New medical application of low-medium glycans in American ginseng and medicinal preparation of low-medium glycans

Abstract

The invention discloses a novel medical application of low-medium glycans in American ginseng and a medicinal preparation of the low-medium glycans in American ginseng. The invention provides low-medium glycans separated from American ginseng. The low-medium glycans are mainly formed by polymerizing 2-15 hexose molecules. The hexose molecule is composed of glucose and fructose. The American ginseng low-medium glycan component provided by the invention not only can down-regulate the expression level of PD-L1 in tumor cells, but also can reduce the expression level of PD-1 in immune cells such as T cells, effectively activate the T cells and hinder tumor immune escape, has the application of preparing a PD-L1 inhibitor, a PD-1 inhibitor, a PD-L1 and PD-1 dual inhibitor or an anti-tumor drug, and can be used for preparing the anti-tumor drug. The compound can be used for immunotherapy of various cancers such as lung cancer, breast cancer, colon cancer, melanoma and the like, and has an application prospect in the aspect of tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to an effective component isolated from traditional Chinese medicinal materials and its application, in particular to a new medical use of low-mid polysaccharides isolated from Panax quinquefolium and its pharmaceutical preparation, and belongs to the field of low-mid polysaccharides isolated from Panax quinquefolium and its medical application. BACKGROUND

[0002] PD-L1 and PD-1 are important immune checkpoints, and their interaction can negatively regulate the activation and proliferation of T cells, and is also an important way for tumor cells to achieve immune escape. Blocking the binding of PD-L1 and PD-1 can relieve the activation and proliferation of T cells, so that tumor-specific T cells are in an activated state, thereby restoring the killing function of T cells. The currently approved PD-L1 inhibitors include atezolizumab, durvalumab, avelumab, enfortumab vedotin, and atezolizumab. Atezolizumab is mainly used for non-small cell lung cancer, durvalumab is mainly used for non-small cell lung cancer and small cell lung cancer, avelumab is mainly used for first-line treatment of metastatic non-squamous non-small cell lung cancer patients, and enfortumab vedotin is mainly used for treating unresectable or metastatic solid tumors. The currently approved PD-1 inhibitors include pembrolizumab, nivolumab, camrelizumab, toripalimab, sintilimab, tislelizumab, pidilizumab, peficitinib, spartalizumab, and sasanlimab. Among them, pembrolizumab and nivolumab are imported inhibitors, mainly used for treating non-small cell lung cancer and head and neck squamous cell carcinoma. These inhibitors are suitable for a small number of cancer types, have high prices, high incidence of adverse reactions, and cannot be used alone and need to be used with other targeted drugs. Therefore, it is necessary to develop a low-cost targeted drug suitable for most tumor patients, which has tumor inhibition and immune enhancement.

[0003] Panax quinquefolium L., belonging to the perennial herb of Araliaceae Panax genus. The dry root of commonly used American ginseng is used as the medicinal part of American ginseng. Traditional Chinese medicine theory believes that American ginseng is bitter, sweet, cool, thick in taste, and thin in gas, has the functions of tonifying the spleen and reducing fire, and producing body fluid, and has been used as medicinal materials in China for more than three hundred years. In China, it is planted in northeast China, Shandong, Shaanxi. At present, American ginseng is widely used as an immunomodulator, which can activate immune cells, improve immune cell activity, and enhance the non-specific and specific immune function of the body. The current patents, such as CN202310691591.0 A kind of traditional Chinese medicine composition for nourishing yin and invigorating deficiency and anti-fatigue and its preparation method and application, CN202410881133.8 A preparation method of American ginseng fermented beverage and the obtained product, all study the efficacy of American ginseng as a compound component. However, so far no literature has shown that low-mid polysaccharide components from American ginseng have the potential to develop as PD-L1 and PD-1 double inhibitors. SUMMARY

[0004] One of the purposes of the present application is to provide low-mid polysaccharides isolated from American ginseng;

[0005] The second purpose of the present application is to use the low-mid polysaccharides isolated from American ginseng to prepare PD-L1 and PD-1 double inhibitors or to prepare antitumor drugs.

[0006] To achieve the above-mentioned purpose, the main technical solutions adopted by the present application include:

[0007] One aspect of the present application is to provide low-mid polysaccharides isolated from American ginseng, which are mainly composed of 2 to 15 hexose molecules; wherein the hexose molecules are composed of glucose and fructose, and the molar ratio of glucose to fructose is 65:35.

[0008] Further, the representative structure of the low-mid polysaccharide is 1,4-Glcp connection, and the representative 1,4-Glcp connection structure is as follows:

[0009]

[0010] Another aspect of the present application is to provide a method for preparing low-mid polysaccharides from American ginseng, comprising:

[0011] (1) using water as the extraction solvent, the American ginseng water extract is obtained by heating reflux extraction method;

[0012] (2) the American ginseng water extract is adsorbed by macroporous resin column, and after removing pigments and proteins by resin adsorption, the American ginseng macroporous resin water eluate is obtained by elution with eluent;

[0013] (3) the macroporous resin water eluate of Panax quinquefolium is subjected to ultrafiltration by an ultrafiltration column or dialysis by a dialysis bag to obtain refined low and medium polysaccharides;

[0014] (4) the refined low and medium polysaccharides of Panax quinquefolium are subjected to nanofiltration by a nanofiltration column and then desalination to obtain a low and medium polysaccharide component of Panax quinquefolium.

[0015] In a preferred embodiment of the present application, the macroporous resin column in step (2) is an AB-8 type macroporous resin; the eluent in step (2) is distilled water, the distilled water elution volume is 5 times the column volume, and the end point of elution is detected by a phenol-sulfuric acid method.

[0016] In a preferred embodiment of the present application, the ultrafiltration column used in step (3) has a molecular weight cut-off of 3 KDa, and the collected component is an effluent with a molecular weight less than 3 KDa; wherein the sample concentration is 8:1 (water volume mL: medicinal material weight g), and the ultrafiltration pressure is 1 MPa.

[0017] In a preferred embodiment of the present application, the nanofiltration column used in step (4) has a molecular weight cut-off of 500 Da, and the refined low and medium polysaccharides of Panax quinquefolium are subjected to nanofiltration to obtain a low and medium polysaccharide component of Panax quinquefolium; wherein the sample concentration is 10:1 (water volume mL: medicinal material weight g), and the nanofiltration pressure is 2 MPa.

[0018] In still another aspect of the present application, there is provided a use of the low and medium polysaccharide component of Panax quinquefolium in the preparation of a PD-L1 inhibitor, a PD-1 inhibitor, a PD-L1 and PD-1 dual inhibitor, or an anti-tumor drug.

[0019] In order to verify whether the low and medium polysaccharide component isolated from Panax quinquefolium has an inhibitory effect on the expression and function of PD-L1 protein after binding to the PD-L1 protein, a cell toxicity experiment was first performed by a CCK-8 method, and the results showed that the proliferation of six different tumor cells was not significantly inhibited at a low dose (500 μg / mL), indicating that the low and medium polysaccharide component of Panax quinquefolium itself does not have cytotoxicity at a low dose. Subsequently, the PD-L1 expression levels of six different tumor cells, including human non-small cell lung cancer A549, human non-small cell lung adenocarcinoma NCI-H157, human breast cancer MCF-7, mouse melanoma B16, mouse myeloid breast cancer E0771, and human colon cancer HT-29, were detected by a Western blot method.

[0020] Further research found that the low-mid polysaccharide of Panax quinquefolium has a down-regulation effect on PD-L1 of four kinds of tumor cells with high expression of PD-L1, among which the down-regulation trend of non-small cell lung cancer NCI-H157 cells and melanoma B16 cells is the most obvious; and the down-regulation effect on the tumor cell surface PD-L1 is concentration-dependent; when the concentration of the drug is 500 μg / mL, the down-regulation of PD-L1 of the tumor cells is the most significant.

[0021] The present application uses 15 ng / mL of interferon IFN-γ to induce non-small cell lung cancer A549 cells and human breast cancer cells MCF-7 cells with high expression of PD-L1, and after the low-mid polysaccharide of Panax quinquefolium acts on the cells, the experimental results show that the low-mid polysaccharide of Panax quinquefolium can obviously down-regulate the expression of PD-L1, which proves that the low-mid polysaccharide of Panax quinquefolium has a down-regulation effect on the tumor cells with high expression of PD-L1 and the induced tumor cells with high expression of PD-L1, especially the inhibition effect on the expression of PD-L1 of lung cancer cell lines is particularly significant.

[0022] The present application uses flow cytometry to detect the influence of the low-mid polysaccharide of Panax quinquefolium on the expression abundance of PD-L1 of lung cancer cells, and the results show that with the increase of the concentration, the fluorescence intensity of the two kinds of human lung cancer cells gradually weakens, which further verifies that the low-mid polysaccharide of Panax quinquefolium has a down-regulation effect on the expression of PD-L1 of non-small cell lung cancer and metastatic lung cancer cell lines. Moreover, the Western Blot results show that the down-regulation effect of the low-mid polysaccharide of Panax quinquefolium on the expression of PD-L1 of the two kinds of human lung cancer cells is also time-dependent, and with the increase of the administration time, the expression amount of PD-L1 significantly decreases.

[0023] In order to verify the inhibition effect of the low-mid polysaccharide of Panax quinquefolium on PD-1 of immune cells, the expression levels of PD-1 of mononuclear macrophage leukemia RAW264.7 cells and peripheral blood leukemia T cells are detected. The present application uses 50 μg / mL of lipopolysaccharide LPS to induce RAW264.7, and uses 1 ng / mL of phorbol ester PMA to induce Jurkat T cells to highly express PD-1, and the low-mid polysaccharide of Panax quinquefolium acts on the cells, and the experimental results prove that the low-mid polysaccharide of Panax quinquefolium has a significant down-regulation effect on the PD-1 protein of the immune cells with high expression of PD-1.

[0024] On this basis, the present application further detects whether the down-regulation of PD-L1 mediated by Panax quinquefolius low-mid polysaccharides affects the level of the combination of tumor cell PD-L1 and T cell PD-1 protein. According to the experimental results, it is found that Panax quinquefolius low-mid polysaccharides dose-dependently block the interaction of PD-1 / PD-L1; further, the killing effect of Jurkat T cells on lung cancer cells mediated by Panax quinquefolius low-mid polysaccharides is verified by the method of co-culturing tumor cells and T cells, and it is found that Panax quinquefolius low-mid polysaccharides mediate Jurkat T cells to play a tumor killing effect, block the interaction of PD-L1 / PD-1, and thus help to restore the biological function of immune checkpoints of the body.

[0025] The present application also constructs a lung cancer transplanted tumor model to treat solid tumors in mice by oral administration of Panax quinquefolius low-mid polysaccharides; according to the experimental results, it can be seen that when the dosage is 200mg / kg, Panax quinquefolius low-mid polysaccharides can significantly inhibit the proliferation of tumor cells in vivo of mice, the tumor morphology changes obviously, the tumor volume gradually becomes smaller, and the effect is obviously better than that of the positive drug. And the Western Blot results confirm that Panax quinquefolius low-mid polysaccharides can significantly inhibit the expression of cancer cell PD-L1 and immune infiltrating cell PD-1 in solid tumors. In addition, the results of mouse body weight and serum biochemical indicators show that Panax quinquefolius low-mid polysaccharides have no obvious toxic and side effects in the dose range of 0-200mg / kg, and are significantly better than the positive drugs (i.e. PD-L1 monoclonal antibody and paclitaxel).

[0026] Therefore, the present application provides the use of Panax quinquefolius low-mid polysaccharides in the preparation of PD-L1 inhibitors, PD-1 inhibitors, PD-L1 and PD-1 dual inhibitors or antitumor drugs; the inhibitors, dual inhibitors or drugs are composed of an effective amount of Panax quinquefolius low-mid polysaccharides and pharmaceutically acceptable adjuvants or carriers.

[0027] Preferably, the tumor is a tumor with high expression of PD-L1 protein, including but not limited to non-small cell lung cancer, colon cancer, breast cancer or melanoma, etc.

[0028] The skilled person in the art can prepare the Panax quinquefolius low-mid polysaccharides into corresponding pharmaceutical preparations according to the conventional methods in the field of pharmaceutical preparations. The dosage form of the pharmaceutical preparation can be in the form of solid, semi-solid or liquid; preferably, it is a lyophilized powder, a tablet, a capsule, a soft capsule, a granule, a pill, an oral solution, a dry suspension, a dripping pill, a dry extract, an injection or an infusion. The administration mode of the pharmaceutical preparation is oral administration or injection administration.

[0029] The adjuvant or carrier in the present application refers to the conventional adjuvant or carrier in the pharmaceutical field, for example: diluent, disintegrant, lubricant, excipient, binder, glidant, filler, surfactant and the like; in addition, other auxiliary agents such as flavoring agents and sweetening agents can also be added in the composition. The diluent can be one or more components that increase the weight and volume of the tablet, and the commonly used diluents include lactose, starch, pregelatinized starch, microcrystalline cellulose, sorbitol, mannitol and inorganic calcium salt and the like; the disintegrant can be one or a mixture of two or more of cross-linked polyvinylpyrrolidone (2-6% by weight), cross-linked sodium carboxymethyl cellulose (2-6% by weight), alginic acid (2-5% by weight) and microcrystalline cellulose (5-15% by weight). The lubricant includes one or a mixture of two or more of stearic acid, sodium stearate, magnesium stearate, calcium stearate, polyethylene glycol, talc and hydrogenated vegetable oil. The amount of the lubricant ranges from 0.10 to 1% by weight, and the general amount is 0.25-0.75%. The binder can be one or more components that facilitate granulation; it can be starch paste (10-30% by weight), hydroxypropyl methyl cellulose (2-5% by weight), polyvinylpyrrolidone (2-20% by weight), and the ethanol aqueous solution of polyvinylpyrrolidone is preferred. The glidant can be one or a mixture of two or more of microcrystalline silica, talc and magnesium trisilicate. The surfactant can be one or more components that can improve wettability and increase drug dissolution, and sodium dodecyl sulfate is commonly used (commonly used range is 0.2-6% by weight).

[0030] The preparation method of the low-mid glycan component provided by the present application has the characteristics of being suitable for large-scale preparation, low cost, high yield and the like. The improved low-mid glycan component of American ginseng can not only down-regulate the expression level of PD-L1 in tumor cells, but also can reduce the expression level of PD-1 in immune cells such as T cells, thereby effectively activating T cells and hindering tumor immune escape. As a natural glycan PD-L1 and PD-1 dual inhibitor, it can be used for immunotherapy of lung cancer, breast cancer, colon cancer, melanoma and other cancers, has no toxic side effects, low price and other advantages, and has a broad application prospect in tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure 1 is the ion chromatogram of the monosaccharide composition of the low-mid glycan component of American ginseng; (A), the UPLC-QTOF-MS of the low-mid glycan component of American ginseng E Total ion current chromatogram; (B), ion chromatogram of low-mid glycan component of American ginseng; (C) determination results of compound types contained in low-mid glycan component of American ginseng using high-performance anion exchange chromatography equipped with a pulsed amperometric detector (HPAEC-PAD).

[0032] Figure 2 Structure of m / z 827.2612 and its fragmentation pathway.

[0033] Figure 3 Structure of m / z 827.2612 and its fragmentation pathway. 1 H-NMR and 13 C-NMR.

[0034] Figure 4 Results of CCK-8 method for detecting the changes in cell proliferation of lung cancer A549 cells, NCI-H157 cells, melanoma B16 cells, breast cancer MCF-7 cells, E0771 cells and colon cancer HT-29 cells after 24 h of treatment with different concentrations of American ginseng low-molecular polysaccharides.

[0035] Figure 5 Results of Western blotting analysis of the effects of American ginseng low-molecular polysaccharides on the membrane epitope PD-L1 expression levels of wild-type PD-L1 highly-expressing lung cancer NCI-H157 cells (A), colon cancer HT-29 cells (B), melanoma B16 cells (C) and breast cancer E0771 cells (D); the experimental results were statistically analyzed, where *P<0.05; **P<0.01; ***P<0.001; note: XYS-G in the figure refers to American ginseng low-molecular polysaccharides.

[0036] Figure 6 Results of screening for the optimal induction PD-L1 expression conditions of interferon gamma (INF-γ)-induced PD-L1 low-expressing A549 and MCF-7 cells; (A) effects of American ginseng low-molecular polysaccharides on the PD-L1 abundance of induced-type PD-L1 highly-expressing cells; (B) the experimental results were statistically analyzed, where *P<0.05; **P<0.01; note: XYS-G in the figure refers to American ginseng low-molecular polysaccharides.

[0037] Figure 7 Results of flow cytometry detection of the inhibitory effects of American ginseng low-molecular polysaccharides on lung cancer cell PD-L1 expression; flow cytometry verification of the effects of different concentrations of American ginseng low-molecular polysaccharides on the membrane surface PD-L1 expression of lung cancer NCI-H157 (A) and A549 cells (B); effects of American ginseng low-molecular polysaccharides with different action times on the membrane surface PD-L1 expression of lung cancer NCI-H157 (C) and A549 cells (D); the experimental results were statistically analyzed, where ***P<0.001; note: XYS-G in the figure refers to American ginseng low-molecular polysaccharides.

[0038] Figure 8The survival rate of low-molecular polysaccharides of Panax quinquefolium on RAW264.7 (A) and Jurkat T cells (B); the influence of low-molecular polysaccharides of Panax quinquefolium on the expression level of PD-1 in RAW2647 (C) and Jurkat cells (D); the experimental results were statistically analyzed, wherein *P<0.05; **P<0.01;

[0039] ***P<0.001; Note: XYS-G in the figure is low-molecular polysaccharides of Panax quinquefolium.

[0040] Figure 9 The verification results of low-molecular polysaccharides of Panax quinquefolium mediated Jurkat T cells killing lung cancer cells A549; the experimental results were statistically analyzed, wherein ***P<0.001; Note: XYS-G in the figure is low-molecular polysaccharides of Panax quinquefolium.

[0041] Figure 10 The verification results of low-molecular polysaccharides of Panax quinquefolium on tumor-bearing mice anti-tumor effect and biological safety; (A) mouse transplanted tumor experiment design; (B) mouse transplanted tumor morphology observation; (C) mouse transplanted tumor weight analysis; (D) mouse body weight change analysis during drug administration; (E) mouse transplanted tumor volume change analysis during drug administration; (F) mouse transplanted tumor PD-L1 and PD-1 protein expression level analysis; (G) mouse biochemical index detection; the experimental results were statistically analyzed, wherein ***P<0.001; Note: XYS-G in the figure is low-molecular polysaccharides of Panax quinquefolium.

[0042] Figure 11 The in vivo direct anti-tumor effect detection of low-molecular polysaccharides of Panax quinquefolium on nude mice LLC transplanted tumor model; (A) nude mice lung cancer xenotransplanted tumor experiment design, (B) mouse tumor size observation, (C) mouse tumor weight analysis, wherein ns represents no significant difference; Note: XYS-G in the figure is low-molecular polysaccharides of Panax quinquefolium. DETAILED DESCRIPTION

[0043] The advantages and characteristics of the present application will become more apparent from the following detailed description. However, it should be understood that the described embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and such modifications or substitutions all fall within the protection scope of the present application.

[0044] Example 1 Preparation of low-molecular polysaccharides of Panax quinquefolium

[0045] About 5 kg of American ginseng medicinal materials were cut into 1-5 mm slices, 250 g each time, 8 L distilled water was added, 3 hours each time, the residue was filtered with gauze, the residue was repeatedly extracted 2 times, and the American ginseng water extract was combined. The water extract was adsorbed by AB-8 type macroporous resin for 12 hours and eluted with distilled water until the eluate was light yellow by phenol sulfuric acid method. The macroporous adsorption resin water eluate was further purified by ultrafiltration chromatographic column with a molecular weight cut-off of 3 KDa to obtain American ginseng refined low-mid polysaccharides, and then the American ginseng refined low-mid polysaccharides were further desalted and refined by nanofiltration chromatographic column with a molecular weight cut-off of 500 Da to obtain American ginseng low-mid polysaccharide components.

[0046] The monosaccharide composition was analyzed by high performance anion exchange chromatography (HPAEC-PAD) using trifluoroacetic acid hydrolysis Figure 1 A), by comparing with standard samples, it was determined that American ginseng low-mid polysaccharides were mainly composed of 2 to 15 hexose molecules; the hexose molecules were composed of glucose and fructose, and the molar ratio of glucose and fructose was 65:35.

[0047] In order to further determine the chemical structure of American ginseng low-mid polysaccharides, UPLC-QTOF-MS E was used for detection, and the total ion chromatogram Figure 1 B) was obtained. As shown in Table 1, a total of 23 peaks were identified and their structures were characterized. According to the monosaccharide composition results, it was mainly neutral low-mid polysaccharides. Peak 13 was taken as an example for analysis. The retention time t R of peak 13 was 17.36 min, and its molecular weight was determined to be 828 by [M-H] - , indicating that its degree of polymerization was 5, composed of 5 six-carbon sugars. According to Figure 2 shown, the deprotonated precursor ion at m / z 827.2612 was cleaved by MS 2 to obtain neutral loss C1-C4 and B1-B4 ions, and cross-ring cleavage ions: 0,2 A1, 0,2 A2- 0,2 A5, 2,4 A2- 2,4 A5 series of ions, deducing its structure as Hex 1 → 4 Hex 1 → 4 Hex 1 → 4 Hex 1 → 4 Hex.

[0048]

[0049]

[0050] By Figure 3 It can be seen that the m / z 827.2612 is 1 Four low-field anomeric hydrogen end group protons were observed in the H-NMR spectrum, which were δH5.25 (1H, s), 5.07 (1H, s), 4.80 (1H, s), 4.49 (1H, s), respectively, which can be attributed to 1,4-Glcp, T-Glcp, 1-Glcp and T-Glcp. Their configurations are α, α, α, β, respectively.

[0051] In addition, the determination of Panax ginseng low and medium polysaccharides was carried out using high-performance anion exchange chromatography equipped with a pulse amperometric detector (HPAEC-PAD) (C). Figure 1 The results showed that there were 23 different compounds in Panax ginseng low and medium polysaccharides.

[0052] Experimental Example 1 Panax ginseng low and medium polysaccharides as PD-L1 and PD-1 double inhibitors

[0053] (1) In vitro cell viability detection

[0054] CCK-8 method was used to detect the effect of Panax ginseng low and medium polysaccharides on the cell activity of six kinds of cancer cells. Six kinds of cancer cells were inoculated in 96-well cell culture plates at a concentration of 1×10 4 / mL. Gradient concentrations of Panax ginseng low and medium polysaccharides were prepared with culture medium, and CCK-8 solution was added in 96-well cell culture plates after 24h. The OD value was measured at 450nm by enzyme-linked immunoassay instrument. The experimental results showed that with the increase of the concentration gradient of Panax ginseng low and medium polysaccharides, the cell viability of six kinds of cancer cells was not significantly inhibited, and Panax ginseng low and medium polysaccharides had no obvious cytotoxicity to six kinds of cancer cells (see Figure 4 ). It is proved that Panax ginseng low and medium polysaccharides have no cytotoxicity and do not inhibit the expression of PD-L1 in tumor cells by direct cytotoxicity mechanism.

[0055] (2) Effect of Panax ginseng low and medium polysaccharides on PD-L1 expression level of wild type PD-L1 high expression tumor cells

[0056] Panax ginseng low and medium polysaccharides were treated with concentration gradient respectively on four kinds of tumor cell lines with high expression of PD-L1, and Western blot method was used to detect the total protein of four kinds of cells, BCA method was used to detect the protein concentration, and SDS polyacrylamide gel electrophoresis (SDS-PAGE) was used after loading, and blocking and immune reaction were carried out after membrane transfer, and ECL chemiluminescence solution was used for development. The exposure results were processed and analyzed by gray value on ImageJ software.

[0057] The experimental results show that the low-molecular polysaccharides of Panax quinquefolium have a significant down-regulation effect on the abundance of PD-L1 in tumor cell lines with high expression of PD-L1 (see Figure 5 ), among which the down-regulation trend of non-small cell lung cancer NCI-H157 and melanoma B16 cells is the most obvious. The above results provide a basis for the development and utilization of low-molecular polysaccharides of Panax quinquefolium as PD-L1 immunosuppressants.

[0058] (3) Effect of low-molecular polysaccharides of Panax quinquefolium on IFN-γ-induced PD-L1 expression in tumor cells

[0059] Interferon IFN-γ is an important factor for inducing PD-L1 expression in the tumor immune microenvironment. In this experiment, low-expression PD-L1 tumor cells (non-small cell lung cancer A549 cells and breast cancer MCF-7 cells) were used, and low-molecular polysaccharides of Panax quinquefolium were co-treated with 15 ng / mL of IFN-γ in the cells. The expression abundance was detected by Western blot method, and the experimental method is the same as shown in (2) above.

[0060] The experimental results show that low-molecular polysaccharides of Panax quinquefolium significantly down-regulate the expression of PD-L1 in A549 and MCF-7 cells with high expression of PD-L1 induced by IFN-γ (see Figure 6 B). It is proved that low-molecular polysaccharides of Panax quinquefolium also have a significant inhibitory effect on the expression of PD-L1 in A549 and MCF-7 cells with high expression of PD-L1 induced by IFN-γ.

[0061] (4) Flow cytometry detection of the inhibitory effect of low-molecular polysaccharides of Panax quinquefolium on PD-L1 expression in lung cancer cells

[0062] NCI-H157 or A549 cells in 6-well plates were treated with different doses of low-molecular polysaccharides of Panax quinquefolium at 0, 31.25, 62.5, 125, 250, and 500 μg / mL for corresponding time, and then stained with fluorescent antibodies in the cells. The fluorescence intensity was detected by flow cytometry.

[0063] The experimental results further verify the conclusion obtained by Western blot: low-molecular polysaccharides of Panax quinquefolium inhibit the expression of PD-L1 in a concentration-dependent manner, which is directly manifested as a gradient decrease in the fluorescence intensity of PD-L1 membrane epitope in NCI-H157 and A549 lung cancer cells (see Figure 7 A and 7B). Moreover, the inhibitory effect of low-molecular polysaccharides of Panax quinquefolium on PD-L1 expression in NCI-H157 and A549 lung cancer cells is time-dependent, and the expression of PD-L1 in cancer cells decreases significantly with the increase of treatment time (see Figure 7The down-regulation of PD-L1 was most significant at a drug concentration of 500 pg / mL and a drug administration time of 24 h. This indicates that the inhibition of lung cancer cell PD-L1 by Panax notoginseng low-molecular polysaccharides is both concentration-dependent and time-dependent. This further proves the specificity of the effect of Panax notoginseng low-molecular polysaccharides on lung cancer cells.

[0064] (5) Effect of Panax notoginseng low-molecular polysaccharides on PD-1 expression in immune cells

[0065] To verify whether Panax notoginseng low-molecular polysaccharides have an inhibitory effect on PD-1 protein expression and function in immune cells, CCK8 detection found that Panax notoginseng low-molecular polysaccharides had no obvious cytotoxicity to the two types of immune cells (see Figure 8 A and 8B).

[0066] Western blot was used to treat activated immune cells RAW264.7 cells stimulated by 50 pg / ml of LPS and Jurkat T cells activated by 50 ng / mL of PMA at a concentration gradient. The Western blot results show that Panax notoginseng low-molecular polysaccharides have a significant inhibitory effect on PD-1 expression in the two types of immune cells (see Figure 8 C and 8D).

[0067] (6) Panax notoginseng low-molecular polysaccharides mediate Jurkat T cell killing of lung cancer cells

[0068] To further evaluate the anti-tumor effect of Panax notoginseng low-molecular polysaccharides in tumor cell and T cell co-culture experiments, A549 cells were seeded in a 24-well culture plate, treated with a gradient of the drug for the required time, and co-cultured with stimulated Jurkat T cells for 24 h. After 4% paraformaldehyde fixation, crystal violet staining was performed, and the survival of tumor cells was observed under a microscope. The specific manifestation is the difference in staining depth in different dose groups of Panax notoginseng low-molecular polysaccharides.

[0069] The experimental results are as follows: A549 cells treated with Panax notoginseng low-molecular polysaccharides are more sensitive to Jurkat T cell killing (see Figure 9 ), which further proves that Panax notoginseng low-molecular polysaccharides mediate Jurkat T cell infiltration to kill tumor cells, down-regulate the expression of cell surface PD-L1, and inhibit the binding of tumor cell PD-L1 and Jurkat T cell PD-1 in a concentration-dependent manner, thereby blocking tumor cell immune escape.

[0070] (7) Application of Panax notoginseng low-molecular polysaccharides as a PD-L1 and PD-1 dual inhibitor

[0071] To explore the anti-tumor effect of Panax notoginseng low-molecular polysaccharides in vivo, a lung cancer xenograft model in mice was established (seeFigure 10 A), the specific method comprises: 56 C57BL / 6 mice are randomly divided into seven groups, 8 mice in each group. The group setting is blank group, model group, low-mid-polymer of American ginseng low-dose group (50 mg / kg), low-mid-polymer of American ginseng middle-dose group (100 mg / kg), low-mid-polymer of American ginseng high-dose group (200 mg / kg), positive drug PD-L1 targeted inhibitor group (PD-L1-IN-10, 10 mg / kg), positive drug PD-1 targeted inhibitor group (BMS-1, 10 mg / kg).

[0072] Select LLC cells in the logarithmic growth phase and in good condition, digest the cells with trypsin, resuspend with PBS after centrifugation, adjust the cell concentration to 1×10 6 6 / mL, inoculate the cell suspension 200 μL in the axillary region of the mouse. After inoculation, observe the growth of the tumor every day, and start dosing when the tumor volume reaches 50-150 mm 3 3. The low-mid-polymer of American ginseng is administered by gavage once a day, for a total of 14 days, and the blank group and the model group are given the same amount of PBS solution. The positive drug PD-L1-IN-10 is administered by gavage once every three days, and the positive drug BMS-1 is administered by intraperitoneal injection once every three days.

[0073] The experimental results show that the tumor growth in the model mice treated with 200 mg / kg of low-mid-polymer of American ginseng is effectively inhibited (see Figure 10 B). Specifically, as the dosing time accumulates, the overall growth of the tumor volume in the mice of the low-mid-polymer of American ginseng dosing group is slow, showing good anti-tumor activity (see Figure 10 C).

[0074] To further verify that it can be used as a PD-L1 and PD-1 dual inhibitor, Western blotting confirmed that the low-mid-polymer of American ginseng can significantly reduce the expression of PD-L1 protein in the isolated tumor of the mouse and the PD-1 protein level in the immune microenvironment of the tumor.

[0075] The specific method comprises the following steps: logarithmic phase growth of cells is taken, and then the cells are inoculated in 6 cm culture dishes respectively, and then the cells are treated with different concentrations of American ginseng low and medium polysaccharides for corresponding time, and then the cells are collected into centrifuge tubes, and then appropriate RIPA lysis solution, protease inhibitor and phosphatase inhibitor (100:1:1) are added, and then the cells are lysed for 30 min, and then the cells are vortexed for lysis every 5 min. The supernatant is collected by centrifugation at 13000 rpm for 30 min; according to the number of samples, appropriate BCA working solution is prepared, and then the BCA working solution is fully mixed. The standard sample is diluted according to the instructions. Appropriate volume of sample is added to the sample hole of the 96-well plate. If the sample is less than 20 μL, the standard sample diluent is added to make up to 20 μL. 200 μL of BCA working solution is added to each hole, and then the 96-well plate is placed at 37 ℃ for 15 min. The absorbance of the sample is measured by an enzyme-labeled instrument at OD = 562 nm, and then the protein concentration of the sample is calculated according to the standard curve and the volume of the sample used. According to the calculated protein concentration, pure water and 5x buffer are added to prepare the sample, and then the sample is heated at 100 ℃ for 10 min to denature the protein; prepare the gel: prepare the separation gel (select appropriate concentration according to the molecular weight of the target protein), prepare the concentrated gel, insert the comb, and then remove the comb after the gel is polymerized; electrophoresis: place the gel into the electrophoresis tank, add the electrophoresis buffer, and then load (20 μg per hole), and then run the concentrated gel at 80 V for about 15 min, and then run the separation gel at 120 V for about 30 min until the target protein is well separated; PVDF membrane is selected as the material for membrane transfer, and then the membrane is wetted with methanol for 30 s, and then the gel and the membrane are assembled in the membrane transfer clamp in the order of “filter paper-membrane-gel-filter paper”, and then the membrane transfer clamp is placed in the membrane transfer tank, and then the pre-cooled membrane transfer buffer is added, and then ice plates are placed on both sides, and then the membrane is transferred at a voltage of 100 V for 60 min; the membrane after the transfer is placed in the blocking solution (5% BSA), and then the membrane is blocked on a shaking table at room temperature for 1.5 h; the membrane is placed in the diluted primary antibody solution according to the instructions, and then the membrane is incubated in a refrigerator at 4 ℃ overnight; the membrane is washed with 1x TBST for 5 times, and each time for 6 min, and then the membrane is placed in the diluted secondary antibody, and then the membrane is incubated at room temperature for 2 h; the membrane is washed with 1x TBST for 5 times, and each time for 6 min, and then the prepared ECL luminescent solution is evenly dropped on the surface of the membrane, and then the membrane is exposed by an imaging instrument, and then the band of the target protein is recorded; the band gray value is measured by using the image analysis software Image J, and then the ratio of the target protein to the internal reference GAPDH is calculated. The expression results are shown in Figure 10 F.

[0076] There is no obvious difference in the weight of the mice in each group (see Figure 10 D); the serum indexes of the mice in the experimental group at a dose of 200 mg / kg are consistent with those of the control group (see Figure 10 G), which indicates that American ginseng low and medium polysaccharides have no obvious toxic and side effects on the mice at the used dose.

[0077] Example 2: Experiment of killing tumor by American ginseng low and medium polysaccharides

[0078] To explore the immune anti-tumor effect of low-mid polysaccharides in vivo, a lung cancer cell LLC transplanted tumor model in nude mice was established (see Figure 11 A).

[0079] Specific methods include: A549 cells were injected into the armpit of nude mice (1 x 10 7 cells were dissolved in 200 μL of PBS solution). After 7 days of cell injection, when the tumor was clearly visible, the mice were randomly divided into 2 groups (n = 3). The administration group was treated with American ginseng low-mid polysaccharides (200 mg / kg) by gavage, and the model group was given the same amount of PBS solution. All experiments were carried out in SPF level experimental environment and under the condition of sufficient water and food. After 14 days of treatment, the mice were euthanized with sodium pentobarbital (60 mg / kg), and organs and tumors were collected for subsequent experiments.

[0080] The experimental results show that: in the model mice treated with 200 mg / kg of American ginseng low-mid polysaccharides, the tumor growth was not effectively inhibited (see Figure 11 B). Specifically, as the accumulation of drug administration time, there was no significant difference in tumor weight between the American ginseng low-mid polysaccharide administration group and the model group (see Figure 11 C). It shows that American ginseng low-mid polysaccharides at a dose of 200 mg / kg do not rely on direct killing to inhibit the growth and proliferation of tumors.

Claims

1. A oligosaccharide isolated from American ginseng, characterized in that, The oligosaccharides are mainly composed of 2 to 15 hexose molecules; wherein the hexose molecules are composed of glucose and fructose, and the molar ratio of glucose to fructose is 65:

35.

2. The oligosaccharide according to claim 1, characterized in that, A representative structure in the oligosaccharides is the 1,4-Glcp linker, and the structure of the representative 1,4-Glcp linker is as follows:

3. A method for preparing the oligosaccharide of claim 1, characterized in that, include: (1) Aqueous extract of American ginseng was obtained by heating and reflux extraction using water as the extraction solvent; (2) The American ginseng water extract was adsorbed by a macroporous resin column. After removing pigments and proteins by resin adsorption, it was eluted with an eluent to obtain American ginseng macroporous resin water eluent. (3) The water eluent of American ginseng macroporous resin is ultrafiltered using an ultrafiltration column or dialyzed using a dialysis bag to obtain refined oligosaccharides. (4) The refined oligosaccharides of American ginseng were subjected to nanofiltration column treatment and then desalted to obtain the American ginseng oligosaccharide components.

4. The method according to claim 3, characterized in that, The macroporous resin column mentioned in step (2) is AB-8 type macroporous resin.

5. The method according to claim 3, characterized in that, The eluent in step (2) is distilled water, and the amount of distilled water used for elution is 5 times the column volume. The elution endpoint is detected by the phenol-sulfuric acid method.

6. The method according to claim 3, characterized in that, The ultrafiltration column used in step (3) has a molecular weight cutoff of 3 kDa, and the collected components are effluent with a molecular weight cutoff of less than 3 kDa; wherein, the ratio of water to medicinal materials is 8:1 based on mL:g, and the ultrafiltration pressure is 1 MPa.

7. The method according to claim 3, characterized in that, In step (4), a nanofiltration column is used to retain products with a molecular weight cutoff of 500 Da. The refined ginseng oligosaccharide is obtained after being treated by nanofiltration column. The ratio of water to medicinal material is 10:1 and the nanofiltration pressure is 2 MPa.

8. Use of the oligosaccharide of claim 1 in the preparation of PD-L1 inhibitors, PD-1 inhibitors, dual inhibitors of PD-L1 and PD-1, or antitumor drugs.

9. The use according to claim 8, characterized in that, The PD-L1 inhibitor, the PD-1 inhibitor, the PD-L1 and PD-1 dual inhibitor, or the antitumor drug consists of an effective amount of American ginseng oligosaccharides and pharmaceutically acceptable excipients or carriers.

10. The use according to claim 8, characterized in that, The tumors mentioned are tumors in which tumor cells highly express PD-L1 protein, including but not limited to non-small cell lung cancer, colon cancer, breast cancer, or melanoma.

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