Preparation method and application of polygonatum cyrtonema polysaccharide liposome with immunomodulatory effect

Abstract

The invention discloses polygonatum cyrtonema polysaccharide lipidosome with an immunoregulation effect as well as a preparation method and application of the polygonatum cyrtonema polysaccharide lipidosome. The polygonatum cyrtonema polysaccharide lipidosome is mainly prepared from polygonatum cyrtonema polysaccharide, soybean lecithin and cholesterol. The polygonatum cyrtonema polysaccharide lipidosome with the immunoregulation function is good in stability, capable of slowly releasing polygonatum cyrtonema polysaccharide and easy to prepare in an enlarged mode. Experiments prove that the polygonatum cyrtonema polysaccharide lipidosome can promote proliferation of phagocytes of mice and increase spleen and thymus indexes of immunosuppressive mice, and has an immunoregulation effect.

Description

[0001] This application is a divisional application. The original application's invention title is "Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect, its preparation method and application", the application number is 202311141476.2, and the application date is September 6, 2023. Technical Field

[0002] The present invention relates to the field of pharmaceutical preparations, and specifically relates to a Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect, its preparation method and application. Background Art

[0003] Polygonatum cyrtonema Hua is a perennial herb of the genus Polygonatum in the family Asparagaceae and is one of the three major sources of Polygonatum medicinal materials. Polygonatum cyrtonema Hua is a traditional medicinal and edible plant. Its rhizome can be used as medicine and has the effects of invigorating qi, nourishing yin, strengthening the spleen and moistening the lungs. Polygonatum cyrtonema Hua contains rich chemical components such as polysaccharides, saponins, flavonoids and alkaloids. Among them, polysaccharides are its main active components. So far, it has been reported that Polygonatum cyrtonema Hua polysaccharides have a variety of pharmacological activities, including anti-fatigue, hypoglycemic, hypolipidemic, anti-tumor, antibacterial and antiviral effects. Especially in terms of immunomodulation, Polygonatum cyrtonema Hua polysaccharides show good effects and have become a research hotspot of natural medicines. However, most traditional Chinese medicine polysaccharides have disadvantages such as poor stability, low bioavailability, easy degradation and weak cell penetration, which affect the efficacy of polysaccharides.

[0004] In recent years, nano-drug delivery systems have attracted much attention because of their targeted drug delivery, slow release of drugs to extend the drug action time, improve the bioavailability of drugs, and enhance the stability of drugs in vitro and in vivo. As an important drug delivery system, liposomes have broad prospects and have been the object of in-depth research for more than 50 years and are widely used in the pharmaceutical and other fields. Many liposome drugs have been marketed, such as doxorubicin liposome (Doxil caelyx8), amphotericin B liposome (AmBisome8), vincristine liposome (Marqibo8). Therefore, it is of great significance to develop Polygonatum cyrtonema Hua polysaccharide liposomes to improve the stability and bioavailability of Polygonatum cyrtonema Hua polysaccharides. Summary of the Invention

[0005] Object of the Invention: Based on the existing objective problems, the present invention provides a Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect, its preparation method and application, thereby solving the disadvantages of poor stability, low bioavailability, easy degradation and weak cell penetration of Polygonatum cyrtonema Hua polysaccharides, enhancing immune activity and improving the medical value of Polygonatum cyrtonema Hua.

[0006] Technical Solution: In order to achieve the above object of the invention, the technical solutions adopted by the present invention are as follows:

[0007] A polygonatum cyrtonema polysaccharide liposome, wherein the polygonatum cyrtonema polysaccharide liposome is mainly a liposome prepared from polygonatum cyrtonema polysaccharide, soybean lecithin and cholesterol.

[0008] Preferably, the mass ratio of soybean lecithin to cholesterol is 4:1 - 10:1; the mass ratio of polygonatum cyrtonema polysaccharide to cholesterol is 1:12.5 - 1:1.

[0009] Preferably, the polygonatum cyrtonema polysaccharide is mainly prepared by the following method:

[0010] Take the dried product of the rhizome of polygonatum cyrtonema, reflux extract with petroleum ether, extract the residue with an ethanol aqueous solution, and then reflux extract the residue with pure water. Concentrate the pure water extract, precipitate with alcohol, remove protein, dialyze, and freeze-dry to obtain the polygonatum cyrtonema polysaccharide.

[0011] More preferably, the time for reflux extraction with petroleum ether is 1 - 3 h; the concentration of the ethanol aqueous solution is 85 - 95%, and the extraction time is 1 - 3 h; for the reflux extraction with pure water, the solid-liquid ratio is 1:(25 - 35), and the reflux extraction is carried out 1 - 3 times, each time for 1 - 3 h; protein removal is carried out using Sevage reagent; dialysis is carried out using a dialysis bag with a molecular weight cut-off of 3000D - 4000D, with pure water as the medium, and the dialysis time is 36 - 60 h.

[0012] The present invention also provides a method for preparing the polygonatum cyrtonema polysaccharide liposome, comprising the following steps:

[0013] 1) Dissolve the polygonatum cyrtonema polysaccharide in a buffer solution to form an aqueous phase;

[0014] 2) Dissolve soybean phospholipid and cholesterol in an organic solvent to form an organic phase;

[0015] 3) Mix the aqueous phase and the oil phase, perform ultrasonic treatment, and evaporate the organic solvent under reduced pressure to reach a colloidal state;

[0016] 4) Add a buffer solution, ultrasonicate to hydrate the colloidal liposome, add a surfactant, and perform ultrasonic emulsification to prepare the polygonatum cyrtonema polysaccharide liposome.

[0017] Preferably, in step 1), the buffer solution is selected from PBS buffer solution; in the aqueous phase, the concentration of polygonatum cyrtonema polysaccharide is 1 - 20 mg / mL.

[0018] Preferably, in step 2), the organic solvent is selected from ethanol; in the organic phase, the concentration of soybean phospholipid is 40 - 60 mg / mL, and the concentration of cholesterol is 10 - 15 mg / mL.

[0019] Preferably, in step 3), the conditions for ultrasonic treatment are as follows: ultrasonic treatment for 2 s, with a gap of 3 s, a power of 220 - 260 W, and a total time of 1.5 - 2.5 min until a stable W / O emulsion is formed.

[0020] Preferably, in step 4), the buffer solution is selected from PBS buffer solution; the surfactant is selected from Tween 80; the conditions for ultrasonic emulsification are as follows: ultrasonic treatment for 2 s, with a gap of 3 s, a power of 220 - 260 W, and a total time of 1.5 - 2.5 min.

[0021] Finally, the present invention provides the use of the Polygonatum cyrtonema Hua polysaccharide liposome in the preparation of an immunomodulatory drug, an immune enhancing drug or an immune adjuvant.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0023] 1. The Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect provided by the present invention has good stability and a relatively high encapsulation rate. In particular, through multiple ultrasonic treatments, the encapsulation rate of the molecular weight heterogeneous Polygonatum cyrtonema Hua polysaccharide can be further improved; in addition, it can slowly release the Polygonatum cyrtonema Hua polysaccharide and is easy to scale up for preparation.

[0024] 2. The present invention also uses a specific method to extract Polygonatum cyrtonema Hua polysaccharide, including first reflux extraction and leaching with an organic solvent, then reflux extraction with water, and finally alcohol precipitation. Compared with the traditional method, the obtained Polygonatum cyrtonema Hua polysaccharide has a higher purity.

[0025] 3. The Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect provided by the present invention can promote the proliferation of mouse phagocytes, increase the spleen and thymus indices of immunosuppressed mice, and has an immunomodulatory effect. Description of the Drawings

[0026] Figure 1 : Particle size diagram and transmission electron microscope diagram of the Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect in Example 1;

[0027] Figure 2 : Stability of the Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect in Example 2;

[0028] Figure 3 : Performance of the slow release of Polygonatum cyrtonema Hua by the Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect in Example 3;

[0029] Figure 4 : Stimulation of the proliferation of mouse phagocytes by the Polygonatum cyrtonema Hua polysaccharide liposome with immunomodulatory effect in Example 4;

[0030] Figure 5: Effects of Polygonatum cyrtonema Hua polysaccharide liposomes with immunomodulatory effects on the spleen and thymus indices of immunosuppressed mice in Example 5; Detailed implementation method

[0031] The present invention will be further described below by way of examples, but the following examples do not limit the scope of the patent rights.

[0032] Example 1: Preparation and particle size determination of Polygonatum cyrtonema Hua polysaccharide liposomes with immunomodulatory effects

[0033] First, prepare Polygonatum cyrtonema Hua polysaccharide, which is prepared by the following method:

[0034] Extraction: Fresh rhizomes of Polygonatum cyrtonema Hua are dried to a constant weight in an oven at 60 °C, pulverized into powder and stored in a drying oven. Weigh 34.87 g, reflux extract with petroleum ether for 2 h, pour the residue after evaporating petroleum ether into 90% ethanol for extraction for 1 h, evaporate the remaining ethanol in the residue, and then reflux extract with pure water at a solid-liquid ratio of 1:30 for 2 h. Filter the residue and reflux extract it in the same way. Filter and collect the filtrate, and combine the filtrates collected twice;

[0035] Concentration: Pour the filtrate into a round-bottom flask and concentrate it to a certain volume with a rotary evaporator at 65 °C and 75 rpm / min;

[0036] Alcohol precipitation: Slowly add 4 times the volume of 95% ethanol to the concentrated solution and stir continuously. Place it in a 4 °C refrigerator for alcohol precipitation overnight. Take out the water extract after alcohol precipitation overnight, recover the ethanol in the supernatant, collect the bottom precipitate, filter it to obtain polysaccharide, wash it with ethanol, and after evaporating the ethanol, add an appropriate amount of pure water to dissolve the precipitate to form a polysaccharide solution for standby;

[0037] Protein removal: Pour the polysaccharide solution into a separatory funnel, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1), shake it for a period of time and then place it on an iron stand. After standing for 20 min, there are obvious polysaccharide solution layer, protein layer and Sevage reagent layer. Discard the Sevage reagent layer and protein layer from the lower outlet. Repeat this operation three times. In the third time, there is no obvious protein layer. Discard the lower layer of reagent and pour out the polysaccharide solution from the upper mouth;

[0038] Dialysis: Pour the polysaccharide solution after protein removal into a pre-treated 3500D dialysis bag, clamp both ends of the dialysis bag, use pure water as the medium, dialyze for 48 h, and change the water every 4 - 8 h;

[0039] Freeze-drying: Take the polysaccharide solution after dialysis, centrifuge it at 4000 r / min for 10 min, take the supernatant, freeze it overnight at 20 °C and then in a freeze dryer (-80 °C, 0.290 Pa), and freeze-dry it into powder for storage for standby, with a purity reaching 92.8%.

[0040] In this example, the reverse evaporation method was used to prepare the polysaccharide liposome of Polygonatum cyrtonema Hua. An appropriate amount of the polysaccharide of Polygonatum cyrtonema Hua was weighed and dissolved in PBS buffer solution to prepare a polysaccharide solution of Polygonatum cyrtonema Hua. 500 mg of soybean phospholipid and 125 mg of cholesterol were weighed in a beaker and dissolved in 10 mL of absolute ethanol. The aqueous phase (10 mL) and the oil phase were mixed and ultrasonically treated for 2 min (ultrasonic for 2 s, interval for 3 s, power 240 W) until a stable W / O emulsion was formed. The organic solvent was removed by evaporation under reduced pressure. After reaching the colloidal state, PBS buffer solution was added, and the colloidal liposome was hydrated by ultrasonic treatment. 1.25 mL of 10% Tween 80 was added, and the volume was fixed to 25 mL with PBS, and then ultrasonic emulsification was carried out for 2 min (ultrasonic for 2 s, interval for 3 s, power 240 W) once to obtain the polysaccharide liposome of Polygonatum cyrtonema Hua. The encapsulation efficiency of the polysaccharide liposome of Polygonatum cyrtonema Hua was measured by the anthrone-sulfuric acid method and reached 38.03% ± 0.050. The particle size and appearance of the polysaccharide liposome of Polygonatum cyrtonema Hua were observed by dynamic light scattering and transmission electron microscopy. The results showed that the hydrated particle size of the prepared liposome was 146 nm, the particle size distribution index was 0.227, and the electron microscopy results showed that the particle size of the liposome loaded with polysaccharide was smaller than the hydrated particle size, as Figure 1 shown.

[0041] Example 2: In vitro stability evaluation of the polysaccharide liposome of Polygonatum cyrtonema Hua with immunomodulatory effect

[0042] 1 mL of the polysaccharide liposome sample of Polygonatum cyrtonema Hua prepared above was taken in a dish, and the particle size and PDI of the polysaccharide liposome of Polygonatum cyrtonema Hua were measured by a dynamic light scattering instrument at the established time points. The results showed that within 21 days, the particle size of the polysaccharide liposome of Polygonatum cyrtonema Hua was between 150 - 183 nm, and the PDI fluctuated between 0.210 and 0.240, with little change, indicating that the polysaccharide liposome of Polygonatum cyrtonema Hua had good stability, as Figure 2 shown.

[0043] Example 3: Evaluation of the release of the polysaccharide of Polygonatum cyrtonema Hua from the polysaccharide liposome with immunomodulatory effect

[0044] In vitro drug release was analyzed using the dialysis method. Neutral PBS (pH 7.4) was selected as the release medium, and 2 mL of free polygonatum polysaccharide and polygonatum polysaccharide liposomes were respectively added into a dialysis bag (1000 kD). Then, both ends of the dialysis bag were sealed tightly, and it was placed in a beaker containing 40 mL of release medium, sealed with plastic wrap, and placed in a constant temperature oscillator (37 °C, 100 rpm) to simulate the body fluid environment. At specific time intervals (1, 2, 4, 6, 8, 10, 12, 24 h...), 0.5 mL of the release medium was taken out each time and placed in a test tube. The samples in the test tubes were quantitatively determined for the amount of polysaccharide using the anthrone-sulfuric acid method (n = 3). The results showed that the free polygonatum polysaccharide had an explosive release in the first 6 h, was basically completely released before 10 h, and there was basically no further release later. However, the release of polygonatum polysaccharide in the polygonatum polysaccharide liposomes had an obvious sustained-release effect and was in a trend of slow and continuous release within 40 h, as Figure 3 shown.

[0045] Example 4: Polygonatum polysaccharide liposomes with immunomodulatory effects stimulate the proliferation of mouse phagocytes.

[0046] Healthy Raw264.7 macrophages with normal passage were selected, the cell density was observed with an optical microscope, adjusted, and measured with a cell counter to make each 1 mL of cell suspension contain 8×10 4 cells. A sterile 96-well plate was taken, and 100 μL of cell suspension was added to each well, and cultured in a cell incubator at 37 °C and 5% CO2 for 24 h. After the cells were completely adherent and grew, the cell culture medium was removed. In the blank group, 100 μL of culture medium was added to each well, and in the experimental group, 100 μL of free polygonatum polysaccharide (PCP), blank liposomes (BL), and polygonatum polysaccharide liposomes (PCPL) solutions with different concentrations (32, 48, 64, 80, 96, 112, 128, 144, 160 μg / mL, the blank liposomes were calculated by liposome concentration, and the free polygonatum polysaccharide and polygonatum polysaccharide liposomes were calculated by polysaccharide concentration) were added to each well. There were 6 parallel experimental wells in each group. After culturing in the cell incubator for 24 h, 10 μL of CCK8 reagent (Cell Counting Kit8) solution was added to each well and continued to be cultured for 1 h, and then the OD value was measured at 450 nm. The results showed that the blank liposomes had little effect on Raw264.7 cells, while the free polygonatum polysaccharide and polygonatum polysaccharide liposomes could promote cell proliferation, up to 160 μg / mL at most, as Figure 4 shown.

[0047] Example 5: Effects of polygonatum polysaccharide liposomes with immunomodulatory effects on the spleen and thymus indices of immunosuppressed mice

[0048] 90 Kunming mice with a body weight of 18 - 22 g were allowed to rest quietly for 1 week and randomly divided into 9 groups of 10 mice each. Group 1 was the blank control group (BC), Group 2 was the cyclophosphamide model group (CTX), Group 3 was the low-dose (10 mg / Kg) polygonatum cyrtonema polysaccharide group (PCP-L), Group 4 was the medium-dose (20 mg / Kg) polygonatum cyrtonema polysaccharide group (PCP-M), Group 5 was the high-dose (40 mg / Kg) polygonatum cyrtonema polysaccharide group (PCP-H), Group 6 was the blank liposome group (BL), Group 7 was the low-dose (10 mg / Kg) polygonatum cyrtonema polysaccharide liposome group (PCPL-L), Group 8 was the medium-dose (20 mg / Kg) polygonatum cyrtonema polysaccharide liposome group (PCPL-M), and Group 9 was the high-dose (40 mg / Kg) polygonatum cyrtonema polysaccharide liposome group (PCPL-H). On the 7th day after rest, except for the blank group which was injected with normal saline, the other groups were intraperitoneally injected with cyclophosphamide (100 mg / Kg) once a day for 3 consecutive days. On the 10th day, the blank group was injected with normal saline via the tail vein, and the other groups were injected with drugs via the tail vein once a day for 5 consecutive days. After the last administration, the mice were fasted but allowed to drink water for 12 hours and then sacrificed to obtain the thymus and spleen. The obtained spleen and thymus were blotted dry with absorbent paper and weighed, and the spleen and thymus indices were calculated. Spleen (thymus) index = spleen weight (thymus) (mg) / body weight (g). The results showed that compared with the model group, all the drug administration groups had an up-regulating effect on the spleen and thymus indices, and it was concentration-dependent, as Figure 5 shown.

[0049] As described above, it is only a general description of the present invention and does not impose any formal restrictions on the present invention. Any changes made by any other relevant person based on the relevant content of the present invention shall fall within the protection scope of the present invention.

Claims

1. A preparation method of polygonatum cyrtonema hua polysaccharide liposome, characterized in that, It includes the following steps: 1) Dissolve polygonatum cyrtonema polysaccharide in a buffer solution to form an aqueous phase; 2) Dissolve soybean phospholipid and cholesterol in an organic solvent to form an organic phase; 3) Mix the aqueous phase and the oil phase, perform ultrasonic treatment, and evaporate the organic solvent under reduced pressure to reach a colloidal state; 4) Add a buffer solution, ultrasonically hydrate the colloidal liposomes, add a surfactant, and perform ultrasonic emulsification to obtain polygonatum cyrtonema polysaccharide liposomes.

2. The preparation method according to claim 1, characterized in that, In step 1), the buffer solution is selected from PBS buffer solution; in the aqueous phase, the concentration of polygonatum cyrtonema polysaccharide is 1 - 20 mg / mL.

3. The preparation method according to claim 1, characterized in that, In step 2), the organic solvent is selected from ethanol; in the organic phase, the concentration of soybean phospholipid is 40 - 60 mg / mL, and the concentration of cholesterol is 10 - 15 mg / mL.

4. The preparation method according to claim 1, wherein In step 3), the conditions for ultrasonic treatment are: ultrasonic for 2 s, interval for 3 s, power of 220 - 260 W, and total time of 1.5 - 2.5 min.

5. The preparation method according to claim 1, wherein In step 4), the buffer solution is selected from PBS buffer solution; the surfactant is selected from Tween 80; the conditions for ultrasonic emulsification are: ultrasonic for 2 s, interval for 3 s, power of 220 - 260 W, and total time of 1.5 - 2.5 min.

6. A polysaccharide liposome of Polygonatum cyrtonema Hua, characterized in that, Prepared by the preparation method according to any one of claims 1 - 5.

7. Use of the polygonatum cyrtonema polysaccharide liposomes according to claim 6 in the preparation of immunomodulatory drugs, immune enhancing drugs or immunoadjuvants.

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