A kind of yellow coral polysaccharide liposome and preparation method thereof

Abstract

The present invention discloses a polysaccharide liposome of yellow coral fungus and a preparation method thereof, belonging to the technical field of polysaccharide liposome materials. 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, and the yellow coral fungus polysaccharide is coated in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. Rotary evaporation is performed to obtain a suspension, and the suspension is subjected to a secondary ultrasonic treatment and filtered to obtain the yellow coral fungus polysaccharide liposome. 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, and reduces the dosage and frequency of medication.

Description

Technical Field

[0001] The present 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] Liposomes are a new type of targeted drug delivery vehicle. Polysaccharide liposomes are typically composed primarily of cholesterol and phosphatidylcholine. They offer multiple advantages as targeted drug formulations, including the ability to delay drug action, increase efficacy, reduce side effects, and improve drug distribution within the body. They are a prime example of targeted drug formulations. Currently, liposomes have become a hot topic of research in the pharmaceutical, food, and cosmetics industries.

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

[0004] The present invention provides a polysaccharide liposome of yellow coral mushroom 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 resulting in poor drug efficacy. The present invention uses the polysaccharide encapsulation rate as an evaluation index to obtain an optimal preparation method of the yellow coral mushroom liposome, and simulates the drug release of the yellow coral mushroom polysaccharide liposome in the human body through an in vitro drug release experiment, thereby providing a yellow coral mushroom 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] Lecithin, cholesterol and lauryl amine are dissolved in a solvent to obtain an organic phase; yellow coral polysaccharide and phosphate buffer are added to the organic phase, and the mixture is ultrasonicated once in an ice-water bath to coat the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation is performed to obtain a suspension, and the suspension is ultrasonicated twice and filtered to obtain yellow coral polysaccharide liposomes.

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

[0008] As a preferred embodiment, the mass ratio of the polysaccharide of Corallii serrata 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 0.45μm and 0.22μm 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 the sonication treatment lasts 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 embodiment, the ultrasonic frequency of the first ultrasound is 16kHz to 22kHz, and the time 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 polysaccharide liposome of yellow coral fungus and a preparation method thereof. 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, and the yellow coral fungus polysaccharide is coated in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The suspension is rotary evaporated to obtain a suspension, and the suspension is subjected to a secondary ultrasonic treatment and filtered to obtain the yellow coral fungus polysaccharide liposome. 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 the body, thereby affecting the efficacy of the polysaccharide liposomes. DETAILED DESCRIPTION

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

[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 the body, etc. when acting on the human body, preparing polysaccharide drugs into polysaccharide liposomes can make up for the shortcomings of yellow coral polysaccharide in the human body to a certain extent, thereby prolonging the retention time of yellow coral polysaccharide in the blood, improving the utilization of yellow coral 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 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 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 Coral Gnaphalium polysaccharide, comprising the following steps:

[0022] Lecithin, cholesterol and lauryl amine are dissolved in a solvent (one or two of chloroform, methanol and ethyl acetate) to obtain an organic phase; yellow coral polysaccharide and phosphate buffer are added to the organic phase, and the mixture is ultrasonicated once in an ice-water bath at 1°C to 4°C to coat the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and rotary evaporation is performed to obtain a suspension, and the suspension is ultrasonicated twice and filtered to obtain yellow coral polysaccharide liposomes.

[0023] In the above technical solution, 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 of the yellow coral fungus in the blood, improving the utilization of the polysaccharide of the yellow coral fungus, 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. When the values ​​are taken within the limited range of the above mass ratios, 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 poor, 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 occur, 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 efficiency 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 yellow coral fungus polysaccharide liposomes, 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 sequence to obtain yellow coral fungus polysaccharide liposomes.

[0028] To further ensure the uniformity of the particle size of the yellow coral polysaccharide liposomes, the second ultrasound is programmed ultrasound using an ultrasonic cell disruptor, with ultrasound at 20kHz to 25kHz 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 treatment 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 liposomes may be destroyed, causing leakage of the drug (yellow coral polysaccharide).

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

[0030] Example 1

[0031] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 65°C water to remove excess chloroform and methanol. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0033] Example 2

[0034] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 1°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0036] Example 3

[0037] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 mixture of 60 mL of methanol to obtain an organic phase; 40 mg of yellow coral polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 22 kHz in a 2°C ice-water bath for 20 min. The yellow coral polysaccharide was coated in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine, and the mixture was rotary evaporated in 62°C water to remove excess methanol. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 22 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 3 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0039] Example 4

[0040] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 18 kHz for 18 min in a 3°C ice-water bath to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 25 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 3 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0042] Example 5

[0043] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 mixture of 72 mL of methanol to obtain an organic phase; 40 mg of yellow coral polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 18 kHz for 20 min in a 4°C ice-water bath to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess methanol. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0045] Example 6

[0046] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 3°C ice-water bath for 16 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess methanol and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 24 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0048] Example 7

[0049] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and ultrasonic treatment was performed at 16 kHz in a 2°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 25 kHz for 5 s, with an interval of 5 s, and ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0051] Example 8

[0052] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 22 kHz in a 4°C ice-water bath for 15 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 3 s, with an interval of 7 s, and ultrasonicated twice for 3 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0054] Example 9

[0055] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 1°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0057] Example 10

[0058] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 1°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0060] Example 11

[0061] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 1°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0063] Example 12

[0064] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 1°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0066] Example 13

[0067] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 1°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0069] Example 14

[0070] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 20 kHz in a 1°C ice-water bath for 18 min to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and ethyl acetate. After the mixture became a colloid, 12 mL of PBS was added and 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 ultrasonicated twice for 4 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0072] In order to further illustrate the technical effects of the present invention, the present invention also provides 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 Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and methanol. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral 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 Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and lauryl amine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and methanol. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0081] Comparative Example 3

[0082] Compared with Example 1, the difference is that laurylamine is not added.

[0083] A method for preparing polysaccharide liposomes of Coral Gnaphalium spp. comprises 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 polysaccharide and 8 mL of phosphate buffer (PBS) were added to the organic phase, and the mixture was ultrasonically treated at 16 kHz for 15 min in a 4°C ice-water bath to encapsulate the yellow coral polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol and laurylamine. The mixture was rotary evaporated in 60°C water to remove excess chloroform and methanol. After the mixture became a colloid, 12 mL of PBS was added and rotary evaporation was continued for 15 min to completely remove the organic solvent to obtain a suspension. The suspension was ultrasonicated at 25 kHz for 3 s, with an interval of 7 s, and ultrasonicated twice for 5 min. The suspension was filtered using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain yellow coral polysaccharide liposomes.

[0085] The performance of the polysaccharide liposomes of the yellow coral fungus prepared in Examples 1 to 14 and Comparative Examples 1 to 3 was tested. After testing, the encapsulation efficiency and drug release of the polysaccharide liposomes of the yellow coral fungus in Examples 1 to 14 of the present invention were similar. Therefore, the polysaccharide liposomes of the yellow coral fungus in 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 the polysaccharide liposomes of the yellow coral fungus by the 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 evenly and let stand. Then, add 3 mL of normal saline thereto, 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 and add 1 mL of normal saline thereto, and determine the polysaccharide content in the sample by the phenol-concentrated sulfuric acid method, 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, the mixture was 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 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. The result was the total polysaccharide content of the yellow coral fungus in the sample. The encapsulation efficiency of the polysaccharide liposome of the yellow coral fungus was calculated as follows. 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 polysaccharide and the fully dissolved yellow coral polysaccharide solution were accurately aspirated and placed in three parallel groups. Each was placed in a dialysis bag and then placed in an Erlenmeyer flask containing 500 mL of phosphate buffer (pH 7.4). The flask was placed in a shaker set at (36 + 0.5) ° C. 5 mL of sample was removed after 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, and 36 h. The phenol-sulfuric acid method was used for determination in a UV spectrophotometer, and the cumulative release percentage of the yellow coral polysaccharide at 12 h and 24 h was calculated. The results are shown in Table 1.

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

[0092] Encapsulation efficiency / % 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 efficiency and drug release performance. The encapsulation efficiency of the polysaccharide liposomes of Examples 1 to 8 of the present invention can reach 78.2%, the in vitro drug release rate of 12 hours is only 8.5%, and the drug release amount of 24 hours is 84.6%. Comparative Examples 1 to 3 are based on Example 1, respectively reducing the amount of lauryl amine from 80 mg to 20 mg (Comparative Example 1), increasing it to 200 mg (Comparative Example 2), and removing lauryl amine (Comparative Example 3), and comparing the drug release properties of the polysaccharide raw material of the polysaccharide raw material of Comparative Example 4. The results show that the encapsulation efficiency of the polysaccharide liposomes of Comparative Examples 1 and Comparative Example 2 is lower than that of Example 1, and the drug release rate of 12 hours is as high as 50.2%. At 24 hours, the drug is basically completely released. When no lauryl amine is added in Comparative Example 3, the polysaccharide drug in the polysaccharide liposomes of the polysaccharide raw material ...

[0094] In summary, the present invention introduces lauryl amine into the polysaccharide liposome structure, thereby effectively regulating the drug release of the yellow coral fungus polysaccharide, prolonging the retention time of the yellow coral fungus polysaccharide in the blood, improving the utilization of the yellow coral fungus polysaccharide, reducing the dosage and frequency of medication, and effectively solving 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 may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing polysaccharide liposomes of Coral Gnaphalium spp., characterized in that: The following steps are involved: Dissolving lecithin, cholesterol, and lauryl amine in a solvent to obtain an organic phase; adding chrysogenum polysaccharide and phosphate buffer to the organic phase, sonicating once in an ice-water bath, and coating the chrysogenum polysaccharide in a liposome encapsulation material formed by lecithin, cholesterol, and lauryl amine to obtain chrysogenum polysaccharide liposomes; the mass ratio of the chrysogenum polysaccharide to lauryl amine is 1:0.6-4.8, and the mass ratio of the chrysogenum polysaccharide to lecithin and cholesterol is 1:10-50:3-6; 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.

2. The preparation method according to claim 1, characterized in that The mass ratio of the polysaccharide of the yellow coral fungus to lauryl amine is 1:1.0~3.

0.

3. 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 using 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain the polysaccharide liposomes of the fungus.

4. The preparation method according to claim 3, characterized in that The second sonication is programmed sonication using an ultrasonic cell disruptor, with sonication at 20 kHz to 25 kHz for 3 seconds to 5 seconds, with an interval of 5 seconds to 7 seconds, and the sonication treatment lasts for 3 minutes to 5 minutes.

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

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

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

8. A polysaccharide liposome prepared by the preparation method according to any one of claims 1 to 7.

Images