Preparation method of a saponin vaccine adjuvant for animals

Abstract

The present application relates to the technical field of vaccine production, in particular to a preparation method of a Sapindus saponin veterinary vaccine adjuvant, comprising: determining the limit concentration of Sapindus saponin with a hemolysis rate lower than 5% and preparing the vaccine adjuvant; determining the limit concentration of Sapindus saponin with a hemolysis rate lower than 5% against red blood cells by spectrophotometry; preparing the vaccine adjuvant by dialysis; and the vaccine adjuvant contains soybean lecithin and cholesterol in a weight ratio of 1:1. The present application first determines the safe use concentration of Sapindus saponin through a standard hemolysis experiment, ensures that the hemolysis rate of the prepared adjuvant meets the safety requirements, and avoids adverse reactions after injection; at the same time, the present application uses soybean lecithin and cholesterol as raw materials, and prepares the nano-liposome adjuvant embedding Sapindus saponin by dialysis, which does not require complex process operation, has low preparation cost, and has safety and practicability, and can effectively assist the vaccine to induce the body to produce stronger immune response and improve the protection effect of the vaccine.

Description

Technical Field

[0001] This invention relates to the field of vaccine production technology, and in particular to a method for preparing a Sapindus mukorossi saponin adjuvant for veterinary vaccines. Background Technology

[0002] In animal husbandry, infectious diseases are a significant factor restricting the industry's development. While vaccines play a crucial role in disease control, some vaccines, when used alone, induce a weak immune response, failing to achieve ideal protective effects. Therefore, they are often used in conjunction with adjuvants to enhance the immune response. Adjuvants are substances that are injected into the body beforehand or simultaneously with the antigen, lacking antigenic properties themselves but enhancing the immune response. Currently, veterinary adjuvants are mainly divided into two categories: aqueous adjuvants and oil-emulsion adjuvants. Among them, liposome adjuvants, as a type of aqueous adjuvant, are composed of phospholipids, cholesterol, etc. They can encapsulate antigens, prevent antigen escape, and efficiently present them to immune cells. They have advantages such as being non-toxic and metabolizable, and have broad application prospects. In traditional Chinese medicine adjuvants, saponins are widely used, such as Quil A from saponin bark. They can activate T cells and antigen-presenting cells, induce immune cell differentiation and maturation, enhance the immune response, and prolong the vaccine's protective period. Immunostimulatory complexes (ISCOMs) formed by the self-assembly of saponins, cholesterol, and lecithin have both adjuvant and antigen-presenting functions, and can induce a comprehensive immune response. They have been used in a variety of vaccines.

[0003] However, the hemolytic effect of saponins limits their development as drugs. High concentrations of saponins can damage red blood cell membranes, triggering hemolytic reactions, leading to inflammation at the injection site and adverse reactions. Therefore, it is necessary to strictly control the dosage of saponins in adjuvants, ensuring the adjuvant's immune-enhancing effect while keeping the hemolysis rate within a safe range. Most existing saponin adjuvant preparation processes do not clearly define the safe concentration range for saponin use and lack standardized methods for measuring hemolysis rates, resulting in adjuvants with unstable safety that can easily affect vaccine efficacy. Furthermore, existing ISCOM-type adjuvant preparation processes are complex and costly, hindering large-scale application in the veterinary vaccine field. Therefore, there is a need to develop a veterinary saponin vaccine adjuvant with a simple preparation process, well-defined safety profile, and low cost. Summary of the Invention

[0004] To address the aforementioned issues, this invention utilizes Sapindus mukorossi saponins to prepare veterinary vaccine adjuvants. First, a spectrophotometric hemolysis experiment is used to determine the safe limit concentration of Sapindus mukorossi saponins with a hemolysis rate not exceeding 5%. Then, using soybean lecithin and cholesterol as lipid raw materials, a nano-liposome-encapsulated Sapindus mukorossi saponin adjuvant is prepared using dialysis. This ensures the immune-enhancing effect of Sapindus mukorossi saponins while controlling the hemolysis rate within a safe range.

[0005] The purpose of this invention is to provide a method for preparing a Sapindus mukorossi saponin adjuvant for veterinary vaccines, in order to solve the problems of existing saponin adjuvants having unclear safe concentration ranges, unstable hemolytic safety, and complex and costly preparation processes for existing ISCOM-type adjuvants.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing a Sapindus mukorossi saponin veterinary vaccine adjuvant includes: determining the limiting concentration of Sapindus mukorossi saponin with a hemolysis rate of less than 5%, and then preparing the vaccine adjuvant based on this concentration; determining the hemolysis rate of Sapindus mukorossi saponin against erythrocytes using spectrophotometry, and screening to obtain the limiting concentration with a hemolysis rate of no more than 5%; and preparing the vaccine adjuvant using dialysis, wherein the vaccine adjuvant contains soybean lecithin and cholesterol in a weight ratio of 1:1.

[0008] Preferably, the red blood cells are selected from rabbit red blood cells, pig red blood cells, bovine red blood cells, and sheep red blood cells.

[0009] Specifically, the preparation method of the Sapindus mukorossi saponin veterinary vaccine adjuvant of the present invention includes the following steps:

[0010] S1, Hemolysis experiment, including: S11, preparation of red blood cell suspension and S12, experiment on the effect of Sapindus mukorossi saponins on hemolysis;

[0011] S2, Preparation of vaccine adjuvants, including: S21, preparation of lipid mixture stock solution and S22, formulation of Sapindus saponin veterinary vaccine adjuvant.

[0012] Further, the operation of S11 is as follows: Weigh 2.70g of sodium chloride and dissolve it in 300mL of ultrapure water to prepare physiological saline for later use; take 10mL of defibrinated blood from a rabbit, add physiological saline, centrifuge at 3100r / min for 4.5min, discard the supernatant, add physiological saline, centrifuge again at 3100r / min for 4.5min, and repeat washing with physiological saline until the supernatant after centrifugation is no longer red; after the supernatant is no longer red, aspirate and discard the upper layer of physiological saline in the centrifuge tube, take 40μL of the lower layer of red blood cell precipitate, add 2mL of physiological saline, and prepare a 2% red blood cell suspension.

[0013] Furthermore, the operation of S12 is as follows:

[0014] (1) Solution preparation

[0015] Accurately measure 1.00 mg of the self-made Sapindus saponin sample, place it in a beaker, dissolve it with 10.0 mL of physiological saline, shake well to obtain a 0.10 mg / mL Sapindus saponin stock solution; take several centrifuge tubes, number them, add different concentrations of Sapindus saponin stock solution to each tube, and then add different concentrations of physiological saline to each tube to prepare gradient solutions of different concentrations.

[0016] (2) Determination of the maximum absorption wavelength of heme solution

[0017] Take 1 mL of distilled water into a test tube, add 1 mL of red blood cell suspension, and mix well; place in a 37℃ constant temperature water bath for 30 min to allow for full hemolysis, and then immediately place in ice water to stop the hemolysis; centrifuge at 3000 r / min for 5 min, and take 100 μL of the supernatant containing heme from each group into a 96-well plate, with 100 μL of physiological saline as a control, and use an ELISA reader to determine the maximum absorption wavelength;

[0018] (3) Spectrophotometric determination of its hemolysis rate

[0019] Take several test tubes and number them, including test tubes containing different concentrations of Sapindus mukorossi saponins and two control tubes. Add 1 mL of 2% red blood cell suspension to each tube first, then add 1 mL of Sapindus mukorossi saponins of different concentrations to each test tube. Add 1 mL of physiological saline to the first control tube and 1 mL of distilled water to the other control tube, so that the total volume of solution in each tube is 2 mL. Mix well and incubate in a water bath at 37°C for 3 hours. After 3 hours, immediately place the solutions in ice water to stop the hemolysis. Centrifuge at 3000 r / min for 5 min, and take 100 μL of the supernatant into a 96-well plate, with 5 replicates per group. Read the OD value of each well at the maximum absorption wavelength on a microplate reader.

[0020] Calculate the hemolysis rate of each well using the formula:

[0021] Hemolysis rate (%) = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100%.

[0022] Further, the operation of S21 is as follows: Weigh 250mg of Mega-10 dry powder, dissolve it in 1mL of distilled water, place it in a 37℃ incubator for 1h to dissolve it, and make up the volume to 1.25mL to prepare a 20% concentration Mega-10 stock solution; add 5mg of soybean lecithin and 5mg of cholesterol to 500μL of the 20% concentration Mega-10 stock solution, place it in a 55℃ water bath for 1h, and perform ultrasonic treatment, ultrasonic work for 3s, stop for 5s, ultrasonic work for a total of 15min, power 40%, while simultaneously using an ice water bath to dissolve it, and after dissolution, place it in a 37℃ incubator overnight to prepare a 1% concentration lipid mixture stock solution.

[0023] Further, the operation in S22 is as follows: Weigh Sapindus mukorossi saponins and dissolve them in sterile PBS solution to obtain a 20 μg / mL Sapindus mukorossi saponin stock solution; take 200 μL of 1% lipid mixture stock solution and add 1300 μL of PBS solution and mix well, add 500 μL of Sapindus mukorossi saponin stock solution, mix thoroughly, and sonicate for 2 seconds, stop for 5 seconds, for a total of 20 minutes of sonication at 20% power, while simultaneously bathing in an ice-water bath; after standing at room temperature for 2 hours, transfer to an 8KD dialysis bag, and dialyze with PBS solution at room temperature for 3 days, changing the dialysate 3 times a day; after dialysis, store at 4℃ to obtain the final product.

[0024] Compared with existing technologies, the beneficial effects of this invention are as follows: First, this invention clarifies the safe concentration of Sapindus saponins through standardized hemolysis experiments, ensuring that the hemolysis rate of the prepared adjuvant meets safety requirements and avoiding adverse reactions after injection; simultaneously, using soybean lecithin and cholesterol as raw materials, a nano-liposome adjuvant encapsulating Sapindus saponins is prepared by dialysis, which does not require complex processes, has low preparation costs, is suitable for large-scale promotion in the field of veterinary vaccines, and combines safety and practicality, effectively assisting vaccines in inducing a stronger immune response in the body and improving the protective effect of vaccines. Attached Figure Description

[0025] Figure 1 Electron microscopy images of NISCOM diluted 10 times, obtained in a specific embodiment of the present invention. Figure 1 ;

[0026] Figure 2 Electron microscopy images of NISCOM diluted 10 times, obtained in a specific embodiment of the present invention. Figure 2 ;

[0027] Figure 3 This is a particle size distribution diagram of NISCOM diluted 10 times according to a specific embodiment of the present invention;

[0028] Figure 4 This is a curve showing the change in mouse body weight;

[0029] Figure 5 The curve showing the change in FPV antibody levels in mouse serum;

[0030] Figure 6 For comparison with ISCOM electron microscopy in relevant literature Figure 1 ;

[0031] Figure 7 For comparison with ISCOM electron microscopy in relevant literature Figure 2 . Detailed Implementation

[0032] The present invention will be described below with reference to examples. These examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0033] A method for preparing a Sapindus mukorossi saponin adjuvant for veterinary vaccines includes the following steps:

[0034] S1, Hemolytic effect experiment of Sapindus mukorossi saponins

[0035] S11. Weigh 2.70g of sodium chloride and dissolve it in 300mL of ultrapure water to prepare physiological saline for later use. Take 10mL of defibrinated blood from a rabbit, add an appropriate amount of physiological saline, centrifuge at 3100r / min for 4.5min, discard the supernatant, add an appropriate amount of physiological saline, and gently blow away the red blood cells at the bottom of the tube with a dropper to prevent the cells from adhering to the centrifuge tube wall. Centrifuge again at 3100r / min for 4.5min, and repeat washing with physiological saline until the supernatant after centrifugation is no longer red. After the supernatant is no longer red, carefully aspirate the upper layer of physiological saline in the centrifuge tube with a dropper and discard it. Take 40μL of the lower layer of red blood cell precipitate, add 2mL of physiological saline, and prepare a 2% red blood cell suspension.

[0036] Experiment on the effect of S12, Sapindus mukorossi saponins on hemolysis

[0037] S121. Solution preparation: Accurately measure 1.00 mg of the self-made Sapindus saponin sample, place it in a beaker, dissolve it in 10.0 mL of physiological saline, shake well to obtain a 0.10 mg / mL Sapindus saponin stock solution; take several centrifuge tubes and number them, add different concentrations of Sapindus saponin stock solution to each tube, and then add different concentrations of physiological saline to each tube to prepare gradient solutions of different concentrations; take 6 centrifuge tubes, numbered 1 to 6, add 37, 75, 225, 375, 525, and 675 μL of Sapindus saponin stock solution to each tube, and then add 1463, 1425, 1275, 1125, 975, and 825 μL of physiological saline to each tube to prepare gradient solutions of 2.5, 5, 15, 25, 35, and 45 μg / mL;

[0038] S122. Determination of the maximum absorption wavelength of heme solution: Take 1 mL of distilled water into a test tube, add 1 mL of red blood cell suspension, and mix well; place in a 37℃ constant temperature water bath for 30 min to allow for complete hemolysis, and then immediately place in ice water to stop the hemolysis; centrifuge at 3000 r / min for 5 min, and take 100 μL of the supernatant containing heme from each group into a 96-well plate, with 100 μL of physiological saline as a control, and use an ELISA reader to determine the maximum absorption wavelength;

[0039] S123. Spectrophotometric determination of hemolysis rate: Take several test tubes and number them, including test tubes containing different concentrations of Sapindus mukorossi saponins, plus two control tubes; take 8 test tubes and number them, where tubes 1-6 contain different concentrations of Sapindus mukorossi saponins, tube 7 is the negative control tube with physiological saline, and tube 8 is the positive control tube with distilled water; add 1 mL of 2% red blood cell suspension to each tube first, then add 1 mL of Sapindus mukorossi saponins of different concentrations to each test tube, add 1 mL of physiological saline to the first control tube, and add 1 mL of distilled water to the other control tube, so that the total volume of solution in each tube is 2 mL, mix well, and incubate at 37℃. Incubate in a water bath for 3 hours; add 1 mL of Sapindus mukorossi saponins (2.5, 5, 15, 25, 35, and 45 μg / mL) to tubes 1-6 respectively, 1 mL of physiological saline to tube 7, and 1 mL of distilled water to tube 8, making the total volume of solution in each tube 2 mL. Mix well and incubate in a water bath at 37°C for 3 hours; after 3 hours, immediately place each tube in ice water to stop hemolysis, centrifuge at 3000 rpm for 5 minutes, and take 100 μL of the supernatant into a 96-well plate, with 5 replicates per group. Read the OD value of each well at the maximum absorption wavelength on a microplate reader and calculate the hemolysis rate of each well using the formula:

[0040] Hemolysis rate (%) = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100%.

[0041] S2. Preparation of Sapindus mukorossi saponins as adjuvants for veterinary vaccines

[0042] S21, Preparation of Mega-10 stock solution: Weigh 250 mg of Mega-10 dry powder, dissolve it in 1 mL of distilled water, place it in a 37℃ incubator for 1 h to dissolve it, and make up to 1.25 mL to prepare a 20% Mega-10 stock solution.

[0043] S22. Preparation of lipid mixture stock solution: Add 5 mg of soybean lecithin and 5 mg of cholesterol to 500 μL of 20% Mega-10 stock solution, place in a 55℃ water bath for 1 hour, and sonicate for 15 minutes at 40% power for 3 seconds followed by 5 seconds of sonication, while simultaneously bathing in an ice-water bath to dissolve the lipids. After dissolution, place in a 37℃ incubator overnight to prepare a 1% lipid mixture stock solution.

[0044] S23. Preparation of Sapindus mukorossi saponin stock solution: Weigh Sapindus mukorossi saponins and dissolve them in sterile PBS solution to obtain a 20 μg / mL Sapindus mukorossi saponin stock solution.

[0045] S24. Preparation of Sapindus saponin veterinary vaccine adjuvant: Take 200 μL of 1% lipid mixture stock solution and add 1300 μL of PBS solution and mix well. Then add 500 μL of Sapindus saponin stock solution and mix thoroughly. Sonicate for 2 seconds, stop for 5 seconds, for a total of 20 minutes at 20% power, while simultaneously bathing in an ice-water bath. After standing at room temperature for 2 hours, transfer to an 8KD dialysis bag and dialyze with PBS solution at room temperature for 3 days, changing the dialysate 3 times a day. After dialysis, store at 4℃. The Sapindus saponin veterinary vaccine adjuvant (NISCOM) is obtained, in which the concentration of Sapindus saponin is 5 μg / mL, cholesterol is 0.1%, and soybean lecithin is 0.1%.

[0046] This invention uses FPV as the antigen, mixed with the obtained NISCOM adjuvant to prepare a vaccine, and compares it with an immunostimulatory complex (ISCOM) as a positive control to detect the immunostimulatory effect of the NISCOM adjuvant. The immunostimulatory complex (ISCOM) is a lipid vesicle with high immunogenicity spontaneously formed by mixing antigenic substances with a mixture of saponin Quil A from saponins, cholesterol, and lecithin in a 1:1:1 ratio. Each vesicle is 40 nm in diameter and contains approximately 10-12 protein molecules. ISCOM is an excellent and novel antigen presentation system with immunostimulatory effects, possessing the dual functions of adjuvant and antigen presenter. It is applied to vaccines against various bacterial, viral, and parasitic diseases, exhibiting the efficacy of generating a "comprehensive" immune response and providing long-term enhancement of specific antibody responses.

[0047] Test content:

[0048] I. Electron Microscopic Observation of NISCOM and Particle Size and Potential Measurement of NISCOM

[0049] ①The steps for NISCOM electron microscopy observation are as follows: After dialysis, take the sample and dilute it 10 times with sterile PBS to prepare TEM sample for delivery.

[0050] ②The steps for NISCOM particle size and potential detection are as follows: After dialysis, take a sample, dilute it 10 times with sterile PBS, and use a particle size analyzer to detect its particle size and potential.

[0051] II. Mouse Experiment

[0052] ① Equilibrate 4-week-old BALB / c mice for 1 week, and immunize them for the first time on day 0 by subcutaneous single-point injection of the prescribed dose of antigen or vaccine prepared by mixing with various adjuvants.

[0053] ② Observe the mice for 3 consecutive days after inoculation for any obvious abnormalities, such as curling up, tearing, or weight loss. Pay special attention to whether there is inflammation, ulcers, or hair loss in the nostrils and mouth, and randomly check the inoculation site for ulcers.

[0054] ③ On days 14 and 21, blood was collected from the eyes of 4 mice in each group. The content of specific antibodies in the serum was detected by ELISA, and the titer of specific antibodies was detected by hemagglutination inhibition test.

[0055] ④ Second immunization on day 21.

[0056] ⑤ On days 28 and 35, blood was collected from the eyes of four mice in each group. The levels of specific antibodies in the serum were detected by ELISA, and the titers of specific antibodies were detected by hemagglutination inhibition assay.

[0057] Experimental results:

[0058] I. Hemolytic Effect Experiment of Sapindus mukorossi Saponins

[0059] Determination of the maximum absorption wavelength of heme solution: The supernatant after adding distilled water to the red blood cell suspension was scanned at 520-570 nm on an ELISA reader, and the maximum absorption wavelength was determined to be 542 nm, which was used as the wavelength for subsequent measurement of the hemolytic effect of Sapindus mukorossi saponins.

[0060] Determination of Sapindus mukorossi saponin concentration: According to Table 1, the experimental results show that when the Sapindus mukorossi saponin concentration is less than or equal to 5 μg / mL, the hemolysis rate is <5%, indicating no hemolysis occurred; when the Sapindus mukorossi saponin concentration is greater than or equal to 15 μg / mL, the hemolysis rate is >5%, indicating hemolysis occurred. Meanwhile, as shown in Table 2, the erythrocyte hemolysis rate exhibits a concentration-dependent relationship; as the Sapindus mukorossi saponin concentration increases, the erythrocyte hemolysis rate also increases. Furthermore, the hemolysis curve clearly shows that the hemolysis process is not uniform; the hemolysis rate exhibits a linear jump when the Sapindus mukorossi saponin concentration is 5–25 μg / mL, and the overall curve shows an "S" shape, as shown in Table 2. Therefore, a Sapindus mukorossi saponin concentration ≤ 5 μg / mL was selected for preparing Sapindus mukorossi saponin veterinary vaccine adjuvant.

[0061] Table 1. Absorbance and hemolysis rate at different concentrations of Sapindus mukorossi saponins

[0062]

[0063] Table 2. Absorbance and hemolysis rate at different saponin concentrations

[0064]

[0065] External characterization of Sapindus saponins as adjuvants in veterinary vaccines:

[0066] The prepared Sapindus mukorossi saponin veterinary vaccine adjuvant, containing Sapindus mukorossi saponin at a concentration of 5 μg / mL, cholesterol at 0.1%, and soybean lecithin at 0.1%, was observed under an electron microscope as follows: Figure 1 and Figure 2As shown, combined with the particle size detection results, it can be seen that NISCOM has a typical honeycomb structure, with relatively uniform size and a diameter of 30 nm. Figure 3 This is the particle size distribution diagram of NISCOM diluted 10 times according to the present invention. This result is consistent with... Figure 6 and Figure 7 The findings are consistent with those in the dissertation "Research on Inactivated Vaccine and Adjuvant for Porcine Mycoplasma Pneumonia" (Shanxi Agricultural University, 2013): the ISCOM-matrix prepared in that dissertation has a typical cage-like structure, is uniform in size, and its diameter is similar to that of ISCOM and its derivatives, all around 30 nm. Potential measurements of NISCOM diluted 10-fold yielded three results of -14.87, -14.39, and -14.93, with an average of -14.73. The negative potential indicates its relative stability.

[0067] Evaluation of Sapindus mukorossi saponins as adjuvants for veterinary vaccines in in vivo:

[0068] After vaccination, there was no significant difference in body weight between the NISCOM group, antigen group, ISCOM group, and PBS group mice. Figure 4 As shown, this indicates that NISCOM adjuvant has a good safety profile. (Combined with...) Figure 5 It can be seen that the FPV antibody levels in each group increased slowly after the first immunization, but increased rapidly after the second immunization. The FPV antibody levels in both the NISCOM and ISCOM groups were higher than those in the antigen group, and the FPV antibody levels in the NISCOM group were also higher than those in the ISCOM group.

[0069] According to the hemagglutination titer test results, as shown in Table 3, it can be seen that 14 days after immunization, FPV antibodies can be detected in the serum of the inactivated FPV, inactivated FPV+ISCOM, and inactivated FPV+NISCOM groups, but the titers are low, at 1:2 respectively. 4 1:2 5 1:2 3 Antibody titers increased significantly on day 28, and the titers in both the inactivated FPV+ISCOM group and the inactivated FPV+NISCOM group reached a ratio of 1:2 35 days post-immunization. 10 The results were largely consistent with the ELISA results, as shown in Table 4. This indicates that the Sapindus mukorossi saponin veterinary vaccine adjuvant (NISCOM) is more effective than ISCOM, significantly increasing the FPV antibody content in the serum of BALB / c immunized mice, demonstrating that the Sapindus mukorossi saponin veterinary vaccine adjuvant has a significant adjuvant effect.

[0070] Table 3. FPV antibody concentration 14 days after immunization

[0071]

[0072] Table 4. FPV antibody concentration 14 days after immunization

[0073]

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a Sapindus mukorossi saponin veterinary vaccine, characterized in that, The method for preparing the Sapindus saponin veterinary vaccine adjuvant includes mixing it with an inactivated FPV. The limiting concentration of Sapindus mukorossi saponins against erythrocyte hemolysis rate of no more than 5% was determined by spectrophotometry. Sapindus mukorossi saponins with a concentration ≤ 5 μg / mL were selected for the preparation of Sapindus mukorossi saponins as adjuvants for veterinary vaccines. Vaccine adjuvants were prepared using dialysis. The vaccine adjuvant contains soy lecithin and cholesterol in a weight ratio of 1:

1.

2. The method according to claim 1, characterized in that, Includes the following steps: S1, Hemolysis experiment, including: S11, preparation of red blood cell suspension and S12, experiment on the effect of Sapindus mukorossi saponins on hemolysis; S2, Preparation of vaccine adjuvants, including: S21, preparation of lipid mixture stock solution and S22, formulation of Sapindus saponin veterinary vaccine adjuvant.

3. The method according to claim 2, characterized in that, The procedure for S11 is as follows: Weigh 2.70g of sodium chloride and dissolve it in 300mL of ultrapure water to prepare physiological saline for later use; take 10mL of defibrinated blood from a rabbit, add physiological saline, centrifuge at 3100r / min for 4.5min, discard the supernatant, add physiological saline, centrifuge again at 3100r / min for 4.5min, and repeat washing with physiological saline until the supernatant after centrifugation is no longer red; after the supernatant is no longer red, aspirate and discard the upper layer of physiological saline in the centrifuge tube, take 40μL of the lower layer of red blood cell precipitate, add 2mL of physiological saline, and prepare a 2% red blood cell suspension.

4. The method according to claim 2, characterized in that, The operation of S12 is as follows: S121, Solution Preparation Accurately measure 1.00 mg of the self-made Sapindus saponin sample, place it in a beaker, dissolve it with 10.0 mL of physiological saline, shake well to obtain a 0.10 mg / mL Sapindus saponin stock solution; take several centrifuge tubes, number them, add different concentrations of Sapindus saponin stock solution to each tube, and then add different concentrations of physiological saline to each tube to prepare gradient solutions of different concentrations. S122. Determination of the maximum absorption wavelength of heme solution Take 1 mL of distilled water into a test tube, add 1 mL of red blood cell suspension, and mix well; place in a 37℃ constant temperature water bath for 30 min to allow for full hemolysis, and then immediately place in ice water to stop the hemolysis; centrifuge at 3000 r / min for 5 min, and take 100 μL of the supernatant containing heme from each group into a 96-well plate, with 100 μL of physiological saline as a control, and use an ELISA reader to determine the maximum absorption wavelength; S123. Spectrophotometric determination of its hemolysis rate Take several test tubes and number them, including test tubes containing different concentrations of Sapindus mukorossi saponins and two control tubes. Add 1 mL of 2% red blood cell suspension to each tube first, then add 1 mL of Sapindus mukorossi saponins of different concentrations to each test tube. Add 1 mL of physiological saline to the first control tube and 1 mL of distilled water to the other control tube, so that the total volume of solution in each tube is 2 mL. Mix well and incubate in a water bath at 37°C for 3 hours. After 3 hours, immediately place the solutions in ice water to stop the hemolysis. Centrifuge at 3000 r / min for 5 min, and take 100 μL of the supernatant into a 96-well plate, with 5 replicates per group. Read the OD value of each well at the maximum absorption wavelength on a microplate reader. Calculate the hemolysis rate of each well using the formula: Hemolysis rate (%) = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100%.

5. The method according to claim 2, characterized in that, The operation of S21 is as follows: Weigh 250 mg of Mega-10 dry powder, dissolve it in 1 mL of distilled water, place it in a 37°C incubator for 1 hour to dissolve it, and make up the volume to 1.25 mL to prepare a 20% Mega-10 stock solution; add 5 mg of soybean lecithin and 5 mg of cholesterol to 500 μL of the 20% Mega-10 stock solution, place it in a 55°C water bath for 1 hour, and perform ultrasonic treatment. The ultrasonic treatment is performed for 3 seconds and then stopped for 5 seconds, for a total of 15 minutes at 40% power. At the same time, it is placed in an ice-water bath to dissolve it. After dissolution, it is placed in a 37°C incubator overnight to prepare a 1% lipid mixture stock solution.

6. The method according to claim 2, characterized in that, The procedure in S22 is as follows: Weigh out Sapindus mukorossi saponins and dissolve them in sterile PBS solution to obtain a 20 μg / mL Sapindus mukorossi saponin stock solution; take 200 μL of 1% lipid mixture stock solution and add 1300 μL of PBS solution and mix well; add 500 μL of Sapindus mukorossi saponin stock solution and mix thoroughly; sonicate for 2 seconds, stop for 5 seconds, for a total of 20 minutes at 20% power, while simultaneously bathing in an ice-water bath; after standing at room temperature for 2 hours, transfer to an 8KD dialysis bag and dialyze with PBS solution at room temperature for 3 days, changing the dialysate 3 times a day; after dialysis, store at 4℃ to obtain the final product.

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