CN111658680A - Application of cordyceps sinensis extract in preparation of immune checkpoint inhibitor - Google Patents

Abstract

The invention provides an application of a cordyceps sinensis extract in preparation of an immune checkpoint inhibitor, wherein the immune checkpoint is PD-1, PD-L1 or TNFR 1. Experimental results show that the cordyceps sinensis extract has stable and high-strength binding capacity with multiple immunoregulation targets (PD-1, PD-L1 and TNFR1), and can effectively block the binding of 293T-h PD-L1 cells and PD-1 protein. Therefore, the cordyceps sinensis extract can be used for preparing immune checkpoint inhibitors including PD-1, PD-L1 and TNFR1, and can also be used for preparing various anti-tumor drugs for treating esophageal squamous cell carcinoma, urothelial cancer and squamous non-small cell lung cancer, and has good application prospect.

Description

Application of cordyceps sinensis extract in preparation of immune checkpoint inhibitor

Technical Field

The invention relates to the field of pharmacy, and in particular relates to application of a cordyceps sinensis extract in preparation of an immune checkpoint inhibitor.

Background

Immunotherapy is an important approach for tumor therapy, and a programmed cell death receptor 1 (DCD 1, PD-1)/programmed cell death receptor ligand 1 (DCD 1ligand 1, DCD1LG1, PD-L1) pathway inhibition drug is a hot spot of current immunotherapy. PD-1 is an immunosuppressive molecule that interacts with the ligands PD-L1 and PD-L2 and causes apoptosis of tumor antigen-specific T cells, thereby allowing tumor cells to escape from the immune surveillance of the body. PD-L1 belongs to a B7-H1 molecular family, the expression of PD-L1 can be detected on the surfaces of a plurality of human tumor cells, such as lung cancer, malignant melanoma, breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma and the like, and the expression level of PD-L1 in a cancer tissue is found to be obviously up-regulated compared with that in a normal tissue. When the high-expression PD-L1 on the tumor cell is combined with the receptor PD-1 on the T cell, a negative regulation signal can be transmitted to induce the T cell to die or cause immune incapacity, so that the tumor cell can escape the immune monitoring and killing of the body. The PD-1/PD-L1 inhibitor can block a PD-1/PD-L1 pathway, promote the activation and proliferation of effector T cells, enhance the cellular immunity, and further recognize and kill tumor tissues. Several studies have shown that PD-1/PD-L1 inhibitors can achieve the goal of treating tumors.

Antibodies targeting PD-1 and PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of a variety of solid tumors. PD-1 inhibitors, such as nivolumab (opdivo), have been approved for the treatment of malignant melanoma and metastatic squamous non-small cell lung cancer, which are unresectable or metastatic and do not respond well to other therapeutic drugs.

The literature, "the clinical research progress of PD-1/PD-L1 inhibitor in the treatment of advanced gastric cancer" discloses the clinical research results of various PD-1/PD-L1 inhibitors in advanced gastric cancer, wherein the PD-L1 inhibitor Avelumab has objective remission rates of 10% and 18.2% in first-line and second-line treatment of patients with positive expression of PD-L1, the objective remission rate of PD-1 inhibitor Pembrolizumab to patients with positive expression of PD-L1 reaches 22.2%, and the objective remission rate of single-drug treatment of PD-L1 inhibitor Nivolumab reaches 14%. In addition, Avelumab and other drugs show tolerable adverse reactions in the current research, the safety of single-drug therapy is also acceptable, and phase III random tests on gastric cancer are about to develop.

The literature "development of PD-1/PD-L1 immune checkpoint inhibitors in lung cancer" discloses that there are 5 main drugs against PD1/PD-L1 targets in the field of lung cancer, including anti-PD-1 monoclonal antibodies (Nivolumab, Pembrolizumab), anti-PD-L1 monoclonal antibodies (Atezolizumab, Durvalumab, Avelumab). Wherein Pembrolizumab exhibits clinical benefit in first-line treatment of advanced non-small cell lung cancer (NSCLC). The research of the KEYNOTE001 in the Ib stage shows that the Pembrolizumab has the advantages of observed antitumor activity in both primary treatment and treated NSCLC patients and controllable safety. For the NSCLC initially treated with positive PD-L1(TPS is more than or equal to 50%), ORR and OS reach 58% and 61% respectively in 24 months. Based on the above results, the KEYNOTE024 study was used for first-line treatment of advanced NSCLC that was PD-L1 (TPS. gtoreq.50%), EGFR/ALK negative. In addition, the successful application of the PD-1/PD-L1 immune checkpoint inhibitor in various clinical trials brings clinical benefits to patients with various pathological lung cancers including squamous carcinoma, adenocarcinoma and small cell lung cancer.

A plurality of documents, namely research progress of PD-1/PD-L1 inhibitors and biomarkers thereof in esophageal squamous cell carcinoma treatment, research progress of PD-1/PD-L1 inhibitors in advanced urothelial cancer, research progress of PD-1/PD-L1 inhibitors in colorectal cancer, research progress of PD-1/PD-L1 inhibitors in lymphoma treatment and research progress of PD-1/PD-L1 pathway inhibitors in digestive system tumor immunotherapy, respectively, disclose that the PD-1/PD-L1 inhibitor has exciting progress in immune drugs for treating esophageal squamous cell carcinoma, advanced urothelial cancer, colorectal cancer, lymphoma, digestive system tumor and other tumors. All, more safe and effective new generation PD-1/PD-L1 inhibitors are researched, and the method has very important significance for preparing various antitumor drugs.

In addition to PD-1, PD-L1, there are a number of other immunomodulatory targets that have received attention, such as the alpha tumor necrosis factor (TNF α) family of protein members that have been identified and developed as regulatory molecules for immunotherapy. TNF α has 2 receptors: TNFR1 and TNFR2, wherein TNFR1 mediates most of the signaling function of TNF α. TNF α, an important inflammatory factor, is widely involved in various physiological actions and plays a key role in immune response in the body, but causes severe toxic and side effects when TNF α therapy is administered. Therefore, the search for new, safer, more effective and specific generation anti-TNF α drugs is receiving extensive attention.

Cordyceps sinensis is a dry complex of Cordyceps sinensis (Berk.) Sacc. parasitic on the stroma and the carcass of insect larvae of Hepialidae family. The cordyceps sinensis is a traditional rare Chinese medicinal material in China, has wide pharmacological actions of immunoregulation, antibiosis, tumor resistance, antioxidation, anti-aging, blood sugar and blood fat reduction and the like, and is called as 'three treasures of traditional Chinese medicine' together with ginseng and pilose antler.

Modern pharmacological studies at home and abroad show that the cordyceps sinensis can play a role in inhibiting tumors by inhibiting the growth and proliferation of B16 melanoma cells, human liver cancer Hep G2 cells, gastric cancer cells, lung cancer cells and other tumor cells. However, Cordyceps sinensis as an immune checkpoint inhibitor for PD-1, PD-L1 or TNFR1 and its use for the treatment of esophageal squamous cell carcinoma, urothelial cancer, squamous non-small cell lung cancer have not been reported.

Disclosure of Invention

In view of the above problems, the present invention aims to provide an application of cordyceps sinensis extract in preparation of an immune checkpoint inhibitor.

The invention provides an application of a cordyceps sinensis extract in preparation of an immune checkpoint inhibitor, wherein the immune checkpoint is PD-1, PD-L1 or TNFR 1.

Further, the immune checkpoint is TNFR 1.

Further, the immune checkpoint inhibitor is capable of blocking the PD-1/PD-L1 signaling pathway.

Further, the inhibitor is an anti-tumor drug.

Further, the anti-tumor drug is a drug for resisting drug-resistant tumor; preferably, the drug for resisting drug-resistant tumors is a tumor immunosuppressant.

Further, the tumor is esophageal squamous cell carcinoma, urothelial carcinoma, squamous non-small cell lung cancer; the squamous non-small cell lung cancer is preferably drug-resistant squamous non-small cell lung cancer.

Further, the cordyceps sinensis extract is prepared by the following method: is prepared from cordyceps through extracting.

Further, the extraction process is as follows: extracting in liquid, dispersing the extract with pure water, extracting with organic solvent, and collecting organic phase and water phase;

preferably, the volume ratio of the organic solvent to pure water is 1: (0.8 to 1.2); the organic solvent is selected from esters and alcohols; the extraction times are 1-5 times;

more preferably, the volume ratio of the organic solvent to pure water is 1: 1; the organic solvent is selected from ethyl acetate and n-butanol; the number of extractions was 3.

Further, the liquid is selected from pure water or 60-80% of methanol; the ratio of material to liquid is 1: (15-25) g/mL; the extraction temperature is 30-45 ℃; the extraction times are 6-12 times; the extraction time is 20-40 min each time;

or the like, or, alternatively,

the extraction mode in the liquid is as follows: firstly, extracting for 4-8 times at 30-45 ℃ for 20-40 min each time in artificial gastric juice according to the feed-liquid ratio of 1 (15-25) g/mL; adding artificial intestinal juice, and extracting for 20-40 min each time for 2-5 times under heat preservation.

Further, the liquid is selected from pure water or 70% methanol; the ratio of material to liquid is 1:20 g/mL; the extraction temperature is 37 ℃; the extraction times are 9 times; the extraction time is 30min each time;

or the like, or, alternatively,

the extraction mode in the liquid is as follows: extracting in artificial gastric juice at 37 deg.C for 30min for 6 times with feed liquid 1:20 g/mL; adding artificial intestinal juice, extracting for 30min for 3 times.

Further, the extract dispersed with pure water is dried extract; and/or the extraction method is ultrasonic extraction.

The invention also provides an anti-tumor medicament which is prepared from the cordyceps sinensis extract and a pharmaceutically acceptable carrier.

Further, the anti-tumor drug is a drug for resisting drug-resistant tumor; preferably, the drug for resisting drug-resistant tumors is an anti-tumor immunosuppressant.

An "immune checkpoint inhibitor" is an agent that promotes T cell activation by antagonizing an immune checkpoint protein, thereby producing an anti-tumor immune effect.

Experiments prove that the cordyceps sinensis extract provided by the invention has stable and high-strength binding capacity with multiple immunoregulation targets (PD-1, PD-L1 and TNFR1), and the cordyceps sinensis extract can also effectively block the binding of 293T-h PD-L1 cells and PD-1 protein and block a PD-1/PD-L1 signal channel. The cordyceps sinensis extract can be used for preparing immune checkpoint inhibitors including PD-1, PD-L1 and TNFR1, and has a good application prospect.

In addition, according to the records of the prior art, the PD-1/PD-L1 immune checkpoint inhibitor can effectively treat various tumors including esophageal squamous cell carcinoma, urothelial carcinoma and squamous non-small cell lung cancer. Therefore, the cordyceps sinensis extract can be used for preparing anti-tumor drugs for treating esophageal squamous cell carcinoma, urothelial cancer and squamous non-small cell lung cancer, and has good application prospect.

In addition, as described in the background, PD-1 inhibitors, such as nivolumab (opdivo), have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic squamous non-small cell lung cancer that is unresectable or metastatic and poorly responsive to other therapeutic drugs. Therefore, the cordyceps sinensis extract disclosed by the invention is used as an immune checkpoint inhibitor and also has good advantages in treating drug-resistant metastatic squamous non-small cell lung cancer.

The preparation method of the cordyceps sinensis extract is simple, low in cost, safe and nontoxic, and is suitable for industrial production.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the results of FACS test of the extract of Cordyceps sinensis of the present invention inhibiting the binding of PD-1 to PD-L1.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Wherein, the cordyceps sinensis can be purchased from Beijing Tongrentang (Guanghua shop) in Chengdu city, and the product specification is as follows: no. 3, net content 10g, product lot No. 20180701. Pulverizing Cordyceps into fine powder with high speed pulverizer.

Example 1 preparation of the extract of Cordyceps sinensis according to the present invention

1. Weighing 2g of Cordyceps powder, taking 70% methanol as solvent, and placing at 37 deg.C for ultrasonic extraction for 9 times (30 min each time) at a material-to-liquid ratio of 1:20 g/mL. Filtering the extract, and concentrating to obtain product S1;

2. weighing 1g of Cordyceps powder, preparing artificial gastric juice and artificial intestinal juice according to Chinese pharmacopoeia, adding artificial gastric juice at 37 deg.C, performing ultrasonic extraction for 6 times (30 min each time) by semi-bionic extraction method at a material-to-liquid ratio of 1:20g/mL, adding artificial intestinal juice, and performing ultrasonic extraction at 37 deg.C for 3 times (30 min each time). Filtering the extract, and concentrating to obtain product S2;

3. weighing 1g of Cordyceps powder, taking pure water as solvent, and placing at 37 deg.C for ultrasonic extraction for 9 times, each time for 30min, with the material-liquid ratio of 1:20 g/mL. Filtering the extract, and concentrating to obtain product S3;

4. dispersing the S1-S3 samples with 75mL of pure water respectively, extracting for 3 times by using ethyl acetate and n-butyl alcohol with equal volumes in sequence, combining the extraction solutions for 3 times, decompressing and evaporating to dryness to obtain an extraction extract, and adding the rest of aqueous phase to obtain 9 samples, wherein the numbers of the samples are respectively S1A, S1B, S1C, S2A, S2B, S2C, S3A, S3B and S3C (A: ethyl acetate extraction phase; B: n-butyl alcohol extraction phase; C: aqueous phase). Respectively transferring six samples of S1A, S1B, S2A, S2B, S3A and S3B into a 2mL centrifuge tube, naturally drying and weighing to obtain weight; the three samples S1C, S2C and S3C were frozen overnight in a refrigerator at-80 ℃ and weighed by freeze-drying the samples using a freeze-dryer. The results are shown in Table 1.

TABLE 1 weight of the obtained Cordyceps sinensis extract

Control example 1 preparation of control sample S4

Taking Cordyceps powder with the same mass as that of the Cordyceps powder used in step 2 in example 1 and artificial gastric juice with the same volume, and performing ultrasonic extraction at 37 deg.C for 6 times (30 min each time), adding artificial intestinal juice, and performing ultrasonic extraction at 37 deg.C for 3 times (30 min each time). The extract was filtered off with suction, concentrated, dried and weighed (see table 1 for weight) as a blank, numbered S4.

The beneficial effects of the Cordyceps sinensis extract of the present invention are described below with reference to the test examples.

Test example 1 in vitro binding affinities of the extract of Cordyceps sinensis according to the present invention to various targets were measured using BIAcore SPR

1. Test method

(1) Preparation of test samples:

respectively drying and weighing 1mg of S1A, S1B, S1C, S2A, S2B, S2C, S3A, S3B and S3C prepared in example 1 and S4 prepared in comparative example 1, dissolving 10 samples in 20 mu L of DMSO, respectively dissolving 1 mu L of samples in 99 mu L of LPBS to prepare a solution with the concentration of 500 mu g/mL, transferring the solution to a centrifuge tube of 1.5mL for later use, and sequentially numbering the samples as CS1-CS 10;

(2) BIAcore SPR assay:

i. preparation of instruments and reagents: the prepared PBS was filtered through a 0.22 μm filter and sonicated for 15min to remove air bubbles, primarily to avoid reagent clogging the channel. The BIAcore instrument and associated software was opened and the line was flushed with filtered PBS. The temperature was set at 25 ℃ and the flow rate was set at 10. mu.L/min.

Formulation of target protein: 50 μ g of PD-1D was dissolved with 200 μ L of pure water to a concentration of 0.25mg/mL, 100 μ gPD-L1 was dissolved with 200 μ L of pure water to a concentration of 0.5mg/mL, 50 μ g of TNFR1 was dissolved with 100 μ L of pure water to a concentration of 0.5mg/mL, and appropriate coupling pH values were found against the isoelectric Points (PI) of the three proteins, PD-1: PI ═ 8.62, PD-L1: PI ═ 6.35, TNFR 1: the PI was 7.29, and thus 6 μ l of each of the three proteins was dissolved in 54 μ l of sodium acetate solution at ph5.5 and ph5.0 in a 1.5mL centrifuge tube for use.

Pre-enrichment of target proteins with chips: when the baseline of the instrument is stable, pre-enrichment sample loading is prepared, and therefore, prepared target protein solutions are respectively taken for sample injection for the three proteins. After a while, the Reaction Unit (RU) value of the target protein and the chip was observed, and finally, by data comparison, the optimum pH of PD-1 was 5.0, that of PD-L1 was 5.5, and that of TNFR1 was 5.5.

Formal coupling of target protein to chip: the formal coupling is divided into three steps. The first step is as follows: and (4) activating. mu.L of NHS and 100. mu.L of EDC were loaded in a centrifuge tube for 7min at a flow rate of 10. mu.L/min to activate dextran on the surface of the CM5 chip. The second step is that: and (3) coupling. And adding 10 mu L of target protein into 90 mu L of sodium acetate solution with the optimal pH value in a 1.5mL centrifuge tube, and after uniformly mixing, loading for 7min at the flow rate of 10 mu L/min. The third step: and (5) sealing. 150 μ L of blocking solution (ETH) was centrifuged in a 1.5mL centrifuge tube for 7min at a flow rate of 10 μ L/min to block the areas of the chip surface that did not bind to the protein. The RU values for PD-1 and chip are 3422.4, PD-L1 and chip are 9927.8, and TNFR1 and chip are 6146.5.

Spr detection: taking the prepared sample, centrifuging to remove bubbles, injecting for 1min, setting the flow rate to be 30 mu L/min, and recording the RU value. To investigate the reproducibility of the experiment, repeated tests were performed on CS1, CS2, CS3, labeled CS1R, CS2R, CS3R, after the end of the sample injection of CS1-CS 10. The magnitude of the RU number can reflect the binding efficiency of the small molecule compound and the target protein in the sample.

Regeneration: when the RU value of the drug and the target protein is larger, the drug and the target protein are combined and then dissociated, Glycine (Glycine) with the pH values of 3.0, 2.5, 2.0 and 1.5 is used for dissociation for 30s, the flow rate is 30 mu L/min, the drug is difficult to dissociate, the drug combined with the target protein is dissociated by 5mM NaOH until the RU value is smaller than 50, and then the next sample detection is carried out.

2. Test results

Ten samples of CS1-CS10 were tested for BIAcore SPR at multiple targets (PD-1, PD-L1, TNFR1) and the experiments were examined for reproducibility. Table 2 shows the in vitro RU values of ten samples (CS1-CS10) in the Cordyceps sinensis extract and the target protein, which are used to show the action strength of the Cordyceps sinensis extract and the target protein; table 3 shows RU values of ten samples (CS1-CS10) of the Cordyceps sinensis extract after two minutes of in vitro binding with the target protein, which are used to show the stability of the Cordyceps sinensis extract in producing in vitro binding with the target protein.

TABLE 2 detection results of SPR detection of multiple target spots of Cordyceps sinensis extract

According to BIAcore SPR detection results in Table 2, the Cordyceps sinensis extract prepared by the invention has in vitro binding affinity to a plurality of target proteins (PD-1, PD-L1 and TNFR 1). Among them, the RU values of the organic phase extracts (CS1, CS2, CS4, CS5, CS7, CS8) were significantly improved compared to the CS10 sample and the aqueous phase extracts (CS3, CS6, CS 9). In particular CS1, CS2, CS4 and CS7, and simultaneously shows excellent in vitro binding capacity to a plurality of target proteins (PD-1, PD-L1 and TNFR 1).

TABLE 3 SPR detection results of the multi-target binding stability of Cordyceps sinensis extract

According to the BIAcore SPR detection result in the table 3, the cordyceps sinensis extract still has good in vitro binding affinity to the target protein after being combined with the target protein in vitro for two minutes. Among them, the RU values of the organic phase extracts (CS1, CS2, CS4, CS5, CS7, CS8) were significantly improved compared to the CS10 sample and the aqueous phase extracts (CS3, CS6, CS 9). Particularly CS1, CS2, CS4 and CS7, simultaneously shows stable and high-strength in vitro binding capacity to a plurality of target proteins (PD-1, PD-L1 and TNFR1), can be used as effective PD-1, PD-L1 and TNFR1 inhibitors, and has very good potential as medicaments for treating tumors through immune regulation.

Test example 2 flow cytometry (FACS) examination of the inhibitory effect of the extract of Cordyceps sinensis of the present invention on PD-1/PD-L1

1. Test method

(1) Materials and sources

Cell line: 293T-h PD-L1, adherent cells; culture medium: DMEM + 10% FBS; fetal bovine serum FBS (Gbico, Cat # 10099-141); DMEM (HyClone, Cat # SH 3002201); PBS (Solarbio, Cat # P1020); pancreatin (Gbico, Cat # 25200072); FACS Buffer (PBS + 1% BSA); PE anti-human IgG Fc Antibody (Biolegend, Cat # 409304); human PD-1 fusion protein (Acro, cat # PD 1-H5257).

(2) Instruments and sources

Biological safety cabinet, Thermo Scientific, Model 1300Series a 2; CO 2₂Incubator, ThermoScientific, Model 3100 Series; inverted microscope, Olympus, CKX41 SF; flow cytometry, Millipore, Guava easyCyte 8 HT; refrigerator, Thermo Scientific, 900 SERIES.

(3) Preparation of samples to be tested

S1A prepared in example 1 was dried, 1mg was weighed, dissolved in DMSO to prepare a 10g/L mother solution, and the sample was named 190228D1 (see Table 5) and was then used for FACS test.

(4) FACS testing procedure

A. Cell collection:

a) cells in logarithmic growth phase were harvested and cell viability was checked by trypan blue exclusion to ensure cell viability above 90%.

b) Centrifuging at 1000r/min for 5min, and removing supernatant;

c) cells were washed once with PBS;

d) resuspend cells using FACS Buffer and count;

e) the density was 6.25 × 10 using FACS Buffer⁶cells/mL of cell suspension;

f) adding 80 mu L of cell suspension into a 96-well plate respectively;

g) cells in 96-well plates were grouped as:

1. blank Control group (Blank Control, without primary antibody, secondary antibody and sample to be tested)

2. Secondary antibody control group (2)^ndAb Control, no primary antibody and sample to be tested, and secondary antibody)

3. Positive control group (PD1-hFC, adding primary antibody and secondary antibody, not adding the sample to be tested)

4. Experimental group (190228D1, plus primary antibody, secondary antibody and sample to be tested).

B. Antibody incubation and detection

a) To the experimental group was added 10. mu.L of 10^×The sample to be tested is subjected to gradient concentration, so that the final concentration is 500 mg/L. Equal volume of FACS Buffer was added to the other control groups;

b) mixing, adding 10 μ L10 into positive control group and experimental group^×PD1-hFC protein at concentrations of, final concentration: 1 mu g/ml; equal volume of FACS Buffer was added to the other control groups;

c) mixing, and incubating at 4 deg.C in dark for 40 min;

d) using FACS Buffer to wash the cells for 3 times, each time 400 uL, centrifuging at the speed of 1000r/min for 5min, and finally using 100 uL FACS Buffer to resuspend the cells;

e) adding 5 mu L of PE-labeled secondary antibody into the experimental group, the secondary antibody control group and the positive group, and adding equal volume of FACS Buffer into the blank control group;

f) mixing, and incubating at 4 deg.C in dark for 40 min;

g) using FACS Buffer to wash the cells for 3 times, each time 400 uL, centrifuging at the speed of 1000r/min for 5min, and finally using 250 uL FACS Buffer to resuspend the cells;

h) flow cytometry detection YEL-HLog (Excitation Laser: 488nm Blue Laser).

C. Data processing

FACS data was analyzed with FlowJo software.

2. Test results

The FACS test result is shown in FIG. 1, and it can be seen that the fluorescence intensity of the experimental group is significantly reduced (P < 0.05) compared with the positive control group, which indicates that the Cordyceps sinensis extract prepared by the invention can effectively block 293T-h PD-L1 cell from binding with PD-1 protein and block PD-1/PD-L1 signal channel.

In conclusion, the invention provides the cordyceps sinensis extract and the preparation method thereof, the cordyceps sinensis extract has stable and high-strength binding capacity with a plurality of immunoregulation targets (PD-1, PD-L1 and TNFR1), and the cordyceps sinensis extract can also effectively block the binding of 293T-h PD-L1 cells and PD-1 protein and block a PD-1/PD-L1 signal channel. The cordyceps sinensis extract can be used for preparing immune checkpoint inhibitors including PD-1, PD-L1 and TNFR1, and can also be used for preparing various anti-tumor drugs for treating esophageal squamous cell carcinoma, urothelial cancer and squamous non-small cell lung cancer, and the application prospect is excellent.

Claims (11)

1. The application of the cordyceps sinensis extract in preparing the immune checkpoint inhibitor is characterized in that: the immune checkpoint is PD-1, PD-L1 or TNFR 1.

2. Use according to claim 1, characterized in that: the immune checkpoint is TNFR 1.

3. Use according to claim 1, characterized in that: the immune checkpoint inhibitor is capable of blocking the PD-1/PD-L1 signaling pathway.

4. Use according to claim 1, characterized in that: the inhibitor is an anti-tumor drug.

5. Use according to claim 4, characterized in that: the anti-tumor drug is a drug resistant tumor drug; preferably, the drug for resisting drug-resistant tumors is a tumor immunosuppressant.

6. Use according to claim 4 or 5, characterized in that: the tumors are esophageal squamous cell carcinoma, urothelial carcinoma and squamous non-small cell lung cancer; the squamous non-small cell lung cancer is preferably metastatic squamous non-small cell lung cancer.

7. Use according to claims 1-6, characterized in that: the cordyceps sinensis extract is prepared by the following method: is prepared from cordyceps through extracting.

8. Use according to claim 7, characterized in that: the extraction process comprises the following steps: extracting in liquid, dispersing the extract with pure water, extracting with organic solvent, and collecting organic phase and water phase;

preferably, the volume ratio of the organic solvent to pure water is 1: (0.8 to 1.2); the organic solvent is selected from esters and alcohols; the extraction times are 1-5 times;

more preferably, the volume ratio of the organic solvent to pure water is 1: 1; the organic solvent is selected from ethyl acetate and n-butanol; the number of extractions was 3.

9. Use according to claim 8, characterized in that: the liquid is selected from pure water or 60-80% of methanol; the ratio of material to liquid is 1: (15-25) g/mL; the extraction temperature is 30-45 ℃; the extraction times are 6-12 times; the extraction time is 20-40 min each time;

or the like, or, alternatively,

the extraction mode in the liquid is as follows: firstly, extracting for 4-8 times at 30-45 ℃ for 20-40 min each time in artificial gastric juice according to the feed-liquid ratio of 1 (15-25) g/mL; adding artificial intestinal juice, and extracting for 20-40 min each time for 2-5 times under heat preservation.

10. Use according to claim 9, characterized in that: the liquid is selected from pure water or 70% methanol; the ratio of material to liquid is 1:20 g/mL; the extraction temperature is 37 ℃; the extraction times are 9 times; the extraction time is 30min each time;

or the like, or, alternatively,

the extraction mode in the liquid is as follows: extracting in artificial gastric juice at 37 deg.C for 30min for 6 times with feed liquid 1:20 g/mL; adding artificial intestinal juice, extracting for 30min for 3 times.

11. Use according to any one of claims 8 to 10, characterized in that: the extract dispersed by pure water is dried extract; and/or the extraction method is ultrasonic extraction.

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