Commetaria botrytis polysaccharide lipidosome and preparation method thereof

Abstract

The invention discloses a Poulenghuajouanghua polysaccharide liposome and a preparation method thereof, and belongs to the technical field of polysaccharide liposome materials.The preparation method comprises the steps that lecithin, cholesterol and lauryl amine are dissolved in a solvent, and an organic phase is obtained; the preparation method comprises the following steps: preparing an organic phase, adding the Poulenghuangtaria paniculata polysaccharide and a phosphate buffer solution into the organic phase, carrying out ultrasonic treatment in an ice-water bath, coating the Poulenghuangtaria paniculata polysaccharide in a liposome encapsulating material formed by lecithin, cholesterol and lauryl amine, carrying out rotary evaporation to obtain a suspension, and carrying out secondary ultrasonic treatment and filtration on the suspension to obtain the Poulenghuangtaria paniculata polysaccharide liposome. According to the preparation method, lauryl amine is introduced into a polysaccharide liposome structure, so that the drug release property of the

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide liposome materials, and in particular to a polysaccharide liposome of Psoralea corylifolia and a preparation method thereof. Background Art

[0002] Liposome is a new targeted drug delivery carrier. The main substances that make up polysaccharide liposome are cholesterol and lecithin. It has multiple advantages of targeted drug preparations, and can delay the action time of drugs, improve efficiency, reduce side effects, and improve the distribution of drugs in the body. It is one of the typical representatives of targeted drug preparations. At present, liposome has become one of the hot issues in the medical field, food and cosmetics industry.

[0003] Polysaccharide drugs have problems such as poor oral absorption, low bioavailability, and short half-life in vivo when acting on the human body, and polysaccharides are prepared to obtain polysaccharide liposomes. Not only can the polysaccharide be wrapped inside the liposome to protect it from the influence of the external environment such as enzymes and pH changes, and the stability of the polysaccharide is increased, but also the slow release of the polysaccharide can be achieved, the action time can be prolonged, and the drug effect persistence can be improved. By modifying the liposome, the targeted release of specific tissues or cells can be achieved, the therapeutic effect of the polysaccharide drug can be improved, and the side effects can be reduced. Therefore, liposomes are used as a drug delivery system, and the yellow coral fungus polysaccharide is loaded therein to prepare the yellow coral fungus polysaccharide liposomes. With the advantages of liposomes, the shortcomings of the yellow coral fungus polysaccharide in the human body can be compensated, thereby prolonging the retention time of the yellow coral fungus polysaccharide in the blood, improving the utilization of the yellow coral fungus polysaccharide, and reducing the dosage and frequency of medication. The encapsulation rate of polysaccharide drugs in polysaccharide liposomes is crucial to the release and retention time of polysaccharide drugs in vivo. However, the encapsulation rate of existing polysaccharide liposomes cannot be effectively controlled, which affects the release and efficacy of polysaccharide drugs. Summary of the invention

[0004] The invention provides a polysaccharide liposome of yellow coral fungus and a preparation method thereof, which effectively solves the technical problem that the existing polysaccharide liposome has a poor drug encapsulation rate, thereby affecting the release and retention time of the polysaccharide drug in the body and causing poor drug efficacy. The invention uses the polysaccharide encapsulation rate as an evaluation index to obtain an optimal preparation method of the yellow coral fungus liposome, and simulates the drug release of the yellow coral fungus polysaccharide liposome in the human body through an in vitro drug release experiment, thereby providing a yellow coral fungus polysaccharide liposome with good in vivo absorption, high bioavailability and long in vivo half-life.

[0005] The first object of the present invention is to provide a method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0006] Phosphatidylcholine, cholesterol and lauryl amine are dissolved in a solvent to obtain an organic phase; polysaccharide of yellow coral fungus and phosphate buffer are added to the organic phase, and the mixture is ultrasonicated once in an ice-water bath to coat the polysaccharide of yellow coral fungus in a liposome encapsulation material formed by phosphatidylcholine, cholesterol and lauryl amine, and the mixture is rotary evaporated to obtain a suspension, and the suspension is ultrasonicated twice and filtered to obtain polysaccharide liposomes of yellow coral fungus.

[0007] As a preferred embodiment, the mass ratio of the yellow coral fungus polysaccharide to lauryl amine is 1:0.6-4.8, and the mass ratio of the yellow coral fungus polysaccharide, lecithin and cholesterol is 1:10-50:3-6.

[0008] As a preferred embodiment, the mass ratio of the polysaccharide of Coral Gnaphalium to lauryl amine is 1:1.0-3.0.

[0009] As a preferred embodiment, Tween-80 is further added to the solvent, and the mass ratio of Tween-80 to the polysaccharide of Coral Gnaphalium is 0.2-1.25:1.

[0010] As a preferred embodiment, after ultrasonication in an ice-water bath, a suspension is obtained, and rotary evaporation is performed at 60°C to 65°C to remove the solvent to obtain a suspension. The suspension is ultrasonicated again and filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain the polysaccharide liposomes of the yellow coral fungus.

[0011] As a preferred embodiment, the second sonication is programmed sonication using an ultrasonic cell disruptor, with sonication at 20kHz to 25kHz for 3s to 5s, with an interval of 5s to 7s, and sonication for 3min to 5min.

[0012] As a preferred embodiment, the solvent is chloroform, methanol or ethyl acetate.

[0013] As a preferred embodiment, the temperature of the ice water bath is 1°C to 4°C.

[0014] As a preferred implementation, the ultrasound frequency of the first ultrasound is 16kHz to 22kHz, and the duration is 15min to 20min.

[0015] The second object of the present invention is to provide a polysaccharide liposome prepared by the above-mentioned preparation method.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a kind of yellow coral fungus polysaccharide liposome and its preparation method, lecithin, cholesterol and lauryl amine are dissolved in a solvent to obtain an organic phase; yellow coral fungus polysaccharide and phosphate buffer are added to the organic phase, ultrasonicated in an ice water bath, yellow coral fungus polysaccharide is coated in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, rotary evaporated to obtain a suspension, the suspension is subjected to secondary ultrasonic treatment, filtered, and yellow coral fungus polysaccharide liposome is obtained. The present invention effectively regulates the drug release of yellow coral fungus polysaccharide by introducing lauryl amine into the polysaccharide liposome structure, prolongs the retention time of yellow coral fungus polysaccharide in the blood, improves the utilization of yellow coral fungus polysaccharide, reduces the dosage and frequency of medication, and effectively solves the problem that the encapsulation rate of polysaccharide substances in traditional polysaccharide liposomes is poor, resulting in the inability of polysaccharide substances to be stably and slowly released in vivo, thereby affecting the efficacy of polysaccharide liposomes. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific examples, but the examples are not intended to limit the present invention. The following test methods and detection methods, unless otherwise specified, are conventional methods; the reagents and raw materials, unless otherwise specified, are commercially available.

[0019] In view of the problems mentioned in the background technology of the present invention: polysaccharide drugs have poor oral absorption, low bioavailability, short half-life in vivo, etc. when acting on the human body, preparing polysaccharide drugs into polysaccharide liposomes can make up for the shortcomings of yellow coral fungus polysaccharide in the human body to a certain extent, thereby prolonging the retention time of yellow coral fungus polysaccharide in the blood, improving the utilization of yellow coral fungus polysaccharide, and reducing the dosage and frequency of medication. The encapsulation rate of polysaccharide drugs in polysaccharide liposomes is crucial for the release and retention time of polysaccharide drugs in the body. However, the encapsulation rate of existing polysaccharide liposomes cannot be effectively controlled, thereby affecting the release and efficacy of polysaccharide drugs. In view of the above technical problems, the present invention provides a yellow coral fungus polysaccharide liposome and a preparation method thereof.

[0020] The technical solution of the present invention is analyzed and explained in detail below.

[0021] The present invention provides a method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0022] Dissolve lecithin, cholesterol and lauryl amine in a solvent (one or two of chloroform, methanol and ethyl acetate) to obtain an organic phase; add yellow coral fungus polysaccharide and phosphate buffer to the organic phase, perform ultrasonic treatment once in an ice water bath at 1°C to 4°C, coat the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, perform rotary evaporation to obtain a suspension, perform a second ultrasonic treatment on the suspension, and filter to obtain yellow coral fungus polysaccharide liposomes.

[0023] In the above technical scheme, when the polysaccharide liposomes of the yellow coral fungus are adsorbed by plasma proteins, the introduction of lauryl amine into the polysaccharide liposome structure enhances the "invisible" state of the polysaccharide liposomes of the yellow coral fungus in the blood, making it difficult to be recognized and cleared by macrophages, thereby promoting the drug release of the polysaccharide liposomes of the yellow coral fungus, prolonging the retention time of the polysaccharide in the blood, improving the utilization of the polysaccharide, and reducing the dosage and frequency of medication.

[0024] In order to achieve a better sustained-release effect of the yellow coral fungus polysaccharide, the mass ratio of the yellow coral fungus polysaccharide and lauryl amine is 1:0.6-4.8, and the mass ratio of the yellow coral fungus polysaccharide, lecithin and cholesterol is 1:10-50:3-6. Within the limited range of the above mass ratio, the sustained-release effect of the yellow coral fungus polysaccharide is better. If the amount of lauryl amine is too small, the drug release effect of the obtained yellow coral fungus polysaccharide liposome is not good, and it is impossible to extend the effective retention time of the yellow coral fungus polysaccharide in the blood, so it cannot achieve a better therapeutic effect. If the amount of lauryl amine is too large, the problem of excessive polysaccharide encapsulation rate will appear, the release rate of the yellow coral fungus polysaccharide is slow, and a better medication effect cannot be achieved.

[0025] In order to further achieve the best sustained-release effect of the yellow coral fungus polysaccharide, the mass ratio of the yellow coral fungus polysaccharide to lauryl amine is 1:1.0-3.0.

[0026] In order to further improve the encapsulation rate of the polysaccharide of the yellow coral fungus, Tween-80 is also added to the solvent, and the mass ratio of the Tween-80 to the polysaccharide of the yellow coral fungus is 0.2-1.25:1.

[0027] In order to prepare liposomes with uniform particle size and improve the stability of polysaccharide liposomes from yellow coral fungus, a suspension was obtained after ultrasonication in an ice-water bath, and the solvent was removed by rotary evaporation at 60°C to 65°C to obtain a suspension. The suspension was ultrasonicated again and filtered using 0.45μm and 0.22μm microporous filter membranes in turn to obtain polysaccharide liposomes from yellow coral fungus.

[0028] In order to further ensure the uniformity of the particle size of the yellow coral fungus polysaccharide liposome, the second ultrasound is a program ultrasound performed using an ultrasonic cell disruptor, with 20kHz to 25kHz ultrasound for 3s to 5s, an interval of 5s to 7s, and an ultrasound treatment of 3min to 5min; the ultrasound frequency of the first ultrasound is 16kHz to 22kHz, and the time is 15min to 20min. If the ultrasound treatment time is too short, the liposome particle size will be too large and the distribution will be uneven. If the treatment time is too long, the liposome may be destroyed, causing the drug (yellow coral fungus polysaccharide) to leak.

[0029] The technical effects of the present invention are described below in conjunction with specific embodiments and comparative examples.

[0030] Example 1

[0031] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0032] 400 mg of lecithin, 133 mg of cholesterol, 80 mg of lauryl amine and 50 mg of Tween-80 were dissolved in a mixture of 8 mL of chloroform and 16 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 65°C water to remove excess chloroform and methanol. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0033] Example 2

[0034] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0035] 800 mg of lecithin, 180 mg of cholesterol, 120 mg of lauryl amine and 8 mg of Tween-80 were dissolved in a mixture of 16 mL of chloroform and 32 mL of ethyl acetate to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz for 18 min in a 1°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and the mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate, and 12 mL of PBS was added after the mixture became a jelly, and the rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 20 kHz for 5 s, with an interval of 5 s, and the suspension was ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0036] Example 3

[0037] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0038] 1000 mg of lecithin, 160 mg of cholesterol, 40 mg of lauryl amine and 30 mg of Tween-80 were dissolved in a mixed solution of 60 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 22 kHz for 20 min in a 2° C. ice water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 62° C. water to remove excess methanol, and 12 mL of PBS was added after the mixture became a jelly, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 22 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 3 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0039] Example 4

[0040] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0041] 600 mg of lecithin, 200 mg of cholesterol, 24 mg of lauryl amine and 20 mg of Tween-80 were dissolved in a mixture of 12 mL of chloroform and 24 mL of ethyl acetate to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 18 kHz for 18 min in a 3°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 25 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 3 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0042] Example 5

[0043] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0044] 1200 mg of lecithin, 140 mg of cholesterol, 192 mg of lauryl amine and 40 mg of Tween-80 were dissolved in a mixed solution of 72 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 18 kHz for 20 min in a 4° C. ice water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60° C. water to remove excess methanol, and 12 mL of PBS was added after the mixture became a jelly, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 20 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0045] Example 6

[0046] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0047] 1600 mg of lecithin, 220 mg of cholesterol, 60 mg of lauryl amine and 10 mg of Tween-80 were dissolved in a mixture of 64 mL of methanol and 32 mL of ethyl acetate to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 20 kHz for 16 min in a 3°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess methanol and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 24 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0048] Example 7

[0049] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0050] 2000 mg of lecithin, 240 mg of cholesterol, 100 mg of lauryl amine and 25 mg of Tween-80 were dissolved in 120 mL of chloroform to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 16 kHz for 18 min in a 2° C. ice water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60° C. water to remove excess chloroform, and 12 mL of PBS was added after the mixture became a jelly, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 25 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0051] Example 8

[0052] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0053] 1800 mg of lecithin, 120 mg of cholesterol, 160 mg of lauryl amine and 45 mg of Tween-80 were dissolved in 108 mL of ethyl acetate to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 22 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess ethyl acetate, and 12 mL of PBS was added after the mixture became a jelly, and the rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 20 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 3 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0054] Example 9

[0055] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0056] 900 mg of lecithin, 150 mg of cholesterol, 90 mg of lauryl amine and 15 mg of Tween-80 were dissolved in a mixture of 18 mL of chloroform and 36 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz for 18 min in a 1°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and the mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and the rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 20 kHz for 5 s, with an interval of 5 s, and the suspension was ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0057] Example 10

[0058] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0059] 700 mg of lecithin, 170 mg of cholesterol, 140 mg of lauryl amine and 35 mg of Tween-80 were dissolved in a mixture of 14 mL of chloroform and 28 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 20 kHz for 18 min in a 1°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 20 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0060] Embodiment 11

[0061] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0062] 1500 mg of lecithin, 190 mg of cholesterol, 180 mg of lauryl amine and 40 mg of Tween-80 were dissolved in a mixture of 30 mL of chloroform and 60 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz for 18 min in a 1°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and the mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and the rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonically treated at 20 kHz for 5 s, with an interval of 5 s, and the suspension was ultrasonically treated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0063] Example 12

[0064] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0065] 500 mg of lecithin, 210 mg of cholesterol, 50 mg of lauryl amine and 20 mg of Tween-80 were dissolved in a mixture of 10 mL of chloroform and 20 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 20 kHz for 18 min in a 1°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 20 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0066] Embodiment 13

[0067] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0068] 600 mg of lecithin, 130 mg of cholesterol, 100 mg of lauryl amine and 20 mg of Tween-80 were dissolved in a mixture of 12 mL of chloroform and 24 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz for 18 min in a 1°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and the mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and the rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 20 kHz for 5 s, with an interval of 5 s, and the suspension was ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0069] Embodiment 14

[0070] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0071] 1200 mg of lecithin, 175 mg of cholesterol, 130 mg of lauryl amine and 40 mg of Tween-80 were dissolved in a mixture of 24 mL of chloroform and 48 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 20 kHz for 18 min in a 1°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 20 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 4 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0072] In order to further illustrate the technical effect of the present invention, the present invention also sets a comparative example, which is as follows:

[0073] Comparative Example 1

[0074] Compared with Example 1, the difference is that the amount of lauryl amine is reduced from 80 mg to 20 mg.

[0075] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0076] 400 mg of lecithin, 133 mg of cholesterol, 20 mg of lauryl amine and 50 mg of Tween-80 were dissolved in a mixture of 8 mL of chloroform and 16 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess chloroform and methanol. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0077] Comparative Example 2

[0078] Compared with Example 1, the difference is that the amount of lauryl amine is increased from 80 mg to 200 mg.

[0079] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0080] 400 mg of lecithin, 133 mg of cholesterol, 200 mg of lauryl amine and 50 mg of Tween-80 were dissolved in a mixture of 8 mL of chloroform and 16 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess chloroform and methanol. After the mixture became a jelly, 12 mL of PBS was added, and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0081] Comparative Example 3

[0082] Compared with Example 1, the difference is that no lauryl amine is added.

[0083] A method for preparing polysaccharide liposomes of Pseudomonas aeruginosa, comprising the following steps:

[0084] 400 mg of lecithin, 133 mg of cholesterol and 50 mg of Tween-80 were dissolved in a mixture of 8 mL of chloroform and 16 mL of methanol to obtain an organic phase; 40 mg of yellow coral fungus polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral fungus polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation was performed in 60°C water to remove excess chloroform and methanol. After the mixture became a jelly, 12 mL of PBS was added, and the rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension, and the suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min, and filtered with 0.45 μm and 0.22 μm microporous filter membranes in turn to obtain yellow coral fungus polysaccharide liposomes.

[0085] The performance of the yellow coral fungus polysaccharide liposomes prepared in the above Examples 1 to 14 and Comparative Examples 1 to 3 was tested. After testing, the encapsulation efficiency and drug release of the yellow coral fungus polysaccharide liposomes of Examples 1 to 14 of the present invention were similar. Therefore, the yellow coral fungus polysaccharide liposomes of Examples 1 to 8 are used as examples to illustrate the effects. The specific process and results are as follows:

[0086] 1. Determination of the encapsulation efficiency of polysaccharide liposomes of Coral Gnaphalium spp. by protamine method

[0087] Accurately measure several 100 μL portions of protamine solution (10 mg / mL), and add them to 100 μL of the yellow coral fungus polysaccharide liposomes prepared in Examples 1 to 8 and Comparative Examples 1 to 2 and the blank liposomes of Comparative Example 3, respectively, mix well, let stand, and then add 3 mL of normal saline thereto respectively, centrifuge at 4000 r / min and 4°C for 30 min, collect the supernatant of the sample after centrifugation, take out 1 mL of the supernatant respectively and add 1 mL of normal saline thereto, and use the phenol-concentrated sulfuric acid method to determine the polysaccharide content in the sample, which is the free yellow coral fungus polysaccharide content. At the same time, 100 μL of the polysaccharide solution of the yellow coral fungus was taken, 10% Triton-100 was added to break the emulsion, mixed and allowed to stand for 3 minutes, 3 mL of normal saline was added thereto, the supernatant was collected, 1 mL of the sample was taken out from the supernatant and 1 mL of normal saline was added thereto, and the polysaccharide content was determined by the phenol-concentrated sulfuric acid method. At this time, the result was the total polysaccharide content of the yellow coral fungus in the sample. The encapsulation rate of the polysaccharide liposome of the yellow coral fungus was calculated by the following formula. The results are shown in Table 1.

[0088]

[0089] 2. Investigation of drug release in vitro from polysaccharide liposomes of Psoralea corylifolia

[0090] 10 mL of the liposome solution of the yellow coral fungus polysaccharide and the solution of the fully dissolved yellow coral fungus polysaccharide were accurately drawn, and 3 groups were set in parallel, and placed in dialysis bags, respectively, and then placed in a conical flask containing 500 mL of phosphate buffer with pH = 7.4, and the placed conical flask was placed in a shaking table set at (36 + 0.5) ° C. 5 mL of the sample was taken out after 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h and 36h. The phenol-sulfuric acid method was used to measure in an ultraviolet spectrophotometer, and the cumulative drug release percentages of the yellow coral fungus polysaccharide at 12h and 24h were calculated, respectively, and the results are shown in Table 1.

[0091] Table 1 Encapsulation efficiency and drug release of the polysaccharide liposome of the present invention

[0092] Encapsulation rate / % 12h drug release rate / % 24h drug release rate / % Example 1 78.2 8.5 84.6 Example 2 75.4 9.7 88.2 Example 3 77.3 8.9 85.3 Example 4 73.8 10.5 90.1 Example 5 72.8 10.8 91.7 Example 6 74.2 10.0 89.4 Example 7 77.1 8.8 85.0 Example 8 73.0 10.2 88.6 Comparative Example 1 65.6 50.2 98.5 Comparative Example 2 68.2 46.7 96.8 Comparative Example 3 54.3 98.0 100

[0093] As shown in Table 1, the polysaccharide liposomes of the present invention have a good encapsulation rate and drug release performance. The encapsulation rate of the polysaccharide liposomes of the present invention in Examples 1 to 8 can reach 78.2%, the in vitro drug release rate of 12h is only 8.5%, and the drug release amount of 24h is 84.6%. Comparative Examples 1 to 3 are based on Example 1, the dosage of lauryl amine is reduced from 80mg to 20mg (Comparative Example 1), increased to 200mg (Comparative Example 2), and lauryl amine is removed (Comparative Example 3), and the drug release of the polysaccharide raw material of the polysaccharide of the polysaccharide raw material of Comparative Example 4 is compared. The results show that the encapsulation rate of the polysaccharide liposomes of the polysaccharide liposomes of Comparative Examples 1 and Comparative Example 2 is lower than that of Example 1, the drug release rate of 12h is as high as 50.2%, and the drug is basically completely released at 24h. When the polysaccharide drug in the polysaccharide liposomes of ...

[0094] In summary, the present invention introduces lauryl amine into the structure of polysaccharide liposomes to effectively regulate the drug release of the yellow coral fungus polysaccharide, prolong the retention time of the yellow coral fungus polysaccharide in the blood, improve the utilization of the yellow coral fungus polysaccharide, reduce the dosage and frequency of medication, and effectively solve the problem that the encapsulation rate of polysaccharides in traditional polysaccharide liposomes is poor, resulting in the inability to stably and slowly release the polysaccharides in the body, thereby affecting the efficacy of the polysaccharide liposomes.

[0095] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing polysaccharide liposomes of Coral Glehnia littoralis, characterized in that: The following steps are involved: Phosphatidylcholine, cholesterol and lauryl amine are dissolved in a solvent to obtain an organic phase; polysaccharide of yellow coral fungus and phosphate buffer are added to the organic phase, and the mixture is ultrasonicated once in an ice water bath to coat the polysaccharide of yellow coral fungus in a liposome encapsulation material formed by phosphatidylcholine, cholesterol and lauryl amine to obtain polysaccharide liposome of yellow coral fungus.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the yellow coral fungus polysaccharide to lauryl amine is 1:0.6-4.8, and the mass ratio of the yellow coral fungus polysaccharide, lecithin and cholesterol is 1:10-50:3-6.

3. The preparation method according to claim 2, characterized in that The mass ratio of the polysaccharide of Psoralea corylifolia to lauryl amine is 1:1.0-3.

0.

4. The preparation method according to claim 1, characterized in that: Tween-80 is also added to the solvent, and the mass ratio of Tween-80 to the polysaccharide of Pseudomonas aeruginosa is 0.2-1.25:

1.

5. The preparation method according to claim 1, characterized in that: After ultrasonication in an ice-water bath, a suspension was obtained, and rotary evaporation was performed at 60° C. to 65° C. to remove the solvent to obtain a suspension. The suspension was ultrasonicated again and filtered with 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain the polysaccharide liposomes of the yellow-branched coral fungus.

6. The preparation method according to claim 5, characterized in that: The second ultrasonic treatment is programmed ultrasonication using an ultrasonic cell disruptor, with ultrasonication at 20kHz to 25kHz for 3s to 5s, with an interval of 5s to 7s, and ultrasonic treatment for 3min to 5min.

7. The preparation method according to claim 1, characterized in that: The solvent is one or two of chloroform, methanol and ethyl acetate.

8. The preparation method according to claim 1, characterized in that: The temperature of the ice water bath is 1°C to 4°C.

9. The preparation method according to claim 1, characterized in that: The ultrasonic frequency of the first ultrasound is 16kHz to 22kHz, and the time is 15min to 20min.

10. A polysaccharide liposome prepared by the preparation method according to any one of claims 1 to 9.

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