Preparation process of southwest panax quinquefolium uniform immunomodulatory polysaccharide and application thereof in preparation of anti-melanoma drugs and immunomodulators

Abstract

This invention discloses a preparation process for a homogeneous immunomodulatory polysaccharide from *Gynostemma pentaphyllum* and its application in the preparation of anti-melanoma drugs and immunomodulators, relating to the field of pharmaceutical manufacturing technology. The preparation process includes four steps: compound enzyme-ultrasound synergistic extraction, fractional alcohol precipitation to remove proteins, DEAE-52 ion exchange column chromatography, and Sephacryl S-400 gel permeation chromatography purification. The resulting homogeneous polysaccharide (GOP-3) has a weight-average molecular weight of 12±1 kDa and is composed of mannose, glucose, and galactose in a specific ratio. The GOP-3 of this invention has a weight-average molecular weight (Mw) of 12.8 kDa, a dispersion (Mw / Mn) < 1.2, a uronic acid content of 23.5%, a well-defined structure, and is suitable for drug application. Furthermore, GOP-3 can significantly promote the secretion of IL-12 by dendritic cells and induce the differentiation of naive T cells into Th1 cells. Simultaneously, compared with a full-dose PD-1 monoclonal antibody, the combination of GOP-3 and a half-dose monoclonal antibody not only reduces the risk of immune-mediated pneumonia caused by antibody drugs but also increases the tumor inhibition rate by more than 40%.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical manufacturing technology, specifically to a preparation process of a homogeneous immunomodulatory polysaccharide from *Gynostemma pentaphyllum* and its application in the preparation of anti-melanoma drugs and immunomodulators. Background Technology

[0002] Although the polysaccharides of Codonopsis pilosula from Southwest China are known to have immunomodulatory activity, the traditional water extraction and alcohol precipitation method yields a mixture of polysaccharides (crude polysaccharides) with a wide molecular weight distribution (from several thousand to several million Da), poor batch-to-batch stability, and difficulty in quality control. Existing technologies only vaguely mention "enhancing immunity" without specifying whether they exert anti-tumor effects by regulating dendritic cell (DC) maturation or T cell infiltration. There are no reports of using Southwest Ginseng Polysaccharide to reverse PD-1 inhibitor resistance. Clinically, the efficacy rate of PD-1 monoclonal antibody monotherapy for melanoma is only 30%-40%, highlighting the urgent need for safe and effective combination therapy regimens. Therefore, a preparation process for homogeneous immunomodulatory polysaccharides from *Gynostemma pentaphyllum* is proposed, and its application in the preparation of anti-melanoma drugs and immunomodulators is described. Summary of the Invention

[0003] The purpose of this invention is to provide a preparation process for homogeneous immunomodulatory polysaccharides from *Gynostemma pentaphyllum* and their application in the preparation of anti-melanoma drugs and immunomodulators, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a preparation process for homogeneous immunomodulatory polysaccharides from *Gynostemma pentaphyllum* and its application in the preparation of anti-melanoma drugs and immunomodulators, comprising the following steps: S1 complex enzyme-ultrasound synergistic extraction: Take the crude powder of Southwest Chinese ginseng, add 20 times the amount of citrate buffer (pH=5.0), add cellulase and papain (mass ratio 2:1, total enzyme amount is 1.5% of the mass of Southwest Chinese ginseng crude powder), extract for 40 minutes under ultrasonic conditions at 55℃ and 300W power, and inactivate the enzyme in boiling water bath for 10 minutes after enzymatic hydrolysis. S2 fractionation and deproteinization: The extract obtained from S1 was centrifuged. After centrifugation, the supernatant was concentrated to ¼ of the original volume. Anhydrous ethanol was slowly added to a final concentration of 40%. The mixture was allowed to stand overnight at 4°C. The precipitate was discarded (to remove macromolecular starch). Anhydrous ethanol was added to a final concentration of 75%. The precipitate was collected to obtain crude polysaccharide. After the crude polysaccharide was reconstituted, it was repeatedly extracted 5 times using the Sevag-n-butanol method (volume ratio 4:1) until there was no protein absorption peak at 280 nm, thus completing the deproteinization process. S3 ion exchange column chromatography (DEAE-52): The deproteinized polysaccharide obtained from S2 was loaded onto a DEAE-52 ion exchange column. Neutral sugars were first removed by elution with deionized water, followed by gradient elution with 0.2M, 0.4M, and 0.6M NaCl solutions. The 0.4M NaCl elution peak fraction was collected (this fraction was screened to have the strongest immunogenicity). S4 Gel permeation chromatography (Sephacryl S-400) purification: The 0.4M NaCl elution peak fraction collected in S3 was loaded onto a Sephacryl S-400 gel column, eluted with 0.1M NaCl solution at a flow rate of 0.5 mL / min, and a single symmetrical elution peak was collected. After dialysis (cutoff of 3500 Da) to remove salt, the sample was freeze-dried to obtain homogeneous immunomodulatory polysaccharide. The homogeneous immunomodulatory polysaccharide has a weight-average molecular weight of 12±1 kDa, a dispersion (Mw / Mn) of <1.2, and is composed of mannose, glucose, and galactose, with a mannose:glucose:galactose ratio of 1.0:2.3:1.8.

[0005] Furthermore, the coarse powder of Southwest Chinese ginseng in S1 is obtained by removing impurities from dried tubers of Southwest Chinese ginseng, washing and drying them, and then crushing them through an 80-100 mesh sieve.

[0006] Furthermore, in S2, the centrifugation conditions are 8000 r / min and 15 min; the concentration is achieved by rotary evaporation at a concentration temperature of 60°C.

[0007] Furthermore, the pretreatment method for the DEAE-52 ion exchange column in S3 is as follows: B1: Soak DEAE-52 in 10 times the amount of distilled water for 12 hours to remove suspended impurities and broken particles; B2: Then, the solution is pretreated in sequence with an alkaline solution, an acidic solution, and another alkaline solution. The alkaline solution is 0.5 mol / L NaOH, and the acidic solution is 0.5 mol / L HCl. B3: Soak in 4 times the volume of the corresponding reagent for half an hour and stir, then filter and wash with distilled water until neutral. B4: After packing the column, equilibrate with the starting buffer until the pH of the elution buffer is consistent with that of the starting buffer.

[0008] Furthermore, in S4, the dialysis time is 24 hours, during which the deionized water is replaced 3-4 times; the freeze-drying temperature is -80℃; the vacuum degree is 10-20 Pa; and the Sephacryl S-400 gel column pretreatment method is as follows: D1: Prepare a gel paste from Sephacryl S-400 gel; D2: Equilibrate with 0.1M NaCl elution buffer, and remove air bubbles from the column after packing.

[0009] Furthermore, the homogeneous immunomodulatory polysaccharide (GOP-3) described above can be used in combination with PD-1 monoclonal antibodies (such as pembrolizumab); Application of Z1 Southwest Ginseng Homogeneous Immunomodulatory Polysaccharide in the Preparation of Drugs that Promote Dendritic Cell Maturation and Increase IL-12 Secretion: This homogeneous polysaccharide can significantly promote the maturation of dendritic cells (DCs) and induce DCs to secrete IL-12 (interleukin-12). IL-12 can further induce naive T cells to differentiate into Th1 cells, enhance the body's cellular immune function, and provide a new active ingredient for the development of immunomodulatory drugs.

[0010] Application of Z2 Southwest Ginseng Homogeneous Immunomodulatory Polysaccharide in the Preparation of Tumor Immunomodulators: This homogeneous polysaccharide can activate DC cells, promote CD8+ T cell infiltration, reshape the tumor immunosuppressive microenvironment, relieve T cell depletion, and enhance the body's immune surveillance and killing ability against tumor cells. It can be used as a tumor immunomodulator to improve the immune function of cancer patients and assist in tumor treatment.

[0011] Application of Z3 Southwest Ginseng Homogeneous Immunomodulatory Polysaccharide in the Preparation of Anti-melanoma Drugs: This homogeneous polysaccharide can be used in combination with PD-1 / PD-L1 immune checkpoint inhibitors (such as pembrolizumab) to prepare anti-melanoma drugs. The usual clinical dose of pembrolizumab for adults is 200 mg every 3 weeks or 400 mg every 6 weeks, administered by intravenous infusion over at least 30 minutes. The preferred combination therapy of this invention is: oral administration of homogeneous immunomodulatory polysaccharide of Codonopsis pilosula at a dose of 50 mg / kg / day; intraperitoneal injection of PD-1 monoclonal antibody at a dose of 50% of the conventional clinical dose (5 mg / kg, once every 3 days). This therapy can effectively reverse PD-1 inhibitor resistance and reduce toxic side effects such as immune pneumonia.

[0012] Compared with the prior art, the beneficial effects of the present invention are: In this application, GOP-3 has a weight-average molecular weight (Mw) of 12.8 kDa, a dispersion (Mw / Mn) of < 1.2, a uronic acid content of 23.5%, a well-defined structure, and is suitable for drug application. Furthermore, GOP-3 can significantly promote the secretion of IL-12 by DC cells and induce the differentiation of naive T cells into Th1 cells. At the same time, compared with the full-dose PD-1 monoclonal antibody, the combination of GOP-3 and half-dose monoclonal antibody not only reduces the risk of immune pneumonia caused by antibody drugs, but also increases the tumor inhibition rate by more than 40%. Detailed Implementation

[0013] The technical solutions in the embodiments of the invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the invention, and not all embodiments.

[0014] Unless otherwise specified in the examples, the experimental methods used are all conventional methods, and the reagents or instruments used are all commercially available products. Unless otherwise specified, the reagents used in the extraction of this invention are all analytical grade reagents.

[0015] Example 1: Preparation process of homogeneous immunomodulatory polysaccharide (GOP-3) from Ginseng from Southwest China S1 Raw Material Pretreatment: Take the dried tubers of Southwest Chinese ginseng, remove impurities and wash them, dry them at 60℃ for 4 hours, crush them and pass them through an 80-100 mesh sieve to obtain Southwest Chinese ginseng coarse powder for later use. S2 complex enzyme-ultrasound synergistic extraction: Weigh 100g of crude ginseng powder from Southwest China and add 2000mL of citrate buffer at pH 5.0. Add cellulase and papain (mass ratio 2:1, total enzyme amount 1.5g) to the solution, stir evenly, and place it in an ultrasonic device at 55℃ and 300W for extraction for 40 minutes. After extraction, inactivate the enzyme by boiling in a water bath for 10 minutes and allow it to cool to room temperature. In the combined enzyme-ultrasound extraction process, cellulase can disrupt the cell wall structure of Codonopsis pilosula, thereby promoting polysaccharide dissolution, while papain can degrade protein impurities in the raw material to reduce the difficulty of subsequent deproteinization steps. At the same time, ultrasound-assisted extraction can further improve extraction efficiency, thereby shortening the extraction time and avoiding the destruction of polysaccharide activity by high temperature, thus achieving efficient and green extraction of polysaccharides.

[0016] S3 Fractional Alcohol Precipitation and Deproteinization: The extract obtained in S2 was placed in a centrifuge tube and centrifuged at 8000 r / min for 15 min to remove the precipitate (impurities). The supernatant after centrifugation was collected and placed in a rotary evaporator. It was concentrated to 1 / 4 of the original volume at 60°C. At the same time, anhydrous ethanol was slowly added to a final concentration of 40%. The mixture was allowed to stand overnight at 4°C and then centrifuged to remove the precipitate (mainly macromolecular starch impurities). Anhydrous ethanol was added to the remaining supernatant to a final concentration of 75%. The mixture was allowed to stand overnight at 4°C and then centrifuged at 8000 r / min for 15 min. The precipitate after centrifugation was collected to obtain the crude polysaccharide of Codonopsis pilosula. The crude polysaccharide was reconstituted with 100 mL of deionized water, and 25 mL of Sevag reagent (chloroform: n-butanol = 4:1) was added. The extraction was repeated 5 times, while vigorous shaking for 10 min and centrifugation at 8000 r / min for 10 min. After centrifugation, the upper aqueous phase was collected until no protein absorption peak was detected at a wavelength of 280 nm. The deproteinization process was completed to obtain a deproteinized polysaccharide solution. Fractional alcohol precipitation can selectively precipitate polysaccharides of different molecular weights by using ethanol of different concentrations, thereby removing macromolecular starch impurities and achieving preliminary purification of polysaccharides. The Sevag-n-butanol method has high deproteinization efficiency and can effectively remove protein impurities from polysaccharides. Moreover, this method has no significant impact on the activity of polysaccharides and can ensure the purity and activity of polysaccharides. S4 ion exchange column chromatography (DEAE-52): Take DEAE-52 and add it to 10 times the volume of distilled water and soak for 12 hours to remove suspended impurities and broken particles. Then soak it in 0.5 mol / L NaOH solution for half an hour with stirring. After filtration, wash it with distilled water until neutral. Then soak it in 0.5 mol / L HCl solution for half an hour with stirring. After filtration, wash it with distilled water until neutral. Finally, soak it in 0.5 mol / L NaOH solution for half an hour with stirring. After filtration, wash it with distilled water until neutral to complete the pretreatment. The pretreated DEAE-52 ion exchange column (2.6cm×60cm) was packed and equilibrated with starting buffer until the pH of the elution buffer was consistent with that of the starting buffer. Then, the deproteinized polysaccharide solution obtained in S3 was loaded onto the DEAE-52 ion exchange column and eluted with deionized water at a flow rate of 1.0mL / min. One fraction was collected every 5mL and the polysaccharide content was determined by the phenol-sulfuric acid method. The neutral sugar fraction was discarded. Finally, gradient elution was performed sequentially with 0.2M, 0.4M, and 0.6M NaCl solutions. Simultaneously, one fraction was collected every 5 mL, and the polysaccharide content of each fraction was detected using the phenol-sulfuric acid method. The fraction corresponding to the 0.4M NaCl elution peak (which, after screening, showed the strongest immunomodulatory activity) was collected, combined, and the combined solution was concentrated to an appropriate volume. S5 Gel Permeation Chromatography (Sephacryl S-400) Purification: Take a Sephacryl S-400 gel and add ¾ volume of gel and ¼ volume of 0.1M NaCl eluent to prepare a gel slurry. Stir the prepared gel slurry thoroughly to avoid the formation of air bubbles. Then, pack it into a chromatography column and remove any air bubbles. Equilibrate the chromatography column with 0.1M NaCl eluent to complete the pretreatment. Next, load the concentrated 0.4M NaCl eluent collected in S4 into the pretreated Sephacryl S-400 gel column (1.6cm × 100cm) and elute with 0.1M NaCl. NaCl solution was used as the eluent, and the flow rate was controlled at 0.5 mL / min. One fraction was collected every 3 mL, and the polysaccharide content was determined using the phenol-sulfuric acid method. The fraction corresponding to a single symmetrical elution peak was collected. The collected fraction was placed in a dialysis bag (with a cutoff of 3500 Da) and dialyzed with deionized water for 24 h. The deionized water was changed 3 times during dialysis. After dialysis, the fraction was freeze-dried at -80 °C to obtain a white, loose powder, which is GOP-3, with a yield of 8.2 g.

[0017] Example 2: Structural characterization of homogeneous immunomodulatory polysaccharide (GOP-3) from Ginseng from Southwest China The structure of GOP-3 prepared in Example 1 was characterized, and the results are shown in the table below:

[0018] The data in the table show that the GOP-3 prepared by this invention has the characteristics of good uniformity, high purity, and well-defined structure, which meets the quality requirements for drug application.

[0019] Example 3: Immunomodulatory Activity Experiment of Uniform Immunomodulatory Polysaccharide (GOP-3) from Ginseng from Southwest China Mouse bone marrow cells were prepared and cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin-dextrin, 20 ng / mL GM-CSF and 10 ng / mL IL-4. The medium was changed every two days during the culture period, and the cells were cultured for a total of seven days to obtain immature dendritic cells. Immature DC cells were divided into four groups: control group, low-dose GOP-3 group (50 μg / mL), medium-dose GOP-3 group (100 μg / mL), and high-dose GOP-3 group (200 μg / mL). Each group had three replicates. During the experiment, an equal volume of culture medium was added to the control group, while each experimental group was given a corresponding concentration of GOP-3, and the mixture was kept at 37°C and 5% concentration. The cells were cultured in an incubator under controlled conditions for 24 hours. During the culture, the supernatant of each experimental group was collected, and the IL-12 content was detected using an ELISA kit. Compared with the control group, all doses of GOP-3 significantly promoted the secretion of IL-12 by DC cells (p<0.001) in a dose-dependent manner. Among them, the high-dose group (200μg / mL) had the highest IL-12 secretion, which was 3.2 times that of the control group. This indicates that GOP-3 has a significant immune-activating effect and can effectively promote the maturation of DC cells.

[0020] Example 4: In vivo anti-melanoma experiment of homogeneous immunomodulatory polysaccharide (GOP-3) from Ginseng from Southwest China. This experiment used the B16-F10 melanoma mouse model to investigate the anti-melanoma effects of GOP-3 alone and in combination with PD-1 monoclonal antibody. First, select 6-8 week old C57BL / 6 mice and subcutaneously inject B16-F10 melanoma cells (1×10⁻⁶) into their backs. (each tumor), until the tumor volume grows to a certain size. When the model was successfully constructed, the mice were randomly divided into 5 groups of 10 mice each. Secondly, the five groups were placed in different environments: model group (saline), GOP-3 monotherapy group, PD-1 monoclonal antibody (full dose) group, GOP-3 + PD-1 monoclonal antibody (half dose) group, and blank control group (no tumor inoculation, saline). Finally, different medication regimens were assigned to each group. Mice in the model group were given oral saline orally and intraperitoneal injection of saline daily (every 3 days); mice in the GOP-3 monotherapy group were given oral GOP-3 (50 mg / kg / day) and intraperitoneal injection of saline daily (every 3 days); mice in the PD-1 monoclonal antibody (full dose) group were given oral saline orally and intraperitoneal injection of PD-1 monoclonal antibody (10 mg / kg, every 3 days); mice in the GOP-3+PD-1 monoclonal antibody (half dose) group were given oral GOP-3 (50 mg / kg / day) and intraperitoneal injection of PD-1 monoclonal antibody (5 mg / kg, every 3 days); and mice in the blank control group were given oral saline orally and intraperitoneal injection of saline daily (every 3 days).

[0021] Twenty-one days after drug administration, tumor volume was measured in each group of mice, and tumor inhibition rate was calculated. Mice were then sacrificed, and tumor tissue was dissected. Flow cytometry was used to detect the proportion of tumor-infiltrating CD8+ T cells, and the results are shown in the table below.

[0022] According to the data in the table, compared with the model group, all treatment groups significantly inhibited tumor growth (p<0.001) and significantly increased the proportion of tumor-infiltrating CD8+ T cells (p<0.001). Among them, the tumor inhibition rate of the group using GOP-3 in combination with half-dose PD-1 monoclonal antibody was the highest (82.7%), which was significantly higher than that of the group using full-dose PD-1 monoclonal antibody (63.2%), and the proportion of tumor-infiltrating CD8+ T cells was the highest. This indicates that the combination of GOP-3 and PD-1 monoclonal antibody has a significant synergistic anti-melanoma effect, can effectively reverse PD-1 inhibitor resistance, and at the same time reduce the dosage of PD-1 monoclonal antibody and reduce its toxic side effects.

[0023] The foregoing has shown and described the basic principles, main features, and advantages of the invention. Those skilled in the art should understand that the invention is not limited to the above embodiments, and various changes and modifications can be made without departing from the spirit and scope of the invention; all such changes and modifications fall within the scope of the invention claims. The scope of the invention claims is defined by the appended claims and their equivalents.

Claims

1. A preparation process for a homogeneous immunomodulatory polysaccharide from *Gynostemma pentaphyllum*, characterized in that, Includes the following steps: S1 complex enzyme-ultrasound synergistic extraction: Take the crude powder of Southwest Chinese ginseng, add 20 times the amount of citrate buffer (pH=5.0), add cellulase and papain (mass ratio 2:1, total enzyme amount is 1.5% of the mass of Southwest Chinese ginseng crude powder), extract for 40 minutes under ultrasonic conditions at 55℃ and 300W power, and inactivate the enzyme in boiling water bath for 10 minutes after enzymatic hydrolysis. S2 fractionation and deproteinization: The extract obtained from S1 was centrifuged. After centrifugation, the supernatant was concentrated to ¼ of the original volume. Anhydrous ethanol was slowly added to a final concentration of 40%. The mixture was allowed to stand overnight at 4°C. The precipitate was discarded (to remove macromolecular starch). Anhydrous ethanol was added to a final concentration of 75%. The precipitate was collected to obtain crude polysaccharide. After the crude polysaccharide was reconstituted, it was repeatedly extracted 5 times using the Sevag-n-butanol method (volume ratio 4:1) until there was no protein absorption peak at 280 nm, thus completing the deproteinization process. S3 ion exchange column chromatography (DEAE-52): The deproteinized polysaccharide obtained from S2 was loaded onto a DEAE-52 ion exchange column. Neutral sugars were removed by elution with deionized water, followed by gradient elution with 0.2M, 0.4M, and 0.6M NaCl solutions. The 0.4M NaCl elution peak fraction was collected (this fraction was screened to have the strongest immunogenicity). S4 Gel Permeation Chromatography (Sephacryl S-400) Purification: The 0.4M NaCl elution peak collected in S3 was loaded onto a Sephacryl S-400 gel column and eluted with 0.1M NaCl solution at a flow rate of 0.5 mL / min. A single symmetrical elution peak was collected, and after dialysis (with a cutoff of 3500 Da) to remove salt, it was freeze-dried to obtain homogeneous immunomodulatory polysaccharide (GOP-3). The homogeneous immunomodulatory polysaccharide has a weight-average molecular weight of 12±1 kDa, a dispersion (Mw / Mn) of <1.2, and is composed of mannose, glucose, and galactose, with a mannose:glucose:galactose ratio of 1.0:2.3:1.

8.

2. The preparation process according to claim 1, characterized in that, The coarse powder of Southwest Chinese ginseng in S1 is obtained by removing impurities, washing and drying, and crushing the dried tubers of Southwest Chinese ginseng into powder through an 80-100 mesh sieve.

3. The preparation process according to claim 1, characterized in that, The centrifugation conditions in S2 are 8000 r / min and 15 min; the concentration is carried out by rotary evaporation at a concentration temperature of 60℃.

4. The preparation process according to claim 1, characterized in that, The pretreatment method for the DEAE-52 ion exchange column in S3 is as follows: B1: Soak DEAE-52 in 10 times the amount of distilled water for 12 hours to remove suspended impurities and broken particles; B2: Then, the solution is pretreated in sequence with an alkaline solution, an acidic solution, and another alkaline solution. The alkaline solution is 0.5 mol / L NaOH, and the acidic solution is 0.5 mol / L HCl. B3: Soak in 4 times the volume of the corresponding reagent for half an hour and stir, then filter and wash with distilled water until neutral. B4: After packing the column, equilibrate with the starting buffer until the pH of the elution buffer is consistent with that of the starting buffer.

5. The preparation process according to claim 1, characterized in that, The S4 dialysis time is 24 hours, with deionized water replaced 3-4 times during dialysis. The freeze-drying temperature is -80℃, the vacuum degree is 10-20 Pa, and the Sephacryl S-400 gel column pretreatment method is as follows: D1: Prepare a gel paste from Sephacryl S-400 gel; D2: Equilibrate with 0.1M NaCl elution buffer, and remove air bubbles from the column after packing.

6. The preparation process according to any one of claims 1-5, characterized in that, The prepared homogeneous immunomodulatory polysaccharide of Codonopsis pilosula.

7. The homogeneous immunomodulatory polysaccharide of *Gynostemma pentaphyllum* according to claim 6, characterized in that, Application in the preparation of drugs that promote dendritic cell maturation and increase IL-12 secretion.

8. The homogeneous immunomodulatory polysaccharide of *Gynostemma pentaphyllum* according to claim 6, characterized in that, Application in the preparation of tumor immunomodulators.

9. A pharmaceutical composition for treating melanoma, characterized in that, The product comprises a therapeutically effective amount of the homogeneous immunomodulatory polysaccharide of *Codonopsis pilosula* as described in claim 6, and an immune checkpoint inhibitor; wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 monoclonal antibody, preferably pembrolizumab.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage of the homogeneous immunomodulatory polysaccharide of the Southwest Chinese ginseng is 10-100 mg / kg / day, and the dosage of the immune checkpoint inhibitor is 30%-70% of the conventional clinical dose.

11. The application of the homogeneous immunomodulatory polysaccharide of *Gynostemma pentaphyllum* according to claim 6 in the preparation of anti-melanoma drugs, characterized in that, The anti-melanoma drug is a combination of the homogeneous polysaccharide and the PD-1 / PD-L1 monoclonal antibody.

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