg yolk lecithin: mannitol 1: 12: 1.2: 12.
taking 120 parts by mass of beta-cyclodextrin and 10 parts by mass of reduced coenzyme Q10Adding 960 parts by mass of water, mixing, dispersing, stirring to obtain paste, heating to 55 ℃, placing into a colloid mill, circularly grinding for 9 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; taking 12 parts by mass of egg yolk lecithin, adding 960 parts by mass of water, mixing and dispersing uniformly to obtain a phospholipid dispersion liquid for later use; taking 120 parts by mass of mannitol, putting into a fluidized bed as a mother nucleus, spraying the phospholipid dispersion liquid to coat a phospholipid membrane on the surface of mannitol particles, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the fluidized bed, wherein the air inlet temperature of the fluidized bed is 98 ℃, and the air outlet temperature of the fluidized bed is 38 ℃, thus obtaining the reduced coenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 5
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including oxidized coenzyme Q10: beta-cyclodextrin: soybean lecithin: mannitol 1: 15: 1.5: 15.
taking 150 parts by mass of beta-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 750 parts by mass of water, mixing, dispersing uniformly, stirring to form paste, heating to 52 ℃, putting into a colloid mill, circularly grinding for 10 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; taking 15 parts by mass of soybean lecithin, adding 750 parts by mass of water, mixing and dispersing uniformly to obtain a phospholipid dispersion liquid for later use; adding 150 mass parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on surface of mannitol microparticle, and spraying the cyclodextrin coenzyme Q10Grinding the slurry, drying the slurry by fluidization,the air inlet temperature of the fluidized bed is 105 ℃, and the air outlet temperature is 43 ℃, thus obtaining the oxidized coenzyme Q10The inclusion compound self-assembled liposome precursor.
Taking 5mg of the liposome precursor, carrying out thermal analysis DSC measurement, and the result shows that the liposome precursor is different from coenzyme Q10Forms a new phase with the DSC pattern of lecithin, beta-cyclodextrin and the mixture thereof.
Example 6
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including oxidized coenzyme Q10: hydroxypropyl β -cyclodextrin: soybean lecithin: mannitol 1: 5: 1.5: 5.
taking 50 parts by mass of hydroxypropyl beta-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 250 parts by mass of water, mixing and dispersing uniformly, stirring into paste, heating to 50 ℃, putting into a colloid mill, circularly grinding for 5 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 15 parts by mass of soybean lecithin into 250 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; adding 50 weight parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on mannitol microparticle surface, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 100 ℃ and the air outlet temperature of the fluidized bed is 40 ℃, and the oxidized coenzyme Q is obtained10The inclusion compound self-assembled liposome precursor.
Example 7
Reduced coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including reduced coenzyme Q in certain weight proportion10: hydroxypropyl β -cyclodextrin: egg yolk lecithin: mannitol 1: 7: 0.8: 8.
taking 70 parts by mass of hydroxypropyl beta-cyclodextrin and 10 parts by mass of reduced coenzyme Q10Adding 560 parts by mass of water, mixing, dispersing, stirring to paste, heating to 55 ℃, putting into a colloid mill, circularly grinding for 7 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid preparationUsing; adding 8 parts by mass of egg yolk lecithin into 560 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; taking 80 parts by mass of mannitol, placing into a fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat the surface of mannitol particles with phospholipid membrane, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the fluidized bed, wherein the air inlet temperature of the fluidized bed is 97 ℃, and the air outlet temperature of the fluidized bed is 42 ℃ to obtain the reduced coenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 8
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including oxidized coenzyme Q10: hydroxypropyl β -cyclodextrin: soybean lecithin: mannitol 1: 10: 0.8: 10.
taking 100 parts by mass of hydroxypropyl beta-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 600 parts by mass of water, mixing, dispersing, stirring to obtain paste, heating to 50 ℃, placing into a colloid mill, circularly grinding for 8 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 8 parts by mass of soybean lecithin into 600 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; adding 100 mass parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on mannitol microparticle surface, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 101 ℃ and the air outlet temperature of the fluidized bed is 41 ℃, and the oxidized coenzyme Q is obtained10The inclusion compound self-assembled liposome precursor.
Example 9
Reduced coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including reduced coenzyme Q in certain weight proportion10: hydroxypropyl β -cyclodextrin: egg yolk lecithin: mannitol 1: 12: 1.2: 12.
taking 120 parts by mass of hydroxypropyl beta-cyclodextrin and 10 parts by mass of reduced coenzyme Q10Adding 840 parts of water by mass, mixing and dispersing uniformly, stirring into paste, heating to 54 ℃, putting into a colloid mill, and circularly grinding 6Secondly, adding water to dilute until the concentration of solid matters is 10 percent to obtain the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 12 parts by mass of egg yolk lecithin into 840 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; taking 120 parts by mass of mannitol, putting into a fluidized bed as a mother nucleus, spraying the phospholipid dispersion liquid to coat a phospholipid membrane on the surface of mannitol particles, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the fluidized bed, wherein the air inlet temperature of the fluidized bed is 99 ℃, and the air outlet temperature of the fluidized bed is 36 ℃, thus obtaining the reduced coenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 10
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including oxidized coenzyme Q10: hydroxypropyl β -cyclodextrin: soybean lecithin: mannitol 1: 15: 1.5: 15.
taking 150 parts by mass of hydroxypropyl beta-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 800 parts by mass of water, mixing, dispersing uniformly, stirring to form paste, heating to 50 ℃, putting into a colloid mill, circularly grinding for 10 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; taking 15 parts by mass of soybean lecithin, adding 800 parts by mass of water, mixing and dispersing uniformly to obtain a phospholipid dispersion liquid for later use; adding 150 mass parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on surface of mannitol microparticle, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 100 ℃ and the air outlet temperature of the fluidized bed is 44 ℃, and the oxidized coenzyme Q is obtained10The inclusion compound self-assembled liposome precursor.
Taking 5mg of the liposome precursor, carrying out thermal analysis DSC measurement, and the result shows that the liposome precursor is different from coenzyme Q10Forms a new phase with the DSC pattern of lecithin, hydroxypropyl beta-cyclodextrin and the mixture thereof.
Example 11
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of the inclusion compound is prepared by the following raw materials according to the weight ratio,oxidized coenzyme Q10: gamma-cyclodextrin: soybean lecithin: mannitol 1: 5: 0.5: 15.
taking 50 parts by mass of gamma-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 350 parts by mass of water, mixing, dispersing uniformly, stirring to form paste, heating to 54 ℃, putting into a colloid mill, circularly grinding for 5 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 5 parts by mass of soybean lecithin into 350 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; adding 150 mass parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on surface of mannitol microparticle, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 96 ℃, and the air outlet temperature of the fluidized bed is 40 ℃, thus obtaining the oxidized coenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 12
Reduced coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including reduced coenzyme Q in certain weight proportion10: gamma-cyclodextrin: egg yolk lecithin: mannitol 1: 10: 0.8: 8.
100 parts by mass of gamma-cyclodextrin and 10 parts by mass of reduced coenzyme Q are taken10Adding 650 parts by mass of water, mixing and dispersing uniformly, stirring into paste, heating to 51 ℃, putting into a colloid mill, circularly grinding for 7 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 8 parts by mass of egg yolk lecithin into 650 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; taking 80 parts by mass of mannitol, placing into a fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat the surface of mannitol particles with phospholipid membrane, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the fluidized bed, wherein the inlet air temperature of the fluidized bed is 102 ℃, and the outlet air temperature of the fluidized bed is 37 ℃ to obtain the reduced coenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 13
The oxidized coenzyme Q of the invention10Inclusion compound self-assembled lipidsA precursor prepared from oxidized coenzyme Q10: gamma-cyclodextrin: soybean lecithin: mannitol 1: 10: 1.5: 10.
taking 100 parts by mass of gamma-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 700 parts by mass of water, mixing and dispersing uniformly, stirring into paste, heating to 60 ℃, putting into a colloid mill, circularly grinding for 8 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; taking 15 parts by mass of soybean lecithin, adding 700 parts by mass of water, mixing and dispersing uniformly to obtain a phospholipid dispersion liquid for later use; adding 100 mass parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on mannitol microparticle surface, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 97 ℃ and the air outlet temperature of the fluidized bed is 37 ℃, and the oxidized coenzyme Q is obtained10The inclusion compound self-assembled liposome precursor.
Example 14
Reduced coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including reduced coenzyme Q in certain weight proportion10: gamma-cyclodextrin: egg yolk lecithin: mannitol 1: 12: 0.9: 7.
taking 120 parts by mass of gamma-cyclodextrin and 10 parts by mass of reduced coenzyme Q10Adding 900 parts by mass of water, mixing, dispersing, stirring to obtain paste, heating to 55 ℃, putting into a colloid mill, circularly grinding for 9 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; adding 9 parts by mass of egg yolk lecithin into 900 parts by mass of water, and uniformly mixing and dispersing to obtain a phospholipid dispersion liquid for later use; taking 70 parts by mass of mannitol, putting into a fluidized bed as a mother nucleus, spraying the phospholipid dispersion liquid to coat a phospholipid membrane on the surface of mannitol particles, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the fluidized bed, wherein the air inlet temperature of the fluidized bed is 103 ℃, and the air outlet temperature of the fluidized bed is 42 ℃, thus obtaining the reduced coenzyme Q10The inclusion compound self-assembled liposome precursor.
Example 15
The oxidized coenzyme Q of the invention10The self-assembled liposome precursor of inclusion compound is prepared with the material including oxidized coenzyme Q10: gamma-cyclodextrin: soybean lecithin: mannitol 1: 15: 1.5: 15.
taking 150 parts by mass of gamma-cyclodextrin and 10 parts by mass of oxidized coenzyme Q10Adding 750 parts by mass of water, mixing, dispersing uniformly, stirring to form paste, heating to 50 ℃, putting into a colloid mill, circularly grinding for 10 times, adding water to dilute until the solid concentration is 10%, and obtaining the cyclodextrin coenzyme Q10Grinding fluid is reserved; taking 15 parts by mass of soybean lecithin, adding 750 parts by mass of water, mixing and dispersing uniformly to obtain a phospholipid dispersion liquid for later use; adding 150 mass parts of mannitol into fluidized bed as mother nucleus, spraying the phospholipid dispersion liquid to coat phospholipid membrane on surface of mannitol microparticle, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 100 ℃ and the air outlet temperature of the fluidized bed is 40 ℃, and the oxidized coenzyme Q is obtained10The inclusion compound self-assembled liposome precursor.
Taking 5mg of the liposome precursor, carrying out thermal analysis DSC measurement, and the result shows that the liposome precursor is different from coenzyme Q10Forms a new phase with the DSC pattern of lecithin, gamma-cyclodextrin and the mixture thereof.
Test example 1 measurement of coenzyme Q in clathrate by high performance liquid chromatography10
Taking the coenzyme Q prepared in the corresponding embodiment of the invention10Adding a small amount of water into a proper amount of the self-assembly liposome precursor of the inclusion compound for dissolving, heating and ultrasonically treating by using ethanol, cooling and fixing the volume to a specified concentration, filtering, taking a subsequent filtrate as a test solution, injecting a sample, and performing chromatographic conditions: coenzyme Q10: and (3) a C18 column, wherein the mobile phase is acetonitrile, tetrahydrofuran and water (volume ratio) is 55: 40:5, the detection wavelength is 280nm, and the quantification is carried out by an external standard method.
Test example 2 measurement of encapsulation efficiency of clathrate self-assembled liposome precursor
Dispersing appropriate amount of clathrate self-assembly liposome precursor sample in appropriate amount of cholic acid-containing artificial intestinal fluid, dissolving thoroughly, filtering with 0.45 μm filter membrane, collecting filtrate as sample solution, injecting, and measuringCoenzyme Q without free drug in clathrate self-assembly liposome precursor10Content (c); taking another appropriate amount of self-assembled liposome precursor sample of clathrate, adding a small amount of water for dissolving, heating with ethanol for ultrasonic treatment, cooling to constant volume to specified concentration, filtering, taking the subsequent filtrate as sample solution, injecting sample, and measuring coenzyme Q in the self-assembled liposome precursor of clathrate10The total content, calculated encapsulation efficiency, results are shown in table 1.
Test example 3 measurement of particle diameter and Zeta potential of clathrate self-assembled liposome precursor after coating with water
Taking coenzyme Q10The self-assembly inclusion compound liposome precursor is dispersed by using a proper amount of cholic acid-containing artificial intestinal juice, the test temperature is 25 ℃, the test times are 3 times, and the particle size and the Zeta potential are measured by using a dynamic laser light scattering particle size tester, and the results are shown in Table 1.
TABLE 1 coenzyme Q10Encapsulation efficiency, particle size and potential of
Test example 4 solubility measurement
The coenzyme Q prepared in the embodiments 1 to 15 of the invention are respectively taken10Clathrate self-assembly liposome precursor, clathrate, simple liposome and coenzyme Q10Adding appropriate amount of cyclodextrin lecithin into 100ml of pure water respectively to obtain supersaturated solution, stirring in water bath at 37 deg.C for 24 hr, and filtering. Taking 1ml of filtrate, diluting to 10ml with anhydrous ethanol, performing ultrasound at 50 deg.C for 20min, cooling to room temperature, filtering with 0.45um filter membrane, and measuring coenzyme Q by high performance liquid chromatography10The results are shown in Table 1. The results show that the coenzyme Q in the clathrate self-assembly liposome precursor prepared in the embodiments 1-15 of the invention10The solubility is 174-; coenzyme Q10Coenzyme Q in clathrate10The solubility of (a) is 21.3. mu.g/ml; coenzyme Q10Coenzyme Q in simple liposomes10Has a solubility of 49.6. mu.g/ml; auxiliary deviceEnzyme Q10Coenzyme Q in physical mixture of cyclodextrin lecithin10The starting material was not detected.
Test example 5 dissolution measurement
The coenzyme Q prepared in the examples 1 to 15 of the invention were weighed separately10Clathrate self-assembly liposome precursor, clathrate, simple liposome and coenzyme Q10Proper amount of physical mixture of cyclodextrin lecithin is dissolved out by a dissolution instrument at different time. 900ml of 0.165 percent sodium taurocholate solution as a medium, 75 r/min of rotation speed, 5ml of timing sampling at 37 +/-0.5 ℃, filtering, and measuring coenzyme Q by adopting a high performance liquid phase method10The contents and results are shown in Table 2. Table 2 shows that the compositions of the invention dissolve more quickly and more.
TABLE 2 coenzyme Q10Solubility and relative dissolution of
Test example 6 measurement of stability of clathrate self-assembled liposome precursor
The coenzyme Q of the invention10Stability test of clathrate self-assembled liposome precursor:
taking the clathrate compound self-assembly liposome precursor, the clathrate compound, the simple liposome and the coenzyme Q prepared by the embodiment of the invention10Proper amount of prototype medicine for influence factor test.
Standing the above materials under 4000LX light at 40 deg.C and relative humidity of 92.5% for 10 days, sampling for measuring coenzyme Q in 0 day, 5 days, and 10 days respectively10The contents and results are shown in Table 3. Table 3 shows that the photostability of the self-assembled liposome precursor of the clathrate compound is superior to that of the clathrate compound, simple liposome and prototype drug.
TABLE 3 Effect test
Example 7 DPPH scavenging free radical method oxidation resistance test:
the test method comprises the following steps: respectively taking a sample to be detected and a blank sample (without coenzyme Q)10Cyclodextrin and phospholipid) was added to 2.85ml of ultrapure water 150. mu.l, i.e., diluted 20 times;
the sample was coenzyme Q prepared according to the examples10Clathrate self-assembly liposome precursor and coenzyme Q10Clathrate compound, coenzyme Q10Simple liposome, coenzyme Q10Prototype drug.
Preparation of DPPH (2, 2-Diphenyl-1-propylhydrzyl) (free radial)) solution: accurately weighing 8mgDPPH powder, adding absolute ethanol to a volumetric flask with a constant volume of 100ml to obtain 2 x 10-4mol/l DPPH solution, and then diluted to 5X 10-5mol/l for standby;
the test tubes are numbered, the samples are added according to the table 4, the mixture is uniformly mixed, the reaction is carried out for 60min at the temperature of 37 ℃ in water bath, and then the absorbance value of the reaction liquid of the sample to be tested and the absorbance value of the blank sample are measured at the wavelength of 517 nm. (zero reference 1ml blank sample mixed with 2ml ultrapure water)
TABLE 4 required amount of each ingredient for DPPH radical scavenging experiment
| Group of | Sample/ml to be tested | Blank sample/ml | DPPH/ml | Ultra pure water/ml |
| ADPPH as sample to be measured | 2.0 | 0 | 4.0 | 0 |
| AWater as sample to be tested | 2.0 | 0 | 0 | 4.0 |
| ABlank DPPH | 0 | 2.0 | 4.0 | 0 |
The inhibition rate was calculated using the following formula:
inhibition rate E (%) - (1- (a)DPPH as sample to be measured-AWater as sample to be tested)/ABlank DPPH)×100％
To coenzyme Q10Clathrate self-assembly liposome precursor and coenzyme Q10Clathrate compound, coenzyme Q10The results of comparing the DPPH radical scavenging ability of the simple liposome and coenzyme Q10 proto-drug are shown in Table 5:
TABLE 5 experimental results of DPPH radical scavenging method
The result shows that the capability of the self-assembled liposome precursor of the coenzyme Q10 clathrate compound of the invention for scavenging free radicals is obviously superior to that of the coenzyme Q10 clathrate compound10Inclusion compound, simple liposome and prototype drug.
Example 8 bioavailability
1. Dosing regimen and rat blood sample collection
The rats are 24 in total, half of the rats are female and half of the rats, and the rats are divided into two groups, 6 rats/group after being adaptively raised for 3 days.
According to coenzyme Q10The daily dose of the drug is 100mg/60kg, the dose of the drug for rats is 10mg/kg, and the preparation concentration is obtained according to the gavage amount. The self-assembly liposome precursor and coenzyme Q of the inclusion compound of example 2 are precisely weighed10Inclusion compound (with coenzyme Q of example 2)10Same content), coenzyme Q10Simple liposome sample (with example 2 coenzyme Q)10The contents are the same), adding water, and stirring in water bath at 37 ℃ to prepare suspension; coenzyme Q is weighed accurately10Prototype drug, adding into 0.5% CMC-Na solution to make into suspension for administration. Adopts one-time oral administration for intragastric administration. Animals were fasted for 12 hours before the test, during which time water was freely available.
After administration, at 0, 2, 4, 6, 7, 8, 9, 10, 12, 24 hours respectively, orbital vein blood sampling is carried out, 0.5ml of blood sample is collected and placed in a heparin sodium anticoagulation tube prepared in advance, centrifugation is carried out for 10min at 4 ℃ and 5000rmp, plasma is separated, freezing and storing are carried out at-20 ℃ for standby, and unfreezing is carried out at room temperature for 2 hours before measurement.
2. Blood sample processing method
100ul of rat plasma is taken and placed in a centrifuge tube, 1ml of extraction solvent (n-hexane: absolute ethyl alcohol: 8:1) is added, vortex extraction is carried out, after centrifugation, supernatant is taken and placed in another centrifuge tube, and vacuum drying is carried out. And adding 100ul of absolute ethyl alcohol into the residue for redissolving, uniformly mixing by vortex, centrifuging, and taking the supernatant for analysis.
3. Conditions of liquid chromatography
A chromatographic column: a C18 column; mobile phase: tetrahydrofuran: acetonitrile: water (40:55: 5); sample introduction volume: 10 ul; flow rate: 1 ml/min; detection wavelength: 280 nm.
4. Standard curve
Coenzyme Q with different concentrations is precisely absorbed10Adding blank plasma 100ul to standard solution 50ul to obtain standard coenzyme Q10The simulated plasma samples with the mass of 0, 1, 2, 4, 8, 16, 32, 64ug/ml were processed in the same way as the extraction solvent under the "blood sample processing method".
5. Recovery rate and precision of the method:
taking 100 mu L of blank plasma, and respectively preparing coenzyme Q with three concentrations of 2, 8 and 32 mu g/mL10Samples and blank samples, 6 samples per concentration were analyzed. Taking coenzyme Q of each concentration10The recovery rate was calculated by the same method as that used in the "blood sample treatment method" from the addition of the extraction solvent, using 50. mu.L of the standard solution. Coenzyme Q10The recovery rates at the low, medium and high concentrations were 90.8%, 92.7% and 88.1%, respectively. Day and day RSD values were calculated by 6 replicates a day and three replicates a three day. The results show that coenzyme Q10Day RSD of<14.6%, daytime RSD<16.4％。
6. Specificity of the method
Respectively sampling blank rat plasma sample, blank rat plasma and reference sample, administered rat plasma sample and reference solution processed under the item of 'blood sample processing method', recording chromatogram, and the result shows that coenzyme Q10It separated well from impurities in plasma and endogenous substances did not interfere with the assay.
7. Blood concentration and time profile
Rats were dosed orally with 10mg/kg of the clathrate self-assembled liposome precursor, clathrate, simple liposome, prototype drug of example 2, and plasma concentration-time curves were generated and fitted with DAS2.0 software and pharmacokinetic parameters were calculated in table 6. AUC according to0-tThe bioavailability of the inclusion compound self-assembly liposome precursor, the inclusion compound and the simple liposome is calculated to be respectively 4.8, 2.7 and 2.9 times of that of the prototype drug. The bioavailability test data show that when SD rats are administrated for 12 hours, the AUC of the clathrate self-assembly liposome precursor0-12141.5 mg. h/L, AUC of clathrate0-1277.05 mg. multidot.h/L, simple lipidAUC of body0-1286.43 mg. h/L, AUC of proto-drug0-12The bioavailability of the clathrate compound self-assembly liposome precursor, the clathrate compound and the simple liposome in 12 hours is equal to 6.8, 3.7 and 4.2 times of that of the prototype drug.
TABLE 6 coenzyme Q after administration to SD rats10Major pharmacokinetic parameters of
Claims (4)
1. Coenzyme Q10The self-assembled liposome precursor of the inclusion compound is characterized by being prepared from the following raw materials in percentage by weight: coenzyme Q10: cyclodextrin: lecithin: mannitol = 1: 5-15: 0.5-1.5: 5-15; the process comprises the following steps: taking cyclodextrin and coenzyme Q according to the weight ratio10Adding water in 5-8 times of cyclodextrin, mixing, stirring to obtain paste, heating to 50-60 deg.C, grinding in colloid mill for 5-10 times, adding water, diluting to solid concentration of 10% to obtain cyclodextrin coenzyme Q10Grinding fluid is reserved; taking phospholipid according to the weight ratio, adding water which is 5-8 times of the weight of the cyclodextrin, mixing and dispersing uniformly to obtain phospholipid dispersion liquid for later use; taking mannitol according to the weight ratio, putting the mannitol into a fluidized bed as a mother nucleus, spraying the phospholipid dispersion liquid to coat a phospholipid membrane on the surface of mannitol particles, and spraying the cyclodextrin coenzyme Q10Grinding the slurry to dry the slurry, wherein the air inlet temperature of the fluidized bed is 90-110 ℃, and the air outlet temperature of the fluidized bed is 35-45 ℃ to obtain the coenzyme Q10The inclusion compound self-assembled liposome precursor.
2. The coenzyme Q according to claim 110The self-assembled liposome precursor of the inclusion compound is characterized by being prepared from the following raw materials in percentage by weight: coenzyme Q10: cyclodextrin: lecithin: mannitol = 1: 7-10: 0.8-1.5:5-10。
3. the coenzyme Q according to claim 1 or 210The inclusion compound self-assembly liposome precursor is characterized in that the cyclodextrin is gamma-cyclodextrin, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin.
4. The coenzyme Q according to claim 1 or 210The self-assembled liposome precursor of the inclusion compound is characterized in that the lecithin is soybean lecithin or egg yolk lecithin.
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