Use of aurelia extract in cancer treatment

Abstract

The present disclosure relates to the field of pharmaceuticals, and in particular to the use of an Aurelia extract in cancer treatment. The Aurelia extract provided by the present disclosure, or the Aurelia extract obtained by a preparation method disclosed in the present disclosure demonstrates significant inhibitory effects on various tumors including intestinal cancer and lung cancer. The inhibitory effects on tumors comprise killing tumor cells, alleviating inflammation caused by tumors, and reducing tumor-induced organ lesions. Therefore, the Aurelia extract disclosed in the present disclosure can be used for preparing products for treating and preventing tumors, including but not limited to various antitumor drugs with different administration routes.

Description

Use of jellyfish extract in cancer treatment

[0001] Priority claim

[0002] The present disclosure claims priority to Chinese patent application No. 2024104429804, filed on April 12, 2024. The present disclosure incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0003] The present disclosure relates to the field of pharmacy, in particular to the use of jellyfish extract in cancer treatment. BACKGROUND

[0004] Jellyfish have important values in cosmetics, food and scientific research. Jellyfish gel is rich in collagen and elastin, which are two of the most important components in the skin, and can make the skin more firm and elastic, reduce the appearance of wrinkles and fine lines. Jellyfish is also a nutritious food, which is low in fat and calories, 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 values 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 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 extract is a substance from jellyfish, which has various bioactive components, including proteins, gelatin and mucin (glycosylated proteins), etc., which endow jellyfish extract with potential application values. For example, in whitening, relieving fatigue and enhancing immunity.

[0007] However, the application of jellyfish extract in cancer treatment still needs further research and development. SUMMARY

[0008] The present disclosure aims to provide a jellyfish extract, a preparation method of jellyfish extract, and its use in treating or preventing tumors.

[0009] In a first aspect, the present disclosure provides the use of jellyfish extract in the preparation of a product for treating or preventing tumors, optionally, the tumors include solid tumors.

[0010] In an alternative embodiment, the jellyfish is selected from the Order Semaeostomeae. Alternatively, the jellyfish is at least one selected from the Family Cyaneidae, the Family Pelagiidae, and the Family Ulmaridae.

[0011] The Family Cyaneidae includes Cyanea capillata, Cyanea ferruginea, Cyanea nozakii, and Cyanea purpurea. The Family Pelagiidae includes Chrysaora helvola, Pelagia noctiluca, and Sanderia malayensis. The Family Ulmaridae includes Aurelia coerulea.

[0012] In an alternative embodiment, the jellyfish is Aurelia coerulea, or alternatively, the jellyfish includes Aurelia aurita and at least one other jellyfish selected from the Order Semaeostomeae.

[0013] In an alternative embodiment, the jellyfish extract is extracted from a whole jellyfish or a certain or certain parts, organs or tissues of the jellyfish, but at least includes the umbrella of the jellyfish.

[0014] In an alternative embodiment, the method for preparing the jellyfish extract includes enzymatic hydrolysis of the dehydrated and desalted whole jellyfish or certain parts of the jellyfish, and the obtained enzymatic hydrolysate is filtered to obtain the jellyfish extract. The enzymes used in the enzymatic hydrolysis include alkaline protease and pepsin.

[0015] In an alternative embodiment, the liquid-solid volume mass ratio in the enzymatic hydrolysis step is 5-10:1, and is alternatively 6:1. Alternatively, the temperature of the enzymatic hydrolysis step is 30-70°C, and is preferably 50°C. Alternatively, the pH of the enzymatic hydrolysis step is 5-9, and is preferably 6-8.5.

[0016] In an alternative embodiment, the jellyfish is clean and free of attachments. Alternatively, after the jellyfish extract is obtained by filtration, a jellyfish extract forming step is further included.

[0017] In an alternative embodiment, the dosage form of the jellyfish extract includes a powder, granules or a liquid.

[0018] In an alternative embodiment, the dosage form of the tumor treatment or prevention product includes an oral preparation.

[0019] In an alternative embodiment, the tumor includes advanced or terminal intestinal cancer, lung cancer or prostate cancer.

[0020] The second aspect of the present disclosure provides a method for preparing a jellyfish extract, which includes subjecting a dehydrated and desalted whole jellyfish or a specific part of the jellyfish to enzymatic hydrolysis, and obtaining the jellyfish extract by filtering the obtained enzymatic hydrolysate; the enzymes used in the enzymatic hydrolysis include alkaline protease and pepsin; the jellyfish extract can be used for treating or preventing tumors; optionally, the tumor includes a solid tumor.

[0021] In an alternative embodiment, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, and optionally 6:1; the temperature of the enzymatic hydrolysis step is 30-70°C, and optionally 50°C; the pH of the enzymatic hydrolysis step is 5-9, and optionally 6-8.5.

[0022] In an alternative embodiment, the jellyfish is clean and free of attachments.

[0023] In an alternative embodiment, after the filtering to obtain the jellyfish extract, a jellyfish extract forming step is further included.

[0024] In an alternative embodiment, the dosage form of the jellyfish extract includes a powder, granules or a liquid.

[0025] The third aspect of the present disclosure provides a jellyfish extract for treating or preventing tumors, which is obtained by the preparation method of the second aspect.

[0026] The fourth aspect of the present disclosure provides a medicine for treating or preventing tumors, which contains a jellyfish extract; the preparation method of the jellyfish extract includes subjecting a dehydrated and desalted whole jellyfish or a specific part of the jellyfish to enzymatic hydrolysis, and obtaining the jellyfish extract by filtering the obtained enzymatic hydrolysate; the enzymes used in the enzymatic hydrolysis include alkaline protease and pepsin; optionally, the tumor includes a solid tumor.

[0027] In an alternative embodiment, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, and optionally 6:1; the temperature of the enzymatic hydrolysis step is 30-70°C, and optionally 50°C; the pH of the enzymatic hydrolysis step is 5-9, and optionally 6-8.5.

[0028] In an alternative embodiment, the dosage form of the medicine includes an oral preparation.

[0029] The fifth aspect of the present disclosure provides a method for preparing the medicine of the fourth aspect, comprising concentrating the jellyfish extract obtained by the method of the second aspect of the present disclosure to obtain a concentrated jellyfish extract, and then performing a molding step to obtain the oral medicine.

[0030] The sixth aspect of the present disclosure provides a method for treating or preventing a tumor, comprising administering an effective amount of the medicine of the fourth aspect of the present disclosure to an individual to be intervened; optionally, the tumor comprises a solid tumor.

[0031] The seventh aspect of the present disclosure provides a use of the jellyfish extract in the preparation of a medicine for treating a disease associated with overexpression of gene A or underexpression of gene B, wherein the gene A is selected from one or more of the following: CYP4F11, FTL, HMOX1, GCLC, TXNRD1, GCNT3, or CYP4F3, and the gene B is selected from one or more of the following: CEMIP, NR4A3, ISG15, PCDH7, EGR1, ASPM, DUSP2, PRR11, EGR2, SACS, PTPN14, SYNE2, IFIT1, CYP4F11, or FTL.

[0032] In an optional embodiment, the method for preparing the jellyfish extract comprises enzymatic hydrolysis of the whole jellyfish or a specific part of the jellyfish after dehydration and desalination, and the obtained enzymatic hydrolysis solution is filtered to obtain the jellyfish extract; the enzymes used in the enzymatic hydrolysis include alkaline protease and pepsin.

[0033] In an optional embodiment, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, and optionally 6:1.

[0034] The temperature in the enzymatic hydrolysis step is 30-70°C, and optionally 50°C.

[0035] The pH in the enzymatic hydrolysis step is 5-9, and optionally 6-8.5.

[0036] In an optional embodiment, the disease comprises a tumor, which can be a solid tumor, and further can be intestinal cancer, lung cancer, or prostate cancer.

[0037] It has been verified that the jellyfish extract provided by the present disclosure, or the jellyfish extract obtained by the preparation method provided by the present disclosure, has a significant inhibitory effect on various tumors including intestinal cancer and lung cancer, and the inhibitory effect on tumors includes killing tumor cells, reducing inflammation and organ lesions caused by tumors. Therefore, the jellyfish extract provided by the present disclosure can be used to prepare tumor treatment or tumor prevention products, including but not limited to various anti-tumor drugs with different administration routes. BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 is a preparation flow of the jellyfish extract in Example 1.

[0039] Figure 2 is a surface structure diagram of the solidified object obtained by light-curing 3D printing in Example 2;

[0040] Figure 3 is a relative cell viability change curve of different drugs in the LC-S T21 experimental group in Example 2;

[0041] Figure 4 is a relative cell viability change curve of different drugs in the LC-S T15 experimental group in Example 2;

[0042] Figure 5 is a flow cytometry histogram of PBMC-Hycells immune regulation detection in Example 3;

[0043] Figure 6 is a flow cytometry histogram of PBMC-HB immune regulation detection in Example 3;

[0044] Figure 7 is a flow cytometry histogram of LC-PE T13 immune regulation detection in Example 3;

[0045] Figure 8 is a flow cytometry histogram of LC-PE T14 immune regulation detection in Example 3;

[0046] Figure 9 is a comparison of the inhibitory effect of jellyfish extract on T cells and lung cancer pleural effusion;

[0047] Figure 10 is the relief effect of jellyfish extract on DSS-induced weight loss in mice;

[0048] Figure 11 is the relief effect of jellyfish extract on DSS-induced colon shortening in mice;

[0049] Figure 12 is the relief effect of jellyfish extract on DSS-induced spleen enlargement in mice;

[0050] Figure 13 is the repair effect of jellyfish extract on DSS-induced colon structure damage in mice;

[0051] Figure 14 is the effect of jellyfish extract on the expression of rectal verification factors in IBD mice;

[0052] Figure 15 is the effect of jellyfish extract on the activity of intestinal cancer cells HCT116;

[0053] Figure 16 is the effect of jellyfish extract on the migration of intestinal cancer cells HCT116;

[0054] Figure 17 is a visual diagram of the reduction of tumor number in CRC mice by jellyfish extract;

[0055] Figure 18 is a statistical result of the reduction of tumor number and the reduction of tumor malignancy in CRC mice by jellyfish extract;

[0056] Figure 19 is the inhibitory effect of jellyfish extract on weight loss in CRC mice;

[0057] Figure 20 is the effect of jellyfish extract on the reduction of the spleen weight of CRC mice;

[0058] Figure 21 is the relative cell viability curve of different drugs in the CRC230511T1 experimental group;

[0059] Figure 22 is the relative cell viability curve of different drugs in the CRC230601T1 experimental group;

[0060] Figure 23 is the relative cell viability curve of different drugs in the CRC221123T3 experimental group;

[0061] Figure 24 is the relative cell viability curve of different drugs in the intestinal cancer cell HCT116 experimental group;

[0062] Figure 25 is the comparison of the number of significantly up-regulated and down-regulated expression genes of jellyfish extract;

[0063] Figure 26 is the classification chart of significantly up-regulated and down-regulated expression genes of jellyfish extract;

[0064] Figure 27 is the expression genes regulated by jellyfish extract significantly related to colon cancer;

[0065] Figure 28 is the GO signal pathway significantly enriched by jellyfish extract;

[0066] Figure 29 is the DO signal pathway significantly enriched by jellyfish extract;

[0067] Figure 30 is the Reactome signal pathway significantly enriched by jellyfish extract;

[0068] Figure 31 is the KEGG signal pathway significantly enriched by jellyfish extract;

[0069] Figure 32 is the effect of jellyfish extract on the cell cycle of intestinal cancer cell HCT116. DETAILED DESCRIPTION

[0070] According to the above content of the present disclosure, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present disclosure, according to the ordinary technical knowledge and common practice in the art.

[0071] I. DEFINITIONS

[0072] Unless otherwise clearly indicated, throughout the specification and claims, the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element or group of elements but not the exclusion of any other element or group of elements.

[0073] 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 with accession number CB0961824 (JELLYFISH EXTRACT).

[0074] 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.

[0075] 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 tumors. By early detection of tumors, early diagnosis, and further administration of an appropriate dose of jellyfish extract, changes in the preclinical or early clinical stage of tumors can be prevented, so that the disease can be detected and treated in the early stage, avoiding or reducing complications.

[0076] As described herein, the term "Order Semaeostomeae" is the most common order in the subphylum Cnidaria, class Scyphozoa. The umbrella is bowl-shaped, butterfly-shaped, with 8 to many notches in the umbrella margin, and tentacle pockets in the notches. The number, distribution, and shape of tentacles vary with species. There are oral arms with ciliated grooves, complex radial tubes, and gastric pouches extending out of the umbrella. 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, and subsequent new discoveries, or those already 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 extract varies slightly among different orders of jellyfish, and there are slight differences in anti-tumor effects. At the same time, some jellyfish can secrete jellyfish toxins, and the jellyfish extract obtained from such jellyfish needs to be investigated for safety and strictly controlled for additive dosage when used for the preparation of anti-tumor drugs. Among them, Aurelia coerulea is a non-toxic jellyfish, and the jellyfish extract of this type of jellyfish has a clear advantage in safety. As for toxic jellyfish, the tentacles or tentacles can be stripped off, and the non-toxic umbrella part can be retained for the preparation of the extract.

[0077] As described herein, the term "alkaline protease", also known as serine protease, is a high alkaline (pH 9-10) proteinase with serine as the active center, which can hydrolyze the peptide chain of protein molecules to form polypeptides or amino acids, and has strong ability to decompose proteins. It is widely present in bacteria such as Bacillus licheniformis, B. amyloliquefaciens, B. pumilus, B. alcalophilus, Streptomyces griseus, S. fradiae and some Fusarium spp., actinomycetes and fungi. Its activity is in the range of pH 7-11. In addition to hydrolyzing peptide bonds, the enzyme also has the ability to hydrolyze ester bonds and amide bonds, as well as the ability to transfer esters and peptides.

[0078] As described herein, the term "flavor protease" is obtained by fermentation of Aspergillus oryzae, advanced extraction process microfiltration, ultrafiltration, vacuum freeze-drying technology, and strict control of the number of microorganisms, which can reach food grade standards. Flavor protease can make the hydrolysate form a unique flavor and reduce the bitterness caused by hydrolysis.

[0079] As described herein, the term "oral preparation" refers to a dosage form that enters the gastrointestinal tract after oral administration and exerts a local or systemic effect by absorption. Drugs that are easily destroyed by acids or enzymes in the gastrointestinal tract generally cannot use such simple dosage forms.

[0080] As described herein, the term "drug" means a composition comprising the jellyfish extract described in the present disclosure, and at least one pharmaceutically acceptable ingredient selected from the group consisting of carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, flavorings, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesion agents, stabilizers, suspending agents, etc. depending on the nature of the administration mode and the dosage form.

[0081] II. DETAILED DESCRIPTION

[0082] In a first aspect of the present disclosure, the use of a jellyfish extract in the preparation of a product for treating or preventing tumors is provided, optionally, the tumors include solid tumors.

[0083] In optional embodiments, the jellyfish is selected from the Order Semaeostomeae. Optionally, at least one of Family Cyaneidae, Family Pelagiidae, Family Ulmaridae.

[0084] wherein 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.

[0085] In optional embodiments, the jellyfish is Aurelia coerulea, or the jellyfish includes Aurelia aurita and at least one other jellyfish selected from the Order Semaeostomeae. Exemplary combinations include, but are not limited to, (1) only Aurelia coerulea, (2) Aurelia coerulea and Sanderia malayensis, (3) Aurelia coerulea and Pelagia noctiluca, (4) Aurelia coerulea and Chrysaora helvola, (5) Aurelia coerulea and Cyanea purpurea, (5) Aurelia coerulea and Cyanea nozakii, (6) Aurelia coerulea and Cyanea ferruginea, (7) Aurelia coerulea and Cyanea capillata, (8) Aurelia coerulea, one Family Cyaneidae, and one Family Pelagiidae, (9) Aurelia coerulea, one Family Cyaneidae, and two Family Pelagiidae, (10) Aurelia coerulea, two Family Cyaneidae, and one Family Pelagiidae, and other combinations including Aurelia coerulea and at least one other jellyfish selected from the Order Semaeostomeae.

[0086] In an alternative embodiment, the jellyfish extract is extracted from a whole jellyfish or a certain or certain parts, organs or tissues of a jellyfish, but at least including the umbrella of a jellyfish. The jellyfish extraction site exemplarily includes, but is not limited to, a whole Aurelia sp., an umbrella of Aurelia sp., an umbrella of Aurelia sp. and other at least one jellyfish selected from the Order Semaeostomeae, a whole Aurelia sp. and an umbrella of Aurelia sp. and other at least one jellyfish selected from the Order Semaeostomeae.

[0087] 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 solution is filtered to obtain the jellyfish extract. It can be understood that when the extract is extracted from the specific parts of the jellyfish, the specific parts of the jellyfish can be separated first in the preparation method, and then dehydrated and desalted, or the whole jellyfish can be dehydrated and desalted first, and then the specific parts of the jellyfish are separated. The purpose of the filtration in the preparation method is to remove solid impurities.

[0088] In an alternative embodiment, two or more enzymes are used for the enzymatic hydrolysis; preferably, the enzymes include alkaline protease and pepsin, and flavor protease can also be added without affecting the activity. It should be noted that the optional enzymatic hydrolysis sequence in the enzymatic hydrolysis step includes (1) alkaline protease hydrolysis- pepsin hydrolysis, or (2) pepsin hydrolysis-alkaline protease hydrolysis, or additional flavor protease hydrolysis step after the above enzymatic hydrolysis sequence (1) or (2).

[0089] In an alternative embodiment, the liquid-solid volume mass ratio in the enzymatic hydrolysis step is 5-10:1, which can be 6:1; optionally, the temperature of the enzymatic hydrolysis step is 30-70℃, such as 30℃, 35℃, 40℃, 45℃ or 50℃, which can be 50℃; optionally, the pH of the enzymatic hydrolysis step is 5-9, which can be 6-8.5, such as 6.5, 7, 7.5, 8 or 8.5.

[0090] The parameter combination of the optional enzymatic hydrolysis step can be: alkaline protease hydrolysis-pepsin hydrolysis, liquid-solid volume mass ratio: 6:1, enzymatic hydrolysis temperature: constant temperature 50℃, enzymatic hydrolysis pH: alkaline protease hydrolysis-8.5, pepsin hydrolysis-6.5.

[0091] In an alternative embodiment, the jellyfish is clean and free of attachments to avoid introducing impurities or degradation products of other organisms.

[0092] In an optional embodiment, after the jellyfish extract is filtered, a jellyfish extract forming step is further included. The forming step and method can be selected and optimized according to the actual requirements of subsequent preparation of the anti-tumor product, the dosage form or administration mode of the anti-tumor product, and the like.

[0093] In an optional embodiment, the dosage form of the jellyfish extract includes a powder, a granule, or a liquid. It should be noted that, in the embodiments of the present disclosure, cell experiments and animal experiments can prove that the jellyfish extract provided by the present disclosure can achieve good anti-tumor effect after injection or oral administration, and therefore, the dosage form of the jellyfish extract that can be administered orally or by injection should belong to the protection scope of the present disclosure. In addition, according to the description of the present disclosure, it can be explicitly inferred that other effective dosage forms should also belong to the protection scope of the present disclosure.

[0094] In an optional embodiment, the dosage form of the anti-tumor product includes an oral preparation. It should be noted that, when the jellyfish extract provided by the present disclosure is applied to the development and preparation of the anti-tumor product, it is easy for a person skilled in the art to develop a more suitable dosage form for administration for different cancer types.

[0095] In an optional embodiment, the tumor includes intestinal cancer, lung cancer, or prostate cancer in an advanced 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. The lung cancer includes, but is not limited to, small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, or large cell carcinoma. The prostate cancer includes adenocarcinoma, ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, or adenosquamous carcinoma.

[0096] The second aspect of the present disclosure provides a preparation method of a jellyfish extract for treating or preventing a tumor, including subjecting a dehydrated and desalted whole jellyfish or a specific part of the jellyfish to enzymatic hydrolysis, and obtaining the jellyfish extract by filtering the obtained enzymatic hydrolysate. The enzymes selected for the enzymatic hydrolysis include alkaline protease and pepsin. The jellyfish extract can be used for treating or preventing a tumor. Optionally, the tumor includes a solid tumor.

[0097] In an optional embodiment, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, and can be 6:1. The temperature of the enzymatic hydrolysis step is 30-70°C, such as 30°C, 35°C, 40°C, 45°C, or 50°C, and can be 50°C. Optionally, the pH of the enzymatic hydrolysis step is 5-9, and can be 6-8.5, such as 6.5, 7, 7.5, 8, or 8.5.

[0098] In an optional embodiment, the jellyfish is clean and free of attachments.

[0099] In an alternative embodiment, the jellyfish extract is obtained by the filtration step.

[0100] In an alternative embodiment, the dosage form of the jellyfish extract includes a powder, a granule or a liquid.

[0101] In an alternative embodiment, the tumor includes an advanced or terminal stage of intestinal cancer, lung cancer or prostate cancer.

[0102] A third aspect of the present disclosure provides a jellyfish extract for treating or preventing a tumor, which is obtained by the preparation method of the second aspect.

[0103] A fourth aspect of the present disclosure provides a medicine for treating or preventing a tumor, which contains a jellyfish extract, wherein the preparation method of the jellyfish extract includes enzymatic hydrolysis of a whole jellyfish or a specific part of a jellyfish after dehydration and desalination, and the obtained enzymatic hydrolysate is filtered to obtain the jellyfish extract; and the enzymes used in the enzymatic hydrolysis include alkaline protease and pepsin; and optionally, the tumor includes a solid tumor.

[0104] In an alternative embodiment, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, and optionally 6:1; the temperature of the enzymatic hydrolysis step is 30-70°C, such as 30°C, 35°C, 40°C, 45°C or 50°C, and optionally 50°C; and the pH of the enzymatic hydrolysis step is 5-9, and optionally 6-8.5, such as 6.5, 7, 7.5, 8 or 8.5.

[0105] In an alternative embodiment, the dosage form of the medicine includes an oral preparation.

[0106] In an alternative embodiment, the medicine is used for treating or preventing lung cancer, and the effective dosage of the jellyfish extract in the medicine is 10-100 mg / ml, and optionally 10-40 mg / ml, or 40-70 mg / ml, including but not limited to 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml or 100 mg / ml.

[0107] In an alternative embodiment, the medicament is used for treating or preventing intestinal cancer, and the effective dose of jellyfish extract in the medicament is 10 mg / ml to 100 mg / ml, alternatively 10 mg / ml to 40 mg / ml, or 40 mg / ml to 70 mg / ml, including but not limited to 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml or 100 mg / ml.

[0108] The fifth aspect of the present disclosure provides a method for preparing the medicament of the fourth aspect, comprising concentrating the jellyfish extract obtained by the method of the second aspect of the present disclosure to obtain a concentrated jellyfish extract, and then performing a shaping step to obtain an oral preparation, an injection preparation, a respiratory tract administration preparation, a mucosal administration preparation, a skin administration preparation or a cavity administration preparation.

[0109] The sixth aspect of the present disclosure provides a method for treating or preventing tumors, comprising administering an effective dose of the medicament of the fourth aspect of the present disclosure to an individual to be intervened; alternatively, the tumor comprises a solid tumor.

[0110] The seventh aspect of the present disclosure provides use of a jellyfish extract in the preparation of a medicament for treating a disease associated with overexpression of gene A or underexpression of gene B, wherein the gene A is selected from one or more of the group consisting of CYP4F11, FTL, HMOX1, GCLC, TXNRD1, GCNT3 or CYP4F3, and the gene B is selected from one or more of the group consisting of CEMIP, NR4A3, ISG15, PCDH7, EGR1, ASPM, DUSP2, PRR11, EGR2, SACS, PTPN14, SYNE2, IFIT1, CYP4F11 or FTL.

[0111] The gene A includes a combination of genes, which can be selected from two, three, four, five, six or seven genes of CYP4F11, FTL, HMOX1, GCLC, TXNRD1, GCNT3 or CYP4F3, for example, CYP4F11 and FTL; CYP4F11, FTL and HMOX1; CYP4F11, FTL, HMOX1 and GCLC; CYP4F11, FTL, HMOX1, GCLC and TXNRD1; CYP4F11, FTL, HMOX1, GCLC, TXNRD1 and GCNT3; CYP4F11, FTL, HMOX1, GCLC, TXNRD1, GCNT3 and CYP4F3; or other combinations.

[0112] The gene B includes a combination of genes, which can be selected from two to fifteen genes of CEMIP, NR4A3, ISG15, PCDH7, EGR1, ASPM, DUSP2, PRR11, EGR2, SACS, PTPN14, SYNE2, IFIT1, CYP4F11 or FTL, for example, CEMIP and NR4A3; CEMIP, NR4A3 and ISG15; CEMIP, NR4A3, ISG15 and PCDH7; CEMIP, NR4A3, ISG15, PCDH7 and EGR1; CEMIP, NR4A3, ISG15, PCDH7, EGR1 and ASPM; CEMIP, NR4A3, ISG15, PCDH7, EGR1, ASPM and DUSP2, etc.

[0113] In an alternative embodiment, the jellyfish extract is prepared by subjecting the dehydrated and desalted whole jellyfish or a specific part of the jellyfish to enzymatic hydrolysis, and then filtering the obtained enzymatic hydrolysate to obtain the jellyfish extract; the enzyme used in the enzymatic hydrolysis includes alkaline protease and pepsin.

[0114] In an alternative embodiment, the liquid-to-solid volume-to-mass ratio in the enzymatic hydrolysis step is 5-10:1, which can be 6:1.

[0115] The temperature in the enzymatic hydrolysis step is 30-70°C, for example, 30°C, 35°C, 40°C, 45°C or 50°C, which can be 50°C.

[0116] The pH in the enzymatic hydrolysis step is 5-9, which can be 6-8.5, for example, 6.5, 7, 7.5, 8 or 8.5.

[0117] In an alternative embodiment, the disease includes a tumor, which can be a solid tumor, further including intestinal cancer, lung cancer or prostate cancer.

[0118] Abbreviation Table:

[0119] LC: lung adenocarcinoma;

[0120] CTG cell viability assay reagent: CellTiter-Glo 3D Cell Viability Assay;

[0121] PBMC: peripheral blood mononuclear cell;

[0122] IBD: inflammatory bowel disease;

[0123] CRC: colorectal cancer;

[0124] III. Examples

[0125] The present disclosure is further illustrated by reference to the examples that follow. The description of the specific exemplary embodiments of the present disclosure is intended for purposes of illustration and example only. It is not intended to be limiting of the present disclosure, which is defined solely by the claims as issued by the Patent Office. Obviously, many modifications and variations of this disclosure can be effected without departing from the scope of the novel concepts of the disclosure.

[0126] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0127] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0128] Example 1 Preparation of jellyfish extract

[0129] In this example, umbrella sections of Aurelia coerulea, which are free of sand and do not spoil, are used as raw materials. After cleaning, dehydration, desalination, homogenization, and stepwise addition of three proteases, the materials are subjected to enzymatic hydrolysis at 50°C for 8 hours, enzyme inactivation and sterilization at 100°C for 20 minutes, filtration, concentration, drying, and packaging to produce jellyfish extract, as shown in FIG. 1.

[0130] The specific steps are as follows:

[0131] 1.1 Collection of live Aurelia coerulea

[0132] In June and July, Aurelia coerulea with umbrella sections having a diameter of 25 cm or more are collected along the coast. The oral arms and nematocysts under the umbrella sections are removed cleanly, and only the umbrella sections are retained as raw materials for the preparation of jellyfish extract.

[0133] 1.2 Cleaning of the umbrella sections of Aurelia coerulea

[0134] Put the umbrella of Aurelia coerulea into the disinfected water tank and soak it in clean water for 30 minutes, the ratio of clean water to umbrella is 5:1, then wash away the impurities and sand, take out the umbrella of Aurelia coerulea, put it into another disinfected water tank, repeat the above cleaning method for several times until there is no sand and no impurities attached.

[0135] 1.3 Dehydration and desalination of the umbrella of Aurelia coerulea

[0136] After cleaning, take out the umbrella of Aurelia coerulea, cut it into thin strips of 5 cm wide, place it in a water leakage container for preliminary autolysis dehydration, when the weight is about 60% of the original weight, put the thin strip-shaped umbrella into a gauze bag and press it with a heavy equipment to further expel water, a large amount of inorganic salt will be brought out during the process of water expulsion, when the weight is about 20% of the original weight, remove the heavy pressing equipment, put the umbrella of Aurelia coerulea into pure water for cleaning, the ratio of umbrella of Aurelia coerulea to pure water is 3:1, after cleaning, pack it according to 50 kg per portion and store it in a-20℃ freezer.

[0137] 1.4 Enzymatic hydrolysis of the umbrella of Aurelia coerulea

[0138] Take the degree of hydrolysis as the index, first select the appropriate enzyme for enzymatic hydrolysis from food additive enzymes, then preliminarily determine the enzyme dosage, enzymatic hydrolysis temperature, enzymatic hydrolysis time, pH value of the enzymatic hydrolysis system, and solid-liquid ratio through single factor experiment, and then determine the optimal enzymatic hydrolysis conditions through orthogonal experiment. The umbrella of Aurelia coerulea is enzymatically hydrolyzed according to the determined optimal enzymatic hydrolysis steps, and the specific steps of the optimized enzymatic hydrolysis are as follows:

[0139] Take 1 portion of the packed umbrella of Aurelia coerulea (50 kg) and homogenize it, put the homogenate into an enzymatic hydrolysis tank, add ultrapure water according to the ratio of 6:1 (volume to mass), add 100 g of alkaline protease (enzyme activity 2×10 6 U / g), set the enzymatic hydrolysis temperature to 50℃, adjust the pH value of the enzymatic hydrolysis system to 8.5 with 2 mol / L sodium hydroxide, after 2 hours of enzymatic hydrolysis, adjust the pH value of the enzymatic hydrolysis system to 6.5 with 2 mol / L hydrochloric acid, then add 200 g of pepsin (enzyme activity 2×10 5U / g), after 3 hours of enzymolysis, the pH value of the enzymolysis system was adjusted to 6.0 by 2 mol / L hydrochloric acid, and then the temperature was increased to 100℃ for enzyme inactivation and sterilization, and the time was 20 minutes.

[0140] 1.5 Filtration of the umbrella enzymolysis liquid of Aurelia coerulea

[0141] After the temperature of the umbrella enzymolysis liquid of Aurelia coerulea was reduced to below 60℃, the enzymolysis liquid was discharged from the enzymolysis tank for cooling, and after cooling to room temperature, filtration was performed. The enzymolysis liquid was placed in a filter for multiple filtrations, and the supernatant was collected.

[0142] 1.6 Concentration of the umbrella enzymolysis liquid of Aurelia coerulea

[0143] The supernatant was poured into an evaporator for concentration, and the evaporation temperature was 75℃. When the volume of the enzymolysis liquid was 1 / 3 of the original, the concentration was completed.

[0144] 1.7 Freeze-drying of the umbrella enzymolysis liquid of Aurelia coerulea

[0145] The concentrated enzymolysis liquid was poured into a freeze dryer tray for freeze-drying, and after 48 hours, a milky white powder-like jellyfish extract was obtained.

[0146] 1.8 Packaging and storage of the jellyfish extract

[0147] The jellyfish extract was packaged in a double-layer vacuum aluminum film bag in a sterile environment with constant temperature and humidity, and then stored in the warehouse.

[0148] In addition, in the enzymolysis step, after the alkaline protease and pepsin enzymolysis was completed, before the temperature was increased for inactivation, 200 g of flavor protease (enzyme activity 3×10 5 U / g), after 3 hours of enzymolysis.

[0149] Example 2: Inhibition of lung cancer cell proliferation experiment

[0150] In this example, a primary lung adenocarcinoma model based on patient tissue was constructed by light-cured biological 3D printing technology. The primary samples used included lung adenocarcinoma samples numbered LC-S T15 and LC-S T21. Docetaxel (Selleck MW: 807.88) was used as a control to investigate the ability of the jellyfish extract obtained in Example 1 to inhibit lung cancer cell proliferation in this model.

[0151] 2.1 Cell recovery and 3D microtumor construction

[0152] 1) Take out the cryopreserved, patient-derived lung adenocarcinoma (LC) surgical specimen-derived cells (LC-S T15 or LC-S T21) from the liquid nitrogen tank, quickly shake in a 37°C water bath to melt the liquid in the cryovial, add 9 ml of LC complete medium for resuspension and dilution, centrifuge at 350g for 5 min, discard the supernatant, resuspend with LC complete medium and count;

[0153] 2) Mix the cell suspension with the photocurable bio-ink (Shanghai Yujie Technology Co., Ltd., YJP0101) at a volume ratio of 1:1, and inoculate 20,000 cells per well into a 96-well plate;

[0154] 3) Use a high-throughput photocurable 3D printer (Cyberiad, Biocube-96) for curing, and the microscopic image of the cured product is shown in FIG. 2. After curing with the high-throughput photocurable 3D printer, all the printed products are regular cylinders, and the LC cells can be seen to proliferate in the bio-ink material. Then, the cured product is placed in a cell incubator (37°C, 5% CO2) for 24 h.

[0155] 2.2 Preparation of mother liquor of test drug

[0156] 1) Preparation of mother liquor of jellyfish extract: weigh 300 mg of the jellyfish extract powder prepared in Example 1, dissolve in a certain volume of LC complete medium to make the final concentration 100 mg / ml, and filter sterilize with 0.22 μm to obtain the mother liquor of jellyfish extract;

[0157] 2) Preparation of mother liquor of docetaxel: add 3 μl of 100 mM docetaxel DMSO stock solution to 3 ml of LC complete medium, blow and ultrasonic for 10 minutes, and the whole process is carried out under sterile conditions.

[0158] 2.3 Drug treatment:

[0159] 1) The experimental concentrations of the jellyfish extract in the test group are 100 mg / ml, 75 mg / ml, 50 mg / ml, 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, 3.13 mg / ml, 1.56 mg / ml, 0.78 mg / ml and 0.39 mg / ml, and the experimental concentrations of docetaxel in the positive control group are 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, 0.39 μM and 0.20 μM. Dilute the drug mother liquor to the experimental concentration in turn;

[0160] 2) After 24 h of stable culture of the 3D printed LC cells, replace the culture medium with the corresponding drug-containing medium, set 10 concentration gradients for the same drug, and set 3 replicate wells for the same concentration.

[0161] 2.4 Drug efficacy assay:

[0162] 1) After adding the drug, the 96-well culture plate was placed in the cell culture box for incubation. After the cells were incubated in the drug-containing medium for 48 h or 72 h, CTG cell activity assay reagent (Promega G9683) was added, and the plate was shaken on a shaker (room temperature, dark) at 350 rpm for 30 min. After incubation, 100 μL of supernatant was transferred to a white opaque 96-well plate.

[0163] 2) The luminescence value was determined by an enzyme-labeled instrument, and compared with the blank control without drug, to calculate the relative cell viability (%).

[0164] 2.5 Assay results:

[0165] In the LC-S T21 experimental group, the change curve of the relative cell viability with increasing drug concentration under the conditions of jellyfish extract-48 h, docetaxel-48 h, jellyfish extract-72 h, and docetaxel-72 h is shown in FIG. 3.

[0166] In the LC-S T15 experimental group, the change curve of the relative cell viability with increasing drug concentration under the conditions of jellyfish extract-48 h, docetaxel-48 h, jellyfish extract-72 h, and docetaxel-72 h is shown in FIG. 4.

[0167] From FIGS. 3 and 4, the IC50 results of each experimental group are as follows:

[0168] From this, the following conclusions can be drawn:

[0169] ① In the 48 h test time, the IC50 of the experimental drug jellyfish extract in the two samples was less than 10 mg / ml, and the experimental group with jellyfish extract > 12.5 mg / ml had a significant inhibitory effect on the tested lung adenocarcinoma sample. 50

[0170] ② In the 72 h test time, the IC50 of the experimental drug jellyfish extract in the two samples was slightly increased, both in the range of 10 mg / ml to 20 mg / ml, and the experimental group with jellyfish extract > 25 mg / ml had a significant inhibitory effect on the tested lung adenocarcinoma sample. 50

[0171] Example 3 Lung cancer microtumor drug efficacy experiment

[0172] In this example, the efficacy of jellyfish extract on lung cancer tissue was investigated using lung cancer patient surgical samples, with docetaxel as a control sample. The immune regulation effect of jellyfish extract was evaluated by detecting peripheral blood mononuclear cells (PBMC) and lung cancer malignant pleural effusion samples.

[0173] ​​The cell samples used include peripheral blood mononuclear cells PBMC-Hycells and PBMC-HB, lung cancer malignant pleural effusion samples LC-PE T13 and LC-PE T14 (shown in Figure 9).

[0174] 3.1 Regulation effect of jellyfish extract on T cell functionality of PBMC activation

[0175] 3.1.1 PBMC resuscitation and T cell activation

[0176] 1) Take out the cryopreserved PBMC (PBMC-Hycells or PBMC-HB) extracted from patient-derived blood from the liquid nitrogen tank, quickly shake in a 37°C water bath to melt the liquid in the cryopreservation tube, add 9ml TC complete medium for resuspension and dilution, centrifuge at 400g for 10min, discard the supernatant, resuspend with TC complete medium and count;

[0177] 2) Resuspend the PBMC at a density of 1×10 6 / ml, add Anti-CD3 / CD28 T Cell Activation (STEMCELL, 10971), evenly distribute the cells in a 10cm dish, and place it in a cell incubator (37°C, 5% CO2);

[0178] 3) Collect the cells every 2-3 days, resuspend at a density of 1×10 6 / ml, and continue to culture in a new 10cm dish after expansion;

[0179] 4) Culture to the 14th day, T cell activation is complete, and all cells are counted.

[0180] 3.1.2 Jellyfish extract mother liquor preparation and drug treatment

[0181] 1) Jellyfish extract mother liquor preparation: weigh the jellyfish extract powder prepared in Example 1, dissolve in a certain volume of TC complete medium to make the final concentration 100mg / ml, and filter sterilize with 0.22μm to obtain the jellyfish extract mother liquor;

[0182] 2) The concentration of jellyfish extract in the test group is 50mg / ml and 10mg / ml, and 2ml of each drug at the double experimental concentration is prepared;

[0183] 3) Resuspend the activated PBMC in a 24-well plate at a density of 1.5×10 6 / ml, add an equal volume of TC complete medium or corresponding concentration of drug, and set up one duplicate well for the blank control group and two duplicate wells for the drug-treated group.

[0184] 3.1.3 Drug efficacy determination:

[0185] 1) After adding drugs, place the 24-well culture plate in the cell culture box for culture. After the cells are incubated in the drug-containing medium for 48 h, collect the cells for flow cytometry sample preparation. The flow cytometry sample preparation is performed according to the steps of the antibody instruction manual;

[0186] 2) Analyze the antibody expression of T cells by flow cytometry. Compare the blank control group, the 50 mg / ml jellyfish extract group and the 10 mg / ml jellyfish extract group, and summarize the regulatory effect of the jellyfish extract on the functionality of T cells in blood.

[0187] The flow cytometry histograms of PBMC-Hycells and PBMC-HB are shown in FIGS. 5 and 6, respectively, and the expression intensity of each epitope is summarized as follows. It can be seen that, within the 48 h test time, the 50 mg / ml jellyfish extract shows a significant inhibitory effect on the PBMC sample, and this effect is not obvious at 10 mg / ml. At both concentrations, the activation effect of T cells is not significantly affected.

[0188] (1) PBMC-Hycells

[0189] (2) PBMC-HB

[0190] 3.2 Regulatory effect of jellyfish extract on the functionality of T cells in lung cancer malignant pleural effusion

[0191] 3.2.1 Resuscitation and drug treatment of lung cancer malignant pleural effusion

[0192] 1) Take the cryopreserved cells (LC-PE T13 and LC-PE T14) extracted from the lung cancer malignant pleural effusion from the patient source from the liquid nitrogen tank, quickly shake in a 37°C water bath to melt the liquid in the cryopreservation tube, add 9 ml of TC complete medium for resuspension and dilution, centrifuge at 400g for 10 min, discard the supernatant, resuspend in TC complete medium and count;

[0193] 2) Jellyfish extract stock solution preparation: weigh the jellyfish extract powder, dissolve in a certain volume of TC complete medium to make the final concentration 100 mg / ml, and filter sterilize with 0.22 μm to obtain the jellyfish extract stock solution;

[0194] 3) The experimental concentrations of the jellyfish extract in the test groups are 50 mg / ml and 10 mg / ml, and 2 ml of each drug at the double test concentration is prepared;

[0195] 4) Resuspend the lung cancer malignant pleural effusion cells in the 24-well plate at a density of 1.5 x 10 6 / ml, add an equal volume of TC complete medium or the corresponding test concentration of drug, set one duplicate well for the blank control group and two duplicate wells for the drug-treated group.

[0196] 3.2.2 Pharmacodynamic Assay:

[0197] 1) After adding the drug, the 24-well culture plate was placed in the cell culture box for culture. After the cells were incubated in the drug-containing medium for 48 h, the cells were collected for flow cytometry sample preparation. The flow cytometry sample preparation was performed according to the steps of the antibody instruction manual;

[0198] 2) The antibody expression of T cells was analyzed by flow cytometry. The blank control group, 50 mg / ml jellyfish extract group and 10 mg / ml jellyfish extract group were compared, and the regulation effect of jellyfish extract on the functionality of T cells in lung cancer malignant pleural effusion was summarized.

[0199] The flow cytometry histograms of LC-PE T13 and LC-PE T14 are shown in FIGS. 7 and 8, respectively, and the epitope expression intensity is summarized as follows. It can be seen that, within the 48 h test time, the 50 mg / ml jellyfish extract showed a significant inhibitory effect on the lung cancer malignant pleural effusion sample (mainly immune cells), and the effect was not obvious at 10 mg / ml.

[0200] (1) LC-PE T13

[0201] (2) LC-PE T14

[0202] Example 4 IBD (colorectal cancer inflammation) mouse experiment

[0203] This example constructs an animal model of inflammation caused by IBD. The anti-intestinal cancer effect of the jellyfish extract (JP) provided in Example 1 of the disclosure is evaluated by multiple indicators such as animal weight, colon length, and organ damage.

[0204] 4.1 Experimental methods include:

[0205] 4.1.1 Cell recovery

[0206] (1) Take the frozen B16F10 cells from the liquid nitrogen tank and thaw in a 37°C water bath. You can shake it a little to speed up the thawing process.

[0207] (2) Centrifuge at 1000 rpm for 3 min;

[0208] (3) Discard the supernatant, and blow the cells with 1 mL of 1640 medium containing 10% serum until they are evenly distributed. Transfer the cell suspension to a 6 cm culture dish, add medium to 3 mL, and shake crossly. Shake it evenly and put it in the incubator (37°C, 5% CO2) for culture.

[0209] (4) After 24h, observe the growth under microscope. When the cells grow to a certain density, carry out the following experiments, such as cell plate, passage, etc.

[0210] 4.1.2 Cell passage

[0211] (1) Take out the cells from the incubator, and remove the old culture medium, and wash once with 1 mL of phosphate buffered saline (PBS) to wash away the culture medium that is not completely absorbed, so as to prevent the culture medium from affecting the digestion ability of trypsin;

[0212] (2) Then add 1 mL of trypsin, and the cells are digested and rounded. Blow the cells to detach them from the bottom of the dish;

[0213] (3) Stop the digestion with 1 mL of complete medium, resuspend the cells, and transfer them to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 3 min;

[0214] (4) Discard the supernatant. Normally, 1 mL of 1640 medium is used to blow the cells until they are uniform. If the color of the melanocytes needs to be changed to gray or black, DMEM medium should be used to culture the melanocytes, and the melanocytes should be gradually "acclimated".

[0215] According to the required density of the subsequent experiment, the cells are divided and the corresponding medium is added, and then gently shaken and placed in the incubator (37°C, 5% CO2) for culture.

[0216] 4.1.3 CCK-8 method for detecting cell activity

[0217] Cell Counting Kit-8 (CCK-8) is an indicator of redox reaction. In the presence of electron carrier 1-Methoxy PMS, dehydrogenase in living cells catalyzes the generation of formazan dye from WST-8, and the amount of formazan dye generated is linearly related to the number of living cells.

[0218] (1) Inoculate 100 μL of cell suspension in a 96-well plate, and culture in a cell incubator (37°C, 5% CO2);

[0219] (2) When the cell density grows to about 30%, add 100 μL of drug at a concentration of 1×10 8 , 1×10 9 , 1×10 10 / mL (5% serum DMEM medium dilution) per well, and culture in the incubator for 48 h;

[0220] (3) Take out the cells to be detected, add 1 / 10 volume of CCK-8 directly into the cell culture solution, mix thoroughly, and ensure uniform color in the hole, but no bubbles are generated. For a 96-well plate, add 10 μL of detection reagent to 100 μL of culture medium;

[0221] (4) Continue to incubate in the cell incubator for about 40 min;

[0222] (5) Before reading the enzyme-labeled instrument, place the hole plate on the shaking bed for about 1 min to ensure uniform color of the hole plate;

[0223] (6) Read the 450 nm light absorption value with the enzyme-labeled instrument, and calculate the cell activity.

[0224] Note: Each experimental group is designed with 6 replicates.

[0225] 4.1.4 Construction of mouse CAC model and treatment

[0226] Colorectal cancer can be divided into two types: colitis-associated colorectal cancer (CAC) and sporadic colorectal cancer. The former is mainly caused by inflammation, and the latter is related to genetic factors. At present, the animal model used most frequently and with good repeatability for CAC research is the mouse CAC model induced by azoxymethane (AOM) and DSS. We constructed this model to study the anti-colorectal cancer effect of jellyfish extract.

[0227] (1) Selection and grouping of experimental animals: 8-week-old, 20-22 g male C57BL / 6 mice were selected and randomly divided into five groups (control group, model group, PTX (3 mg / kg) treatment group, low concentration JP (37.5 mg / kg) treatment group, and high concentration JP (75 mg / kg) treatment group) after recording the initial body weight, with 6 mice in each group for CAC model construction.

[0228] (2) AOM and DSS induced CAC: The carcinogen AOM was dissolved in PBS to prepare a 200 μg / ml solution, and on the first day of modeling, mice except the control group were injected with AOM solution (10 mg / kg) intraperitoneally, and the control group was injected with the corresponding amount of PBS. At weeks 2, 4, and 6 of modeling, DSS was dissolved in the drinking water of mice at a ratio of 2%, and fed to the remaining four groups of mice except the control group to simulate the process of repeated onset and long-term non-healing of colitis, and induce the occurrence of CAC; the control group of mice drank blank drinking water.

[0229] (3) Drug treatment: Carboxymethylcellulose sodium was dissolved in 1xPBS at a ratio of 0.5% to be used as a solvent for the drug. From the first day of modeling, the mice were given the corresponding drug by gavage every day for 7 weeks. Control and model groups: blank carboxymethylcellulose sodium solution; PTX (3 mg / kg) treatment group: 0.6 mg / mL PTX solution; low concentration JP (37.5 mg / kg) treatment group: 7.5 mg / mL JP solution; high concentration JP (75 mg / kg) treatment group: 15 mg / mL JP solution. After the end of drug administration, the mice were not treated in the 8th week.

[0230] (4) Intestine and spleen: After the end of the 8th week, the body weight of the mice was measured and the change from the initial body weight was calculated. The method of taking the intestines was the same as in Chapter 2. The spleen of the mice was separated from other tissues, washed thoroughly in PBS and the adipose tissue was removed and weighed, and photographed.

[0231] (5) Colon treatment: The method of treating the colon was the same as in the previous examples. After the colon tissue was unfolded, it was photographed, the number of tumors formed in the colon was counted, and the diameter of the tumors was measured.

[0232] 4.1.5 Construction of UC model in mice and drug treatment

[0233] (1) Selection and grouping of experimental animals: 8-week-old, 20-22 g male C57BL / 6 mice were selected, the initial body weight was recorded, and then randomly divided into five groups (control group, model group, 5-ASA (100 mg / kg) treatment group, low concentration JP (37.5 mg / kg) treatment group, high concentration JP (75 mg / kg) treatment group), 3 mice in each group, for the construction of UC model.

[0234] (2) DSS-induced UC: From the first day of modeling, DSS was dissolved in the drinking water of the mice at a ratio of 3%, and fed to the remaining four groups of mice except the control group for 5 days to induce UC; the control group of mice drank blank drinking water.

[0235] (3) Drug treatment: Carboxymethylcellulose sodium was dissolved in 1xPBS at a ratio of 0.5% to be used as a solvent for the drug. From the first day of modeling, the mice were given the corresponding drug by gavage every day for 10 days. Control and model groups: blank carboxymethylcellulose sodium solution; 5-ASA (100 mg / kg) treatment group: 20 mg / mL 5-ASA solution; low concentration JP (37.5 mg / kg) treatment group: 7.5 mg / mL JP solution; high concentration JP (75 mg / kg) treatment group: 15 mg / mL JP solution.

[0236] (4) Taking the intestine: On the 11th day after the end of modeling, the body weight of the mice was measured. The mice were sacrificed by cervical dislocation, and the skin of the mouse's abdomen and the ophthalmic scissors and forceps were disinfected with alcohol. The mouse's abdominal skin and peritoneum were cut open, and the mouse's colon was cut off at the upper end of the ileum and the lower end of the anus, so that it was separated from other organs in the abdomen.

[0237] (5) Colon treatment: The isolated colon was washed in pre-cooled PBS, the mesentery and adipose tissue were removed, and the length of the colon was measured with a ruler. The blade of the ophthalmic scissors was inserted into the anus, and the colon was cut open from the anus upwards and unfolded in PBS, and the feces were thoroughly washed to remove the feces for paraffin section or extraction of protein and RNA.

[0238] 4.1.6 Mouse Disease Activity Index (DAI) Score

[0239] After the end of modeling, the feces of the mice were observed, and the disease activity index (DAI) score was given from three dimensions of the proportion of body weight loss, the degree of fecal viscosity, and blood in the stool. The specific scoring criteria are shown in the table below.

[0240] Fecal occult blood was determined by o-tolidine method, and the determination method was as follows: 25 μl of o-tolidine solution was added dropwise in a 96-well plate, and 25 μl of hydrogen peroxide solution was added dropwise in the same well, and the mouse feces were picked and added to the well, and the color change of the reagent was observed. The judgment criteria are as follows:

[0241] (1) Negative: no obvious change within 3 minutes;

[0242] (2) Weak positive: blue-green color appeared within 1 minute;

[0243] (3) Positive: blue-green color appeared immediately;

[0244] (4) Strong positive: deep blue color appeared immediately.

[0245] 4.1.7 Western Blot

[0246] (1) Tissue protein extraction: RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor was prepared in advance and added to a 1.5 mL EP tube, which was pre-cooled on ice. The separated and unfolded colon tissue was added to the lysis buffer, which was cut on ice and homogenized with a tissue homogenizer to completely lyse the tissue. Centrifuge at 4°C, 12000 rpm for 10 min to precipitate the tissue fragments, and transfer the supernatant to a new EP tube, taking care not to suck the precipitate and suspended fat.

[0247] (2) Protein quantification: Protein quantification was performed according to the instructions of the BCA protein quantification kit. According to the quantification results, the protein concentration was uniformized with RIPA lysis buffer. 5xLoading Buffer (final concentration 1x) and 1% β-mercaptoethanol were added, vortexed, and then placed in a 100°C metal bath for 10 min to denature the protein. Finally, the protein sample was centrifuged and stored at -80°C, and then taken out for Western blotting when needed.

[0248] (3) SDS-PAGE gel configuration: The gel plate was cleaned and soaked with pure water, the long plate and short plate were assembled and fixed on the gel holder, and the water was checked for leakage. After the leakage check was completed, the water was poured out and inverted on the absorbent paper, and the separation gel was configured according to the formula, among which 10% APS and TEMED were coagulants and were added last. The prepared separation gel was added to the gel plate, and anhydrous ethanol was sealed. After 30-40 min, the separation gel and ethanol were clearly separated. The concentrated gel was configured according to the formula and added to the gel plate. The appropriate comb was inserted, and the concentrated gel was allowed to solidify for 20-30 min.

[0249] (4) Running gel: The gel plate was fixed in the gel clamp and clamped tightly, 1xRunning Buffer was added to cover the short plate, and the comb was carefully removed to avoid deformation of the hole. The protein sample was vortexed and mixed, and then a certain amount of sample was added to the hole according to the requirements, and pre-stained protein markers were added to the holes on both sides. After the sample was added, 1xRunning Buffer was added to the gel clamp, and the gel was placed in the electrophoresis tank. The electrophoresis instrument was turned on, and the gel was run at 60V until the marker was completely separated. The voltage was adjusted to 90V, and the gel was continued to run until the bromophenol blue was close to the bottom of the gel plate.

[0250] (5) Transmembrane: 10xTrans Buffer, methanol and ddH2O were mixed in a ratio of 1:2:7 to prepare 1xTrans Buffer, and placed in the refrigerator for pre-cooling. The transmembrane clamp, sponge pad and filter paper were soaked in 1xTrans Buffer from bottom to top. After the gel was run, the gel plate was taken out, the gel was separated, and the gel was carefully placed on the filter paper and the bubbles were driven away. The NC membrane was covered on the gel, the bubbles were gently scraped off with a spatula, and the filter paper, sponge pad and transmembrane clamp were covered in turn to form a sandwich structure, and then placed in a transmembrane tank containing 1xTrans Buffer. The electrophoresis instrument was turned on, and the transmembrane was carried out at 100V for 90 min in an ice bath.

[0251] (6) Blocking: After transmembrane, the NC membrane was taken out from the transmembrane tank, rinsed with PBS, and soaked in 5% skim milk powder. Shake the clamp for about 1 hour.

[0252] (7) Primary antibody incubation: Rinse the blocking solution with PBS, cut the NC membrane into the size of the strip according to the marker, place it in the antibody incubation box, add the corresponding primary antibody, and incubate overnight on a 4°C shaking table.

[0253] (8) Secondary antibody incubation: Warm the incubation box for 10 min, recover the primary antibody, and wash it with PBST (0.2% Tween 20) on a shaking table for 3 times, 5 min each time. After rinsing with PBS, add the corresponding secondary antibody, and incubate it on a shaking table for 1 h in the dark. Recover the secondary antibody, and wash it with PBST (0.2% Tween 20) on a shaking table for 3 times, 5 min each time.

[0254] (9) Scan the membrane: Scan the NC membrane using the Odyssey membrane scanner, and analyze the results.

[0255] 4.1.8 Preparation of colon tissue paraffin sections

[0256] (1) Colon treatment: Take out the unfolded colon tissue, cut the colon end, rectum, and anus (about 2 cm from the anus) as cancer and paracancer tissues. Place the cut tissue with the intestinal wall inner surface facing down on filter paper, and put it into a 6-well plate with 4% paraformaldehyde at room temperature in the dark overnight (about 24 h).

[0257] (2) Water flushing: After the fixation is completed, take out the tissue with forceps, put it into a plastic embedding clamp, mark it with a pencil, and then place it under running water for overnight washing until the fixative is completely washed away (about 24 h).

[0258] (3) Dehydration, transparency, and wax immersion:

[0259] (4) Embedding: Take the tissue out of the embedding clamp, trim the Swiss roll side flat, and place it with the flat side facing down in a ferrous embedding box. Add fresh melted paraffin, cover the embedding clamp, and place it on ice until it solidifies. After overnight incubation at room temperature, remove the ferrous embedding box, and trim the wax block into the appropriate shape and size. It can be stored in a -20°C refrigerator for long-term storage.

[0260] (5) Sectioning and spreading: Fix the tissue on a microtome, trim it, and cut it into 5-μm-thick, continuous, long strip-shaped wax sections. Carefully transfer the wax sections to water at 42°C using a brush or forceps for spreading.

[0261] (6) Baking: After the wax sections are completely spread, immediately remove them with a glass slide. Shake off and wipe off the excess water on the sections, and place them on a baking machine at 42°C to dry the water. Then adjust the temperature of the baking machine to 62°C, and bake the sections for 2 h. After completion, store the sections at room temperature.

[0262] 4.1.9 HE staining

[0263] (1) Dewaxing: The tissue section was sequentially immersed in xylene I, xylene II for 10 min, xylene: ethanol = 1:1 for 5 min.

[0264] (2) Rehydration: The tissue section was sequentially immersed in anhydrous ethanol, 95% ethanol, 85% ethanol for 3 min each.

[0265] (3) Nucleus staining: The section was washed with pure water, and the excess water was wiped off. The section was immersed in a staining tank containing hematoxylin for staining for about 5 min (the specific staining time was determined according to the staining solution and the tissue).

[0266] (4) Color separation: The tissue section was immersed in 1% hydrochloric acid alcohol (75% ethanol) for about 5 s for color separation.

[0267] (5) Blue return: The section after color separation was placed under running water for 30-60 min. During this period, the section was observed under a microscope, and the nucleus was blue.

[0268] (6) Dehydration and eosin staining: The tissue section was sequentially immersed in 75% ethanol, 85% ethanol, 95% ethanol for 3 min each, and then eosin staining was performed in a staining tank for 5-10 s, followed by dehydration in 95% ethanol and anhydrous ethanol for 2 min each.

[0269] (7) Transparency: The tissue section was sequentially immersed in xylene: ethanol = 1:1, xylene I, xylene II for 5 min each.

[0270] (8) Mounting: The section was taken out, and the excess xylene was wiped off. An appropriate amount of neutral resin was added to the section, and care was taken not to directly drop it on the tissue. The cover glass was carefully placed on the tissue at 45° from one side of the glass slide to avoid air bubbles. After air drying, the section was observed under a microscope and photographed.

[0271] (9) Histological score: The pictures under different fields of view were scored from three dimensions of normal crypt number reduction, mucosal damage and cell infiltration, and the specific scoring criteria were shown in the following table

[0272] 4.1.10 Immunofluorescence staining

[0273] (1) Dewaxing: The steps in HE were followed.

[0274] (2) Rehydration: The steps in HE were followed.

[0275] (3) Antigen repair: The histological box containing 200 mL of EDTA antigen repair solution was placed in a 100°C water bath before use. After dewaxing and rehydration of the section, the section was placed in the histological box, and the antigen was repaired for 30 min. After completion, the histological box was taken out and naturally cooled to room temperature.

[0276] (4) Put the slides on the spring clips in the glass staining jar, add PBST (0.2% Triton X-100) to immerse the tissues, and wash 3 times on a shaker for 3 min each time.

[0277] (5) Blocking: wipe off the excess liquid on the slides, frame the tissues to be dyed on the slides with a histological pen, and add about 100 μl of 1% BSA blocking solution in the frame to cover the tissues, and block at room temperature for 30 min.

[0278] (6) Primary antibody incubation: shake off the blocking solution, add the corresponding primary antibody (1% BSA) in the frame, put it in a wet box, and incubate at 4°C overnight.

[0279] (7) Rewarm for 5-10 min, recover the primary antibody, and wash 3 times with PBST (0.2% Triton X-100) on a shaker for 3 min each time.

[0280] (8) Secondary antibody incubation: wipe off the excess liquid on the slides, add the corresponding secondary antibody (PBS) in the frame, and incubate at room temperature in the dark for 2 h.

[0281] (9) Recover the secondary antibody, and wash 3 times with PBST (0.2% Triton X-100) on a shaker for 3 min each time.

[0282] (10) Nucleus staining: wipe off the excess liquid on the slides, add DAPI staining solution (PBS) in the frame, and incubate at room temperature in the dark for 5 min.

[0283] (11) Recover DAPI, and wash 3 times with PBST (0.2% Triton X-100) on a shaker for 3 min each time.

[0284] (12) Mounting: wipe off the excess liquid on the slides, add an appropriate amount of anti-fluorescence quencher to the slides, and note that it should not be directly added to the tissues. Carefully mount the cover glass on the tissues at 45° from one side of the glass slide to avoid air bubbles. After a period of time, observe and take pictures under a fluorescence microscope.

[0285] 4.1.11 Real-time fluorescent quantitative PCR (RT-qPCR)

[0286] (1) Tissue processing: add the separated and expanded colon tissues to a 1.5 mL EP tube containing Trizol lysis solution, cut them on ice, and homogenize them with a tissue homogenizer to completely lyse the tissues. Centrifuge at 4°C and 12000 g for 10 min to precipitate the tissue fragments, and transfer the supernatant to a new EP tube, noting that the precipitate and suspended fat should be avoided.

[0287] (2) RNA extraction: Add chloroform to the sample according to the ratio of 100 μl chloroform per 500 μl Trizol lysate, shake vigorously for about 15 s, pay attention to avoid using vortex shaker, stand at room temperature for 3 min, centrifuge at 4°C, 12000 g for 15 min. Carefully transfer the supernatant water phase to a new RNase-free EP tube, pay attention to avoid sucking the bottom organic phase and the middle layer. Add an equal volume of isopropanol, mix well by inverting, stand for 10 min, centrifuge at 4°C, 12000 g for 10 min. Discard the supernatant, add 75% ethanol prepared with DEPC water to wash the precipitate, invert to mix, centrifuge at 4°C, 7500 g for 10 min to precipitate the RNA. Discard the supernatant, place the EP tube upside down on the water absorption paper to dry the water, and after instant centrifugation, use a pipette to suck the supernatant, open the lid and stand for 10-15 min to dry the RNA precipitate. Add an appropriate amount of DEPC water to dissolve the RNA on ice for about 30 min, and detect the concentration and purity of the RNA by Nano Drop instrument for subsequent experiments.

[0288] (3) RNA reverse transcription: RNA is easily degraded, so it should be reverse transcribed into stable cDNA as soon as possible after extraction. The RNA reverse transcription program is carried out according to the instructions of the reverse transcription kit, and the specific system and program are shown in the following table:

[0289] After reverse transcription, the concentration of cDNA is detected by Nano Drop instrument, and it is diluted to 350 ng / μl with ddH2O. The cDNA can be stored at -20°C, and the remaining RNA can be stored at -80°C.

[0290] (4) qPCR: Use cDNA as a template, mix with SYBR Mix, primers and ddH2O, configure three repeated amounts for each sample, mix well after configuration, and add 10 μl of the system to each well of the eight-tube tube for qPCR and analysis of the detection results. The qPCR system and program are shown in the following table.

[0291] 4.1.12 Immunohistochemical staining

[0292] (1) The steps of deparaffinization, rehydration and antigen repair are the same as immunofluorescence staining

[0293] (2) Treat the skin sections with freshly prepared 3% hydrogen peroxide-methanol at room temperature for 10-20 min, and wash with PBST (0.2% Triton X-100) on a shaker for 3 min each time.

[0294] (3) Excess liquid on the slice is wiped dry, and the tissue to be treated is framed with a histological pen, and 100 μl or so of 1% BSA blocking solution is added dropwise in the frame to cover the tissue, and the blocking is performed at room temperature for 30 min.

[0295] (4) Primary antibody incubation: The blocking solution is shaken off, and the corresponding primary antibody (1% BSA) is added dropwise in the frame, and the wet box is put in, and the incubation is performed at 4°C overnight.

[0296] (5) Rewarming for 5-10 min, recovering the primary antibody, and washing with PBST (0.2% Triton X-100) on a shaker for 3 times, each for 3 min.

[0297] (6) Secondary antibody incubation: Excess liquid on the slice is wiped dry, and the biotinylated secondary antibody is added dropwise in the frame, and the incubation is performed at room temperature for 20 min.

[0298] (7) Recovering the secondary antibody, and washing with PBST (0.2% Triton X-100) on a shaker for 3 times, each for 3 min.

[0299] (8) Excess liquid on the slice is wiped dry, and streptavidin horseradish peroxidase is added dropwise in the frame, and the incubation is performed at room temperature in the dark for 20 min.

[0300] (9) Washing with PBST (0.2% Triton X-100) on a shaker for 3 times, each for 3 min.

[0301] (10) DAB developing solution is prepared according to the kit instructions under light-proof conditions. Excess liquid on the slice is wiped dry, and the DAB developing solution is added dropwise in the frame, and the developing is performed at room temperature in the dark, and the developing condition is observed under a microscope (5-10 min).

[0302] (11) After the developing is complete, the slice is placed in water and washed for 3 times.

[0303] (12) The subsequent steps such as hematoxylin staining are performed according to the steps in HE staining.

[0304] 4.2 Experimental results

[0305] 4.2.1 Jellyfish extract inhibits weight loss of IBD (colorectal cancer inflammation) mice

[0306] The results are shown in FIG. 10, and it can be seen that the jellyfish extract can reverse the weight loss of mice caused by DSS.

[0307] 4.2.2 Jellyfish extract inhibits colon shortening of IBD (colorectal cancer inflammation) mice

[0308] The results are shown in FIG. 11, and it can be seen that the jellyfish extract can reverse the colon shortening of mice caused by DSS.

[0309] 4.2.3 Jellyfish extract inhibits the enlargement of the spleen of IBD (colorectal inflammatory bowel disease) mice

[0310] The results are shown in Figure 12, and it can be seen that the jellyfish extract can reverse the enlargement of the spleen of mice caused by DSS.

[0311] 4.2.4 Jellyfish extract repairs the colorectal structure of IBD (colorectal inflammatory bowel disease) mice

[0312] The results are shown in Figure 13, and it can be seen that the jellyfish extract can repair the destruction of the colon structure of mice caused by DSS.

[0313] 4.2.5 Jellyfish extract inhibits the expression of colorectal inflammatory factors in IBD (colorectal inflammatory bowel disease) mice

[0314] The results are shown in Figure 14, and it can be seen that the jellyfish extract can down-regulate the expression of colorectal inflammatory factors in mice caused by DSS.

[0315] 4.2.6 Jellyfish extract can inhibit the proliferation of colon cancer cells HCT116, and the results are shown in Figure 15.

[0316] 4.2.7 Jellyfish extract can inhibit the migration of colon cancer cells HCT116, and the results are shown in Figure 16.

[0317] 4.2.8 Jellyfish extract can reduce the number of tumors in CRC mice, and the results are shown in Figures 17 and 18.

[0318] 4.2.9 Jellyfish extract can inhibit the weight loss of CRC mice, and the results are shown in Figure 19.

[0319] 4.2.10 Jellyfish extract can reduce the weight of the spleen of CRC mice, and the results are shown in Figure 20.

[0320] Example 5 Microtumor pharmacodynamic experiment of jellyfish extract inhibiting colorectal cancer proliferation

[0321] This example constructs a primary colorectal cancer microtissue model based on patient-derived cells by light-cured biological 3D printing technology, and a colorectal cancer cell line 3D model based on colorectal cancer cell line HCT116. Through these two models, the inhibitory effect of jellyfish extract on the proliferation of primary colorectal cancer tissue and the effect of jellyfish extract on the proliferation of HCT116 cells are further explored. In addition, the RNA expression and cell cycle changes of the cells before and after treatment are compared to reveal the mechanism of jellyfish extract killing tumor cells.

[0322] 5.1 Inhibitory effect of jellyfish extract on primary human colorectal cancer cells

[0323] 5.1.1 Cell recovery and 3D microtumor construction

[0324] (1) Take out the cryopreserved cells (CRC230511T1, CRC230601T1 or CRC221123T3) extracted from the patient-derived colorectal cancer (CRC) surgical samples from the liquid nitrogen tank, quickly shake in a 37°C water bath to melt the liquid in the cryopreservation tube, add 9ml CRC complete medium for resuspension and dilution, centrifuge at 350g for 5min, discard the supernatant, resuspend with CRC complete medium and count;

[0325] (2) Mix the cell suspension with the bio-ink (Shanghai Yujie Technology Co., Ltd., YJP0101) at a ratio of 1:1, and inoculate 20,000 cells per well into a 96-well plate;

[0326] (3) Use a high-throughput light-cured 3D printer (Cyberiad, Biocube-96) for curing, and then place it in a cell incubator (37°C, 5% CO2) for 48h of culture.

[0327] 5.1.2 Preparation of mother liquor of test drug

[0328] (1) Preparation of jellyfish extract mother liquor: weigh 300mg of jellyfish extract powder prepared in Example 1, dissolve in a certain volume of CRC complete medium to make the final concentration 100mg / ml, and filter sterilize with 0.22μm to obtain the jellyfish extract mother liquor;

[0329] (2) Preparation of paclitaxel mother liquor: add 3μl of 100mM paclitaxel DMSO stock solution to 3ml of CRC complete medium, blow and beat after ultrasonic treatment for 10min, the whole process is carried out under sterile conditions.

[0330] 5.1.3 Drug treatment:

[0331] (1) The test concentrations of jellyfish extract in the test group are 100mg / ml, 50mg / ml, 25mg / ml, 12.5mg / ml, 6.25mg / ml, 3.13mg / ml and 1.56mg / ml, and the test concentrations of paclitaxel in the positive control group are 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.13μM and 1.56μM, and the drug mother liquor is diluted to the test concentration in turn;

[0332] (2) After 48h of culture of the 3D-printed CRC cells, replace the culture medium with the corresponding drug-containing medium, set 7 concentration gradients for the same drug, and set 4 replicate wells for the same concentration.

[0333] 5.1.4 Drug efficacy determination:

[0334] (1) After adding drugs, place the 96-well culture plate in the cell culture box for culture. After the cells are incubated in the drug-containing medium for 48 h, add CTG cell activity assay reagent, place the well plate on a shaker (room temperature, avoid light), 350 g, 30 min, and after the incubation is completed, transfer 100 μL of supernatant to a white non-transparent 96-well plate.

[0335] (2) Measure the luminescence value with a microplate reader, and compare it with the blank control without adding drugs to calculate the relative cell viability (%).

[0336] In the CRC230511 T1 experimental group, under the conditions of jellyfish extract-48 h and paclitaxel-48 h, the change curve of the relative cell viability with the increase of drug concentration is shown in Figure 21.

[0337] In the CRC230601 T1 experimental group, under the conditions of jellyfish extract-48 h and paclitaxel-48 h, the change curve of the relative cell viability with the increase of drug concentration is shown in Figure 22.

[0338] In the CRC221123 T3 experimental group, under the conditions of jellyfish extract-48 h and paclitaxel-48 h, the change curve of the relative cell viability with the increase of drug concentration is shown in Figure 23.

[0339] From Figures 21-23, the IC50 results of each experimental group are as follows:

[0340] It can be concluded that within the 48-hour test time, the jellyfish extract showed a good dose-effect relationship on the primary 3D printed colorectal cancer surgical samples. The test drug jellyfish extract had a significant inhibitory effect on the tested colorectal cancer samples in the experimental group with jellyfish extract ≥ 12.5 mg / ml, and the IC 50 range was 10-20 mg / ml.

[0341] 5.2 Effect of jellyfish extract on HCT116 cell proliferation

[0342] 5.2.1 HCT116 cell recovery and 3D construction

[0343] (1) Take the cryopreserved HCT116 cells from the liquid nitrogen tank, quickly shake in a 37°C water bath to melt the liquid in the cryopreservation tube, add 9 ml of DMEM medium for resuspension and dilution, centrifuge at 200 g for 5 min, discard the supernatant, and after resuspension with complete culture medium, evenly add the cells to a 10 cm dish, and place it in a cell culture box (37°C, 5% CO2); the complete culture medium consists of DMEM + 10% FBS + 1% P / S.

[0344] (2) Change the medium every day, observe the cell density, and when the cells in the 10 cm dish are fully covered, collect the cells and resuspend them at a density of 5 x 10 6 / ml.

[0345] (3) Mix the cell suspension with the bio-ink 1:1 and inoculate into the 96-well plate at a number of 5,000 cells per well;

[0346] (4) Use a high-throughput light-curing 3D printer to solidify, and then place in a cell incubator (37°C, 5% CO2) for 48 hours.

[0347] 5.2.2 Preparation of mother liquor of test drug

[0348] (1) Preparation of mother liquor of jellyfish extract: weigh 300 mg of jellyfish extract powder, dissolve in a certain volume of complete medium to make the final concentration 100 mg / ml, and filter sterilize with 0.22 μm to obtain the mother liquor of jellyfish extract;

[0349] (2) Preparation of mother liquor of paclitaxel: add 3 μl of 100 mM paclitaxel DMSO stock solution to 3 ml of complete medium, blow and ultrasonic for 10 min, and the whole process is carried out under sterile conditions.

[0350] 5.2.3 Drug treatment:

[0351] (1) The test concentrations of jellyfish extract in the test group are 100 mg / ml, 50 mg / ml, 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, 3.13 mg / ml and 1.56 mg / ml, and the test concentrations of paclitaxel in the positive control group are 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM and 1.56 μM, and the drug mother liquor is diluted to the test concentration in turn;

[0352] (2) After the 3D printed HCT116 cells are cultured for 48 h, replace the medium with the corresponding drug-containing medium, set 7 concentration gradients for the same drug, and set 3 replicate wells for the same concentration.

[0353] 5.2.4 Drug efficacy determination:

[0354] (1) After adding the drug, place the 96-well culture plate in the cell incubator for culture, and after the cells are incubated in the drug-containing medium for 48 h, add CTG cell activity assay reagent, and place the plate on a shaker (room temperature, avoid light) at 350 g for 30 min, and after the incubation is completed, transfer 100 μL of supernatant to a white opaque 96-well plate;

[0355] (2) Measure the luminescence value with a microplate reader, and compare it with the blank control without drug, and calculate the relative cell viability (%).

[0356] The relative cell viability curves of the HCT116 experimental groups under the conditions of jellyfish extract-48h and paclitaxel-48h with increasing drug concentration are shown in Figure 24. From Figure 24, the IC50 results of the HCT116 experimental groups are as follows:

[0357] It can be seen that the jellyfish extract showed a good dose-effect relationship on the 3D printed colorectal cancer cell line HCT116 sample within 48 hours of testing time. The test drug jellyfish extract had a significant inhibitory effect on the tested colorectal cancer sample in the experimental group with jellyfish extract ≥ 25 mg / ml, and the IC50 value was 27.81 mg / ml. 50

[0358] 5.3 Mechanism of jellyfish extract killing tumor cells

[0359] 5.3.1 HCT116 cell recovery and 3D construction

[0360] (1) Take the cryopreserved HCT116 cells from the liquid nitrogen tank, quickly shake in a 37°C water bath to melt the liquid in the cryopreservation tube, add 9 ml of DMEM medium for resuspension and dilution, centrifuge at 200g for 5 min, discard the supernatant, resuspend with complete medium, and then evenly add the cells to a 10 cm dish, and place it in a cell culture incubator (37°C, 5% CO2); the composition of the complete medium is DMEM + 10% FBS + 1% P / S;

[0361] (2) Replace the culture medium every day and observe the cell density. After the cells in the 10 cm dish are fully covered, collect the cells and resuspend them at a density of 5 x 10 6 / ml;

[0362] (3) Mix the cell suspension with the bio-ink 1:1 and seed 50,000 cells per well into a 24-well plate;

[0363] (4) Use the light-cured bio-3D printer (550A) to solidify, and then place it in a cell culture incubator (37°C, 5% CO2) for 48h.

[0364] 5.3.2 Jellyfish extract stock solution preparation and drug treatment

[0365] (5) Jellyfish extract stock solution preparation: weigh the jellyfish extract powder, dissolve it in a certain volume of complete medium to make its final concentration 30 mg / ml, and filter sterilize with 0.22 μm;

[0366] (6) After the 3D printed HCT116 cells are cultured for 48h, replace the culture medium with the corresponding drug. Replace the blank control group with fresh complete medium. ​

[0367] 5.3.3 Analysis of pharmacodynamic mechanism:

[0368] (1) After adding drugs, the 24-well culture plate was placed in the cell culture box for culture. After the cells were incubated in the drug-containing medium for 48 hours, the cells were collected for RNA sequencing and cell cycle detection;

[0369] (2) The cycle expression of HCT116 cells was analyzed by flow cytometry. The flow cytometry sample was prepared according to the steps of the cell cycle detection kit;

[0370] (3) Combined with the results of flow cytometry and RNA detection, the killing mechanism of jellyfish extract on tumor cells was analyzed by comparing the blank control group and the 30 mg / ml jellyfish extract group.

[0371] 5.3.4 Experimental results:

[0372] 5.3.4.1 RNA detection results

[0373] (1) After treating HCT116 colorectal cancer cells with jellyfish extract (30 mg / ml), 74 significantly up-regulated genes and 128 significantly down-regulated genes (padj<0.05, |log2FC|>1) were found, as shown in Figure 25.

[0374] (2) After treating HCT116 colorectal cancer cells with jellyfish extract (30 mg / ml), the significantly up-regulated genes included CYP4F1, GCNT3, etc., and the significantly down-regulated genes included CEMIP, NR4A3, EGR1, etc., as shown in Figure 26.

[0375] (3) In the TCGA colon cancer database, it was found that the genes CEMIP, EGR1, SACS, etc. significantly down-regulated after jellyfish extract treatment were associated with poor prognosis of patients, so jellyfish extract may have clinical benefits for colon cancer, as shown in Figure 27.

[0376] (4) The GO, DO pathways significantly enriched after jellyfish extract treatment included receptor binding, response to virus, aging, female reproductive system disease, benign hyperplasia, etc., as shown in Figures 28 and 29.

[0377] (5) The Reactome pathways significantly enriched after jellyfish extract treatment included DNA repair, as shown in Figure 30.

[0378] (6) The KEGG pathways significantly enriched after jellyfish extract treatment included viral carcinogenesis mechanism, as shown in Figure 31.

[0379] (7) The cell cycle detection results are shown in the following table and Figure 32:

[0380] In addition, it has been verified that the jellyfish extract provided by the present disclosure has similar therapeutic or preventive effects on prostate cancer.

[0381] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, and are not intended 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 replacements for part or all of the technical features; and these modifications or replacements 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. Use of a jellyfish extract in preparing a product for treating or preventing tumors; optionally, the tumor includes a solid tumor.

2. The use according to claim 1, wherein The jellyfish is selected from the order Semaeostomeae, and can be selected from at least one of the family Cyaneidae, the family Pelagiidae, and the family Ulmaridae.

3. The use according to claim 1, wherein The jellyfish is selected from at least one of Cyanea capillata, Cyanea ferruginea, Cyanea nozakii, Cyanea purpurea, Chrysaora helvola, Pelagia noctiluca, Sanderia malayensis and Aurelia coerulea.

4. The use according to any one of claims 1 to 3, wherein The jellyfish is Aurelia coerulea, or the jellyfish includes Aurelia aurita and at least one other jellyfish selected from the order Semaeostomeae.

5. The use according to any one of claims 1 to 4, wherein The jellyfish extract is derived from at least the umbrella part of the jellyfish.

6. The use according to any one of claims 1 to 5, wherein The preparation method of the jellyfish extract comprises enzymatically hydrolyzing a dehydrated and desalted whole jellyfish or a specific part of the jellyfish, and filtering the resulting enzymatic hydrolyzate to obtain the jellyfish extract; The enzymes selected for the enzymolysis include alkaline protease and pepsin.

7. The use according to claim 6, wherein The liquid-to-solid volume mass ratio in the enzymatic hydrolysis step is 5 to 10:1, and can be optionally 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.

8. The use according to claim 6 or 7, wherein The jellyfish is clean and has no attachments; Optionally, after filtering to obtain the jellyfish extract, the process further includes a step of shaping the jellyfish extract.

9. The use according to any one of claims 1 to 8, wherein The dosage form of the jellyfish extract includes powder, granule or liquid.

10. The use according to any one of claims 1 to 9, wherein The dosage form of the product for treating or preventing tumors includes oral preparations.

11. The use according to any one of claims 1 to 10, wherein The tumor includes advanced or terminal colorectal cancer, lung cancer or prostate cancer.

12. A method for preparing a jellyfish extract for treating or preventing tumors, comprising enzymatically hydrolyzing a dehydrated, desalted whole jellyfish or a specific portion of a jellyfish, and filtering the resulting enzymatic hydrolyzate to obtain the jellyfish extract; The enzymes selected for enzymolysis include alkaline protease and pepsin; Optionally, the tumor comprises a solid tumor.

13. The preparation method according to claim 12, wherein The liquid-to-solid volume mass ratio in the enzymatic hydrolysis step is 5 to 10:1, and can be optionally 6:1; The temperature of the enzymatic hydrolysis step is 30-70°C, optionally 50°C; The pH of the enzymatic hydrolysis step is 5 to 9, and can be optionally 6 to 8.

5.

14. The preparation method according to claim 12 or 13, wherein The jellyfish is clean and has no attachments; Optionally, after filtering to obtain the jellyfish extract, the process further includes a step of shaping the jellyfish extract.

15. The preparation method according to any one of claims 12 to 14, wherein the dosage form of the jellyfish extract comprises powder, granule or liquid.

16. The jellyfish extract prepared by the preparation method according to any one of claims 12 to 15.

17. A drug for treating or preventing tumors, comprising a jellyfish extract, wherein the tumor comprises a solid tumor; in, The preparation method of the jellyfish extract comprises enzymatically hydrolyzing a dehydrated and desalted whole jellyfish or a specific part of the jellyfish, and filtering the obtained enzymatic hydrolyzate to obtain the jellyfish extract; The enzymes selected for the enzymolysis include alkaline protease and pepsin.

18. The drug according to claim 17, wherein The liquid-to-solid volume mass ratio in the enzymatic hydrolysis step is 5 to 10:1, and can be optionally 6:1; The temperature of the enzymatic hydrolysis step is 30-70°C, optionally 50°C; The pH of the enzymatic hydrolysis step is 5 to 9, and can be optionally 6 to 8.

5.

19. The drug according to claim 17, wherein The dosage form of the drug includes oral preparations.

20. Use of a jellyfish extract in the preparation of a medicament for treating a disease associated with overexpression of gene A or underexpression of gene B, wherein gene A is selected from a combination of one or more of CYP4F11, FTL, HMOX1, GCLC, TXNRD1, GCNT3, or CYP4F3, and gene B is selected from a combination of one or more of CEMIP, NR4A3, ISG15, PCDH7, EGR1, ASPM, DUSP2, PRR11, EGR2, SACS, PTPN14, SYNE2, IFIT1, CYP4F11, or FTL.

21. The use according to claim 20, wherein The preparation method of the jellyfish extract comprises enzymatically hydrolyzing a dehydrated and desalted whole jellyfish or a specific part of the jellyfish, and filtering the obtained enzymatic hydrolyzate to obtain the jellyfish extract; The enzymes selected for the enzymolysis include alkaline protease and pepsin.

22. The use according to claim 21, wherein The liquid-to-solid volume mass ratio in the enzymatic hydrolysis step is 5 to 10:1, and can be optionally 6:1; The temperature of the enzymatic hydrolysis step is 30-70°C, optionally 50°C; The pH of the enzymatic hydrolysis step is 5 to 9, and can be optionally 6 to 8.

5.

23. The use according to claim 20, wherein The disease includes a tumor, which may be a solid tumor, or further may be intestinal cancer.

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