Jellyfish polypeptide, preparation method, and use

Abstract

Provided are a jellyfish polypeptide, and a preparation method therefor and a use thereof. The jellyfish polypeptide provided by the present disclosure or a jellyfish polypeptide obtained by using the preparation method provided by the present disclosure exhibits a significant inhibitory effect on multiple tumors including colorectal cancer, lung cancer and prostate cancer, and also has a regulating function on mammalian immune systems.

Description

Jellyfish polypeptide, preparation method and use

[0001] Priority claim

[0002] The present disclosure claims priority to the Chinese invention patent application No. 2024106698491, filed on May 28, 2024, the whole content of which is incorporated into the present disclosure. TECHNICAL FIELD

[0003] The present disclosure relates to the field of pharmacy, in particular to a jellyfish polypeptide, a preparation method and use thereof. BACKGROUND

[0004] Jellyfish has important value in beauty, food and scientific research. Jellyfish gel is rich in collagen and elastin, which are one of the most important components in the skin, can make the skin more firm and elastic, and reduce the appearance of wrinkles and fine lines. Jellyfish is also a nutritious food, which is a low-fat and low-calorie food, and its gelatin contains rich proteins and various minerals such as calcium, iron, zinc, etc., which have good health care effects on human health. As a very special organism, jellyfish has unique characteristics in body structure and life activity mode, and also has important value in scientific research. At the same time, they are often kept or observed as ornamental fish or marine biological exhibits, bringing people the enjoyment of beauty.

[0005] In addition, jellyfish also has high economic value and ecological value. In some countries and regions, jellyfish is considered as a delicacy, especially in the coastal areas of China. At the same time, jellyfish can absorb and enrich toxic substances and pollutants, so they can be used in ecological restoration and environmental governance. Some small jellyfish can also be used as biological indicators to monitor the health status of marine environment and ecosystem.

[0006] Jellyfish also contains toxic substances, which are collectively referred to as jellyfish toxins, which are a mixture of unique and novel proteins and polypeptides with a wide range of activities, such as hemolysis, antioxidant and other biological activities, and the main components are toxic protein-like toxins and polypeptides, enzymes. However, due to the diversification of its toxic effects, there are potential safety hazards.

[0007] Therefore, in the process of extraction and development of active components from jellyfish, it is necessary to pay attention to non-toxic active products derived from jellyfish. SUMMARY

[0008] The purpose of the present disclosure is to provide a jellyfish polypeptide, a preparation method of the jellyfish polypeptide, and the use of the jellyfish polypeptide in treating or preventing tumors and in preparing immunomodulators.

[0009] In a first aspect of the present disclosure, a jellyfish polypeptide is provided, comprising: a polypeptide fragment with an amino acid residue number selected from 8 to 27 obtained by liquid chromatography-mass spectrometry separation of a jellyfish extract.

[0010] In an optional embodiment, the preparation method of the jellyfish extract comprises: subjecting the dehydrated and desalted whole jellyfish or specific part of the jellyfish to enzymatic hydrolysis, and obtaining the jellyfish extract by filtering the obtained enzymatic hydrolysate;

[0011] The enzyme used in the enzymatic hydrolysis comprises alkaline protease and pepsin.

[0012] Optionally, the liquid-solid volume mass ratio in the enzymatic hydrolysis step is 5-10:1, and optionally 6:1.

[0013] Optionally, the temperature of the enzymatic hydrolysis step is 30-70℃, and preferably 50℃.

[0014] Optionally, the pH of the enzymatic hydrolysis step is 5-9, and preferably 6-8.5.

[0015] In an optional embodiment, the jellyfish is selected from Order Semaeostomeae. Optionally, the jellyfish is at least one selected from Family Cyaneidae, Family Pelagiidae, and Family Ulmaridae.

[0016] The Family Cyaneidae comprises Cyanea capillata, Cyanea ferruginea, Cyanea nozakii, and Cyanea purpurea. The Family Pelagiidae comprises Chrysaora helvola, Pelagia noctiluca, and Sanderia malayensis. The Family Ulmaridae comprises Aurelia coerulea.

[0017] In an optional embodiment, the jellyfish is Aurelia coerulea, or the jellyfish comprises Aurelia aurita and at least one other jellyfish selected from Order Semaeostomeae.

[0018] In an alternative embodiment, the jellyfish polypeptide is extracted from a whole jellyfish or a certain or certain parts, organs or tissues of the jellyfish, but at least including the umbrella of the jellyfish.

[0019] In an alternative embodiment, the jellyfish is clean and free of attachments; optionally, after the jellyfish polypeptide is filtered, a jellyfish polypeptide forming step is further included.

[0020] In an alternative embodiment, the number of amino acid residues of the jellyfish polypeptide is 8; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID Nos: 1 to 3. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 9; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID Nos: 4 to 7. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 10; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No: 8. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 11; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No: 9. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 12; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID Nos: 10 to 13. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 13; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No: 14 or 15. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 14; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No: 16 or 17. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 15; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID Nos: 18 to 20. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 16; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No: 21. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 18; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No: 22 or 23. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 27; optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No: 24.

[0021] In a second aspect, the present disclosure provides a jellyfish polypeptide composition comprising two or more different jellyfish polypeptides selected from the jellyfish polypeptides according to any one of the preceding embodiments.

[0022] In an optional embodiment, the jellyfish polypeptides comprise a total of 21 polypeptides as shown in SEQ ID No: 1-9, SEQ ID No: 11-14, SEQ ID No: 16-19 and SEQ ID No: 21-24. Alternatively, the jellyfish polypeptides comprise a total of 23 polypeptides as shown in SEQ ID No: 1-14 and SEQ ID No: 16-24. Alternatively, the jellyfish polypeptides comprise a total of 23 polypeptides as shown in SEQ ID No: 2-24.

[0023] In a third aspect, the present disclosure provides use of the jellyfish polypeptide according to any one of the embodiments of the first aspect or the jellyfish polypeptide composition according to any one of the embodiments of the second aspect in the preparation of a product for treating or preventing a tumor; optionally, the tumor comprises a solid tumor.

[0024] In an optional embodiment, the tumor comprises an advanced or terminal stage of intestinal cancer, lung cancer or prostate cancer.

[0025] In an optional embodiment, the dosage form of the product for treating or preventing a tumor comprises an oral preparation.

[0026] In a fourth aspect, the present disclosure provides use of the jellyfish polypeptide according to any one of the embodiments of the first aspect or the jellyfish polypeptide composition according to any one of the embodiments of the second aspect in the preparation of an immunomodulator.

[0027] In a fifth aspect, the present disclosure provides a method for preparing a jellyfish polypeptide, comprising subjecting a dehydrated and desalted whole jellyfish or a specific part of a jellyfish to enzymatic hydrolysis, filtering the obtained enzymatic hydrolysate to obtain a jellyfish extract, and subjecting the jellyfish extract to liquid chromatography-mass spectrometry to obtain a polypeptide fragment having a number of amino acid residues selected from 8-27; the enzyme used in the enzymatic hydrolysis comprises alkaline protease and pepsin.

[0028] In an optional embodiment, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, optionally 6:1; the temperature of the enzymatic hydrolysis step is 30-70°C, optionally 50°C; and the pH of the enzymatic hydrolysis step is 5-9, optionally 6-8.5.

[0029] In an optional embodiment, the mobile phase of the liquid chromatography in the liquid chromatography-mass spectrometry comprises an aqueous phase and an organic phase; the aqueous phase comprises an aqueous phase containing formic acid; and the organic phase comprises acetonitrile containing formic acid.

[0030] Optionally, the content of formic acid in the aqueous phase and the organic phase is 0.05% to 0.2%, and optionally 0.1%.

[0031] Optionally, the gradient elution condition of the liquid chromatography comprises:

[0032] 0-45 min, 2%-22% (v / v) of the organic phase;

[0033] 45-50 min, 22%-37% (v / v) of the organic phase;

[0034] 50-55 min, 37%-80% (v / v) of the organic phase;

[0035] 55-60 min, 80% (v / v) of the organic phase.

[0036] In an optional embodiment, the jellyfish is clean and free of attachments.

[0037] In an optional embodiment, after the polypeptide of jellyfish is filtered, a shaping step of the polypeptide of jellyfish is further included.

[0038] The sixth aspect of the present disclosure provides a medicine for treating or preventing tumors, which contains the polypeptide of jellyfish according to any one of the embodiments of the first aspect or the composition of polypeptide of jellyfish according to any one of the embodiments of the second aspect.

[0039] Optionally, the tumors include solid tumors.

[0040] Optionally, the dosage form of the medicine for treating or preventing tumors includes oral preparations.

[0041] The seventh aspect of the present disclosure provides an immunomodulator, which contains the polypeptide of jellyfish according to any one of the embodiments of the first aspect or the composition of polypeptide of jellyfish according to any one of the embodiments of the second aspect.

[0042] The eighth aspect of the present disclosure provides a preparation method of the medicine according to the sixth aspect or the immunomodulator according to the seventh aspect, which comprises concentrating the polypeptide of jellyfish according to any one of the embodiments of the first aspect or the composition of polypeptide of jellyfish according to any one of the embodiments of the second aspect to obtain a concentrated solution of the polypeptide of jellyfish or the composition thereof, and then performing a shaping step to obtain an oral preparation medicine or an immunomodulator.

[0043] The eighth aspect of the present disclosure provides a method for treating or preventing tumors, which comprises administering an effective dose of the medicine according to the sixth aspect of the present disclosure to an individual to be intervened; optionally, the tumors include solid tumors.

[0044] The ninth aspect of the present disclosure provides a method for improving immunity, comprising administering to a subject to be intervened an effective amount of the immune modulator of the seventh aspect of the present disclosure.

[0045] The tenth aspect of the present disclosure provides a method for treating an autoantibody-mediated disease, comprising administering to a subject to be intervened an effective amount of the immune modulator of the seventh aspect of the present disclosure.

[0046] It has been verified that the jellyfish polypeptide or composition thereof provided by the present disclosure, or the jellyfish polypeptide or composition thereof obtained by the preparation method provided by the present disclosure, has a significant inhibitory effect on various tumors including intestinal cancer, lung cancer and prostate cancer, and the inhibitory effect on tumors includes killing tumor cells, reducing inflammation and organ lesions caused by tumors. At the same time, it can improve the immunity of the individual as a whole, and has a relieving and treating effect on autoantibody-mediated diseases. Therefore, the jellyfish polypeptide or composition thereof provided by the present disclosure can be used for preparing a tumor treatment or tumor prevention product, including but not limited to various anti-tumor drugs of different administration routes, or an immune modulator. BRIEF DESCRIPTION OF DRAWINGS

[0047] FIG. 1 is a preparation flow of the jellyfish polypeptide in Example 1;

[0048] FIG. 2 is a distribution diagram of the peptide segment number of the jellyfish polypeptide in Example 1;

[0049] FIG. 3 is a distribution diagram of the molecular weight of the peptide segment of the jellyfish polypeptide in Example 1;

[0050] FIG. 4 is a hierarchical clustering dendrogram of the jellyfish polypeptide compositions SM1, SM2 and SM3 in Example 1;

[0051] FIG. 5 is a sample correlation analysis diagram of the jellyfish polypeptide compositions SM1, SM2 and SM3 in Example 1. DETAILED DESCRIPTION

[0052] According to the above content of the present disclosure, according to the ordinary technical knowledge and means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical ideas of the present disclosure.

[0053] I. DEFINITIONS

[0054] Unless otherwise explicitly indicated, throughout the specification and claims, the term "comprise" or its variants such as "comprises" or "comprising" will be understood to encompass the stated element or components, without excluding the presence of other elements or components.

[0055] As described herein, the term "jellyfish extract" refers to a biologically functional active substance extracted from jellyfish, without limiting the specific jellyfish species and extraction method. In the present disclosure, the jellyfish extract is a mixture containing various active components directly obtained by the extraction method. An exemplary jellyfish extract is registered on Chemical Book under accession number CB0961824 (JELLYFISH EXTRACT). The term "jellyfish polypeptide" as described herein refers to a polypeptide fragment derived from a jellyfish protein contained in the jellyfish extract. The term "jellyfish polypeptide composition" refers to a composition composed of two or more jellyfish polypeptides.

[0056] As described herein, the term "treatment" includes inhibition, alleviation or elimination of one or more symptoms or side effects associated with the disease, disorder or malady being treated. The term "effective amount" (therapeutically effective amount) refers to a dose sufficient to treat, inhibit or alleviate one or more symptoms of the disease state being treated or otherwise provide the desired pharmacologic and / or physiologic effect. The precise dose will vary according to factors such as the subject-dependent variables (e.g., age, immune system health, etc.), the disease or illness, and the treatment administered. The effect of the effective amount can be relative to a control. These controls are known in the art and discussed herein, and can be, for example, the subject's condition prior to administration of the drug or drug combination or without administration, or in the case of a drug combination, the effect of the combination can be compared to the effect of administering only one of the drugs.

[0057] As described herein, the term "prevention" primarily refers to secondary prevention, also known as preclinical prevention (or presymptomatic), i.e., the "three early" prevention measures of early detection, early diagnosis and early treatment in the preclinical stage of the tumor. By early detection of the tumor, early diagnosis, and further administration of an appropriate dose of jellyfish polypeptide, changes in the preclinical or early clinical stage of the tumor are prevented, allowing the disease to be detected and treated at an early stage, avoiding or reducing complications.

[0058] As described herein, the term "Order Semaeostomeae" is the most common order in the subphylum Cnidaria, class Scyphozoa. Umbrella is bowl-shaped, butterfly-shaped, with 8 to 100 notches on the umbrella edge, and tentacle pockets in the notches. The number, distribution, and shape of tentacles vary with species. There are oral arms, and the oral arms have ciliated grooves. The complex radial tube, ring tube, or no ring tube, extends from the gastric sac. Most of them live in coastal areas. Among the currently known jellyfish species, the subcategories include Family Cyaneidae, Family Pelagiidae, and Family Ulmaridae. Therefore, the "Order Semaeostomeae" described herein should not be understood as being limited to the above three families of jellyfish. Subsequent new discoveries, or those that have been discovered but subsequently identified as Order Semaeostomeae, are also within the protection scope of the Order Semaeostomeae described in the present disclosure. Similarly, among the currently known jellyfish classifications, Family Cyaneidae includes Cyanea capillata, Cyanea ferruginea, Cyanea nozakii, and Cyanea purpurea. Family Pelagiidae includes Chrysaora helvola, Pelagia noctiluca, and Sanderia malayensis. Family Ulmaridae currently only includes Aurelia coerulea, as recorded in the Chinese Species List 2023. It should be noted that the composition of jellyfish polypeptides varies slightly among different orders of jellyfish, and there are slight differences in antitumor effects. At the same time, some jellyfish can secrete jellyfish toxins. When jellyfish polypeptides obtained from such jellyfish are used to prepare antitumor drugs, the safety needs to be investigated, and the additive amount needs to be strictly controlled. Among them, Aurelia coerulea is a non-toxic jellyfish, and the jellyfish polypeptides of this type of jellyfish have obvious advantages in safety. As for toxic jellyfish, the tentacles or tentacles can be stripped, and the non-toxic umbrella part can be reserved for the preparation of extracts.

[0059] As described in this article, the term "alkaline protease," also known as serine protease, is a highly alkaline (pH 9-10) protease with serine as its active center. It hydrolyzes protein molecules, specifically peptide chains, to produce polypeptides or amino acids, and possesses a strong ability to break down proteins. It is widely found in bacteria, actinomycetes, and fungi, including *Bacillus licheniformis*, *B. amyloliquefaciens*, *B. pumi Chemicalbooklus*, *B. alcalophilus*, *Streptomyces griseus*, *S. fradiae*, and certain *Fusarium* species. Its activity ranges from pH 7 to 11. In addition to hydrolyzing peptide bonds, this enzyme also has the ability to hydrolyze ester and amide bonds, as well as the ability to transesterify and transpeptide.

[0060] As described in this article, the term "flavor protease" is obtained through fermentation with Aspergillus oryzae, refined using advanced extraction processes such as microfiltration, ultrafiltration, and vacuum freeze-drying. The number of microorganisms is strictly controlled to meet food-grade standards. Flavor proteases can impart a unique flavor to hydrolysates and reduce bitterness caused by hydrolysis.

[0061] As described in this article, the term "oral formulation" refers to a dosage form that, after oral administration, enters the gastrointestinal tract and exerts a local effect or, through absorption, a systemic effect. Drugs that are easily destroyed by acids or enzymes in the gastrointestinal tract generally cannot be used in this type of simple dosage form.

[0062] As described herein, the term "medicine" means a composition comprising the jellyfish polypeptides described in this disclosure, and at least one pharmaceutically acceptable ingredient selected from the following, depending on the manner of administration and the nature of the dosage form: including, but not limited to, carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0063] As described in this article, liquid chromatography-mass spectrometry (LC-MS) refers to the sequential use of liquid chromatography and mass spectrometry to separate mixtures containing multiple components, or specific components. This method combines the ability of liquid chromatography to effectively separate thermally unstable and high-boiling-point compounds with the strong component identification capabilities of mass spectrometry. It is an effective means of separating and analyzing complex organic mixtures.

[0064] As described herein, the term "identity" can be considered equivalent to the term "homology" or "similarity" without affecting the results. The relative sequence identity can be determined by commercially available computer programs that can calculate the percentage of identity between two or more sequences. The jellyfish polypeptide "having 80% to 100% identity" described herein refers to a jellyfish polypeptide variant having a percentage of amino acid sequence identity between 80% to 100% with the jellyfish polypeptide shown in SEQ ID No: 1-23 herein, and having anti-tumor or whitening, anti-wrinkle and other active functions. The identity ratio includes but is not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0065] As described herein, the term "immunomodulator" refers to a substance that has an activating or inhibiting function on the body's immune system, which is divided into two categories: immune enhancers and immune suppressors. Immune enhancers are drugs that enhance immunity, and representatives include immunoglobulin, gamma globulin, complex vitamins, complex trace elements, etc.; Immunosuppressants are therapeutic drugs that inhibit the function of the immune system, such as cyclosporine, azathioprine, methotrexate, cyclophosphamide, etc. Currently, the main clinical use is immune enhancers, some of which also have the function of regulating immune imbalance. The jellyfish polypeptide or jellyfish polypeptide composition provided by the present disclosure has the function of enhancing immunity, and can regulate the immune system and inhibit the activity of certain autoantibodies, hormones or enzymes, thereby achieving the therapeutic function of autologous antibody-mediated diseases.

[0066] II. DETAILED DESCRIPTION

[0067] In a first aspect of the present disclosure, a jellyfish polypeptide is provided, comprising a polypeptide fragment with an amino acid residue number selected from 8 to 27 obtained by liquid chromatography-mass spectrometry separation of a jellyfish extract, the amino acid residue number includes but is not limited to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.

[0068] In an alternative embodiment, the preparation method of the jellyfish extract comprises enzymatic hydrolysis of the dehydrated and desalted whole jellyfish or specific parts of the jellyfish, and the obtained enzymatic hydrolysis liquid is filtered to obtain the jellyfish extract. It can be understood that when the specific part of the jellyfish is used as the raw material for the extraction of the extract, the preparation method can first separate the specific part of the jellyfish, and then dehydrate and desalt, or the whole jellyfish can be dehydrated and desalted first, and then the specific part of the jellyfish is separated. The purpose of the filtration in the preparation method is to remove solid impurities.

[0069] In an alternative embodiment, the enzymatic hydrolysis uses two or more enzymes; preferably, the enzymes include alcalase and pepsin, and a flavourzyme can be added without affecting the activity. It is noted that the alternative enzymatic hydrolysis sequence includes (1) alcalase hydrolysis-pepsin hydrolysis, or (2) pepsin hydrolysis-alcalase hydrolysis, or an additional flavourzyme hydrolysis step after the above enzymatic hydrolysis sequence (1) or (2).

[0070] In an alternative embodiment, the liquid to solid volume mass ratio in the enzymatic hydrolysis step is 5-10:1, optionally 6:1; optionally, the enzymatic hydrolysis step is at a temperature of 30-70°C, such as 30°C, 35°C, 40°C, 45°C or 50°C, optionally 50°C; optionally, the enzymatic hydrolysis step is at a pH of 5-9, optionally 6-8.5, such as 6.5, 7, 7.5, 8 or 8.5.

[0071] An alternative combination of parameters for the enzymatic hydrolysis step can be: alcalase hydrolysis-pepsin hydrolysis, liquid to solid volume mass ratio: 6:1, enzymatic hydrolysis temperature: constant 50°C, enzymatic hydrolysis pH: alcalase hydrolysis-8.5, pepsin hydrolysis-6.5.

[0072] In an alternative embodiment, the jellyfish is selected from Order Semaeostomeae. Optionally, at least one of Family Cyaneidae, Family Pelagiidae, Family Ulmaridae.

[0073] Among them, the Family Cyaneidae includes Cyanea capillata, Cyanea ferruginea, Cyanea nozakii, Cyanea purpurea. The Family Pelagiidae includes Chrysaora helvola, Pelagia noctiluca, Sanderia malayensis. The Family Ulmaridae includes Aurelia coerulea.

[0074] In an alternative embodiment, the jellyfish is Aurelia coerulea, or the jellyfish comprises Aurelia aurita and at least one other jellyfish selected from the Order Semaeostomeae. Exemplary combinations include, but are not limited to, (1) Aurelia aurita alone, (2) Aurelia aurita and Malaiaca, (3) Aurelia aurita and Noctiluca, (4) Aurelia aurita and Chrysaora, (5) Aurelia aurita and Cyanea purpurea, (5) Aurelia aurita and Leucophyllum, (6) Aurelia aurita and Chrysaora brownii, (7) Aurelia aurita and Chrysaora fuscescens, (8) Aurelia aurita, one species of Chrysaorid and one species of Siphonophorid, (9) Aurelia aurita, one species of Chrysaorid and two species of Siphonophorid, (10) Aurelia aurita, two species of Chrysaorid and one species of Siphonophorid, and other combinations comprising Aurelia aurita and at least one other jellyfish selected from the Order Semaeostomeae.

[0075] In an alternative embodiment, the jellyfish polypeptide is extracted from a whole jellyfish or a particular part, organ or tissue of the jellyfish, but at least the umbrella of the jellyfish. Exemplary jellyfish extraction parts include, but are not limited to, a whole Aurelia aurita, an umbrella of Aurelia aurita, an umbrella of Aurelia aurita and at least one other jellyfish selected from the Order Semaeostomeae, a whole Aurelia aurita and an umbrella of at least one other jellyfish selected from the Order Semaeostomeae.

[0076] In an alternative embodiment, the jellyfish is clean and free of attachments to avoid introducing impurities or degradation products from other organisms.

[0077] In an alternative embodiment, after the jellyfish polypeptide is filtered, a shaping step is further included. The shaping step and method can be selected and optimized according to the actual needs of subsequent preparation of anti-tumor products or cosmetics, the dosage form or administration method of the anti-tumor products, etc.

[0078] In an alternative embodiment, the number of amino acid residues of the jellyfish polypeptide is 8; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in any one of SEQ ID Nos: 1 to 3. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 9; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in any one of SEQ ID Nos: 4 to 7. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 10; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in SEQ ID No: 8. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 11; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in SEQ ID No: 9. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 12; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in any one of SEQ ID Nos: 10 to 13. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 13; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in SEQ ID No: 14 or 15. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 14; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in SEQ ID No: 16 or 17. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 15; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in any one of SEQ ID Nos: 18 to 20. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 16; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in SEQ ID No: 21. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 18; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in SEQ ID No: 22 or 23. Alternatively, the number of amino acid residues of the jellyfish polypeptide is 27; alternatively, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity to the amino acid sequence set forth in SEQ ID No: 24.

[0079] Alternatively, the 80% to 100% identity described hereinabove includes, but is not limited to, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0080] In a second aspect of the present disclosure, a jellyfish polypeptide composition is provided, which contains two or more different jellyfish polypeptides selected from the jellyfish polypeptides according to any one of the preceding embodiments.

[0081] In an optional embodiment, the jellyfish polypeptide includes a total of 21 polypeptides as shown in SEQ ID No: 1-9, SEQ ID No: 11-14, SEQ ID No: 16-19 and SEQ ID No: 21-24. Alternatively, the jellyfish polypeptide includes a total of 23 polypeptides as shown in SEQ ID No: 1-14 and SEQ ID No: 16-24. Alternatively, the jellyfish polypeptide includes a total of 23 polypeptides as shown in SEQ ID No: 2-24.

[0082] In a third aspect of the present disclosure, the jellyfish polypeptide according to any one of the embodiments of the first aspect or the jellyfish polypeptide composition according to any one of the embodiments of the second aspect is used for preparing a product for treating or preventing a tumor; optionally, the tumor includes a solid tumor.

[0083] In an optional embodiment, the tumor includes intestinal cancer, lung cancer or prostate cancer in a progressive or terminal stage. The intestinal cancer includes, but is not limited to, colon cancer or rectal cancer, and subtypes such as adenocarcinoma, mucinous adenocarcinoma or squamous cell carcinoma, etc. The lung cancer includes, but is not limited to, small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma or large cell carcinoma, etc. The prostate cancer includes adenocarcinoma, ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma or adenosquamous carcinoma, etc.

[0084] In an optional embodiment, the dosage form of the product for treating or preventing a tumor includes an oral preparation. It should be noted that in the embodiments of the present disclosure, cell experiments and animal experiments can confirm that the jellyfish polypeptides provided by the present disclosure can achieve good anti-tumor effect after oral administration, and therefore, the dosage form of the jellyfish polypeptide that can be administered orally should belong to the protection scope of the present disclosure. In addition, according to the description of the present disclosure, it can be clearly inferred that other effective dosage forms should also belong to the protection scope of the present disclosure.

[0085] In a fourth aspect of the present disclosure, the jellyfish polypeptide according to any one of the embodiments of the first aspect or the jellyfish polypeptide composition according to any one of the embodiments of the second aspect is used for preparing an immunomodulator.

[0086] In a fifth aspect of the present disclosure, a preparation method of a jellyfish polypeptide is provided, which includes subjecting a dehydrated and desalted whole jellyfish or a specific part of the jellyfish to enzymatic hydrolysis, filtering the obtained enzymatic hydrolysate to obtain a jellyfish extract, and subjecting the jellyfish extract to liquid chromatography-mass spectrometry separation to obtain a polypeptide fragment with an amino acid residue number selected from 8-27; the enzymes used for the enzymatic hydrolysis include alkaline protease and pepsin.

[0087] In an optional embodiment, the liquid to solid volume mass ratio in the enzymatic hydrolysis step is 5-10:1, optionally 6:1; the temperature of the enzymatic hydrolysis step is 30-70°C, for example 30°C, 35°C, 40°C, 45°C or 50°C, optionally 50°C; optionally, the pH of the enzymatic hydrolysis step is 5-9, optionally 6-8.5, for example 6.5, 7, 7.5, 8 or 8.5.

[0088] In an optional embodiment, the mobile phase of the liquid chromatography in the liquid chromatography-mass spectrometry comprises an aqueous phase and an organic phase, the aqueous phase comprises an aqueous phase comprising formic acid, and the organic phase comprises acetonitrile comprising formic acid;

[0089] Optionally, the content of formic acid in the aqueous phase and the organic phase is 0.05%-0.2%, including but not limited to 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2%, optionally 0.1%.

[0090] Optionally, the gradient elution condition of the liquid chromatography comprises:

[0091] 0-45 min, 2%-22% (v / v) of the organic phase;

[0092] 45-50 min, 22%-37% (v / v) of the organic phase;

[0093] 50-55 min, 37%-80% (v / v) of the organic phase;

[0094] 55-60 min, 80% (v / v) of the organic phase.

[0095] In an optional embodiment, the jellyfish is clean and free of attachments;

[0096] In an optional embodiment, after the polypeptide of jellyfish is filtered, a polypeptide of jellyfish forming step is further included.

[0097] The sixth aspect of the present disclosure provides a medicine for treating or preventing tumors, the medicine comprising the polypeptide of jellyfish according to any one of the embodiments of the first aspect or the polypeptide of jellyfish composition according to any one of the embodiments of the second aspect.

[0098] In an optional embodiment, the tumor comprises a solid tumor.

[0099] In an optional embodiment, the dosage form of the medicine comprises oral preparations.

[0100] The seventh aspect of the present disclosure provides an immunomodulator comprising the jellyfish polypeptide according to any one of the embodiments of the first aspect or the jellyfish polypeptide composition according to any one of the embodiments of the second aspect.

[0101] The eighth aspect of the present disclosure provides a method for preparing the medicament according to the sixth aspect or the immunomodulator according to the seventh aspect, comprising concentrating the jellyfish polypeptide according to any one of the embodiments of the first aspect or the jellyfish polypeptide composition according to any one of the embodiments of the second aspect to obtain a concentrated solution of the jellyfish polypeptide or the composition thereof, and then performing a shaping step to obtain the oral preparation medicament or the immunomodulator.

[0102] The eighth aspect of the present disclosure provides a method for treating or preventing a tumor, comprising administering to an individual to be intervened an effective dose of the medicament according to the sixth aspect of the present disclosure; optionally, the tumor comprises a solid tumor.

[0103] The ninth aspect of the present disclosure provides a method for improving immunity, comprising administering to an individual to be intervened an effective dose of the immunomodulator according to the seventh aspect of the present disclosure.

[0104] The tenth aspect of the present disclosure provides a method for treating an autoantibody-mediated disease, comprising administering to an individual to be intervened an effective dose of the immunomodulator according to the seventh aspect of the present disclosure.

[0105] III. Examples

[0106] The present disclosure is further illustrated with reference to the following examples. The description of specific exemplary embodiments of the present disclosure is intended for purposes of illustration and example only. These descriptions are not intended to limit the present disclosure to the precise forms set forth and, as such, many modifications and variations will be apparent to those skilled in the art from this teachings. The examples were chosen and described in order to explain the principles of the present disclosure and its practical application and to enable others skilled in the art to best utilize the present disclosure together with various modifications as are suited to the particular

[0107] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0108] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0109] Example 1 Preparation of jellyfish polypeptide

[0110] The umbrella of Aurelia coerulea without silt and corruption was used as raw material, and the product was prepared by the following steps: cleaning, dehydration, desalination, homogenization, adding three proteases step by step, enzymolysis at 50℃ for 8 hours, enzyme inactivation and sterilization at 100℃ for 20 minutes, filtration, concentration, drying, packaging, etc. as shown in Figure 1.

[0111] The specific steps are as follows:

[0112] 1.1 Collection of living Aurelia coerulea

[0113] In June to July, Aurelia coerulea with umbrella diameter of more than 25 cm was collected along the coast, and the oral arms and nematocysts under the umbrella were cut off cleanly, and only the umbrella was reserved as the raw material for preparing jellyfish polypeptide.

[0114] 1.2 Cleaning of Aurelia coerulea umbrella

[0115] The Aurelia coerulea umbrella was placed in a disinfected water tank and soaked in clean water for 30 minutes, with a water to umbrella ratio of 5:1. Then the impurities and silt were washed away, and the Aurelia coerulea umbrella was taken out and placed in another disinfected water tank. The above cleaning method was repeated multiple times until there was no silt and no attached impurities.

[0116] 1.3 Dehydration and desalination of Aurelia coerulea umbrella

[0117] After cleaning, the Aurelia coerulea umbrella was taken out and cut into thin strips 5 cm wide, placed in a water leakage container for preliminary autolysis dehydration. When the weight was about 60% of the original weight, the thin strip-shaped umbrella was placed in a gauze bag and pressed with a heavy equipment to further expel water. During the process of water expulsion, a large amount of inorganic salt was removed, and the weight was about 20% of the original weight. The pressing heavy equipment was removed, and the Aurelia coerulea umbrella at this time was placed in pure water for cleaning, with a ratio of 3:1. After cleaning, it was packed according to 50 kg per portion and stored in a-20℃ freezer.

[0118] 1.4 Enzymolysis of Aurelia coerulea umbrella

[0119] With the degree of hydrolysis as an index, first, suitable enzyme for enzymolysis is screened from enzymes for food additives, then through single factor test, enzyme dosage, enzymolysis temperature, enzymolysis time, pH value of enzymolysis system, and solid-liquid ratio are preliminarily determined, and then through orthogonal test, the optimal enzymolysis conditions are determined. The umbrella of Aurelia coerulea is enzymolyzed by the determined optimal enzymolysis steps, and the specific steps of the optimized enzymolysis are as follows:

[0120] Take 1 part of the homogenate of the umbrella of Aurelia coerulea (50 kg) which is divided into portions, and put the homogenate into an enzymolysis tank, add ultrapure water according to the ratio of 6:1 of liquid to solid, add 100 g of alkaline protease (enzyme activity 2×105 U / g), set the enzymolysis temperature to 50℃, adjust the pH value of the enzymolysis system to 8.5 with 2 mol / L sodium hydroxide, after 2 hours of enzymolysis, adjust the pH value of the enzymolysis system to 6.5 with 2 mol / L hydrochloric acid, then add 200 g of pepsin (enzyme activity 2×105 U / g), after 3 hours of enzymolysis, adjust the pH value of the enzymolysis system to 6.0 with 2 mol / L hydrochloric acid, and then heat to 100℃ for enzyme inactivation and sterilization, and the time is 20 minutes. 6 U / g), set the enzymolysis temperature to 50℃, adjust the pH value of the enzymolysis system to 8.5 with 2 mol / L sodium hydroxide, after 2 hours of enzymolysis, adjust the pH value of the enzymolysis system to 6.5 with 2 mol / L hydrochloric acid, then add 200 g of pepsin (enzyme activity 2×105 U / g), after 3 hours of enzymolysis, adjust the pH value of the enzymolysis system to 6.0 with 2 mol / L hydrochloric acid, and then heat to 100℃ for enzyme inactivation and sterilization, and the time is 20 minutes.

[0121] 1.5 Filtration of the enzymolysis liquid of the umbrella of Aurelia coerulea

[0122] After the temperature of the enzymolysis liquid of the umbrella of Aurelia coerulea is reduced to below 60℃, the enzymolysis liquid is discharged from the enzymolysis tank for cooling, and after cooling to room temperature, filtration is performed. The enzymolysis liquid is placed into a filter for multiple filtrations, and the supernatant is collected.

[0123] 1.6 Concentration of the enzymolysis liquid of the umbrella of Aurelia coerulea

[0124] The supernatant is poured into an evaporator for concentration, the evaporation temperature is 75℃, and when the volume of the enzymolysis liquid is 1 / 3 of the original, the concentration is completed.

[0125] 1.7 Freeze-drying of the enzymolysis liquid of the umbrella of Aurelia coerulea

[0126] The concentrated enzymolysis liquid is poured into a freeze-drying machine tray for freeze-drying, and after 48 hours, it is taken out to obtain a jelly-like jellyfish extract.

[0127] 1.8 LC-MS

[0128] 1.8.1 Pretreatment

[0129] (1) Take 1 mg of the jellyfish extract powder obtained in a 1.5 mL EP tube, add 1 mL of liquid chromatography A phase (aqueous phase) to dissolve thoroughly;

[0130] (2) Oscillation 10 min, ultrasonic water bath 10 min, oscillation 10 min, and centrifugation to the bottom of the tube;

[0131] (3) Take 100 ul of the solution for desalination, and dry the desalinated peptide;

[0132] (4) Use 100 ul of phase A to re-dissolve the desalinated peptide, and perform nanodrop quantification to an appropriate concentration for machine operation.

[0133] 1.8.2 Liquid chromatography-mass spectrometry conditions

[0134] The analysis instrument is a Nanoelute liquid chromatograph coupled with a Tims Tofpro2 mass spectrometer.

[0135] Liquid chromatography conditions:

[0136] The mobile phase A (aqueous phase) is water-formic acid (99.9%:0.1%, v / v); the mobile phase B (organic phase) is acetonitrile-formic acid (99.9:0.1, v / v). The sample is loaded onto an analysis column (75 μm x 15 cm, 1.6 μm, C18, ionopticks) by an automatic sampler for separation.

[0137] The gradient elution conditions are: 0-45 min, 2%-22% B; 45-50 min, 22%-37% B; 50-55 min, 37%-80% B; 55-60 min, 80% B.

[0138] Mass spectrometry conditions:

[0139] The timsTOF Pro2 is used in the positive ion mode to perform data acquisition in the parallel accumulation serial fragmentation (PASEF) DIA mode. The capillary voltage is set to 1400 V, the mass spectrometry primary and secondary scanning range is 100-1700 m / z, and the ion mobility window range (1 / K0) is 0.85-1.3 Vs / cm 2 . The ion accumulation and release time is set to 100 ms to achieve an ion utilization rate of nearly 100%. The fragmentation energy is from 20-59 eV corresponding to the ion mobility 1 / K0=0.6-1.6 Vs / cm 2 .

[0140] 1.9 Packaging and storage of jellyfish polypeptides

[0141] The jellyfish polypeptide composition obtained above is denoted as SM1 and is packaged in a double-layer vacuum aluminum film bag in a sterile environment with constant temperature and humidity, and then stored in a warehouse.

[0142] ​In addition, in the enzymolysis step, after the alkaline protease and pepsin enzymolysis is completed, before the temperature is raised for inactivation, flavor protease 200 g (enzyme activity 3 x 10 5 U / g) can be added, and enzymolysis is performed for 3 hours.

[0143] The above steps are repeated, and only the pH and temperature of the enzymolysis process are adjusted to obtain jellyfish polypeptide compositions SM2 and SM3.

[0144] Example 2 Polypeptide Composition

[0145] In this example, the jellyfish polypeptide compositions SM1, SM2 and SM3 obtained in Example 1 are subjected to component analysis.

[0146] 2.1 Polypeptide Identification Results

[0147] 2.1.1 Polypeptide Component Annotation

[0148] The DIA raw data is processed using Spectronaut Pulsar software, and the search sequence file is uniprot-Scyphozoa_6142_2024_01_30.fasta and NCBI-Cnidaria-sequence.fasta.

[0149] After the jellyfish polypeptide compositions SM1, SM2 and SM3 are detected by LC-MS and searched, a total of 24 peptides are identified, as shown in Table 1.

[0150] Table 1 Amino acid sequences of 24 peptides and protein source information

[0151] 2.1.2 Protein annotation results of polypeptide sources in samples

[0152] The polypeptide-derived proteins are analyzed, and SM1 is annotated to 21 polypeptides, SM2 is annotated to 23 polypeptides, and SM3 is annotated to 23 polypeptides. The annotated protein information is shown in Table 2.

[0153] Table 2 Polypeptide information contained in SM1, SM2 and SM3

[0154] 2.1.3 Amino acid composition and molecular weight distribution of peptides

[0155] Most of the peptides in samples SM1, SM2 and SM3 have a length distribution of 8-9 amino acids and 12-15 amino acids, and a molecular weight of 800-2000 Da, as shown in Figures 2 and 3.

[0156] 2.2 Sample Cluster Analysis

[0157] Cluster analysis is a multivariate statistical method for classifying research objects according to certain characteristics. In this embodiment, hierarchical clustering was performed on samples SM1, SM2 and SM3 according to quantitative data, and the hierarchical clustering tree diagram of the samples is shown in Figure 4. The results showed that the polypeptide identification results of SM2 and SM3 were relatively similar, and there was a certain difference with SM1.

[0158] 2.3 Sample correlation analysis

[0159] Sample correlation analysis is a correlation calculation of research objects according to sample detection characteristics, which can reflect the grouping and repeatability of samples. The correlation results of this embodiment showed that the correlation of SM2 and SM3 was higher, with a correlation coefficient r of 0.96, and the correlation with SM1 sample was low, with r≤0.5 (Figure 5).

[0160] 2.4 Differential polypeptide analysis

[0161] Fold change (FC) is an index used to measure the difference between two groups of data, usually expressed as a comparison of the content of a polypeptide in different groups. Its calculation formula is: Fold change = experimental group average / control group average. Fold change can help find the differential polypeptides between two groups.

[0162] In this embodiment, the differential screening condition is: FC≥1.2 or≤0.83.

[0163] The differential polypeptide analysis results showed that for polypeptides with more than 14 amino acids, 6 polypeptides were higher in SM1, and 1 polypeptide was higher in SM2 compared with SM1; 4 polypeptides were higher in SM1, and 4 polypeptides were higher in SM2 compared with SM1; 6 polypeptides were higher in SM3 compared with SM2. The detailed differential polypeptide screening results are shown in Table 3.

[0164] Table 3 Differential polypeptide analysis results of SM1, SM2 and SM3

[0165] Example 3 Inhibition of intestinal cancer cell proliferation experiment

[0166] In this embodiment, human intestinal cancer cells HCT116 were selected as experimental cells, and HCT116 cells were treated with different concentrations of SM1, SM2 and SM3, and CCK-8 technology was used to detect the effect of JP on the activity of HCT116 cells. The results showed that SM1, SM2 and SM3 had inhibitory effect on the activity of HCT116 cells when reaching a certain concentration.

[0167] Example 4 Inhibition of AOM / DSS-induced weight loss in mice

[0168] The CAC model of mice was constructed in this embodiment, and the positive drug PTX (paclitaxel) was used as a control to investigate the effects of SM1, SM2 and SM3 on inhibiting weight loss in mice. When CAC occurs, the intestinal structure is destroyed, the intestinal digestion and absorption function is reduced, and the body weight of the organism will decrease to a certain extent. Therefore, the weight change of the CAC mice was observed in this embodiment, and the change of the weight at the end of the eighth week from the initial weight was calculated. The results showed that the weight of the mice in the negative control group increased by about 3.2 g in 8 weeks, which was about 14.5% of the initial average weight; while the weight gain of the mice in the model group was almost 0 in 8 weeks, which was significantly lower than that of the control group mice, indicating that the body weight of the mice would significantly decrease with the occurrence of CAC; the body weight of the mice in the different concentrations of SM1, SM2 and SM3 treatment groups increased significantly, which was significantly higher than that of the model group, and the effect was better than that of the positive drug PTX.

[0169] Example 5 Inhibition of AOM / DSS-induced colon tumor formation in mice

[0170] After the end of the experiment in Example 4, the colon tissues of the mice were taken out to observe the tumor formation and count the number of tumors. The results showed that a large number of tumors were formed in the distal sigmoid colon, rectum and anus of the mice in the model group; while the number of tumors formed in the colon of the mice in the SM1, SM2 and SM3 treatment groups was significantly reduced, especially after high concentration treatment, the number of tumors was significantly reduced compared with the model group, indicating that the occurrence of colon cancer was significantly inhibited. The diameter of the tumor can indirectly reflect the malignant degree of the tumor, therefore, in addition to the number of tumors, the diameter of the tumor is also one of the important indicators for detecting the occurrence of cancer. In this embodiment, tumors with a diameter greater than 2 mm were considered as large tumors, and the number of such tumors was counted. In the model group, not only the number of tumors increased significantly, but also the size of the tumors increased significantly, and the number of tumors with a diameter greater than 2 mm increased significantly compared with the control group; however, the number of tumors with a diameter greater than 2 mm in the SM1, SM2 and SM3 treatment groups was significantly reduced compared with the model group. The above results showed that SM1, SM2 and SM3 could effectively inhibit the formation of colon tumors in mice induced by AOM / DSS, and the effect was better than that of the positive drug.

[0171] Example 6 Inhibition of AOM / DSS-induced splenomegaly

[0172] In the process of cancer or tumor occurrence, in addition to directly affecting the cancerous organs, other organs of the body will also be affected, especially the immune system of the body such as lymph nodes, spleen and peripheral blood, etc. Related studies have shown that both the innate and adaptive immune systems are involved in the immune response to tumor tissues in tumor patients, affecting the occurrence of tumors. The spleen is the largest secondary lymphoid organ in the human body, with extensive immune functions, and plays an important role in the regulation of the immune system of the body. When the body develops cancer such as colon cancer, it will cause immune disorders and systemic inflammatory reactions, which will cause pathological enlargement of the spleen.

[0173] Based on this, the changes in the spleen of CAC mice after the end of the experiment in Example 4 were also observed. The results showed that the spleen of the model group mice was significantly larger than that of the control group, and the color was white, indicating that the body may have immune disorders and severe inflammatory reactions; while the spleen of the SM1, SM2 and SM3 treatment group mice was significantly smaller than that of the model group, and did not show a white pathological state, especially the high concentration treatment group, the spleen volume was close to that of the control group mice. The spleen of the mice was weighed, and the results were consistent with the observed phenomena. The spleen weight of the model group mice was significantly higher than that of the control group, while the spleen weight of the high concentration SM1, SM2 and SM3 treatment group mice was significantly lower than that of the model group. This result shows that JP can inhibit the pathological enlargement of the spleen of mice caused by AOM / DSS, and the effect is better than that of the positive drug.

[0174] Example 7 Inhibition of AOM / DSS-induced colon structure destruction

[0175] Cancer cells have the characteristics of unlimited proliferation, so in cancer and cancer-adjacent tissues or tissues that are undergoing carcinogenesis, abnormal cell proliferation will usually be exhibited, leading to changes in tissue structure.

[0176] HE staining and histological scoring were performed on the colon tissues of the mice at the end of the experiment of Example 4 to observe the changes in intestinal structure and abnormal hyperplasia. The staining results showed that the colon tissue structure of the control group mice was complete, without hyperplasia, hyperemia, ulceration and other abnormal phenomena, the number and morphology of crypts in the mucosa layer were normal, arranged closely, the muscle layer thickness was normal, and there was no obvious cell infiltration phenomenon. In the colon tissue of the model group mice, the mucosa layer was obviously thickened, with obvious abnormal hyperplasia, among which the crypts were severely deformed, showing long strip shape, and arranged loosely, and the goblet cells were swollen and necrotic; the muscle layer also showed obvious thickening, and there were a large number of inflammatory cell infiltration phenomena in the muscle layer and mucosa layer. The histological scoring also showed significant differences between the model group and the control group, and the results showed that AOM / DSS induced colon structure damage in mice, with obvious abnormal hyperplasia. Compared with the model group, SM1, SM2 and SM3, especially high concentration, could significantly alleviate the damage of intestinal structure and abnormal hyperplasia. In the mucosa layer, the crypt structure and goblet cell morphology were normal, arranged closely; in the muscle layer, no obvious thickening and inflammatory cell infiltration were observed, and the histological score was also significantly lower than that of the model group. This result showed that SM1, SM2 and SM3 could inhibit the colon structure damage caused by AOM / DSS in mice, and the effect was better than that of the positive drug.

[0177] Example 8 Inhibition of AOM / DSS-induced colon cell abnormal proliferation

[0178] Ki67 is a marker of eukaryotic cell proliferation and division. The effect of SM1, SM2 and SM3 on tumor tissue cell proliferation in Example 4 was further detected by specific staining of Ki67 by immunohistochemistry and counting the number of Ki67 positive cells.

[0179] The results of immunohistochemical staining showed that in the colon tissue of the control group mice, only the cells at the bottom of the crypt were Ki67 positive, because the cells at the bottom of the crypt mainly included intestinal stem cells and transient proliferating cells, which had the ability of proliferation and division; and the cells at the top of the crypt were intestinal epithelial cells, which had lost the ability of proliferation due to high differentiation, and were Ki67 negative. In the colon tissue of the model group mice, a large number of Ki67 positive cells appeared at the top of the crypt, indicating that these cells had canceration and presented abnormal proliferation state, thereby causing tumor formation and tissue abnormal hyperplasia. However, in the SM1, SM2 and SM3 treatment groups, the number of Ki67 positive cells at the top of the crypt was significantly lower than that of the model group. These results showed that SM1, SM2 and SM3 could inhibit the abnormal proliferation of colon cells caused by AOM / DSS, and the effect was better than that of the positive drug.

[0180] Example 9 Inhibition of AOM / DSS-induced up-regulation of inflammatory factor expression

[0181] Inflammation plays an important role in the process of tumor formation and is a hallmark in many cancer diseases. In order to detect the inhibitory effect of SM1, SM2 and SM3 on inflammation in the process of CAC, the levels of inflammatory factors in the intestinal tissues of mice at the end of the experiment in Example 4 were detected by qPCR technology. The experimental results showed that the expression levels of inflammatory factors Il-1β, Tnf-α and Ifn-γ were significantly increased in the model group, which had a significant difference with the control group; however, the expression levels of the three inflammatory factors were significantly reduced after the treatment of SM1, SM2 and SM3 compared with the model group. This result shows that JP can inhibit the up-regulation of intestinal inflammatory factors caused by AOM / DSS, and the effect is better than that of the positive drug.

[0182] Example 10 Inhibition of Macrophage Infiltration in Colon Tissue Caused by AOM / DSS

[0183] Chronic intestinal inflammation also causes the infiltration of inflammatory cells, so the infiltration of macrophages in mice at the end of the experiment in Example 4 was detected in this example. The experimental results showed that there was a large number of macrophage infiltration in the mucosal layer and muscle layer in the model group, and the number of F4 / 80 positive cells was significantly increased compared with the control group, indicating that macrophages had infiltrated into the deep layer of intestinal wall and there was a severe inflammatory reaction in the tissue; however, in the SM1, SM2 and SM3 treatment groups, the infiltration of macrophages was significantly inhibited, and there was almost no macrophage in the muscle layer, only a small amount of macrophage infiltration in the superficial mucosal layer, and the number of F4 / 80 positive cells was also significantly reduced compared with the model group, indicating that the intestinal inflammatory reaction was obviously improved after the treatment of SM1, SM2 and SM3. This result shows that JP can inhibit the infiltration of macrophages in the colon caused by AOM / DSS, and the effect is better than that of the positive drug.

[0184] Example 11 Inhibition of Body Weight Loss in Mice Caused by DSS

[0185] Intestinal barrier is involved in the first line of defense of the body's immune system and is one of the components of epithelial barrier, and its dysfunction can lead to the occurrence of intestinal diseases, such as inflammatory bowel disease (IBD). Ulcerative colitis (UC) is one of IBD, which mainly affects the mucosal layer of colon and rectum, thus easily causing intestinal barrier dysfunction and possibly further causing canceration. UC can be caused by multiple factors such as environmental factors, genetic susceptibility, intestinal barrier dysfunction and intestinal flora disorder, and patients with UC can show symptoms such as diarrhea, hematochezia, colon atrophy, weight loss, anemia and abdominal pain.

[0186] Currently, the most important way to study UC is to use dextran sodium sulfate (DSS) induced mouse ulcerative colitis model. At 6 days and 11 days of modeling, the body weight of mice was recorded and counted, and the body weight change of mice from the end of colitis induction to the end of drug administration was calculated. The results showed that at 6 days after the end of DSS feeding, the average weight of the negative control group mice increased by about 0.4g, while the average weight of the model group mice decreased by about 2.4g, which was significantly lighter than the control group; while the DSS-induced weight loss in SM1, SM2 and SM3 gavage treatment groups was inhibited in a concentration-dependent manner, and the effect was better than that of the positive drug 5-ASA (mesalazine). This result shows that JP can significantly inhibit DSS-induced weight loss in mice, and the effect is better than that of the positive drug 5-ASA.

[0187] Example 12 Inhibition of DSS-induced increase in DAI score in mice

[0188] At 11 days of modeling in Example 11, the disease activity index of mice was scored according to three indicators of blood in stool, stool consistency and body weight loss. The results showed that compared with the negative control group, the model group mice produced severe blood in stool and diarrhea, and the DAI score increased significantly; while SM1, SM2 and SM3 treatment could alleviate the blood in stool, diarrhea and body weight loss of mice in a concentration-dependent manner, and reduce the DAI score. This result shows that JP can inhibit the increase of disease activity index in mice induced by DSS, and the effect is better than that of the positive drug 5-ASA.

[0189] Example 13 Inhibition of DSS-induced DAI colon shortening in mice

[0190] The occurrence of UC can cause the death of intestinal cells, thereby causing colon shortening. At 11 days of modeling in Example 11, the colon tissues of mice were taken out and observed and measured. The results showed that the average colon length of the model group mice was significantly shorter than that of the negative control group, and statistical analysis also showed that the colon length of the model group mice was significantly reduced compared with the negative control group; however, the colon shortening of mice was significantly inhibited after SM1, SM2 and SM3 treatment, especially in the high concentration SM1, SM2 and SM3 treatment groups, the average colon length of mice was significantly increased compared with the model group. This result shows that SM1, SM2 and SM3 can inhibit DSS-induced colon shortening in mice, and the effect is better than that of the positive drug.

[0191] Example 14 Inhibition of DSS-induced colon structure destruction in mice

[0192] The intestinal barrier is mainly composed of crypts, tight junctions between intestinal epithelial cells and mucus layers. As a functional unit of the intestine, the normal number and structure of crypts are the premise for maintaining the integrity of the intestinal barrier and the normal function of the intestine.

[0193] After the colon tissue of mice was removed on the 11th day of Example 11, it was made into a Swiss roll for embedding, sectioning and HE staining to observe the changes of intestinal structure and crypt of mice. The experimental results showed that in the intestinal tissue of control mice, the crypt structure of cup was clearly visible, and the crypt was arranged closely and the intestinal structure was complete; but in the intestinal tissue of model mice, a large number of crypts appeared deformation, the structure was obviously destroyed, and the number of crypts was reduced, the arrangement was also loose, and there were a large number of inflammatory cells infiltrated in the intestinal wall, which indicated that the intestinal structure was significantly destroyed; while in the intestinal tissue of mice in SM1, SM2 and SM3 treatment groups, JP significantly inhibited the phenomena of crypt reduction, ulceration, tissue edema and cell infiltration caused by DSS, and the intestinal structure was obviously repaired. At the same time, this embodiment also scored the intestinal tissue of mice after HE staining from three dimensions of reduction of crypt number, damage of mucosal layer and cell infiltration to judge the severity of colitis. The histological score results showed that compared with the negative control group, the histological score of the model group was significantly increased, while the histological scores of SM1, SM2 and SM3 treatment groups were significantly lower than those of the model group. These results showed that SM1, SM2 and SM3 could inhibit the destruction of intestinal structure caused by DSS, and the effect was better than that of the positive drug.

[0194] Example 15 Inhibition of DSS-induced colon barrier damage in mice

[0195] The changes of intestinal barrier also include the changes of the expression amount of some factors constituting the intestinal barrier, which include tight junction proteins (ZO-1, CLDN1) between intestinal epithelial cells and mucin (MUC2) in intestinal mucus layer.

[0196] After the intestinal tissue was removed on the 11th day of Example 11, the proteins and RNAs therein were extracted, and the expression amount of the related proteins constituting the intestinal barrier was detected by Western Blot technology. The WB results showed that the expression amount of Zo-1, Muc2 and Cldn1 in the model group was significantly down-regulated compared with the control group, while SM1, SM2 and SM3 could increase the expression amount of the three barrier proteins in a concentration-dependent manner. At the same time, the expression amount changes of the mRNA of the three factors were detected at the transcription level by Q-PCR. The results at the mRNA level were consistent with those at the protein level, that is, compared with the model group, the mRNA of the three barrier proteins in SM1, SM2 and SM3 treatment groups was significantly up-regulated, and showed a concentration-dependent increase.

[0197] In addition, matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation and remodeling, and play a key role in tissue remodeling and repair. However, in many inflammatory diseases, MMPs are expressed in an unregulated manner, causing the destruction of tight junctions. For example, MMP9 has been reported to be highly expressed and to destroy the intestinal barrier in the development of IBD. Therefore, the mRNA expression level of Mmp9 in the colon of mice was also detected by qPCR in this embodiment. The results showed that the expression level of Mmp-9 in the model group was significantly higher than that in the negative control group, while the treatment of SM1, SM2 and SM3 inhibited this trend, indirectly reflecting the role of SM1, SM2 and SM3 in repairing the intestinal barrier.

[0198] The above results demonstrate that SM1, SM2 and SM3 can inhibit the down-regulation of the expression of colon barrier proteins Zo-1, Muc2 and Cldn1 and the abnormal increase in the expression of Mmp9 caused by DSS, indicating that SM1, SM2 and SM3 can repair the damaged intestinal barrier in UC.

[0199] Example 16 Inhibition of the up-regulation of inflammatory factors in the colon of mice caused by DSS

[0200] The destruction of the intestinal barrier allows microorganisms, pathogens and their metabolites such as lipopolysaccharide (LPS), a major component of bacterial cell walls, to enter the intestinal tissue or flow to the whole body through the blood vessels, causing inflammation and immune response in the intestine or the body. Chronic intestinal inflammation is characterized by excessive secretion of inflammatory factors and infiltration of immune cells, which further exacerbate the destruction of the intestinal barrier, leading to worsening of colitis and even the occurrence of intestinal cancer.

[0201] Several inflammatory factors that were abnormally highly expressed in ulcerative colitis in Example 11 were detected by qPCR. The results showed that the expression levels of Il-1β, Tnf-α and Ifn-γ were significantly increased in the model group, suggesting that DSS can cause severe inflammation in the colon of mice. However, after treatment with SM1, SM2 and SM3 and 5-ASA, the expression levels of these three inflammatory factors were significantly lower than those in the model group. This result indicates that SM1, SM2 and SM3 can significantly inhibit the up-regulation of the expression of inflammatory factors in the colon of mice caused by DSS, and the effect is better than that of the positive drug.

[0202] Example 17 Inhibition of the infiltration of macrophages in the colon of mice caused by DSS

[0203] In colitis, inflammatory immune cells such as macrophages migrate from the peripheral blood or peripheral lymph nodes under the recruitment of chemotactic factors, so detecting the number and infiltration of macrophages in the colitis tissue can also reflect the severity of inflammation.

[0204] F4 / 80 is a membrane protein, which is one of the markers of mature macrophages in mice. Immunofluorescence staining was performed on the sections of the colon of the mice in Example 11, and F4 / 80 was specifically labeled. The results showed that in the intestines of mice without DSS treatment, only a small amount of F4 / 80 + cells was observed. In the intestines of mice with DSS-induced enteritis, the submucosa and muscle layer of the intestinal wall of mice were significantly thickened, and the number of F4 / 80 + cells increased significantly, indicating that the number of macrophages increased and the inflammatory response was severe; at the same time, the inventors found that F4 / 80 + cells were distributed in the mucosa, submucosa and muscle layer, especially in the submucosa and muscle layer, and the number was very large. This result indicated that with the severe damage of the intestinal barrier, a large number of microorganisms and their metabolites entered the deep layer of the intestinal wall, resulting in a large number of macrophages infiltrating into this place. However, treatment with different doses of SM1, SM2 and SM3 could reduce the number of F4 / 80 + cells in the intestinal wall. These results indicated that SM1, SM2 and SM3 not only inhibited the inflammatory response of the colon tissue in intestinal inflammation, but also repaired the damaged intestinal barrier.

[0205] In order to more intuitively observe the inhibition of the increase and infiltration of macrophages by SM1, SM2 and SM3, the results of immunofluorescence staining were quantitatively counted for the number of F4 / 80 + cells. The quantitative counting results showed that the number of F4 / 80 + cells in the model group increased significantly compared with the negative control group, while the number of F4 / 80 + cells in the SM1, SM2 and SM3 treatment groups decreased significantly compared with the model group. This result further confirmed that SM1, SM2 and SM3 could inhibit the increase and infiltration of macrophages caused by DSS.

[0206] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, and not to limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. A jellyfish polypeptide, wherein the jellyfish polypeptide comprises a polypeptide fragment obtained by separating a jellyfish extract by liquid chromatography-mass spectrometry with an amino acid residue number selected from 8 to 27.

2. The jellyfish polypeptide according to claim 1, wherein, The method for preparing the jellyfish extract includes enzymatic hydrolysis of a dehydrated and desalted intact jellyfish or a specific part of a jellyfish, and the resulting enzymatic hydrolysate is filtered to obtain the jellyfish extract. The enzymes used in the enzymatic hydrolysis include alkaline protease and pepsin. Optionally, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5 to 10:1, and can be 6:

1. Optionally, the temperature of the enzymatic hydrolysis step is 30–70°C, preferably 50°C; Optionally, the pH of the enzymatic hydrolysis step is 5 to 9, preferably 6 to 8.

5.

3. The jellyfish polypeptide according to claim 1, wherein, The jellyfish is selected from the order Semaeostomeae, and may be at least one of the following families: Cyaneidae, Pelagiidae, and Ulmaridae.

4. The jellyfish polypeptide according to claim 1, wherein, The jellyfish is selected from at least one of the following: Cyanea capillata, Cyanea ferruginea, Cyanea nozakii, Cyanea purpurea, Chrysaora helvola, Pelagia noctiluca, Sanderia malayensis, or Aurelia coerulea.

5. The jellyfish polypeptide according to claim 1, wherein, The jellyfish is Aurelia coerulea, or the jellyfish includes Aurelia aurita and at least one other jellyfish selected from the order Semaeostomeae.

6. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide is derived at least from the umbrella part of the jellyfish.

7. The jellyfish polypeptide according to claim 1, wherein, In the liquid chromatography-mass spectrometry (LC-MS) coupling, the mobile phase of the liquid chromatography includes an aqueous phase and an organic phase. The aqueous phase includes an aqueous phase containing formic acid, and the organic phase includes acetonitrile containing formic acid; Optionally, the formic acid content in the aqueous phase and the organic phase is 0.05% to 0.2%, and optionally 0.1%; Optionally, the gradient elution conditions for the liquid chromatography include: 0–45 min, 2%–22% (v / v) organic phase; 45–50 min, 22%–37% (v / v) organic phase; 50–55 min, 37%–80% (v / v) organic phase; 55–60 min, 80% (v / v) organic phase.

8. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 8 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID No: 1 to 3.

9. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 9 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID No:4 to 7.

10. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 10 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No:

8.

11. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 11 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No:

9.

12. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 12 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID No: 10 to 13.

13. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 13 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No:14 or 15.

14. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 14 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No:16 or 17.

15. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 15 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in any one of SEQ ID No: 18 to 20.

16. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 16 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No:

21.

17. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 18 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No:22 or 23.

18. The jellyfish polypeptide according to claim 1, wherein, The jellyfish polypeptide has 27 amino acid residues. Optionally, the amino acid sequence of the jellyfish polypeptide has 80% to 100% identity with the amino acid sequence shown in SEQ ID No:

24.

19. A jellyfish polypeptide composition comprising two or more different jellyfish polypeptides, wherein the jellyfish polypeptides are selected from the jellyfish polypeptides described in any one of claims 1 to 18.

20. The jellyfish polypeptide composition according to claim 19, wherein, The jellyfish polypeptides include a total of 21 polypeptides as shown in SEQ ID No:1~9, SEQ ID No:11~14, SEQ ID No:16~19 and SEQ ID No:21~24.

21. The jellyfish polypeptide composition according to claim 19, wherein, The jellyfish polypeptides include a total of 23 polypeptides as shown in SEQ ID No:1~14 and SEQ ID No:16~24.

22. The jellyfish polypeptide composition according to claim 19, wherein, The jellyfish polypeptides include a total of 23 polypeptides as shown in SEQ ID No:2 to 24.

23. Use of the jellyfish polypeptide of any one of claims 1 to 18 or the jellyfish polypeptide composition of any one of claims 19 to 22 in the preparation of a product for treating or preventing tumors; Optionally, the tumor includes a solid tumor.

24. The use according to claim 23, wherein, The tumors include advanced or terminal colorectal cancer, lung cancer, or prostate cancer.

25. The use according to claim 23 or 24, wherein, The dosage forms of the products used to treat or prevent tumors include oral formulations.

26. Use of the jellyfish polypeptide of any one of claims 1 to 18 or the jellyfish polypeptide composition of any one of claims 19 to 22 in the preparation of an immunomodulator.

27. A method for preparing jellyfish polypeptides, comprising enzymatic hydrolysis of a dehydrated and desalted intact jellyfish or a specific part of a jellyfish, filtering the resulting enzymatic hydrolysate to obtain a jellyfish extract, and separating the jellyfish extract by liquid chromatography-mass spectrometry to obtain a polypeptide fragment with 8 to 27 amino acid residues. The enzymes used in the enzymatic hydrolysis include alkaline protease and pepsin.

28. The preparation method according to claim 27, wherein, The liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, and can be selected as 6:1; The temperature of the enzymatic hydrolysis step is 30–70°C, and can be selected as 50°C; The pH of the enzymatic hydrolysis step is 5 to 9, and can be selected as 6 to 8.

5.

29. The preparation method according to claim 27 or 28, wherein, In the liquid chromatography-mass spectrometry (LC-MS) coupling, the mobile phase of the liquid chromatography includes an aqueous phase and... The organic phase, wherein the aqueous phase comprises an aqueous phase containing formic acid, and the organic phase comprises acetonitrile containing formic acid; Optionally, the formic acid content in the aqueous phase and the organic phase is 0.05% to 0.2%, and optionally 0.1%; Optionally, the gradient elution conditions for the liquid chromatography include: 0–45 min, 2%–22% (v / v) organic phase; 45–50 min, 22%–37% (v / v) organic phase; 50–55 min, 37%–80% (v / v) organic phase; 55–60 min, 80% (v / v) organic phase.

30. A medicament for treating or preventing tumors, comprising a jellyfish polypeptide according to any one of claims 1 to 18 or a jellyfish polypeptide composition according to any one of claims 19 to 22, wherein optionally, the tumor comprises a solid tumor; optionally, the dosage form of the medicament for treating or preventing tumors comprises an oral formulation.

31. An immunomodulatory agent comprising the jellyfish polypeptide of any one of claims 1 to 18 or the jellyfish polypeptide composition of any one of claims 19 to 22; optionally, the immunomodulatory agent is in the form of an oral formulation.

32. A method for preparing the drug of claim 30 or the immunomodulator of claim 31, comprising concentrating the jellyfish polypeptide of any one of claims 1 to 18 or the jellyfish polypeptide composition of any one of claims 19 to 22 to obtain a concentrated solution of the jellyfish polypeptide or its composition, and then proceeding to a molding step to obtain an oral formulation drug or immunomodulator.

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