CN117243896A - Xinhui citrus limonin liposome and preparation method thereof and application of Xinhui citrus limonin liposome in preparation of drugs/health-care products for preventing and treating liver injury - Google Patents

Xinhui citrus limonin liposome and preparation method thereof and application of Xinhui citrus limonin liposome in preparation of drugs/health-care products for preventing and treating liver injury Download PDF

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CN117243896A
CN117243896A CN202311301827.1A CN202311301827A CN117243896A CN 117243896 A CN117243896 A CN 117243896A CN 202311301827 A CN202311301827 A CN 202311301827A CN 117243896 A CN117243896 A CN 117243896A
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limonin
liposome
preparation
liver injury
cholesterol
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Inventor
郑国栋
韦敏燕
陈柏忠
蔡轶
李楚文
杨婉玲
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Guangdong Xinbaotang Biotechnology Co ltd
Guangzhou Medical University
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Guangdong Xinbaotang Biotechnology Co ltd
Guangzhou Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A23L33/11—Plant sterols or derivatives thereof, e.g. phytosterols
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The application belongs to the technical field of natural compounds, and discloses a Xinhui citrus limonin liposome, a preparation method thereof and application thereof in preparation of drugs/health care products for preventing and treating liver injury. The limonin liposome disclosed by the invention comprises the following components: limonin, cholesterol, soybean phospholipids and solvents. Aiming at the problems of low solubility, poor stability, poor bioavailability and quick metabolism of limonin, the liposome has high encapsulation efficiency which can reach more than 90 percent and high drug loading capacity which can reach 4.76 percent, and the obtained liposome has good stability, thereby effectively overcoming the defects of poor water solubility, low bioavailability and the like of limonin. The limonin liposome provided by the invention has the advantages that the concentration in vivo is effectively improved, an obvious liver protection effect is shown, the liver injury can be prevented and/or reduced and/or reversed, the limonin liposome can be applied to the development of medicines/health care products and natural food supplements with functional protection effect on the liver injury, and a basis is provided for the reasonable development and utilization of citrus seed resources.

Description

Xinhui citrus limonin liposome and preparation method thereof and application of Xinhui citrus limonin liposome in preparation of drugs/health-care products for preventing and treating liver injury
Technical Field
The invention belongs to the technical field of natural compounds, and in particular relates to a Xinhui citrus limonin liposome, a preparation method thereof and application thereof in preparation of drugs/health care products for preventing and treating liver injury.
Background
Limonin (LM) is a natural tetracyclic triterpene compound, widely found in citrus seeds. Most citrus in the market is utilized in a manner of leaving skin and discarding meat, which not only causes environmental pollution, but also causes serious resource waste. Studies show that limonin can alleviate lipopolysaccharide-induced liver injury, and has effects of resisting lipid deposition, resisting oxidation and inflammation, and protecting alcoholic fatty liver disease. Drug-induced liver injury is one of the most common and serious adverse drug reactions at present, and serious patients can cause liver failure and even death, especially Acetaminophen (APAP) -induced liver toxicity. Limonin was demonstrated to have good liver protecting activity against APAP-induced hepatotoxicity. However, limonin has low solubility, poor stability, poor bioavailability, fast metabolism, and pharmacokinetic studies indicate that its main metabolic pathways in vivo and in vitro are reduction and hydrolysis, which limits the further broad application of limonin.
High-speed counter-current chromatography (HSCCC) is a rapid separation technique based on continuous liquid-liquid partition chromatography. Compared with the traditional chromatographic technique, the HSCCC technique has higher separation efficiency, no irreversible adsorption and high separation recovery rate. Hsccs have been successfully applied to the enrichment of food active compounds. The HSCCC technology is expected to realize efficient extraction and separation of limonin from citrus seeds, and is favorable for sustainable utilization of the citrus seeds.
Disclosure of Invention
In order to overcome the defects of low solubility, poor stability, poor bioavailability and fast metabolism of limonin in the prior art, the primary aim of the invention is to provide limonin liposome.
The invention also aims at providing a preparation method of the limonin liposome.
The invention also aims to provide the application of the limonin liposome in preparing drugs/health care products for preventing and treating liver injury. In particular to the application in preparing medicines/health care products for preventing and treating drug-induced liver injury.
The aim of the invention is achieved by the following scheme:
a limonin liposome comprising the following components: limonin, cholesterol, soybean phospholipids and solvents.
In some embodiments of the invention, the mass ratio of cholesterol to soybean phospholipids may be 1:3-1:6.
In some embodiments of the invention, the concentration of the soybean phospholipid may be 24-60mg/mL.
In some embodiments of the invention, the limonin: the mass ratio of (cholesterol + soybean phospholipids) may be 1:10-1:160.
In some embodiments of the invention, the limonin: the mass ratio of (cholesterol + soybean phospholipids) may be 1:20-1:80.
In some embodiments of the invention, the solvent is a hydration liquid conventionally used in the art, such as water or a buffer.
In some embodiments of the invention, the limonin may be citrus seed derived limonin.
In some embodiments of the invention, the liposomes have a particle size of 200nm or less.
In some embodiments of the invention, the D50 of the liposome is 70-90nm.
In some embodiments of the invention, the limonin can be extracted and separated from the citrus seed crude extract by hscc technology and recrystallization, and the obtained limonin has high purity. The citrus seed may be from citrus unshiu.
Aiming at the problems of low solubility, poor stability, poor bioavailability and quick metabolism of limonin, the limonin liposome has high encapsulation efficiency which can reach more than 90 percent and high drug loading capacity which can reach 4.76 percent, and meanwhile, the obtained liposome has good stability. The double-layer spherical liposome vesicle of the liposome can provide a system for transferring hydrophobic compounds, a hydrophobic region formed by cross-linking of a phospholipid nonpolar tail part and an aqueous phase core formed by surrounding a phospholipid polar head part, has the capacity of embedding liposoluble and water-soluble active substances at the same time, greatly improves the transfer efficiency of the active substances, and cholesterol improves the stability of a liposome membrane structure by being embedded into a phospholipid bilayer, so that the liposome has an important role in stabilizing the liposome; under the dosage proportion of the invention, the liposome with high encapsulation efficiency, high drug loading and high stability is obtained, and has the advantages of accurate content, small particle size, stable property of functional substances, difficult oxidation, high bioavailability, good slow release property and the like, and the defects of poor water solubility, low bioavailability and the like of limonin are effectively overcome.
The limonin liposome can be prepared by adopting a film hydration-homogenization method.
In some embodiments of the invention, the film may be obtained by dissolving limonin, cholesterol, soybean phospholipids in solvent B in the limonin liposome composition of the invention, and removing solvent B.
In some embodiments of the present invention, the solvent B may be an organic solvent conventionally used in the art, such as ethanol, dichloromethane, etc., or may be a mixture of ethanol and dichloromethane, which is effective in dissolving limonin, cholesterol, and soybean phospholipids.
In some embodiments of the present invention, the solvent B may be a mixture of ethanol and methylene chloride in a volume ratio of 2:3-3:1.
In some embodiments of the invention, the removal solvent B may be removed by spin evaporation.
In some embodiments of the invention, the hydration refers to mixing the solvent in the limonin liposome components of the invention with the film to obtain a hydration liquid.
In some embodiments of the invention, the homogenized pressure may be 750bar or more.
In some embodiments of the invention, the hydration fluid may be sonicated prior to homogenization.
In some embodiments of the invention, the ultrasonic means is processed by a conventional ultrasonic processor.
The invention provides a specific preparation method of the limonin liposome, which comprises the following steps:
(1) Dissolving limonin, cholesterol and soybean lecithin in a solvent B, and removing the solvent B to form a film;
(2) Mixing a solvent with the film to obtain hydration liquid;
(3) Homogenizing the hydration liquid to obtain limonin liposome.
The invention also provides application of the limonin liposome in preparing a medicine/health care product for treating or preventing or reversing or reducing liver injury. In particular to the application in preparing the medicine for preventing and treating the drug-induced liver injury.
In some embodiments of the invention, the liver injury includes liver tissue and cell injury.
In some embodiments of the invention, the liver injury comprises a drug induced liver injury by a chemical hepatotoxic substance.
In some embodiments of the invention, the chemical hepatotoxic substance comprises alcohol, carbon tetrachloride, acetaminophen, acetaldehyde, and the like.
In some embodiments of the invention, the liver injury includes toxic hepatitis, cirrhosis, liver fibrosis, and the like.
In some embodiments of the invention, the same or different drugs/nutraceuticals each comprise a therapeutically effective amount of limonin liposomes.
In some embodiments of the present invention, the same or different drugs/health care products are prepared into various dosage forms by a conventional method, wherein the dosage forms comprise: oral liquid, buccal preparation, suspension, drop and other oral dosage forms, injection and other dosage forms other than oral administration.
In some embodiments of the present invention, the same or different drugs/health care products also contain one or more pharmaceutically acceptable carriers.
The limonin liposome is subjected to physicochemical property characterization, in-vitro release research and stability research, and an APAP-induced liver injury animal model is constructed, so that the limonin liposome is applied to injured liver tissues and cells, and histopathological sections, ALT (alanine aminotransferase), AST (aspartate aminotransferase) and the like are adopted as detection indexes, and the result proves that the limonin liposome effectively improves the in-vivo concentration and shows an obvious liver protection effect, can prevent and/or reduce and/or reverse liver injury, can be applied to development of medicines/health products and natural food supplements with functional protection effect on liver injury, and provides basis for reasonable development and utilization of citrus seed resources.
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 (A) is a HPLC chart of limonin content in citrus seed extract; (B) purity determination HPLC chart of recrystallized product.
FIG. 2 is a chromatographic separation chart of high-speed countercurrent chromatography.
FIG. 3 is a graph showing the change in encapsulation efficiency at various phospholipid concentrations.
Fig. 4 is an electron microscope scan of limonin liposomes.
FIG. 5 is a graph showing the particle size distribution of limonin liposomes.
FIG. 6 is a graph of the solubility of limonin liposomes and free limonin.
FIG. 7 is a graph showing changes in the stability of limonin liposomes.
FIG. 8 is a graph showing ALT and AST activity statistics in mouse serum.
Fig. 9 is a graph showing the area statistics of liver damage in mice.
FIG. 10 is a H & E stained section of mouse liver.
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, a limonin liposome comprises the following components: limonin, cholesterol, soybean phospholipids and solvents.
In some embodiments of the invention, the mass ratio of cholesterol to soybean phospholipids may be 1:3-1:6. In one embodiment, the mass ratio of the cholesterol to the soybean lecithin is 1:3; in another embodiment, the mass ratio of the cholesterol to the soybean phospholipids is 1:6; in yet another embodiment, the mass ratio of cholesterol to soybean phospholipids may be 1:5.
In some embodiments of the invention, the concentration of the soybean phospholipid may be 24-60mg/mL. In one embodiment, the concentration of the soybean lecithin is 24mg/mL; in another embodiment, the concentration of the soybean phospholipid is 60mg/mL; in yet another embodiment, the concentration of the soybean phospholipid is 48mg/mL.
In some embodiments of the invention, the limonin LM: the mass ratio of (cholesterol + soybean phospholipid) L may be 1:10-1:160. In one embodiment, the limonin: the mass ratio of (cholesterol and soybean lecithin) is 1:10; in another embodiment, the limonin: the mass ratio of (cholesterol and soybean lecithin) is 1:40; in yet another embodiment, the limonin: the mass ratio of (cholesterol and soybean lecithin) is 1:160.
In some embodiments of the invention, the limonin: the mass ratio of (cholesterol + soybean phospholipids) may be 1:20-1:80. In one embodiment, the limonin: the mass ratio of (cholesterol and soybean lecithin) is 1:20; in another embodiment, the limonin: the mass ratio of (cholesterol and soybean lecithin) is 1:40; in yet another embodiment, the limonin: the mass ratio of (cholesterol and soybean lecithin) is 1:80.
In some embodiments of the invention, the solvent is a hydration liquid conventionally used in the art, such as water or a buffer. The buffer solution is near neutral buffer solution. The hydration liquid used has no obvious influence on the particle size, encapsulation efficiency and the like of the liposome.
In some embodiments of the invention, the limonin may be citrus limonin from citrus seeds.
In some embodiments of the invention, the limonin is prepared according to existing methods.
In some embodiments of the invention, the limonin is purchased from commercial products.
In some embodiments of the invention, the limonin can be extracted and separated from the citrus seed crude extract by hscc technology and recrystallization, and the obtained limonin has high purity.
In some embodiments of the invention, the citrus seed is from citrus unshiu.
Specifically, the method comprises the following steps: pulverizing citrus seeds, sieving, adding dichloromethane (feed-liquid ratio of 1:3-1:5, w/v), ultrasonic extracting for 25-40min (320 w,40 kHz), filtering, and collecting supernatant; repeating the extraction for three times, mixing the three filtrates, degreasing with petroleum ether (filtrate: petroleum ether=1:3-1:5, v/v), precipitating and volatilizing, adding dichloromethane for redissolving, taking a dichloromethane layer, spin-drying in a rotary evaporator, drying to obtain a limonin crude product, and carrying out HPLC-PDA purity analysis, as shown in fig. 1 (A); preparing limonin monomer by high-speed countercurrent chromatography separation: dissolving the crude product in a mixed solvent, violently oscillating, completely dissolving the crude product, standing, layering up and down, volatilizing the upper phase and the lower phase with equal volumes after two phases are balanced, dissolving the upper phase and the lower phase with equal volumes of methanol, performing HPLC-PDA analysis, and calculating a K value (distribution coefficient); the calculation formula of the distribution coefficient K: k=a u /A L ，A u : peak area of target compound in upper phase, A L : the peak areas of the target compounds in the lower phase are shown in Table 1. Theoretically, the K of the target compound in the solvent system should be between 0.2 and 5.0. In an n-hexane-n-butanol-methanol-water system, the solubility of limonin is good, but a mixed solution of n-butanol and water is easy to emulsify, so that a baseline of high-speed countercurrent chromatography cannot reach an equilibrium state under the experimental flow rate and the rotating speed, and the layering time of a solvent system is long. The methylene dichloride-methanol-water (1.9:1.6:1) system has good solubility to limonin in citrus seeds, simultaneously meets K value (K=0.22), ensures that the fixed retention rate reaches 65.0-75.0%, and finally selects the solvent system to purify the limonin crude extract. The flow rate is 1.8-2.5mL/min, the temperature is set to 20-25 ℃, and the rotating speed is 750-900rpm. After drying, the hscc purification is added with an appropriate amount of dichloromethane: methanol (1:1-1:2.5) was recrystallized and the recrystallized product was subjected to HPLC-PDA purity analysis (HPLC purity analysis chart is shown in FIG. 1 (B); chromatographic separation chart of high-speed countercurrent chromatography is shown in FIG. 2).
TABLE 1
The limonin liposome can be prepared by adopting a film hydration-homogenization method.
In some embodiments of the invention, the film may be obtained by dissolving limonin, cholesterol, soybean phospholipids in solvent B in the limonin liposome composition of the invention, and removing solvent B.
In some embodiments of the present invention, the solvent B is a solvent conventionally used in the art for effectively dissolving limonin, cholesterol, and soybean phospholipids, such as ethanol, dichloromethane, and the like, or may be a mixture of ethanol and dichloromethane. In one embodiment, the solvent B is ethanol; in another embodiment, the solvent B is dichloromethane; in yet another embodiment, the solvent B is a mixture of ethanol and dichloromethane. The mixed solvent can meet the requirements and has better safety.
In some embodiments of the present invention, the solvent B may be a mixture of ethanol and methylene chloride in a volume ratio of 2:3-3:1.
In some embodiments of the invention, the removal solvent B may be removed by spin evaporation.
In some embodiments of the invention, the hydration refers to mixing the solvent in the limonin liposome components of the invention with the film to obtain a hydration liquid.
In some embodiments of the invention, the homogenized pressure may be 750bar or more. The relatively higher homogenization pressure is advantageous for obtaining liposomes with smaller particle sizes. In one embodiment, the homogenized pressure is 750bar; in another embodiment, the homogenized pressure is 900bar; in yet another embodiment, the homogenizing pressure is 850bar.
In some embodiments of the invention, the hydration fluid may be sonicated prior to homogenization.
In some embodiments of the invention, the ultrasonic means is processed by a conventional ultrasonic processor.
In some embodiments of the invention, the liposomes have a particle size of 200nm or less.
In some embodiments of the invention, the D50 of the liposome is 70-90nm.
In some embodiments of the invention, the D90 of the liposome is 100-191nm.
In one embodiment, a specific preparation method of the limonin liposome comprises the following steps:
(1) Dissolving limonin, cholesterol and soybean lecithin in a solvent B, and removing the solvent B to form a film;
(2) Mixing a solvent with the film to obtain hydration liquid;
(3) Homogenizing the hydration liquid to obtain limonin liposome.
In one embodiment, the limonin liposome is applied to preparation of a medicine/health care product for treating or preventing or reversing or reducing liver injury. In particular to the application in preparing the medicine for preventing and treating the drug-induced liver injury.
In some embodiments of the invention, the liver injury includes liver tissue and cell injury.
In some embodiments of the invention, the liver injury comprises a liver injury caused by a chemical hepatotoxic substance.
In some embodiments of the invention, the chemical hepatotoxic substance comprises alcohol, carbon tetrachloride, acetaminophen, acetaldehyde, and the like.
In some embodiments of the invention, the liver injury includes toxic hepatitis, cirrhosis, liver fibrosis, and the like.
In some embodiments of the invention, the same or different drugs/nutraceuticals each comprise a therapeutically effective amount of limonin liposomes.
In some embodiments of the present invention, the same or different drugs/health care products are prepared into various dosage forms by a conventional method, wherein the dosage forms comprise: oral liquid, buccal preparation, suspension, drop and other oral dosage forms, injection and other dosage forms other than oral administration.
In some embodiments of the present invention, the same or different drugs/health care products also contain one or more pharmaceutically acceptable carriers.
Example 1: preparation of Xinhui citrus limonin liposome (Lip-LM)
The membrane hydration-ultrasonic-high pressure homogenization method is adopted to prepare the Xinhui citrus limonin liposome (Lip-LM). Dissolving limonin LM, cholesterol, and soybean phospholipids in ethanol/dichloromethane (2:1, v/v), and vacuum rotary evaporating at 35-40deg.C to obtain dry film. Then, the obtained uniform film is hydrated by PBS buffer solution (pH 7.40), ultrasonic treatment is carried out for 10min (80 w), then homogenizing circulation is carried out for 20 times by a high-pressure homogenizer under high pressure of 900bar, dispersed liposome is obtained, and physicochemical property characterization is carried out on the Lip-LM obtained.
TABLE 2
TABLE 3 Table 3
TABLE 4 Table 4
TABLE 5
As can be seen from tables 2-5 and FIG. 3, the novel citrus limonin liposome provided by the invention has a mass ratio of cholesterol to soybean phospholipids of 1:3-1:6, and the concentration of soybean phospholipids is 24-60mg/mL, and limonin: at a mass ratio of (cholesterol + soybean phospholipids) of 1:20-1:80, lip-LM shows high encapsulation efficiency (over 80%, up to 93%) and good drug loading (> 2.0%, up to 4.76%).
Physical and chemical property examination is carried out on the Xinhui citrus limonin liposome prepared by the formula 11, and the results are shown in fig. 4-5. As can be seen from Table 6, FIG. 4 and FIG. 5, the Lip-LM of the present invention has the characteristics of small particle size, negatively charged surface, good dispersing ability, high encapsulation efficiency, etc.
In addition, the concentration of limonin in Lip-LM and the saturated dissolution concentration of free limonin in water are respectively measured, and as can be seen from figure 6, the limonin in the liposome has large drug-loading rate and high coating rate, the LM concentration can reach 1121.44 +/-18.73 mug/mL, and is 452.19 times of the free limonin concentration 2.48+/-0.09 mug/mL in water, which indicates that the solubility and the dispersity of limonin after liposome encapsulation under the proportion of the invention are obviously improved.
There are studies showing that too high encapsulation efficiency may cause drug leakage during drug storage, which may affect the stability of Lip-LM. The stability of Lip-LM was tested: the Lip-LM of the present invention was stored at 4℃and 25℃for 15 days, respectively. As shown in Table 6 and FIG. 7, the Lip-LM system of the present invention is stable, the particle size, the potential, etc. are hardly changed, sedimentation, etc. are not generated, and the higher encapsulation efficiency (more than 85% in each case) is maintained, which proves that the liposome of the present invention has the advantages of high drug-loading rate, high encapsulation efficiency, excellent stability, and effective product stability and efficacy assurance.
TABLE 6
Example 2: simulated gastrointestinal in vitro release investigation of Xinhui citrus limonin liposome (Lip-LM)
In order to study the release kinetics of Lip-LM, in vitro simulated gastrointestinal release studies were performed. In particular, the in vitro kinetics of limonin release from liposomes was examined by dialysis. Lip-LM and Free-LM suspensions (Free limonin suspensions) were placed in dialysis bags (molecular weight [ Mw ] 8,000-14,000 Da) and PBS (pH 1.50 and pH 6.80) of different pH was used as release medium to simulate release conditions in stomach and intestinal tract, respectively, and magnetically stirred at 37℃for 48 hours to simulate release. At different time points, samples were taken from the release liquid and simultaneously replaced with an equal volume of fresh release medium (n=3). The release of limonin was detected by HPLC-PDA at 209nm and the cumulative release data was fitted by simulation using Origin 9.0 software, the results of which are shown in table 7.
TABLE 7
The table shows that the Lip-LM has obvious slow release effect compared with Free-LM in-vitro simulated release environment under different pH conditions. The liposome prepared by encapsulating limonin by using cholesterol and soybean lecithin can maintain high concentration of limonin in the digestive tract, and can be used for oral absorption without frequent administration. The physical and chemical property characterization and the evidence of in vitro release studies show that Lip-LM has remarkable advantages in oral absorption and has better liver protection function than Free-LM.
Example 3: protective effect of Xinhui citrus limonin liposome on acute liver injury
(1) The experimental object: mouse C57BL/6 (6 weeks old)
(2) Experimental grouping: (1) blank Control group (Control group, normal saline lavage); (2) model group (APAP group, normal saline lavage); (3) free limonin group (Free-LM group, gastric lavage dose: 60 mg/kg); (4) limonin liposome group (Lip-LM group, stomach-filling dose: 60mg/kg of limonin); each group of 6.
(3) The experimental steps are as follows: after continuous administration according to the group for one week, the three groups except for the blank group were subjected to intraperitoneal injection of Acetaminophen (APAP, 350 mg/kg) to induce an acute liver injury animal model, after molding for 48 hours, 4% tribromoethanol was injected for anesthesia, whole blood was taken from the eyeballs, and after standing at room temperature for 2 hours, the whole blood was centrifuged at 3000rpm for 10 minutes to obtain serum, and the levels of alanine Aminotransferase (ALT) and glutamic-oxaloacetic Aminotransferase (AST) in the serum were detected by a biochemical analyzer, and the result is shown in FIG. 8. After mice were sacrificed, livers were taken and placed in 4% paraformaldehyde solution for more than 48H for H & E staining sections, the results are shown in figures 9 and 10.
As can be seen from FIG. 8, the ALT and AST activities of APAP group mice were 908.15 + -172.73U/L and 702.68 + -117.01U/L, respectively; ALT and AST activities of Free limonin group (Free-LM) mice were 792.59 + -212.67U/L and 665.25 + -150.28U/L, respectively; ALT and AST activities of limonin liposome group mice were 609.02 + -53.50U/L and 347.37 + -67.59U/L, respectively. The liver function enzyme activity of the limonin liposome group mice is obviously reduced (p is less than 0.01), which shows that compared with free limonin, lip-LM has better protection effect on acute liver injury induced by APAP.
As can be seen from fig. 9 and 10, compared with the blank control group, the liver cells of the APAP group mice were significantly necrotized, the cell boundaries were blurred and the arrangement was poor, the liver cell structure was loose, and the pathological area was 24.05±2.02%. The Free-LM group showed a decrease in hepatocyte necrosis area, but was not significant (pathological area 21.56±2.20%). The liver cell necrosis and inflammatory cell infiltration area in the Lip-LM group of the invention is greatly reduced (the pathological area is only 12.49+/-2.71%), so that the Lip-LM shows obvious protective effect on liver cells. The results show that the liver protection effect of the Lip-LM on the acute liver injury caused by APAP is superior to that of Free-LM, and the Lip-LM can obviously relieve the acute liver injury induced by APAP.
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. Limonin liposome is characterized by comprising the following components: limonin, cholesterol, soybean phospholipids and solvents.
2. Limonin liposome according to claim 1, characterized in that: the mass ratio of the cholesterol to the soybean phospholipid is 1:3-1:6.
3. Limonin liposome according to claim 1, characterized in that: the concentration of the soybean lecithin is 24-60mg/mL.
4. Limonin liposome according to claim 1, characterized in that: the limonin: the mass ratio of (cholesterol and soybean lecithin) is 1:10-1:160.
5. A method for preparing limonin liposome according to any one of claims 1-4, characterized in that it is prepared by a thin film hydration-homogenization method.
6. The method of manufacturing according to claim 5, wherein: the film is prepared by dissolving limonin, cholesterol and soybean lecithin in a solvent B, and removing the solvent B.
7. The method of manufacturing according to claim 5, wherein: the hydration refers to mixing the solvent in the components with the film to obtain hydration liquid.
8. The method of manufacturing according to claim 5, wherein: the homogenizing pressure is more than or equal to 750bar.
9. Use of limonin liposome according to any one of claims 1-4 for the preparation of a medicament/health care product for treating or preventing or reversing or reducing liver damage.
10. Use of limonin liposome according to any one of claims 1-4 for the preparation of a medicament for the prevention and treatment of pharmaceutical liver damage.
CN202311301827.1A 2023-10-10 2023-10-10 Xinhui citrus limonin liposome and preparation method thereof and application of Xinhui citrus limonin liposome in preparation of drugs/health-care products for preventing and treating liver injury Pending CN117243896A (en)

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