Preparation method and preservation method of Yulangsan polysaccharide liposome

Abstract

The invention relates to the technical field of polysaccharide preparation, in particular to a preparation method and a preservation method of Yulangsan polysaccharide liposome, and the preparation method comprises the following steps: (1) preparing Yulangsan polysaccharide thick paste, (2) refining Yulangsan polysaccharide, and (3) preparing the Yulangsan polysaccharide liposome. The Yulangsan polysaccharide liposome obtained by the method disclosed by the invention is small in particle size and high in encapsulation efficiency, conforms to the state-specified hemolysis standard, and can provide a reference basis for intravenous administration.

Description

[Technical field]

[0001] The invention belongs to the technical field of polysaccharide preparation, and particularly relates to a preparation method of Yulangsan polysaccharide liposome and a preservation method thereof. [Background technology]

[0002] Yulangsan (YLS, also known as Longyanshen), the original plant is verified to be the tuberous root of Millettia sparse-leaved, a plant of Fabaceae, and is a commonly used medicinal material. The present invention has conducted a large number of pharmaceutical and pharmacodynamic studies on Yulangsan. The experiment found that the water extract, alcohol extract and total flavonoids of Yulangsan can permanently and stably reduce essential hypertension, significantly inhibit myocardial contractility, reduce myocardial oxygen consumption, increase coronary flow and reduce myocardial ischemic damage. Yulangsan polysaccharide is an active substance extracted from Yulangsan. Studies have found that Yulangsan polysaccharide has a more obvious anti-senile dementia and obvious inhibition of liver fibrosis, tumors and other effects.

[0003] However, in actual clinical applications, in many cases, polysaccharide drugs cannot achieve the expected ideal pharmacological activity in the body. This is mainly because most polysaccharide drugs have been metabolized before reaching the lesion in the body, and this type of drug does not have targeting to the lesion. Therefore, how to make polysaccharide drugs metabolized as little as possible before reaching the lesion, and how to "target" to the lesion to obtain the ideal clinical effect, has become a common problem that urgently needs to be solved in this technical field.

[0004] Liposome drug delivery systems have attracted widespread attention due to their good effects in clinical applications. However, their preparation, especially the preparation of polysaccharide liposomes with high encapsulation efficiency, is still a recognized problem. It is even more difficult to obtain polysaccharide liposomes with high drug encapsulation efficiency and small particle size.

[0005] The prior art has not yet conducted relevant research on the preparation of Yulangsan polysaccharide liposomes, and the present invention arises at the historic moment. [Summary of the invention]

[0006] In view of the above, it is necessary to provide a preparation method of Yulangsan polysaccharide liposomes and a preservation method thereof. The Yulangsan polysaccharide liposomes obtained by the method of the present invention have a small particle size and a high encapsulation rate, meet the national hemolysis standards, and can provide a reference for intravenous administration.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for preparing Yulangsan polysaccharide liposomes, the method comprising the following steps:

[0009] (1) Preparing a thick paste of Yulangsan polysaccharide: crushing the Yulangsan slices, adding them to water and boiling them for 2-4 hours, then filtering, repeating the above method for 3-4 times, combining the filtrates obtained several times, then letting the filtrates stand and taking the supernatant to concentrate under reduced pressure, then adding ethanol, mixing them thoroughly and letting them stand for 8-12 hours, filtering with suction, taking the precipitate and washing it with ethanol for 2-4 times, then concentrating and drying, and then obtaining a thick paste of Yulangsan polysaccharide;

[0010] (2) Refining of Yulangsan polysaccharide: Take a thick paste of Yulangsan polysaccharide and add it to distilled water to dissolve it, then add papain solution, treat it at 50-60°C for 3-5 hours, inactivate the enzyme in a boiling water bath for 4-6 minutes, then centrifuge to remove the precipitate, centrifuge again, remove free protein from the obtained supernatant, separate the organic phase, centrifuge the aqueous phase, and then take the supernatant and concentrate it under reduced pressure, add ethanol and let it stand for 10-14 hours, filter it, wash the precipitate with ethanol and methanol for 2-3 times each, and dry it to obtain Yulangsan polysaccharide;

[0011] (3) Preparation of Yulangsan polysaccharide liposomes: soybean lecithin, cholesterol and Tween 80 were dissolved in chloroform and methanol solutions, and ultrasonic dissolution was performed to obtain an organic phase. The organic solvent was removed by evaporation under reduced pressure at 35-45°C. After a lipid film was formed, chloroform was added to dissolve the liposomes. Yulangsan polysaccharide was dissolved in phosphate buffer as the aqueous phase. The aqueous phase was then introduced into the organic phase reaction vessel. Ultrasonic emulsification was performed in an ice-water bath to form a stable milky white W / O type emulsion. After evaporation under reduced pressure to form a colloidal state, phosphate buffer was added. The organic solvent was removed by rotary evaporation to form an aqueous suspension. The suspension was then placed in an ultrasonic cell crusher for ultrasonic treatment. The suspension was then extruded through 0.45 μm and 0.22 μm microporous filter membranes to obtain Yulangsan polysaccharide liposomes.

[0012] In the present invention, further, the step (1) of adding ethanol after reduced pressure concentration specifically adds 95% ethanol, and then uses an alcohol meter to adjust the ethanol concentration in the concentrate to 80%.

[0013] In the present invention, further, in the step (2), before adding the thick paste of Yulangsan polysaccharide to distilled water, the thick paste of Yulangsan polysaccharide is first ground and crushed using a mortar.

[0014] In the present invention, further, in the step (2), during the dissolution of the thick paste of Yulangsan polysaccharide in distilled water, the thick paste is heated in a boiling water bath for 0.5-1.5h and ultrasonic dissolution is performed.

[0015] In the present invention, further, the step (2) of adding ethanol and letting it stand specifically includes adding 95% ethanol to make the ethanol concentration be 80%, and then letting it stand for 12 hours.

[0016] In the present invention, further, the ultrasonic cell pulverizer in step (3) performs ultrasonic treatment with an ultrasonic intensity of 62%, 5.0s on, 5.0s off, a temperature of 4°C, and an action time of 5 minutes.

[0017] In the present invention, further, in the step (3), the mass ratio of soybean lecithin to cholesterol is 6:1, the mass ratio of soybean lecithin to Yulangsan polysaccharide is 20:1, and the mass ratio of soybean lecithin to Tween 80 is 4:1.

[0018] In the present invention, further, the ratio of the organic phase to the aqueous phase in step (3) is 5:1.

[0019] In the present invention, further, in step (3), the ice-water bath ultrasonic emulsification is carried out in an ice-water bath at 4° C. for 15 minutes; when the organic solvent is removed by rotary evaporation, the water bath temperature is controlled to be 55° C.

[0020] The present invention also provides a method for preserving Yulangsan polysaccharide liposomes. The method comprises the following steps: preparing the obtained Yulangsan polysaccharide liposomes into Yulangsan polysaccharide freeze-dried liposomes, and specifically preparing the Yulangsan polysaccharide liposomes into a Yulangsan polysaccharide suspension with a concentration of 1 mg / mL, placing lactose in a culture medium, adding the Yulangsan polysaccharide suspension, placing the culture medium in a refrigerator and freezing the culture medium at -20°C for 12 hours, then placing the culture medium in a freeze dryer and freeze drying the culture medium for 48 hours, and then taking the culture medium out and preserving the culture medium at 4°C, wherein the amount of the added lactose accounts for 5% of the volume of the Yulangsan polysaccharide suspension.

[0021] The present invention also provides a preparation method of Yulangsan polysaccharide freeze-dried liposomes, which specifically comprises the following steps: preparing Yulangsan polysaccharide liposomes into a Yulangsan polysaccharide suspension with a concentration of 1 mg / mL, placing lactose in a culture medium, adding the Yulangsan polysaccharide suspension, placing the culture medium in a refrigerator and freezing the culture medium at -20°C for 12 hours, then placing the culture medium in a freeze dryer for freeze drying for 48 hours, taking the culture medium out and storing the culture medium in a refrigerator at 4°C, wherein the amount of lactose added accounts for 5% of the volume of the Yulangsan polysaccharide suspension.

[0022] The present invention has the following beneficial effects:

[0023] The Yulangsan polysaccharide liposomes obtained by the method of the present invention have a small particle size and a high encapsulation rate, and are verified by experiments to meet the national hemolysis standards, which can provide a reference basis for intravenous administration and has a very positive significance.

[0024] In addition, in order to better preserve the obtained Yulangsan polysaccharide liposomes, the applicant further prepared them into Yulangsan polysaccharide freeze-dried liposomes to meet the requirements of long-term stability of drug preparations, and stored them at 4°C. At this time, the particle size, potential, and PDI of the liposomes changed little, and the properties of the liposomes were relatively stable.

Brief Description of the Drawings

[0025] Figure 1 This is a picture of the Yulangsan polysaccharide of Example 2 of the present invention under an optical microscope;

[0026] Figure 2 It is the in vitro release curve of Yulangsan polysaccharide and Yulangsan polysaccharide liposome;

[0027] Figure 3 This is the appearance photo of Yulangsan polysaccharide freeze-dried liposomes;

[0028] Figure 4 This is a photo of the hemolytic test results. [Specific implementation method]

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0030] Embodiment 1:

[0031] This embodiment provides a method for preparing Yulangsan polysaccharide liposomes, which comprises the following steps:

[0032] (1) Preparation of Yulangsan polysaccharide paste: 500 g of Yulangsan slices were taken and pulverized by a high-speed multifunctional pulverizer (30,000 rpm, 2 minutes), added to 5 L of water and boiled (100° C.) for 2 h, then filtered through white gauze, and the residue was repeated 3 times in the above manner, and the filtrates obtained several times were combined to obtain a brown-yellow extraction filtrate. The filtrate was then allowed to stand overnight (12 h), and the extract (obvious stratification phenomenon occurred) was centrifuged at 3900 r / min for 20 min in a centrifuge to obtain a supernatant, and the supernatant was then placed in a rotary evaporator for vacuum distillation to obtain a concentrate accounting for 60% of the volume of the supernatant, cooled, and then 95% ethanol was added, and the ethanol concentration of the concentrate was adjusted to 80% with a (70 degree) alcohol meter, and the mixture was fully stirred and mixed, and allowed to stand for 8 h, and filtered, and the precipitate was washed twice with 95% ethanol, and evaporated in a 70° C. forced air drying oven to obtain a Yulangsan polysaccharide paste;

[0033] (2) Refining of Yulangsan polysaccharide: Take the thick paste of Yulangsan polysaccharide and add it into distilled water to dissolve it. Because the polysaccharide is difficult to dissolve in water, first grind the thick paste of Yulangsan polysaccharide in a mortar, then add distilled water, heat it in a boiling water bath for 0.5 h, and use ultrasound to dissolve it to increase its solubility in water. After the Yulangsan polysaccharide is fully dissolved, let it cool, then add 10g / L papain solution, treat it at 5℃ for 3h, inactivate the enzyme in a boiling water bath for 4min, let it cool, and centrifuge it in a centrifuge at 10000r / min (speed) for 1 minute. 0min, discard the precipitate, centrifuge again, and use the sevag method to remove free protein again in the supernatant, that is, add chloroform-n-butanol (4:1) and reflux in a water bath for 45min, twice in total, separate the organic phase with a separatory funnel, centrifuge the aqueous phase in a centrifuge at 10000r / min for 10min, concentrate the supernatant under reduced pressure, add 95% ethanol to make the alcohol concentration 80%, let stand for 10h, filter, wash the precipitate with 95% ethanol and methanol three times each, and dry at 70℃ to obtain Yulangsan polysaccharide;

[0034] (3) Preparation of Yulangsan polysaccharide liposomes: soybean lecithin, cholesterol and Tween 80 were dissolved in chloroform and methanol solution (mass ratio of 1:2), and the organic phase was obtained by ultrasonic dissolution. The organic solvent was removed by reduced pressure evaporation at 35°C. After the lipid film was formed, a small amount of chloroform (about 2.0 ml for 2 g soybean lecithin) was added to dissolve it; Yulangsan polysaccharide was dissolved in phosphate buffer (PBS: accurately weigh 0.5001 g of PBS dry powder, add 50.0 ml of distilled water and mix well, and adjust the pH to 7.3) as the aqueous phase, wherein the solid-liquid ratio of Yulangsan polysaccharide to phosphate buffer was 1 g: 200 mL; then the aqueous phase was introduced into the organic phase reaction vessel and placed in an ice-water bath at 4°C. Ultrasonic emulsification treatment for 15 minutes forms a stable milky white W / O type emulsion, after reduced pressure evaporation to form a colloidal state, phosphate buffer is added, and the organic solvent is removed by rotary evaporation. The water bath temperature is controlled to be 55°C to form an aqueous suspension, and then placed in an ultrasonic cell crusher for ultrasonic treatment (ultrasonic intensity is 62%, 5.0s on, 5.0s off, temperature 4°C, 5min), and then the suspension is squeezed through 0.45μm and 0.22μm microporous filter membranes respectively to obtain Yulangsan polysaccharide liposomes, which are placed in 10mlPE round bottom centrifuge tubes with lids (centrifuge tubes are made of transparent polypropylene PP, which are widely used for sample preparation, centrifugation and storage), and stored in a refrigerator at 4°C. Above, the mass ratio of soybean lecithin to cholesterol is 6:1, the mass ratio of soybean lecithin to Yulangsan polysaccharide is 20:1, and the mass ratio of soybean lecithin to Tween 80 is 4:1; the organic phase is chloroform and methanol according to the composition, and the ratio of organic phase to aqueous phase is 5:1.

[0035] Embodiment 2:

[0036] This embodiment provides a method for preparing Yulangsan polysaccharide liposomes, which comprises the following steps:

[0037] (1) Preparation of Yulangsan polysaccharide paste: 500 g of Yulangsan slices were taken and pulverized by a high-speed multifunctional pulverizer (30,000 rpm, 2 minutes), added to 5 L of water and boiled (100° C.) for 3 h, then filtered through white gauze, and the filter residue was repeated 3 times in the above manner, and the filtrates obtained several times were combined to obtain a brown-yellow extraction filtrate. The filtrate was then allowed to stand overnight (12 h), and the extract (obvious stratification phenomenon occurred) was centrifuged in a centrifuge at 3900 r / min for 20 min to obtain a supernatant, and the supernatant was placed in a rotary evaporator for vacuum distillation to obtain a concentrate accounting for 62% of the volume of the supernatant, cooled, and then 95% ethanol was added, and the ethanol concentration of the concentrate was adjusted to 80% with an alcohol meter (80 degrees), fully stirred and mixed, and allowed to stand for 10 h, and filtered, and the precipitate was washed 2-4 times with 95% ethanol, and evaporated in a 70° C. forced air drying oven to obtain a Yulangsan polysaccharide paste;

[0038] (2) Refining of Yulangsan polysaccharide: Take the thick paste of Yulangsan polysaccharide and add it into distilled water to dissolve it. Because the polysaccharide is difficult to dissolve in water, first grind the thick paste of Yulangsan polysaccharide into powder using a mortar, then add distilled water, heat it in a boiling water bath for 1 hour, and use ultrasound to dissolve it to increase its solubility in water. After the Yulangsan polysaccharide is fully dissolved, cool it down, then add 10g / L papain solution, treat it at 55℃ for 4 hours, inactivate the enzyme in a boiling water bath for 5 minutes, cool it down, and centrifuge it in a centrifuge at 10000r / min (speed) for 1 minute. 0min, discard the precipitate, centrifuge again, and remove free protein again by the sevag method of the supernatant, that is, add chloroform-n-butanol (4:1) and reflux in a water bath for 45min, twice in total, separate the organic phase with a separatory funnel, centrifuge the aqueous phase in a centrifuge at 10000r / min for 10min, concentrate the supernatant under reduced pressure, add 95% ethanol to make the alcohol concentration 80%, let stand for 12h, filter, wash the precipitate with 95% ethanol and methanol 3 times each, and dry at 70℃ to obtain Yulangsan polysaccharide;

[0039] (3) Preparation of Yulangsan polysaccharide liposomes: soybean lecithin, cholesterol and Tween 80 were dissolved in chloroform and methanol solution (mass ratio of 1:2), and the organic phase was obtained by ultrasonic dissolution. The organic solvent was removed by reduced pressure evaporation at 40°C. After the lipid film was formed, a small amount of chloroform (about 2.0 ml for 2 g soybean lecithin) was added to dissolve it; Yulangsan polysaccharide was dissolved in phosphate buffer (PBS: accurately weigh 0.5001 g of PBS dry powder, add 50.0 ml of distilled water and mix well, and adjust the pH to 7.3) as the aqueous phase, wherein the solid-liquid ratio of Yulangsan polysaccharide to phosphate buffer was 1 g: 200 mL; then the aqueous phase was introduced into the organic phase reaction container and ultrasonically heated in an ice-water bath at 4°C. The sonic emulsification treatment was carried out for 15 minutes to form a stable milky white W / O type emulsion, and a phosphate buffer was added after the reduced pressure evaporation formed a colloidal state, and the organic solvent was removed by rotary evaporation. The water bath temperature was controlled to be 55°C to form an aqueous suspension, and then placed in an ultrasonic cell crusher for ultrasonic treatment (ultrasonic intensity was 62%, 5.0s on, 5.0s off, temperature 4°C, and 5min), and then the suspension was squeezed through 0.45μm and 0.22μm microporous filter membranes respectively to obtain Yulangsan polysaccharide liposomes, which were placed in a 10ml PE round bottom centrifuge tube with a lid (the centrifuge tube was made of transparent polypropylene PP, which was widely used for sample preparation, centrifugation and storage), and stored in a refrigerator at 4°C. In the above, the mass ratio of soybean lecithin to cholesterol was 6:1, the mass ratio of soybean lecithin to Yulangsan polysaccharide was 20:1, and the mass ratio of soybean lecithin to Tween 80 was 4:1; the organic phase was chloroform and methanol according to the composition, and the ratio of the organic phase to the aqueous phase was 5:1.

[0040] Embodiment 3:

[0041] This embodiment provides a method for preparing Yulangsan polysaccharide liposomes, which comprises the following steps:

[0042] (1) Preparation of Yulangsan polysaccharide paste: 500 g of Yulangsan slices were taken and pulverized by a high-speed multifunctional pulverizer (30,000 rpm, 2 minutes), added to 5 L of water and boiled (100° C.) for 4 h, then filtered through white gauze, and the residue was repeated 4 times in the above manner, and the filtrates obtained several times were combined to obtain a brown-yellow extraction filtrate. The filtrate was then allowed to stand overnight (12 h), and the extract (obvious stratification phenomenon occurred) was centrifuged at 3900 r / min for 20 min to obtain a supernatant, and the supernatant was placed in a rotary evaporator for reduced pressure distillation to obtain a concentrate accounting for 65% of the volume of the supernatant, cooled, and then 95% ethanol was added, and the ethanol concentration of the concentrate was adjusted to 80% with a (90 degree) alcohol meter, and the mixture was fully stirred and mixed, and allowed to stand for 12 h, and filtered, and the precipitate was washed 4 times with 95% ethanol, and evaporated in a 70° C. forced air drying oven to obtain a Yulangsan polysaccharide paste;

[0043] (2) Refining of Yulangsan polysaccharide: Take the thick paste of Yulangsan polysaccharide and add it into distilled water to dissolve it. Because the polysaccharide is difficult to dissolve in water, first grind the thick paste of Yulangsan polysaccharide in a mortar and then add distilled water, heat it in a boiling water bath for 1.5 hours, and use ultrasound to dissolve it to increase its solubility in water. After the Yulangsan polysaccharide is fully dissolved, cool it down, then add 10g / L papain solution, treat it at 60℃ for 5 hours, inactivate the enzyme in a boiling water bath for 6 minutes, cool it down, and centrifuge it in a centrifuge at 10000r / min (speed) for 10 minutes. , discard the precipitate, centrifuge again, and use the sevag method to remove free protein again in the supernatant, that is, add chloroform-n-butanol (4:1) and reflux in a water bath for 45 minutes, twice in total, separate the organic phase with a separatory funnel, centrifuge the aqueous phase in a centrifuge at 10000r / min for 10 minutes, concentrate the supernatant under reduced pressure, add 95% ethanol to make the alcohol concentration 80%, let stand for 14 hours, filter, wash the precipitate with 95% ethanol and methanol three times each, and dry at 70°C to obtain Yulangsan polysaccharide; use an optical microscope (such as Figure 1 As shown, it was observed under a 40×0.65x inverted microscope. Under the microscope, the liposomes were round or oval in shape, with a transparent aperture on the outer ring. The liposomes were light yellow emulsions, evenly dispersed, and without flocculation.

[0044] (3) Preparation of Yulangsan polysaccharide liposomes: soybean lecithin, cholesterol and Tween 80 were dissolved in chloroform and methanol solution (mass ratio of 1:2), and the organic phase was obtained by ultrasonic dissolution. The organic solvent was removed by reduced pressure evaporation at 45°C. After the lipid film was formed, a small amount of chloroform (about 2.0 ml for 2 g soybean lecithin) was added to dissolve it; Yulangsan polysaccharide was dissolved in phosphate buffer (PBS: accurately weigh 0.5001 g of PBS dry powder, add 50.0 ml of distilled water and mix well, and adjust the pH to 7.3) as the aqueous phase, wherein the solid-liquid ratio of Yulangsan polysaccharide to phosphate buffer was 1 g: 200 mL; then the aqueous phase was introduced into the organic phase reaction container and ultrasonically heated in an ice-water bath at 4°C. The sonic emulsification treatment was carried out for 15 minutes to form a stable milky white W / O type emulsion, and a phosphate buffer was added after the reduced pressure evaporation formed a colloidal state, and the organic solvent was removed by rotary evaporation. The water bath temperature was controlled to be 55°C to form an aqueous suspension, and then placed in an ultrasonic cell crusher for ultrasonic treatment (ultrasonic intensity was 62%, 5.0s on, 5.0s off, temperature 4°C, and 5min), and then the suspension was squeezed through 0.45μm and 0.22μm microporous filter membranes respectively to obtain Yulangsan polysaccharide liposomes, which were placed in a 10ml PE round bottom centrifuge tube with a lid (the centrifuge tube was made of transparent polypropylene PP, which was widely used for sample preparation, centrifugation and storage), and stored in a refrigerator at 4°C. In the above, the mass ratio of soybean lecithin to cholesterol was 6:1, the mass ratio of soybean lecithin to Yulangsan polysaccharide was 20:1, and the mass ratio of soybean lecithin to Tween 80 was 4:1; the organic phase was chloroform and methanol according to the composition, and the ratio of the organic phase to the aqueous phase was 5:1.

[0045] Embodiment 4:

[0046] The present embodiment provides a method for preserving Yulangsan polysaccharide liposomes, the method comprising preparing the Yulangsan polysaccharide liposomes obtained in any one of Examples 1-3 into Yulangsan polysaccharide freeze-dried liposomes, specifically comprising: preparing the Yulangsan polysaccharide liposomes into a Yulangsan polysaccharide suspension with a concentration of 1 mg / mL, placing lactose in a culture medium, adding the Yulangsan polysaccharide suspension, placing the culture medium in a refrigerator and freezing it at -20°C for 12 hours, then placing the culture medium in a freeze dryer and freeze drying for 48 hours, taking the culture medium out and storing the culture medium at 4°C, wherein the amount of lactose added accounts for 5% of the mass of the Yulangsan polysaccharide suspension.

[0047] Test example:

[0048] In order to illustrate the effect of this application, the applicant conducted the following experiments:

[0049] Experiment 1: Testing the effect of Yulangsan polysaccharide liposomes under different preparation methods

[0050] 1. Raw materials, reagents and instruments:

[0051] Refined Yulangsan polysaccharide (obtained in the manner of Example 2); methanol (AR, analytical grade, Chengdu Cologne Chemicals Co., Ltd., batch number 2022030702); chloroform (AR, Chengdu Cologne Chemicals Co., Ltd., batch number 2021080901); Tween 80 (AR, Solarbio, batch number T8360-100ml); soybean lecithin (AR, Shanghai McLean Biochemical Technology Co., Ltd., batch number L812367-25g); cholesterol (AR, Shanghai Aladdin Biochemical Co., Ltd., batch number C104028-25g); 10% ammonium sulfate (Solarbio, batch number A1030-1ml); PBS (AR, Solarbio, batch number P1010); dialysis bag 8000-14000 (Shanghai Yuanye Biotechnology Co., Ltd., batch number MD1425-5m).

[0052] Instruments: Desktop high-speed refrigerated centrifuge H2050R (Xiangyi); ZetasizerNano nanoparticle size analyzer, Malvern Instruments Ltd., UK; SCIENTZ-ⅡD ultrasonic cell crusher, Ningbo Xinzhi Biotechnology Co., Ltd.; rotary evaporator RE-52AA, Shanghai Xiande Experimental Instrument Co., Ltd.; YRDLSB-6\20 low-temperature coolant circulation pump, Shanghai Yarong Biochemical Instrument Factory; ZRD-5210 fully automatic new type blast drying oven, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; inverted microscope (10×0.25, 40×0.65) Olympus (China) Co., Ltd.; SB-120D ultrasonic cleaning machine, Ningbo Xinzhi Biotechnology Co., Ltd.

[0053] 2. Preparation method:

[0054] 2.1 Thin film dispersion method

[0055] Most of the steps are the same as those in Example 2, except that: the organic phase is placed in a 250 mL round-bottom flask, and the organic solvent is removed by reduced pressure evaporation at 40° C. When a uniform thin film is formed on the wall of the flask, 5.0 mL of a PBS (pH 7.3) solution containing 2 mg / mL Yulangsan polysaccharide is introduced to elute the thin film on the wall of the flask; hydration at 25° C. for 2 h; ultrasonic treatment with a probe (intensity 62%, 5.0 s on, 5.0 s off, 4° C., action 5 min) is performed to fully and uniformly integrate the Yulangsan polysaccharide into the liposome, and also to make the particle size of the Yulangsan polysaccharide liposome more uniform, thereby obtaining a Yulangsan polysaccharide liposome suspension; and then extruding it through a microporous filter membrane with a pore size of 0.45 μm and 0.22 μm, and repeating three times to obtain a Yulangsan polysaccharide liposome solution.

[0056] 2.2 Ethanol injection method

[0057] Most of the steps are the same as those in Example 2, except that: 5.0 mL of PBS (pH 7.3) solution containing 2 mg / mL Yulangsan polysaccharide is slowly introduced into the organic phase under the shaking of a 50° C. water bath shaker, and the shaking is continued at 50° C. for 30 min; the mixture is placed in a 250 mL round-bottom flask, and anhydrous ethanol is removed by evaporation under reduced pressure at 50° C., and the mixture is ultrasonically treated with a probe (intensity 62%, 5.0 s on, 5.0 s off, 4° C., and 5 min) to fully and evenly integrate the Yulangsan polysaccharide into the liposomes, and also to make the particle size of the Yulangsan polysaccharide liposomes more uniform, thereby obtaining a Yulangsan polysaccharide liposome suspension; the mixture is then extruded through a microporous filter membrane with a pore size of 0.45 μm, and the reaction is repeated three times to obtain a Yulangsan polysaccharide liposome solution.

[0058] 2.3 Ultrasonic dispersion method

[0059] Most of the steps are the same as those in Example 2, except that the aqueous phase is added dropwise to the organic phase, the organic solvent is removed by rotary evaporation, and then the liposomes are treated with ultrasound for 30 minutes to obtain the polysaccharide liposomes of Yulangsan, and then filtered with a 0.45 μm microporous filter membrane to obtain the polysaccharide liposome suspension of Yulangsan. Ultrasonic instrument, specifications, performance, etc.: SB-120D ultrasonic cleaning machine, Ningbo Xinzhi Biotechnology Co., Ltd., ultrasonic frequency: 40Khz, ultrasonic power: 120W, voltage: 220V / 50Hz.

[0060] 2.4 Ammonium sulfate gradient method

[0061] Most of the steps are the same as those in Example 2, except that: under the shaking of a 50°C water bath shaker, the organic phase is introduced into 5.0 mL of a 10% ammonium sulfate solution, and then placed in a 250 mL round-bottom flask, and the organic solvent is removed by reduced pressure evaporation at 50°C; dialyzed in PBS (pH 7.3) for 24 h; under the shaking of a 50°C water bath shaker, 5.0 mL of a PBS (pH 7.3) solution containing 2 mg / mL Yulangsan polysaccharide is slowly introduced into the organic phase, and the shaking is continued at 50°C for 30 min; then placed in an ultrasonic cell crusher for ultrasonic treatment (intensity 62%, 5.0 s on, 5.0 s off, 0°C, action 30 min), so that the Yulangsan polysaccharide is fully and evenly integrated into the liposome, and the particle size of the Yulangsan polysaccharide is also made more uniform, thereby obtaining a Yulangsan polysaccharide liposome suspension; then extruded through a microporous filter membrane with a pore size of 0.45 μm, repeated three times, to obtain a Yulangsan polysaccharide liposome solution.

[0062] 3. Experiment

[0063] 3.1 Morphological observation: Observe using an optical microscope;

[0064] 3.2 Particle size and its distribution: Malvern nanoparticle size analyzer was used to measure the potential, particle size and polydispersity index (PDI) of the obtained liposomes;

[0065] 3.3 Determination of encapsulation efficiency by high-speed centrifugation: Take 1.0 mL of the prepared Yulangsan polysaccharide liposomes, centrifuge at 13100 r / min for 1 h, set the temperature to 4°C, take 0.1 ml of the supernatant and dilute to 6.0 mL with normal saline, take 1.0 ml and use the phenol-sulfuric acid method to determine the absorbance of free drugs, which is 0.1571, then calculate the mass of free drugs according to the standard curve, and then calculate the encapsulation efficiency according to the formula.

[0066] Blank liposomes were treated in the same manner as above and used as blank control.

[0067] Blank liposomes: 0.4162g soybean lecithin, 0.0531g cholesterol and 0.1001g Tween 80 were dissolved in 12mL chloroform and methanol (1:2) solution, and ultrasonicated for 15min to fully dissolve the organic phase; the organic phase was placed in a 250mL round-bottom flask, and the organic solvent was removed by evaporation at 30℃ under reduced pressure. When a uniform film was formed on the wall of the flask, 20mL PBS (pH7.3) solution was added dropwise to form a two-phase system, and ultrasonicated in an ice-water bath (4℃) to form a stable W / O emulsion, and evaporated at 50℃ under reduced pressure. After the colloid was formed, 10ml The PBS solution was rotary evaporated for 15 min to remove the organic solvent, and then placed in an ultrasonic cell disruptor for ultrasonic treatment (intensity 62%, 5.0 s on, 5.0 s off, 0°C, 30 min) to make the liposome particle size uniform, thereby obtaining a blank liposome suspension; then it was extruded through a microporous filter membrane with a pore size of 0.45 μm, and repeated three times to obtain a blank liposome solution.

[0068] Standard curve: C = 102.66A + 2.083;

[0069] Encapsulation efficiency = ((W 总 -C 游离 ×V×D) / W 总 )×100%;

[0070] C 游离 is the free polysaccharide content; W 总 is the total dosage for preparing liposomes; V is the measurement volume; D is the dilution multiple;

[0071] The appearance, encapsulation efficiency, particle size and other indicators of the polysaccharide liposomes prepared in the above-mentioned methods were compared. The results are shown in Table 1:

[0072] Table 1

[0073]

[0074] Comparing the data obtained by each method, the ammonium sulfate gradient method has the highest encapsulation rate of 89.10%, but its particle size is larger; the reverse evaporation method of Example 2 of the present invention has an encapsulation rate of 84.25%, but its particle size is the smallest. In addition, the ammonium sulfate gradient method is time-consuming, consumes more materials, and has a more complicated process. Considering the cost, effect and convenience of experimental operation, the reverse evaporation method of Example 2 of the present invention is selected to prepare Yulangsan polysaccharide liposomes.

[0075] Experiment 2: The impact of different factors on the results

[0076] 1. Comparison of encapsulation efficiency and drug loading under different raw material composition ratios

[0077] Drug loading W% = (mass of encapsulated Yulangsan polysaccharide liposomes / the sum of the masses of soybean lecithin and cholesterol × 100%;

[0078] 1.1 Comparison of appearance, encapsulation efficiency and drug loading under different ratios of soybean lecithin and cholesterol. Only the cholesterol content was changed according to the ratio. Other methods were the same as Example 2. The results are shown in Table 2:

[0079] Table 2

[0080]

[0081] The results in Table 2 show that too large or too small a ratio of soybean lecithin to cholesterol will reduce the encapsulation efficiency. When the ratio of soybean lecithin to cholesterol is 6:1, the encapsulation efficiency is the highest.

[0082] 1.2 Comparison of the appearance, encapsulation efficiency and drug loading of soybean lecithin and Yulangsan polysaccharide at different ratios. Only the content of Yulangsan polysaccharide was changed according to the ratio. The other methods were the same as those in Example 2. The results are shown in Table 3:

[0083] Table 3

[0084]

[0085] The results in Table 3 show that the encapsulation efficiency will be reduced when the ratio of Yulangsan polysaccharide to soybean lecithin is too large or too small. The encapsulation efficiency is the highest when the ratio of Yulangsan polysaccharide to soybean lecithin is 1:20.

[0086] 2. Comparison of the effects of ultrasound and water bath parameters on the results

[0087] 2.1 Comparison of appearance, encapsulation efficiency and drug loading under different ultrasonic time, wherein the ultrasonic time is the ultrasonic emulsification treatment time in a 4°C ice water bath. Except for the ultrasonic time, the other methods are the same as those in Example 2. The results are shown in Table 4:

[0088] Table 4

[0089]

[0090] The results in Table 4 show that the encapsulation efficiency is the highest when the ultrasound time is 15 min.

[0091] 2.2 Comparison of appearance, encapsulation efficiency and drug loading at different water bath temperatures. The water bath temperature is the water bath temperature for removing the organic solvent by rotary evaporation. Except for the water bath temperature, the other methods are the same as those in Example 2. The results are shown in Table 5:

[0092] Table 5

[0093]

[0094] The results in Table 4 show that a water bath temperature that is too high or too low will reduce the encapsulation efficiency. When the water bath temperature is 55°C, the encapsulation efficiency is the highest.

[0095] Experiment 3: Burst release and in vitro release of Yulangsan polysaccharide and Yulangsan polysaccharide liposomes

[0096] Take Yulangsan polysaccharide liposome YLSPL (mass 1 mg) and Yulangsan polysaccharide YLSPS (1 mg / ml) solution and add them to the dialysis bag respectively to simulate the body fluid environment, and measure the polysaccharide release curve within 96 hours (select neutral PBS (same as above) (pH 7.4) as the release medium, add 3 mL of Yulangsan polysaccharide liposome and Yulangsan polysaccharide solution to the dialysis bag (8000-14000D) (Shanghai Yuanye Biotechnology Co., Ltd., batch number MD1425-5m), and then seal the dialysis bag. The two ends were placed in a conical flask containing 30 mL of release medium and the experiment was carried out in a constant temperature oscillator (37°C, 100 rpm) to simulate the body fluid environment. At the set time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96 h), 3 mL of release solution was extracted into a 10 mL centrifuge tube, and an equal amount of new release medium was added to the conical flask in time. The amount of polysaccharide in the extract was determined using the phenol concentrated sulfuric acid method. (n = 3) Er = cumulative drug release / drug mass × 100%;

[0097] Comparing the cumulative release rate / % of the two, the results are as follows Figure 2 As shown in the figure, there are generally three situations in which drugs are contained in liposomes, namely adsorption, inclusion and embedding. In the in vitro release test, the drug adsorbed on the surface will be released quickly, which is called the burst release effect. The pharmacopoeia requires that the release amount within the first 0.5 hours should be less than 40%. In this experiment, the release rate of Yulangsan polysaccharide liposomes in 0.5 hours was 11.86%, which was less than 40% and met the pharmacopoeia quality standards.

[0098] Depend on Figure 2 The in vitro release curve shows that the Yulangsan polysaccharide solution (YLSPS) is released explosively in the first 6 hours, and the release is complete, and no more release is performed in the later stage. The release of the drug in the Yulangsan polysaccharide liposome (YLSPL) solution has an obvious sustained release effect, and is in a trend of slow and sustained release within 96 hours. Therefore, the present application prepares the Yulangsan polysaccharide liposome, which has positive significance.

[0099] Experiment 4: Preservation of Yulangsan polysaccharide liposomes

[0100] According to the method of Example 4, Yulangsan polysaccharide liposomes were prepared into Yulangsan polysaccharide freeze-dried liposomes, and the effects of the following different raw materials as protective agents were compared, including mannitol, lactose, sucrose, and maltose (all added in an amount of 5% (w / v)), and a blank control group was set up.

[0101] The results are shown in Table 6:

[0102] Table 6

[0103]

[0104]

[0105] It can be seen that the color and properties of the Yulangsan polysaccharide freeze-dried liposomes prepared with lactose as a protective agent did not change. The freeze-dried liposomes were light yellow sponge-like, fluffy and full, with good redispersibility, a small particle size of 114.67±0.68nm, and a high encapsulation rate of 84.56%, so lactose was selected as a protective agent.

[0106] The effects of lactose addition of 0%, 2.5%, 5%, 10% and 15% on the appearance, redispersibility, particle size, PDI, Zeta potential and encapsulation efficiency of the freeze-dried samples were further investigated. The results are shown in Table 7:

[0107] Table 7

[0108]

[0109] It can be seen that when 5% lactose is used as the single freeze-dried protective agent, the freeze-dried product has a fluffy and full appearance (such as Figure 3 As shown), the redispersibility is good, the particle size is small, which is 110.73±0.45nm, and the encapsulation efficiency is high, which is 85.00%, so 5% lactose is selected as the protective agent.

[0110] The freeze-dried liposomes were stored in a refrigerator (4°C) and at room temperature (25°C) in the dark, and samples were taken at 0d, 30d, 60d, and 125d, and the liposomes were reconstituted, the appearance was observed, and the particle size, PDI, Zeta potential, and encapsulation efficiency were measured. The results are shown in Table 8:

[0111] Table 8

[0112]

[0113] According to the results in Table 8, the particle size, potential and PDI of the liposomes changed little at 4°C, and the properties of the liposomes were relatively stable, so the storage condition of freeze-dried liposomes was selected as refrigeration at 4°C.

[0114] Test 5: Hemolytic test

[0115] 1. Preparation of 2% red blood cell suspension

[0116] Take 10mL of healthy blood (blood from the blood bank of the First Affiliated Hospital of Guangxi Medical University) and place it in a centrifuge tube soaked with heparin, centrifuge at 3000rpm for 10min to obtain 8ml of red blood cell sediment, add about 5 times the amount of normal saline, shake and centrifuge, discard the supernatant, repeat this method 4 to 5 times, and wash until the supernatant is colorless and transparent. Suspend 2ml of red blood cells again in 0.9% sodium chloride (2%, v / v) injection to prepare 100ml of red blood cell suspension, and store it in a 4℃ refrigerator for use.

[0117] 2. 1% Triton-X-100 configuration: Triton X-100 (polyethylene glycol octylphenyl ether) is a non-ionic surfactant (or detergent). Its Chinese name is: Triton X-100. It can dissolve lipids to increase the permeability of cell membranes to antibodies. Dilute to the required concentration before use: Take 1 ml of Triton X-100 and mix it with PBS to 100 ml, place it in a 37℃~40℃ water bath for 2~3 hours to fully dissolve and mix.

[0118] 3. Absorption wavelength selection

[0119] The hemolysis rate determination method specified in the fourth part of the 2020 edition of the Chinese Pharmacopoeia can only qualitatively identify the hemolysis of drugs, but cannot quantitatively determine the size of the drug hemolysis rate. In this regard, this study selected the ultraviolet spectrophotometry method to determine the hemolysis rate of red blood cells in vitro through screening, and its maximum absorption wavelength was the key parameter. The determination steps were as follows: 2 mL of human red blood cell suspension was added to 2 mL of normal saline as the negative control group, and 2 mL of human red blood cell suspension was added to 2 mL of pure water as the positive control group; 4 mL of the prepared Yulangsan polysaccharide liposome was taken as the test solution as the sample group. Take each group of solutions, shake them well, place them in a 37℃ constant temperature water bath for 1h, take them out and centrifuge them at 3000r / min for 10min, take the supernatant, take the negative group as the blank control, and perform spectral scanning on the positive control group and the sample group at a wavelength of 200-800nm. The results showed that its maximum absorption wavelength was 545nm, and the sample group had no interfering absorption at this wavelength. Therefore, the wavelength of 545 nm was selected as the measurement wavelength of hemolysis rate.

[0120] 4. Experimental steps

[0121] 0.5mL Yulangsan polysaccharide prepared by Example 2, 0.5mL Yulangsan polysaccharide liposome, 0.5mL Yulangsan polysaccharide liposome lyophilized solution (concentration is 1mg / ml) prepared by Example 4 are mixed with isopykalemic red blood cell suspension respectively, in addition 0.5mL 1%Triton-X-100 is mixed with isopykalemic red blood cell suspension as positive control group, 0.5mL0.9% sodium chloride injection is mixed with red blood cell suspension as negative control group. Sample is placed in 37 ℃ of water baths and incubated for 1h, 3000rpm centrifugal 10min, supernatant is taken to measure the absorbance of 545nm, and the hemolysis rate of each administration group is calculated. Generally speaking, the hemolysis rate of the positive control group is 100%, and the hemolysis rate of the negative control group is 0%.

[0122] Hemolysis rate (%) = (A 给药组 -A 阴性对照组 ) / (A 阳性对照组 -A 阴性对照组 )×100%;

[0123] The results are shown in Table 9:

[0124] Table 9

[0125]

[0126] The hemolytic test is one of the factors that can be used to determine whether nanomedicine can be safely used for intravenous injection. The test results are shown in Table 9 and Figure 4 As shown: It can be seen that the hemolysis rates of the Yulangsan polysaccharide liposome freeze-dried, Yulangsan polysaccharide liposome and Yulangsan polysaccharide groups are all less than 5%, which meets the national hemolysis standards and can provide a reference for intravenous administration.

[0127] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing Yulangsan polysaccharide liposomes, characterized in that: The steps include: (1) Preparing a thick paste of Yulangsan polysaccharide: crushing the Yulangsan slices, adding them to water and boiling them for 2-4 hours, then filtering, repeating the above method for 3-4 times, combining the filtrates obtained several times, then letting the filtrates stand and taking the supernatant to concentrate under reduced pressure, then adding ethanol, mixing them thoroughly and letting them stand for 8-12 hours, filtering with suction, taking the precipitate and washing it with ethanol for 2-4 times, then concentrating and drying, and then obtaining a thick paste of Yulangsan polysaccharide; (2) Refining of Yulangsan polysaccharide: Take a thick paste of Yulangsan polysaccharide and add it to distilled water to dissolve it, then add papain solution, treat it at 50-60°C for 3-5 hours, inactivate the enzyme in a boiling water bath for 4-6 minutes, then centrifuge to remove the precipitate, centrifuge again, remove free protein from the obtained supernatant, separate the organic phase, centrifuge the aqueous phase, and then take the supernatant and concentrate it under reduced pressure, add ethanol and let it stand for 10-14 hours, filter it, wash the precipitate with ethanol and methanol for 2-3 times each, and dry it to obtain Yulangsan polysaccharide; (3) Preparation of Yulangsan polysaccharide liposomes: soybean lecithin, cholesterol and Tween 80 were dissolved in chloroform and methanol solutions, and ultrasonic dissolution was performed to obtain an organic phase. The organic solvent was removed by evaporation under reduced pressure at 35-45°C. After a lipid film was formed, chloroform was added to dissolve it. Yulangsan polysaccharide was dissolved in phosphate buffer as the aqueous phase. The aqueous phase was introduced into the organic phase reaction vessel, and ultrasonic emulsification was performed in an ice-water bath to form a stable milky white W / O type emulsion. After evaporation under reduced pressure to form a colloidal state, phosphate buffer was added, and the organic solvent was removed by rotary evaporation to form an aqueous suspension. The suspension was then placed in an ultrasonic cell crusher for ultrasonic treatment, and the suspension was extruded through 0.45μm and 0.22μm microporous filter membranes to obtain Yulangsan polysaccharide liposomes.

2. The method according to claim 1, characterized in that In the step (1), ethanol is added after reduced pressure concentration, specifically 95% ethanol is added, and then the ethanol concentration in the concentrated solution is adjusted to 80% using an alcohol meter.

3. The method according to claim 1, characterized in that In the step (2), before adding the thick paste of Yulangsan polysaccharide to distilled water, the thick paste of Yulangsan polysaccharide is firstly ground and crushed using a mortar.

4. The method according to claim 1, characterized in that: In the step (2), during the dissolution of the thick paste of Yulangsan polysaccharide in distilled water, the thick paste is heated in a boiling water bath for 0.5-1.5 h and ultrasonic dissolution is performed.

5. The method according to claim 1, characterized in that The step (2) of adding ethanol and letting it stand specifically includes adding 95% ethanol to make the ethanol concentration be 80%, and then letting it stand for 12 hours.

6. The method according to claim 1, characterized in that The ultrasonic cell pulverizer in step (3) performs ultrasonic treatment with an ultrasonic intensity of 62%, 5.0s on, 5.0s off, a temperature of 4°C, and a duration of 5 minutes.

7. The method according to claim 1, characterized in that In the step (3), the mass ratio of soybean lecithin to cholesterol is 6:1, the mass ratio of soybean lecithin to Yulangsan polysaccharide is 20:1, and the mass ratio of soybean lecithin to Tween 80 is 4:

1.

8. The method according to claim 1, characterized in that The ratio of the organic phase to the aqueous phase in step (3) is 5:

1.

9. The method according to claim 1, characterized in that: In the step (3), the ice-water bath ultrasonic emulsification is carried out in an ice-water bath at 4° C. for 15 minutes; when the organic solvent is removed by rotary evaporation, the water bath temperature is controlled to be 55° C.

10. The method for preserving Yulangsan polysaccharide liposomes according to claims 1-9, characterized in that: The method is to prepare the obtained Yulangsan polysaccharide liposomes into Yulangsan polysaccharide freeze-dried liposomes, specifically: the Yulangsan polysaccharide liposomes are prepared into a Yulangsan polysaccharide suspension with a concentration of 1 mg / mL, lactose is placed in a culture medium, the Yulangsan polysaccharide suspension is added, and the culture medium is placed in a refrigerator and frozen at -20°C for 12 hours, and then placed in a freeze dryer for freeze drying for 48 hours, and then taken out and stored in a refrigerator at 4°C, wherein the amount of lactose added accounts for 5% of the volume of the Yulangsan polysaccharide suspension.

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