CN116077441A - Sichuan citrin solid dispersion and application thereof in preparation of drugs for preventing and treating liver injury - Google Patents

Abstract

The invention belongs to the technical field of natural products, and particularly relates to a nobiletin solid dispersion, a preparation method thereof and application thereof in preparing a medicine for preventing and treating liver injury. The invention relates to a nobiletin solid dispersion, which comprises the following components in percentage by mass: 70-80% of Soluplus, 10-15% of PVP/VA, and 10-15% of nobiletin. The invention also provides a preparation method of the dispersion. The nobiletin solid dispersion is compounded by nobiletin and a carrier material Soluplus, PVP/VA64, and has the advantages of accurate content, small particle size, stable property of functional substances, difficult oxidation, high bioavailability and the like. The nobiletin solid dispersion of the invention has obvious liver protection effect, can prevent and/or reduce and/or reverse liver injury, and can be applied to preparation of drugs/health care products for treating or preventing or reversing or reducing liver injury.

Description

Sichuan citrin solid dispersion and application thereof in preparation of drugs for preventing and treating liver injury

Technical Field

The invention belongs to the technical field of natural products, and particularly relates to a nobiletin solid dispersion, a preparation method thereof and application thereof in preparing a medicine for preventing and treating liver injury.

Background

Nobiletin (NOB) is a low-polarity polymethoxy flavonoid natural product with a planar structure, has higher content in traditional Chinese medicines such as citrus peel and pummelo peel of Rutaceae, and has better oxidation resistance. Therefore, the development of the nobiletin as a liver protection drug for preventing and treating liver injury is presumed to have strong feasibility. However, since NOB is a nonpolar molecule, there are problems of small water solubility, poor oral absorption, low bioavailability (only about 4%), and the like, so that the clinical application of NOB is greatly limited. In order to improve the antioxidant property of nobiletin, the solubility of nobiletin needs to be improved by adopting a proper preparation technology so as to better exert the effect of preventing and treating liver injury of NOB.

Amorphous solid dispersion (Amorphous solid dispersion, ASD) technology is a novel technique for highly dispersing a drug in a solid carrier, and is an effective method for improving the solubility of poorly soluble drugs. The poorly soluble drug is prepared as a solid dispersion, and the drug can be highly dispersed in the carrier in a molecular, colloidal, microcrystalline or amorphous state. The high dispersibility of the medicine and the high solubility of the carrier can effectively improve the solubility and the dissolution rate of the insoluble medicine, thereby improving the bioavailability of the insoluble medicine and enhancing the pharmacological activity of the insoluble medicine.

Hot melt extrusion technology (Hot melt extrusion, HME) is a formulation technology that sustainably prepares solid dispersions. The solid dispersion is prepared by adopting the HME technology, the medicine and the carrier are uniformly mixed in a molten state, and the mixture is extruded and molded by utilizing a rotary screw rod, cooled and granulated. Compared with the preparation technology of other solid dispersions, the HME technology integrates multiple unit operations such as solid conveying, material melting, shearing mixing, melt conveying, extrusion molding and the like, and has the remarkable advantages of no organic solvent residue, sustainable industrial production and the like.

The liver is the main organ of metabolism of human body, and many factors such as drugs, poison, ethanol and the like can induce liver injury. Severe or sustained liver injury will lead to various chronic liver diseases, up to liver failure. Liver injury can be divided into two major categories, chemical liver injury and immune liver injury, wherein chemical liver injury can be caused by a variety of chemical liver toxic substances, including carbon tetrachloride, acetaminophen, acetaldehyde, and the like. Among them, acetaminophen is a classical hepatophilic poison, which can cause acute liver injury. In recent years, the incidence rate of acute liver injury is increased year by year, and various liver diseases seriously threaten the health of human beings, so that the research on natural products or medicines for preventing and treating the liver injury has very important significance.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the primary purpose of the invention is to provide a nobiletin solid dispersion.

The invention also aims at providing a preparation method of the nobiletin solid dispersion.

The invention further aims at providing application of the nobiletin solid dispersion in preparing medicines for preventing and treating liver injury.

The aim of the invention is achieved by the following scheme:

a nobiletin solid dispersion (NOB/SD) comprises the following components in percentage by mass: 70-80% of Soluplus, 10-15% of PVP/VA, and 10-15% of nobiletin.

In one embodiment of the present invention, the nobiletin is citrus-derived nobiletin such as dried orange peel.

As an aspect of the invention, the PVP/VA64 and Soluplus are in a mass ratio of 1:8-3:14. The invention adopts the combination of the Soluplus, PVP/VA64 and the nobiletin to obtain the nobiletin solid dispersion, which has high solubility, large drug-loading capacity and good stability; adding the nobiletin solid dispersion into water, wherein the particle size of the nobiletin solid dispersion is less than or equal to 200nm; the equilibrium solubility of the preparation is 5.8 times of that of the free NOB (Crystalline NOB), and the accumulated dissolution rate is more than 70%, so that the solubility, bioavailability and bioactivity of the nobiletin are effectively improved, and the technical problems of poor water solubility, low bioavailability and the like of the nobiletin in the prior art are effectively solved.

The invention also provides a preparation method of the nobiletin solid dispersion, which is a hot-melt extrusion method, and specifically comprises the steps of uniformly mixing all the components in proportion and extruding the mixture by a hot-melt extruder.

As an aspect of the present invention, the extrusion temperature is 170 ℃ or less.

As one scheme of the invention, the feeding speed of the hot melt extruder is less than or equal to 50mg/min.

As an aspect of the present invention, the screw speed of the hot melt extruder may be 40-80rpm.

As an aspect of the present invention, the indoor humidity during the preparation is preferably 40-55%.

In one embodiment of the present invention, the solid nobiletin dispersion obtained by extrusion is in the form of a wire, and a powdery product can be obtained by a method such as pulverization.

As an aspect of the present invention, the pulverized material may be further processed by sieving.

The nobiletin solid dispersion is prepared by compounding nobiletin and a carrier material Soluplus and PVP/VA64 through a hot melt extrusion method, and has the advantages of accurate content, small particle size, stable property of functional substances, difficult oxidation, high bioavailability and the like.

The invention also provides a preparation method of the nobiletin solid dispersion, which is a solvent volatilization method, and specifically comprises the steps of respectively dissolving all the components in a solvent according to a proportion, uniformly mixing, and removing the solvent to obtain a product.

As an aspect of the present invention, the solvent may be absolute ethanol, water, methanol, or the like.

As one scheme of the invention, the dissolution speed can be accelerated by adopting methods such as ultrasonic treatment, stirring and the like.

As one scheme of the invention, the uniform mixing can be realized by stirring, ultrasonic and other methods.

As an aspect of the present invention, the solvent may be removed by heating, spin-drying, vacuum drying, or the like.

As an aspect of the present invention, the solvent-removed product may also be dried in a drying oven to remove the effects of moisture and the like.

The invention also provides application of the nobiletin solid dispersion in preparing a medicine for preventing and treating liver injury.

The invention also provides application of the nobiletin solid dispersion in preparing medicines for treating or preventing or reversing or reducing liver injury.

The invention also provides application of the nobiletin solid dispersion in preparing health care products for treating or preventing or reversing or reducing liver injury.

As one aspect of the present invention, the liver injury includes liver tissue and cell injury.

As one aspect of the present invention, the liver injury is caused by a chemical hepatotoxic substance.

As an aspect of the present invention, the chemical hepatotoxic substance includes alcohol, carbon tetrachloride, acetaminophen, acetaldehyde, and the like.

As one aspect of the present invention, the liver injury includes toxic hepatitis, cirrhosis, liver fibrosis, and the like.

The medicaments are the same or different and respectively comprise a therapeutically effective amount of a solid dispersion of nobiletin.

The medicines which are the same or different can be respectively prepared into various medicinal dosage forms by adopting a conventional method, and the dosage forms comprise: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, medicinal granules, pills, pellets, suspensions, wines, tinctures, drops and other oral administration forms, and injection and other oral administration forms, such as injection and the like.

The medicines are the same or different and respectively can also contain one or more than one pharmaceutically acceptable carrier or excipient.

The excipient may include diluents, wetting agents, lubricants, fillers, preservatives, and the like.

As an aspect of the present invention, the nobiletin solid dispersion includes the following components in percentage by mass: 70-80% of Soluplus, 10-15% of PVP/VA, and 10-15% of nobiletin.

As one scheme of the invention, the nobiletin solid dispersion is applied to damaged liver tissues and cells, and adopts histopathological sections, ALT, AST and the like as detection indexes, so that the protective effect of the nobiletin solid dispersion on liver injury is verified, the obvious liver protective effect is shown, and the liver injury can be prevented and/or reduced and/or reversed.

Compared with the prior art, the invention has the following advantages:

(1) The nobiletin solid dispersion has high solubility, large drug loading, good stability, balanced solubility of 5.8 times of free NOB, and accumulated dissolution rate of more than 70%, effectively improves the solubility, bioavailability and bioactivity of nobiletin, and effectively solves the technical problems of poor water solubility, low bioavailability and the like of nobiletin in the prior art;

(2) The nobiletin solid dispersion has the advantages of simple formula, convenient preparation process, accurate content, small particle size, stable property of functional substances, difficult oxidization, high bioavailability and the like.

(3) The nobiletin solid dispersion of the invention has obvious liver protection effect, can prevent and/or reduce and/or reverse liver injury, and can be applied to the preparation of medicaments/health care products for treating or preventing or reversing or reducing liver injury.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing particle size distribution of nobiletin solid dispersion and free nobiletin.

FIG. 2 is a scanning electron microscope image of a solid dispersion of nobiletin and free nobiletin.

Fig. 3 is a DSC profile, FTIR profile, and XRD profile of a nobiletin solid dispersion.

FIGS. 4 to 6 are dissolution charts of nobiletin solid dispersion.

FIG. 7 is a DSC curve of a solid dispersion of nobiletin after 3 months of storage.

FIG. 8 is a graph showing ALT and AST activity statistics in mouse serum.

FIG. 9 is a graph of liver HE staining sections of mice.

Fig. 10 is a graph of liver damage area of mice.

FIG. 11 is a graph showing the blood concentration versus time for solid dispersions of nobiletin and free nobiletin.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. The materials referred to in the examples below are available commercially unless otherwise specified. The method is conventional unless otherwise specified.

In one embodiment, the nobiletin solid dispersion comprises the following components in percentage by mass: 70-80% of Soluplus, 10-15% of PVP/VA, and 10-15% of nobiletin.

In one embodiment, the nobiletin is nobiletin from wide orange peel.

In one embodiment, the nobiletin: soluplus: the PVP/VA64 mass ratio was 1:5:1.

In another embodiment, the nobiletin: soluplus: the PVP/VA64 mass ratio was 3:15:2.

In another embodiment, the nobiletin: soluplus: the PVP/VA64 mass ratio was 3:14:3.

In yet another embodiment, the nobiletin: soluplus: the PVP/VA64 mass ratio was 2:15:3.

In one embodiment, the PVP/VA64 and Soluplus are present in a mass ratio of 1:8 to 3:14.

The invention adopts the Soluplus, PVP/VA64 and nobiletin in the specific proportion to compound to obtain the nobiletin solid dispersion, which has high solubility, large drug-loading capacity and good stability; adding the nobiletin solid dispersion into water, wherein the particle size of the nobiletin solid dispersion is less than or equal to 200nm; the equilibrium dissolution rate is 5.8 times of Free NOB, the cumulative dissolution rate is more than 70%, the solubility, bioavailability and bioactivity of the nobiletin are effectively improved, and the technical problems of poor water solubility, low bioavailability and the like of the nobiletin in the prior art are effectively solved.

In one embodiment, the nobiletin solid dispersion of the invention is added into water, and the particle size is less than or equal to 200nm.

In another embodiment, the D50 of the nobiletin solid dispersion of the invention is 60-98nm.

In yet another embodiment, the D90 of the nobiletin solid dispersion of the invention is 32-156nm.

In another embodiment, the preparation method of the nobiletin solid dispersion is a hot-melt extrusion method, and specifically comprises the steps of uniformly mixing the components in proportion and extruding the mixture through a hot-melt extruder.

In one embodiment, the extrusion temperature is 170 ℃ or less.

In one embodiment, the feed rate of the hot melt extruder is less than or equal to 50mg/min.

In one embodiment, the screw speed of the hot melt extruder may be 40-80rpm.

In one embodiment, the indoor humidity in the preparation process is preferably less than or equal to 40-55%.

In one embodiment, the solid nobiletin dispersion obtained by extrusion is in a wire shape, and a powdery product can be obtained by a method such as crushing.

In one embodiment, the crushed materials can be further processed by sieving.

The nobiletin solid dispersion is prepared by compounding nobiletin and a carrier material Soluplus and PVP/VA64 through a hot melt extrusion method, and has the advantages of accurate content, small particle size, stable property of functional substances, difficult oxidation, high bioavailability and the like.

In another embodiment, the preparation method of the nobiletin solid dispersion is a solvent volatilization method, and specifically comprises the steps of respectively dissolving all the components in a solvent according to a proportion, uniformly mixing, and removing the solvent to obtain a product.

In one embodiment, the solvent is absolute ethanol, water, methanol, or the like.

In one embodiment, the dissolution is accelerated by ultrasonic or stirring.

In one embodiment, the uniform mixing is performed by stirring or ultrasonic method.

In one embodiment, the removal solvent is removed by heating, spin-steaming, vacuum drying, or the like.

In one embodiment, the solvent-removed product may also be dried in a dry box to remove moisture and the like.

In one embodiment, the application of the nobiletin solid dispersion in preparing the medicine for preventing and treating liver injury is provided.

In another embodiment, the use of a solid dispersion of nobiletin in the manufacture of a medicament for treating or preventing or reversing or reducing liver damage.

In yet another embodiment, the use of a solid dispersion of nobiletin in the preparation of a health care product for treating or preventing or reversing or reducing liver damage.

In one embodiment, the liver injury includes liver tissue and cell injury.

In one embodiment, the liver injury is caused by a chemical hepatotoxic substance.

In one embodiment, the chemical hepatotoxic substances include alcohol, carbon tetrachloride, acetaminophen, acetaldehyde, and the like.

In one embodiment, the liver injury includes toxic hepatitis, cirrhosis, liver fibrosis, and the like.

In one embodiment, the medicaments are the same or different and each comprise a therapeutically effective amount of a solid dispersion of nobiletin.

In one embodiment, the same or different drugs can be prepared into various pharmaceutical dosage forms by conventional methods, and the dosage forms comprise: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, medicinal granules, pills, pellets, suspensions, wines, tinctures, drops and other oral administration forms, and injection and other oral administration forms, such as injection and the like.

In one embodiment, the same or different drugs may also contain one or more pharmaceutically acceptable carriers or excipients.

In one embodiment, the excipient may include diluents, wetting agents, lubricants, fillers, preservatives, and the like.

In one embodiment, the nobiletin solid dispersion comprises the following components in percentage by mass: 70-80% of Soluplus, 10-15% of PVP/VA, and 10-15% of nobiletin.

In one embodiment, the nobiletin solid dispersion is applied to damaged liver tissues and cells, and adopts histopathological sections, ALT, AST and the like as detection indexes, so that the protection effect of the nobiletin solid dispersion on liver injury is verified, the obvious liver protection effect is shown, and the liver injury can be prevented and/or reduced and/or reversed.

Example 1

The nobiletin solid dispersion comprises the following components in percentage by mass: 70-80% of Soluplus, 10-15% of PVP/VA, and 10-15% of nobiletin.

Specifically, the nobiletin: soluplus: PVP/VA64 mass ratio may be 1:5:1;

or, the nobiletin: soluplus: PVP/VA64 mass ratio may be 3:15:2;

or, the nobiletin: soluplus: PVP/VA64 mass ratio may be 3:14:3;

or, the nobiletin: soluplus: PVP/VA64 mass ratio may be 2:16:2;

or, the nobiletin: soluplus: the PVP/VA64 mass ratio may be 2:15:3.

Example 2: preparation of nobiletin solid dispersion

Hot melt extrusion process: mixing the components in proportion, adding into a hot-melt extruder, extruding at a feeding speed of less than or equal to 50mg/min and a screw rotation speed of 40-80rpm at an extrusion temperature of less than or equal to 170 ℃ to obtain linear nobiletin solid dispersion, crushing and sieving with a 40-mesh sieve to obtain powdery nobiletin solid dispersion (NOB/SD) for standby. Wherein, with nobiletin: soluplus: PVP/VA64 with the mass ratio of 1:5:1 is prepared into a product by adopting a hot melt extrusion method, and the product is marked as NOB/SD I; the following formula of nobiletin: soluplus: PVP/VA64 with the mass ratio of 3:15:2 is prepared into a product by adopting a hot melt extrusion method, and the product is marked as NOB/SD II;

solvent evaporation method: weighing nobiletin bulk drugs and carriers according to a proportion, respectively dissolving NOB and carriers in a proper amount of absolute ethyl alcohol, stirring and carrying out ultrasonic treatment for 5min to enable the NOB and the carriers to be fully dissolved, uniformly mixing the NOB and the carriers and carrying out ultrasonic treatment for 10min, continuously stirring and heating the NOB and the carriers in a constant-temperature water bath kettle at 80 ℃ until the absolute ethyl alcohol is completely evaporated, rapidly transferring the NOB and the carriers to a refrigerator at-20 ℃ for cooling and solidifying for 12h, transferring the NOB and the carriers to a drying oven, and drying the NOB and the carriers for 48h to obtain the nobiletin solid dispersion. Wherein, with nobiletin: soluplus: PVP/VA64 mass ratio is 1:5:1, and the product is prepared by adopting a solvent evaporation method and is marked as NOB/SD III.

The solid dispersion prepared by the grinding method is used as a control group, and can be prepared by the following steps: mixing the components at a certain ratio, adding into a ball mill, and grinding (revolution: 30-200rpm, rotation: 60-200 rpm) for 20min to obtain a Mixture (NOB+Soluplus+PVP/VA 64, or mixing NOB). Wherein, with nobiletin: soluplus: PVP/VA64 in a mass ratio of 1:5:1 was prepared to give the product, labeled as mix NOB I.

Example 3

(1) Characterization of physicochemical Properties

The NOB/SD was analyzed using a particle size analyzer, a Scanning Electron Microscope (SEM), a Differential Scanning Calorimeter (DSC), a Fourier transform infrared spectrometer (FTIR), a polycrystal diffractometer (XRD) or the like to evaluate whether the NOB/SD was formed and to determine whether the dispersed state of the NOB/SD promoted the presence of nobiletin in an amorphous state in the matrix. The results are shown in FIGS. 1-3.

FIG. 1 is a graph showing particle size distribution of nobiletin solid dispersion NOB/SD I and free nobiletin. As can be seen from the graph, the solid dispersion of nobiletin of the invention has a particle size of 200nm or less, a D50 of 60-98nm and a D90 of 32-156nm when added into water. The free nobiletin has extremely poor water solubility and the particle size distribution of 1000-10000nm.

FIG. 2 is a scanning electron microscope image of a solid dispersion of nobiletin and free nobiletin. As can be seen, the free nobiletin exists in a Crystalline state (crystal NOB); the nobiletin in the Mixture of NOB+Soluplus+PVP/VA64, the Mixture of MIXURE NOB I, is attached to the surface of the carrier in a crystal form; in the nobiletin solid dispersions NOB/SD I and NOB/SD III, the nobiletin is uniformly dispersed in the matrix in an amorphous form.

FIG. 3 is a DSC curve, FTIR curve and XRD curve of nobiletin solid dispersion NOB/SD I. In the DSC curve (see FIG. 3A), both the free NOB and the simply mixed NOB matrix components have sharp melting peaks at 140 ℃; whereas the melting peak of NOB/SD at 140℃completely disappeared, indicating that the preparation of NOB as a solid dispersion alters its crystalline form in the matrix. FTIR curves (see FIG. 3B) show free NOB and simple mixing of NOB matrix components at 1646cm ^-1 There was a c=o telescopic shock absorption peak at each location, with no significant spike in NOB/SD, indicating that the NOB and matrix formed an amorphous state. XRD curves (see FIG. 3C) showed complete disappearance of the peak of NOB/SD at 5-30, indicating that the compound was transformed into an amorphous complexAnd (3) an object.

The test results of the nobiletin solid dispersion with other proportions are similar to or identical to those described above, and are not described in detail herein.

(2) In vitro release

Dissolution experiments were performed with PBS as the release medium. NOB/SD with the drug content of 54mg and 54mg of free NOB are respectively put into 900mL of PBS release medium with pH of 6.5, and placed on a constant temperature shaking table at 37 ℃. At various time points, 4mL of assay and an equal volume of PBS (n=3) was taken from the release medium. After passing through a 0.45 μm microporous filter membrane, the NOB concentration was measured at 330nm by an ultraviolet-visible spectrophotometer, and the dissolution rate was calculated, and the results are shown in FIGS. 4 and 5. FIG. 4 is a dissolution rate diagram of NOB/SD I. FIG. 5 shows the dissolution profiles of NOB/SD I and NOB/SD II.

As can be seen from the dissolution profile (FIG. 4), the cumulative maximum dissolution of NOB/SD I was 94.35%, the equilibrium solubility was 71.65.+ -. 6.79%, and an increase of 5.8 times over the free NOB. As can be seen from FIG. 4, NOB/SD I rapidly diffused in PBS release medium within 5min with release of 94.35+ -1.39% and release of free NOB of only 1.21+ -0.10%, NOB/SD was released 78 times as much as free NOB; NOB/SD I release behavior was reduced within 2h, with a release of 71.65 + -6.79% and free NOB release gradually increased, eventually 12.28+ -2.5%. NOB/SD I was 5.8 times more soluble than free NOB at 2h overall. These phenomena indicate that the NOB/SD release of the present invention has a significantly rapid release effect compared to free NOB, and the release amount is large and the total amount is high. Evidence of physicochemical characterization and in vitro release studies indicate that NOB/SD of the present invention has significant advantages in terms of oral absorption.

As can be seen from FIG. 5, the highest dissolution rate of NOB/SD II is 87.20+ -10.92%, the dissolution rate at 2h is 72.34 + -3.91%, and is 5.9 times of the maximum release rate of free NOB, and the dissolution behavior of NOB/SD is similar to that of NOB/SD I, which shows that NOB/SD with different proportions has higher release rate than free medicine.

The test results of the nobiletin solid dispersion with other proportions are similar to or identical to those described above, and are not described in detail herein.

The results of the release experiments for NOB/SD I and NOB/SD III are shown in FIG. 6. As can be seen from the graph, the maximum release degree of the nobiletin solid dispersion prepared by the hot-melt extrusion method is 94.35+/-1.39%, and the maximum release degree of the solid dispersion prepared by the solvent evaporation method is 66.24 +/-2.20%, which shows that the dispersion degree of the solid dispersion prepared by the hot-melt extrusion method is higher than that prepared by the solvent evaporation method, and the release effect is better. After the product is stored for 3 months at normal temperature under sealing, the release degree experiment is carried out, and the result is shown in figure 6B, the release degree of NOB/SD I still reaches 69.05 +/-5.71% when the product is released for 2 hours, and NOB: soluplus=1: the release degree of the composition 6 is only 41.61 +/-2.03 percent. As can be seen by combining the DSC curve of FIG. 7, after 3 months of storage at sealed normal temperature, NOB/SD I (FIG. 7A) of the present invention has no obvious endothermic peak of nobiletin at 140 ℃; whereas NOB/SD (NOB: soluplus=1:6) (FIG. 7B) showed an endothermic peak of nobiletin, indicating the presence of nobiletin crystals, and the solid dispersion aged to cause precipitation of a part of nobiletin crystals. The result shows that the solid dispersion can effectively slow down the aging degree of the solid dispersion by adopting the compound dispersion of the solution plus PVP/VA 64.

Example 4: protective effect of nobiletin solid dispersion on acute liver injury in vivo

(1) The experimental object: c57 mice

(2) Experimental grouping: (1) blank control group (normal saline lavage); (2) model group (normal saline lavage); (3) free NOB group (lavage dose: 50 mg/kg); (4) NOB/SD group (gastric lavage dose: NOB amount 50 mg/kg); 3 in each group. NOB/SD group experiments were performed with NOB/SD I.

(3) The experimental steps are as follows: continuous administration is carried out for one week according to groups, and acetaminophen (350 mg/kg) is injected into the abdominal cavity to induce an acute liver injury animal model, and a blank control group is not injected; injecting 4% tribromoethanol into abdominal cavity after 48h for anesthesia, collecting whole blood from eyeball, standing at room temperature for 2h, centrifuging at 3000rpm for 10min, collecting serum, and detecting alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels in rat serum by biochemical analyzer. The results are shown in FIG. 8.

After the mice were sacrificed by cervical pulling, the livers were taken and placed in 4% paraformaldehyde solution for fixation for more than 48h for HE staining sections. The results are shown in FIGS. 9-10.

As can be seen from FIG. 8, ALT and AST activities of Free NOB mice were 819.22 + -39.2 and 535.5+ -60.42U/L, respectively; ALT and AST activities in NOB/SD mice were 253.88 + -135.98 and 131.36 + -26.08U/L, respectively. The liver function enzyme activity of mice in NOB/SD group was significantly reduced (p < 0.05), indicating that the bioavailability of NOB/SD was higher than that of free NOB.

As can be seen from fig. 9 to 10, the liver surface of mice in the blank group (CTL) was smooth and fine, while the liver (normal saline) surface of the model group was rough, the whole color was whitened, and a large number of granular white lipid droplets and punctiform red inflammatory lesions were present. The free NOB group had improved liver surface roughness compared to the model group, but still had granular white lipid droplets and punctiform red inflammatory lesions. And the liver surface of NOB/SD group tends to be smooth, and the granular white lipid drop and punctiform red inflammatory lesion surface level are obviously reduced. The results of the HE staining and slicing of the liver of the mice show that the liver cells of the mice in the model group have loose structures, obviously necrotic liver cells and abnormally proliferated more fibrous connective tissue, and the tissues can be obviously infiltrated by inflammatory cells (the pathological area is 41.82+/-4.86). The free NOB group has reduced hepatocyte necrosis area, reduced inflammatory cell infiltration area, and improved liver injury (26.03% + -8.02 pathological area). And the area of hepatocyte necrosis and inflammatory cell infiltration in the NOB/SD group is greatly reduced (pathological area is 6.91% +/-1.33), so that obvious protective effect on the hepatocytes is shown. The above results indicate that NOB/SD of the present invention exhibits excellent liver protecting effect in treating acute liver injury caused by acetaminophen.

Example 5: in vivo pharmacokinetic study of nobiletin solid dispersions

(1) The experimental object: male Sprague-Dawley (SD) rats (SPF, 200-220 g) were supplied by Beijing Warcon Biotechnology Co., ltd (Beijing, china).

(2) Experimental grouping: (1) NOB/SD group (aqueous solution, lavage dose: 50 mg/kg); (2) free NOB group (aqueous solution, lavage dose: 50 mg/kg); 3 in each group; the NOB concentration in both sets of solutions was 2.5. Mu.g/mL. NOB/SD group, nobiletin: soluplus: PVP/VA64 is prepared by a hot melt extrusion method, wherein the mass ratio of PVP to VA64 is 1:5:1.

(3) The experimental steps are as follows: rats were fed and maintained at room temperature, 50±5% humidity and constant conditions of 12h light/dark cycle. All rats fasted for 12 hours during the experiment and were free to drink water. Following dosing, 200 μl blood samples were collected from the tail vein of each rat at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8 and 12h, respectively, and placed in heparinized tubes. Immediately centrifuging the sample at 6000rpm for 10min, taking 100 mu L of plasma, placing the plasma into a 1.5mL sterile centrifuge tube, adding 10 mu L of hesperetin internal standard working solution (20 mu g/mL), adding 250 mu L of acetonitrile precipitated protein, performing vortex oscillation for 2min, centrifuging at 13000rpm for 10min at 4 ℃, and taking supernatant for sample injection detection analysis. The results are shown in FIG. 11.

As can be seen from FIG. 11, the peak time for blood concentration of NOB/SD group NOB was 0.67.+ -. 0.14h, the peak blood concentration was 7.23.+ -. 1.62. Mu.g/mL, and the maximum blood concentration of free NOB reached at 0.83.+ -. 0.14h, which was 1.26.+ -. 0.51. Mu.g/mL. The highest blood concentration of NOB/SD is 5.74 times higher than that of free NOB. After one oral administration, the AUC0-12 of NOB/SD was 9.68+ -1.73 μg.h/mL, whereas the free NOB was only 2.89+ -1.28 μg.h/mL. The relative bioavailability of oral NOB/SD is 3.25 times that of free NOB. After administration, the NOB blood concentration of the NOB/SD group is rapidly increased, and a higher effective blood concentration level can be realized, so that the effect of rapid and effective treatment is realized, and in the first 3 hours, the NOB blood concentration of the NOB/SD group is obviously higher than that of the free NOB group, which indicates that the NOB/SD group has higher bioavailability.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. The nobiletin solid dispersion is characterized by comprising the following components in percentage by mass: 70-80% of Soluplus, 10-15% of PVP/VA, and 10-15% of nobiletin.

2. The nobiletin solid dispersion according to claim 1, characterized in that the mass ratio of PVP/VA64 and Soluplus is 1:8-3:14.

3. A process for preparing the solid dispersion of nobiletin as claimed in claim 1, which is characterized by hot-melt extrusion, specifically comprising the steps of uniformly mixing the components in proportion and extruding the mixture by a hot-melt extruder.

4. A method of preparation according to claim 3, characterized in that: the extrusion temperature is less than or equal to 170 ℃.

5. A process for preparing the solid dispersion of nobiletin as claimed in claim 1, which is characterized by solvent volatilizing method, specifically comprising the steps of respectively dissolving each component in solvent according to a certain proportion, uniformly mixing, and removing solvent to obtain the product.

6. The method of manufacturing according to claim 5, wherein: the solvent comprises at least one of absolute ethyl alcohol, water and methanol.

7. Application of nobiletin solid dispersion in preparing medicine for preventing and treating liver injury is provided.

8. Application of nobiletin solid dispersion in preparing medicine for treating, preventing, reversing or reducing liver injury is provided.

9. The use according to any one of claims 7 to 8, wherein the medicaments, identical or different, each further comprise one or more pharmaceutically acceptable carriers or excipients.

10. Application of nobiletin solid dispersion in preparing health care products for treating or preventing or reversing or reducing liver injury.

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